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PHARMACEUTICAL MANUFACTURING HANDBOOK
Production and Processes
SHAYNE COX GAD, PH.D., D.A.B.T.
Gad Consulting Services
Cary, North Carolina
CONTRIBUTORS
Susanna Abrahms e n - Alami, AstraZeneca R & D Lund, Lund, Sweden, Oral
Extended - Release Formulations
James Agalloco, Agalloco & Associates, Belle Mead, New Jersey, Sterile Product
Manufacturing
Fakhrul Ahsan, Texas Tech University, Amarillo, Texas, Nasal Delivery of Peptide
and Nonpeptide Drugs
James Akers, Akers Kennedy & Associates, Kansas City, Missouri, Sterile Product
Manufacturing
Raid G. Alany, The University of Auckland, Auckland, New Zealand, Ocular Drug
Delivery; Microemulsions as Drug Delivery Systems
Monique Alric, Universit e d ’ Auvergne, Clermont - Ferrand, France, Recombinant
Saccharomyces Cerevisiae as New Drug Delivery System to Gut: In Vitro Validation
and Oral Formulation
Sacide Alsoy Altinkaya, Izmir Institute of Technology, Urla - Izmir, Turkey, Controlled
Release of Drugs from Tablet Coatings
Maria Helena Amaral, University of Porto, Porto, Portugal, Vaginal Drug
Delivery
Anil Kumar Anal, Living Cell Technologies (Global) Limited, Auckland, New
Zealand, Controlled - Release Dosage Forms
Gavin Andrews, Queen ’ s University Belfast, Belfast, Northern Ireland, Effects of
Grinding in Pharmaceutical Tablet Production
Sophia G. Antimisiaris, School of Pharmacy, University of Patras, Rio, Greece,
Liposomes and Drug Delivery
vi CONTRIBUTORS
Robert D. Arnold, The University of Georgia, Athens, Georgia, Biotechnology -
Derived Drug Product Development
C. Scott Asbill, Samford University, Birmingham, Alabama, Transdermal Drug
Delivery
Maria Fernanda Bahia, University of Porto, Porto, Portugal, Vaginal Drug
Delivery
Bernard Bataille, University of Montpelier 1, Montpellier, France, Tablet Design
Gerald W. Becker, SSCI, West Lafayette, Indiana, Biotechnology - Derived Drug
Product Development; Regulatory Considerations in Approval of Follow - On
Protein Drug Products
B. Wayne Bequette, Rensselaer Polytechnic Institute, Troy, New York, From Pilot
Plant to Manufacturing: Effect of Scale - Up on Operation of Jacketed Reactors
Erem Bilensoy, Hacettepe University Faculty of Pharmacy, Ankara, Turkey, Cyclodextrin
- Based Nanomaterials in Pharmaceutical Field
St e phanie Blanquet, Universit e d ’ Auvergne, Clermont - Ferrand, France, Recombinant
Saccharomyces Cerevisiae as New Drug Delivery System to Gut: In Vitro
Validation and Oral Formulation
Gary W. Bumgarner, Samford University, Birmingham, Alabama, Transdermal
Drug Delivery
Isidoro Caraballo, University of Sevilla, Seville, Spain, Tablet Design
Stephen M. Carl, Purdue University, West Lafayette, Indiana, Biotechnology -
Derived Drug Product Development; Regulatory Considerations in Approval of
Follow - On Protein Drug Products
Sudhir S. Chakravarthi, University of Nebraska Medical Center, College of Pharmacy,
Omaha, Nebraska, Biodegradable Nanoparticles
D.F. Chowdhury, University of Oxford, Oxford, United Kingdom, Pharmaceutical
Nanosystems: Manufacture, Characterization, and Safety
Barbara R. Conway, Aston University, Birmingham, United Kingdom, Solid Dosage
Forms
Jos e das Neves, University of Porto, Porto, Portugal, Vaginal Drug Delivery
Osama Abu Diak, Queen ’ s University Belfast, Belfast, Northern Ireland, Effects
of Grinding in Pharmaceutical Tablet Production
Brit S. Farstad, Instititue for Energy Technology, Isotope Laboratories, Kjeller,
Norway, Radiopharmaceutical Manufacturing
Dimitrios G. Fatouros, School of Pharmacy and Biomedical Sciences, Portsmouth,
England, Liposomes and Drug Delivery
Jelena Filipovi c - Gr i , Faculty of Pharmacy and Biochemistry, University of
Zagreb, Zagreb, Croatia, Nasal Powder Drug Delivery
c c
CONTRIBUTORS vii
Eddy Castellanos Gil, Center of Pharmaceutical Chemistry and University of
Havana, Havana, Cuba; University of Sevilla, Seville, Spain; University of Montpelier
1, Montpellier, France, Tablet Design
Anita Hafner, Faculty of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia, Nasal Powder Drug Delivery
A. Atilla Hincal, Hacettepe University Faculty of Pharmacy, Ankara, Turkey,
Cyclodextrin - Based Nanomaterials in Pharmaceutical Field
Michael Hindle, Virginia Commonwealth University, Richmond, Virginia, Aerosol
Drug Delviery
Bhaskara R. Jasti, University of the Pacifi c, Stockton, California, Semisolid Dosages:
Ointments, Creams, and Gels
Yiguang Jin, Beijing Institute of Radiation Medicine, Beijing, China, Nanotechnology
in Pharmaceutical Manufacturing
David Jones, Queen ’ s University Belfast, Belfast, Northern Ireland, Effects of
Grinding in Pharmaceutical Tablet Production
Anne Juppo, University of Helsinki, Helsinki, Finland, Oral Extended - Release
Formulations
Paraskevi Kallinteri, Medway School of Pharmacy, Universities of Greenwich and
Kent, England, Liposomes and Drug Delivery
Gregory T. Knipp, Purdue University, West Lafayette, Indiana, Biotechnology -
Derived Drug Product Development; Regulatory Considerations in Approval of
Follow - On Protein Drug Products
Anette Larsson, Chalmers University of Technology, G o teborg, Sweden, Oral
Extended - Release Formulations
Beom - Jin Lee, Kangwon National University, Chuncheon, Korea, Pharmaceutical
Preformulation: Physiochemical Properties of Excipients and Powders and Tablet
Characterization
Xiaoling Li, University of the Pacifi c, Stockton, California, Semisolid Dosages:
Ointments, Creams, and Gels
David J. Lindley, Purdue University, West Lafayette, Indiana, Biotechnology -
Derived Drug Product Development
Roberto Londono, Washington State University, Pullman, Washington, Liquid
Dosage Forms
Ravichandran Mahalingam, University of the Pacifi c, Stockton, California, Semisolid
Dosages: Ointments, Creams, and Gels
Kenneth R. Morris, Purdue University, West Lafayette, Indiana, Biotechnology -
Derived Drug Product Development; Regulatory Considerations in Approval of
Follow - On Protein Drug Products
Erin Oliver, Rutgers, The State University of New Jersey, Piscataway, New Jersey,
Biotechnology - Derived Drug Product Development; Regulatory Considerations
in Approval of Follow - On Protein Drug Products
viii CONTRIBUTORS
Iv a n Pe n uelas, University of Navarra, Pamplona, Spain, Radiopharmaceutical
Manufacturing
Omanthanu P. Perumal, South Dakota State University, Brookings, South Dakota,
Role of Preformulation in Development of Solid Dosage Forms
Katharina M. Picker - Freyer, Martin - Luther - University Halle - Wittenberg, Institute
of Pharmaceutics and Biopharmaceutics, Halle/Saale, Germany, Tablet Production
Systems
Satheesh K. Podaralla, South Dakota State University, Brookings, South Dakota,
Role of Preformulation in Development of Solid Dosage Forms
Dennis H. Robinson, University of Nebraska Medical Center, College of Pharmacy,
Omaha, Nebraska, Biodegradable Nanoparticles
Arcesio Rubio, Caracas, Venezuela, Liquid Dosage Forms
Maria V. Rubio - Bonilla, Research Associate, College of Pharmacy, Washington
State University, Pullman, Washington, Liquid Dosage Forms
Ilva D. Rupenthal, The University of Auckland, Auckland, New Zealand, Ocular
Drug Delivery
Maria In e s Rocha Miritello Santoro, Department of Pharmacy, Faculty of Pharmaceutical
Sciences, University of S a o Paulo, S a o Paulo, Brazil, Packaging and
Labeling
Helton M.M. Santos, University of Coimbra, Coimbra, Portugal, Tablet
Compression
Raymond K. Schneider, Clemson University, Clemson, South Carolina, Clean -
Facility Design, Construction, and Maintenance Issues
Anil Kumar Singh, Department of Pharmacy, Faculty of Pharmaceutical Sciences,
University of S a o Paulo, S a o Paulo, Brazil, Packaging and Labeling
Jo a o J.M.S. Sousa, University of Coimbra, Coimbra, Portugal, Tablet
Compression
Shunmugaperumal Tamilvanan, University of Antwerp, Antwerp, Belgium, Progress
in Design of Biodegradable Polymer - Based Microspheres for Parenteral
Controlled Delivery of Therapeutic Peptide/Protein; Oil - in - Water Nanosized
Emulsions: Medical Applications
Chandan Thomas, Texas Tech University, Amarillo, Texas, Nasal Delivery of
Peptide and Nonpeptide Drugs
Gavin Walker, Queen ’ s University Belfast, Belfast, Northern Ireland, Effects of
Grinding in Pharmaceutical Tablet Production
Jingyuan Wen, The University of Auckland, Auckland, New Zealand, Microemulsions
as Drug Delivery Systems
Hui Zhai, Queen ’ s University Belfast, Belfast, Northern Ireland, Effects of Grinding
in Pharmaceutical Tablet Production
ix
CONTENTS
PREFACE xiii
SECTION 1 MANUFACTURING SPECIALTIES 1
1.1 Biotechnology-Derived Drug Product Development 3
Stephen M. Carl, David J. Lindley, Gregory T. Knipp, Kenneth R. Morris,
Erin Oliver, Gerald W. Becker, and Robert D. Arnold
1.2 Regulatory Considerations in Approval on Follow-On Protein
Drug Products 33
Erin Oliver, Stephen M. Carl, Kenneth R. Morris, Gerald W. Becker, and
Gregory T. Knipp
1.3 Radiopharmaceutical Manufacturing 59
Brit S. Farstad and Ivan Penuelas
SECTION 2 ASEPTIC PROCESSING 97
2.1 Sterile Product Manufacturing 99
James Agalloco and James Akers
SECTION 3 FACILITY 137
3.1 From Pilot Plant to Manufacturing: Effect of Scale-Up on
Operation of Jacketed Reactors 139
B. Wayne Bequette
x CONTENTS
3.2 Packaging and Labeling 159
Maria Ines Rocha Miritello Santoro and Anil Kumar Singh
3.3 Clean-Facility Design, Construction, and Maintenance Issues 201
Raymond K. Schneider
SECTION 4 NORMAL DOSAGE FORMS 233
4.1 Solid Dosage Forms 235
Barbara R. Conway
4.2 Semisolid Dosages: Ointments, Creams, and Gels 267
Ravichandran Mahalingam, Xiaoling Li, and Bhaskara R. Jasti
4.3 Liquid Dosage Forms 313
Maria V. Rubio-Bonilla, Roberto Londono, and Arcesio Rubio
SECTION 5 NEW DOSAGE FORMS 345
5.1 Controlled-Release Dosage Forms 347
Anil Kumar Anal
5.2 Progress in the Design of Biodegradable Polymer-Based
Microspheres for Parenteral Controlled Delivery of Therapeutic
Peptide/Protein 393
Shunmugaperumal Tamilvanan
5.3 Liposomes and Drug Delivery 443
Sophia G. Antimisiaris, Paraskevi Kallinteri, and Dimitrios G. Fatouros
5.4 Biodegradable Nanoparticles 535
Sudhir S. Chakravarthi and Dennis H. Robinson
5.5 Recombinant Saccharomyces cerevisiae as New Drug Delivery
System to Gut: In Vitro Validation and Oral Formulation 565
Stephanie Blanquet and Monique Alric
5.6 Nasal Delivery of Peptide and Nonpeptide Drugs 591
Chandan Thomas and Fakhrul Ahsan
5.7 Nasal Powder Drug Delivery 651
Jelena Filipovi -Gr i and Anita Hafner
5.8 Aerosol Drug Delivery 683
Michael Hindle
5.9 Ocular Drug Delivery 729
Ilva D. Rupenthal and Raid G. Alany
5.10 Microemulsions as Drug Delivery Systems 769
Raid G. Alany and Jingyuan Wen
c c c
CONTENTS xi
5.11 Transdermal Drug Delivery 793
C. Scott Asbill and Gary W. Bumgarner
5.12 Vaginal Drug Delivery 809
Jose das Neves, Maria Helena Amaral, and Maria Fernanda Bahia
SECTION 6 TABLET PRODUCTION 879
6.1 Pharmaceutical Preformulation: Physicochemical Properties of
Excipients and Powers and Tablet Characterization 881
Beom-Jin Lee
6.2 Role of Preformulation in Development of Solid Dosage Forms 933
Omathanu P. Perumal and Satheesh K. Podaralla
6.3 Tablet Design 977
Eddy Castellanos Gil, Isidoro Caraballo, and Bernard Bataille
6.4 Tablet Production Systems 1053
Katharina M. Picker-Freyer
6.5 Controlled Release of Drugs from Tablet Coatings 1099
Sacide Alsoy Altinkaya
6.6 Tablet Compression 1133
Helton M. M. Santos and Joao J. M. S. Sousa
6.7 Effects of Grinding in Pharmaceutical Tablet Production 1165
Gavin Andrews, David Jones, Hui Zhai, Osama Abu Diak, and
Gavin Walker
6.8 Oral Extended-Release Formulations 1191
Anette Larsson, Susanna Abrahmsen-Alami, and Anne Juppo
SECTION 7 ROLE OF NANOTECHNOLOGY 1223
7.1 Cyclodextrin-Based Nanomaterials in Pharmaceutical Field 1225
Erem Bilensoy and A. Attila Hincal
7.2 Nanotechnology in Pharmaceutical Manufacturing 1249
Yiguang Jin
7.3 Pharmaceutical Nanosystems: Manufacture, Characterization,
and Safety 1289
D. F. Chowdhury
7.4 Oil-in-Water Nanosized Emulsions: Medical Applications 1327
Shunmugaperumal Tamilvanan
INDEX 1367
xiii
PREFACE
This Handbook of Manufacturing Techniques focuses on a new aspect of the drug
development challenge: producing and administering the physical drug products
that we hope are going to provide valuable new pharmacotherapeutic tools in medicine.
These 34 chapters cover the full range of approaches to developing and producing
new formulations and new approaches to drug delivery. Also addressed are
approaches to the issues of producing and packaging these drug products (that is,
formulations). The area where the most progress is possible in improving therapeutic
success with new drugs is that of better delivery of active drug molecules to the
therapeutic target tissue. In this volume, we explore current and new approaches to
just this issue across the full range of routes (oral, parenteral, topical, anal, nasal,
aerosol. ocular, vaginal, and transdermal) using all sorts of forms of formulation.
The current metrics for success of new drugs in development once they enter the
clinic (estimated at ranging from as low as 2% for oncology drugs to as high as 10%
for oral drugs) can likely be leveraged in the desired direction most readily by
improvements in this area of drug delivery.
The Handbook of Manufacturing Techniques seeks to cover the entire range of
available approaches to getting a pure drug (as opposed to a combination product)
into the body and to its therapeutic tissue target. Thanks to the persistent efforts of
Michael Leventhal, these 34 chapters, which are written by leading practitioners in
each of these areas, provide coverage of the primary approaches to these fundamental
problems that stand in the way of so many potentially successful pharmacotherapeutic
interventions.
MANUFACTURING SPECIALTIES
SECTION 1
3
1.1
BIOTECHNOLOGY - DERIVED DRUG
PRODUCT DEVELOPMENT
Stephen M. Carl, 1 David J. Lindley, 1 Gregory T. Knipp, 1
Kenneth R. Morris, 1 Erin Oliver, 2 Gerald W. Becker, 3 and
Robert D. Arnold 4
1 Purdue University, West Lafayette, Indiana
2 Rutgers, The State University of New Jersey, Piscataway, New Jersey
3 SSCI, West Lafayette, Indiana
4 The University of Georgia, Athens, Georgia
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
Contents
1.1.1 Introduction
1.1.2 Formulation Assessment
1.1.2.1 Route of Administration and Dosage
1.1.2.2 Pharmacokinetic Implications to Dosage Form Design
1.1.2.3 Controlled - Release Delivery Systems
1.1.3 Analytical Method Development
1.1.3.1 Traditional and Biophysical Analytical Methodologies
1.1.3.2 Stability - Indicating Methodologies
1.1.3.3 Method Validation and Transfer
1.1.4 Formulation Development
1.1.4.1 Processing Materials and Equipment
1.1.4.2 Container Closure Systems
1.1.4.3 Sterility Assurance
1.1.4.4 Excipient Selection
1.1.5 Drug Product Stability
1.1.5.1 Defi ning Drug Product Storage Conditions
1.1.5.2 Mechanisms of Protein and Peptide Degradation
1.1.5.3 Photostability
1.1.5.4 Mechanical Stress
1.1.5.5 Freeze – Thaw Considerations and Cryopreservation
1.1.5.6 Use Studies
1.1.5.7 Container Closure Integrity and Microbiological Assessment
1.1.5.8 Data Interpretation and Assessment
4 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
1.1.6 Quality by Design and Scale - Up
1.1.6.1 Unit Operations
1.1.6.2 Bioburden Considerations
1.1.6.3 Scale - Up and Process Changes
1.1.7 Concluding Remarks
References
1.1.1 INTRODUCTION
Although the origins of the fi rst biological and/or protein therapeutics can be
traced to insulin in 1922, the fi rst biotechnology - derived pharmaceutical drug
product approved in the United States was Humulin in 1982. In the early stages
of pharmaceutical biotechnology, companies that specialized primarily in the development
of biologicals were the greatest source of research and development in
this area. Recent advances in molecular and cellular biological techniques and
the potential clinical benefi ts of biotechnology drug products have led to a substantial
increase in their development by biotechnology and traditional pharmaceutical
companies . In terms of pharmaceuticals, the International Conference on
Harmonization (ICH) loosely defi nes biotechnology - derived products with biological
origin products as those that are “ well - characterized proteins and polypeptides,
their derivatives and products of which they are components, and which are
isolated from tissues, body fl uids, cell cultures, or produced using rDNA technology
” [1] . In practical terms, biological and biotechnology - derived pharmaceutical
agents encompass a number of therapeutic classes, including cytokines,
erythropoietins, plasminogen activators, blood plasma factors, growth hormones
and growth factors, insulins, monoclonal antibodies, and vaccines [1] . Additionally,
short interfering and short hairpin ribonucleic acids (siRNA, shRNA) and antisense
oligonucleotide therapies are generally characterized as biotechnology -
derived products.
According to the biotechnology advocacy group, The Biotechnology Industry
Organization (BIO), pharmaceutical - based biotechnology represents over a $ 30
billion dollar a year industry and is directly responsible for the production of
greater than 160 drug therapeutics and vaccines [2] . Furthermore, there are more
than 370 biotechnology - derived drug products and vaccines currently in clinical
trials around the world, targeting more than 200 diseases, including various cancers,
Alzheimer ’ s disease, heart disease, diabetes, multiple sclerosis, acquired immunodefi
ciency syndrome (AIDS), and arthritis. While the clinical value of these
products is well recognized, a far greater number of biotechnology - derived drug
products with therapeutic potential for life - altering diseases have failed in
development.
As the appreciation of the clinical importance and commercial potential for biological
products grows, new challenges are arising based on the many technological
limitations related to the development and marketing of these complex agents.
Additionally, the intellectual property protection of an associated agent might not
provide a suffi cient window to market and regain the costs associated with the discovery,
research, development, and scale - up of these products. Therefore, to properly
estimate the potential return on investment, a clear assessment of potential
therapeutic advantages and disadvantages, such as the technological limitations in
the rigorous characterization required of these complex therapeutic agents to gain
Food and Drug Administration (FDA) approval, is needed prior to initiating
research. Clearly, research focused on developing methodologies to minimize these
technological limitations is needed. In doing so we hypothesize the attrition rate
can be reduced and the number of companies engaged in the development of biotechnology
- derived products and diversity of products will continue to expand.
Technological limitations have limited the development of follow - on, or generic
biopharmaceutical products that have lost patent protection. In fact, the potential
pitfalls associated with developing these compounds are so diverse that regulatory
guidance concerning follow - on biologics is relatively obscure and essentially notes
that products will be assessed on a case - by - case basis. The reader is encouraged to
see Chapter 1.2 for a more detailed discussion concerning regulatory perspectives
pertaining to follow - on biologics.
Many of the greatest challenges in producing biotechnology - derived pharmaceuticals
are encountered in evaluating and validating the chemical and physical nature
of the host expression system and the subsequent active pharmaceutical ingredient
(API) as they are transferred from discovery through to the development and marketing
stages. Although this area is currently a hotbed of research and is progressing
steadily, limitations in analytical technologies are responsible for a high degree of
attrition of these compounds. The problem is primarily associated with limited
resolution of the analytical technologies utilized for product characterization. For
example, without the ability to resolve small differences in secondary or tertiary
structure, linking changes to product performance or clinical response is impossible.
The biological activity of traditional small molecules is related directly to their
structure and can be determined readily by nuclear magnetic resonance (NMR),
X - Ray crystallography (X - ray), mass spectrometry (MS), and/or a combination of
other spectroscopic techniques. However, methodologies utilized for characterizing
biological agents are limited by resolution and reproducibility. For instance, circular
dichroism (CD) is generally considered a good method to determine secondary
structural elements and provides some information on the folding patterns (tertiary
structure) of proteins. However, CD suffers from several limitations, including a
lower resolution that is due in part to the sequence libraries used to deconvolute
the spectra. To improve the reliability of determining the secondary and tertiary
structural elements, these databases need to be developed further. An additional
example is the utility of two - dimensional NMR (2D - NMR) for structural determination.
While combining homonuclear and heteronuclear experimental techniques
can prove useful in structural determination, there are challenges in that 2D - NMR
for a protein could potentially generate thousands of signals. The ability to assign
specifi c signals to each atom and their respective interactions is a daunting task.
Resolution between the different amino acids in the primary sequence and their
positioning in the covalent and folded structures become limited with increasing
molecular weight. Higher dimensional techniques can be used to improve resolution;
however, the resolution of these methods remains limited as the number of
amino acids is increased.
INTRODUCTION 5
6 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
Understanding the limitations of the analytical methodologies utilized for product
characterization has led to the development of new experimental techniques as well
as the refi ned application of well - established techniques to this emerging fi eld. Only
through application of a number of complementary techniques will development
scientists be able to accurately characterize and develop clinically useful products.
Unfortunately, much of the technology is still in its infancy and does not allow for
a more in - depth understanding of the subtleties of peptide and protein processing
and manufacturing. For instance, many of the analytical techniques utilized for
characterization will evaluate changes to product conformation on the macroscopic
level, such as potential denaturation or changes in folding, as observed with CD.
However, these techniques do not afford the resolution to identify subtle changes
in conformation that may either induce chemical or physical instabilities or unmask
antigenic epitopes.
Further limiting successful product development is a lack of basic understanding
as to critical manufacturing processes that have the potential to affect the structural
integrity and activity of biopharmaceuticals. As with traditional small molecules,
stresses associated with the different unit operations may affect biopharmaceutical
products differently. In contrast to traditional small molecules, there is considerable
diffi culty in identifying potentially adverse affects, if any, that a particular unit operation
may have on the clinically critical structural elements of a drug. Considering
that many proteins exhibit a greater potential for degradation from shear stress, it
is particularly important to assess any negative effects of mixing, transport through
tubing, fi ltration, and fi lling operations. Essentially all unit operations for a given
manufacturing process could create enough shear stress to induce minor structural
changes that could lead to product failure. The diffi culty is establishing what degree
of change will have an impact on the stability, bioactivity, or immunogenic potential
of the compound. Unfortunately, unless exhaustive formulation development studies
are conducted, coupled with a comprehensive spectrum of analytical methodologies,
these effects may not be readily evident until after scale - up of the manufacturing
process or, worse yet, in the clinical setting. Moreover, modeling shear and stress
using fl uid dynamic structurally diverse molecules is a foreboding task. Extending
these models to validate process analytical technologies (PAT) and incorporate
critical quality by design (QbD) elements in the development process for a collection
of biopharmaceuticals would be largely hindered by the daunting nature of the
task at hand.
The use of biological systems to produce these agents results in additional variability.
Slight changes in nutrient profi le could affect growth patterns and protein
expression of cultured cells. Furthermore, microbial contamination in the form of
viruses, bacteria, fungi, and mycoplasma can be introduced during establishment of
cell lines, cell culture/fermentation, capture and downstream processing steps, formulation
and fi lling operations, or drug delivery [3] . Therefore, establishing the
useful life span of purifi cation media and separation columns remains a critical issue
for consistently producing intermediates and fi nal products that meet the defi ned
quality and safety attributes of the product [4] . In short, understanding the proper
processability and manufacturing controls needed has been a major hurdle that has
kept broader development of biopharmaceutical products relatively limited.
Notwithstanding the many technological hurdles to successfully develop a pharmaceutically
active biotechnology product, they offer many advantages in terms of
therapeutic potency, specifi city, and target design (not generally limited to a particular
class or series of compounds). This is an iterative approach, whereby every new
approved compound, new lessons, and applications to ensure successful product
development are realized, thereby adding to our knowledge base and facilitating
the development of future products. This chapter will discuss some of the fundamental
issues associated with successful biopharmaceutical drug product development
and aims to provide an understanding of the subtleties associated with their
characterization, processing, and manufacturing.
1.1.2 FORMULATION ASSESSMENT
In order to select the most appropriate formulation and route of administration for
a drug product, one must fi rst assess the properties of the API, the proposed therapeutic
indication, and the requirements/limitations of the drug and the target patient
population. Development teams are interdisciplinary comprised of individuals with
broad expertise, for example, chemistry, biochemistry, bioengineering, and pharmaceutics,
that can provide insight into the challenges facing successful product development.
As such, knowledge gained through refi nement of the API manufacturing
process through to lead optimization is vital to providing an initial starting point
for success. Information acquired, for example, in the way of analytical development
and API characterization, during drug discovery or early preclinical development
that can be applied to fi nal drug product development may contribute to shorter
development times of successful products.
The host system utilized for API production is critical to the production of the
fi nal product and will determine the basic and higher order physicochemical characteristics
of the drug. Typically biopharmaceuticals are manufactured in Escherichia
coli as prokaryotic and yeast and Chinese hamster ovary (CHO) cells as eukaryotic
expression systems [5] . While general procedures for growth condition optimization
and processing and purifi cation paradigms have emerged, differences in posttranslational
modifi cations and host – system related impurities can exist even with relatively
minor processing changes within a single production cell line [5] . Such changes
have the potential to alter the biopharmaceutical properties of the active compound,
its bioactivity , and its potential to elicit adverse events such as immunogenic reactions.
These properties will be a common theme as they could potentially play a
major role in both analytical and formulation development activities.
During the process of lead optimization, characterization work is performed that
would include a number of parameters that are critical to formulation and analytical
development scientists. The following information is a minimalist look at what
information should be available to support product development scientists:
• Color
• Particle size and morphology (for solid isolates)
• Thermoanalytical profi le (e.g., Tg for lyophiles)
• Hygroscopicity
• Solubility with respect to pH
• Apparent solution pH
FORMULATION ASSESSMENT 7
8 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
• Number and p Ka of ionizable groups
• Amino acid sequence
• Secondary and tertiary structural characteristics
• Some stability parameters with respect to
pH
Temperature
Humidity
Light
Mechanical stress
Oxygen sensitivity
• Impurity profi le
Misfolded/misaligned active
Potential isoforms
Expression system impurities
• Potency [median inhibitory concentration (IC 50 )]
• Animal Pharmacokinetic/Pharmacodynamic (PK/PD) and Tox profi les
All of the above information will prove invaluable in determining the potential
methods for rational drug delivery. Particular attention should be paid to the relative
hygroscopicity of the API, of course, any stability information, as well as the
impurity profi le and ADMET (absorption, distribution, metabolism, excretion, and
toxicity) information. In short, the more information that is available when development
activities are initiated, the easier it is to avoid common pitfalls and make
development decisions more rationally.
1.1.2.1 Route of Administration and Dosage
Biologics are traditionally very potent molecules that may require only picomolar
blood concentrations to elicit a therapeutic effect. Given that the amount of drug
required per dosage will be commensurate with the relative potency of the molecule,
small concentrations are generally required for any unit dose. Biopharmaceuticals
typically have large molecular weights relative to conventional pharmaceutical
agents, which may be increased further by posttranslational modifi cations. The pharmacokinetics
(ADMET) of biotechnology products have been reviewed elsewhere
[6] , but generally they have short circulating half - lives [7] . As such, biological products
are most often delivered parenterally and formulated as solutions, suspensions,
or lyophilized products for reconstitution [8, 9] . However, one must fi rst ascertain
the potential physiological barriers to drug delivery and effi cacy before assessing
potential routes of administration. These barriers may include actual physical barriers,
such as a cell membrane, that could restrict the drug from reaching its site of
action or chemical barriers, including pH or enzymatic degradation. Based on
current drug delivery approaches, the proteinaceous nature of biological products
limits their peroral delivery due to their susceptibility to proteases and peptidases
present in the gastrointestinal tract as well as size limitations for permeating through
absorptive enterocytes [10] .
Diffi culties in peroral delivery have stimulated researchers to explore alternate
delivery mechanisms for biologics, such as through the lungs or nasal mucosa [11,
12] . Further, advances in technology and our understanding of the mechanisms
limiting oral delivery of biotechnology products have led to innovative drug delivery
approaches to achieve suffi cient oral bioavailability. However, no viable products
have successfully reached the market [13] . As a result of the technological limitations
inherent in biopharmaceutical delivery, these compounds are largely delivered
parenterally through an injection or implant.
When assessing the potential routes of administration, one must consider the
physicochemical properties of the drug, its ADMET properties, the therapeutic
indication, and the patient population, some of which are discussed below. Table 1
provides a list of some of those factors that must be addressed when determining
the most favorable route of administration and the subsequent formulation for
delivery. Ideally the route of administration and subsequent formulation will be
optimized after identifying critical design parameters to satisfy the needs of patients
and health care professionals alike while maintaining the safety and effi cacy of the
product.
Parenteral administration is the primary route of delivering biopharmaceutical
agents (e.g., insulin); however, issues associated with patient compliance with administration
of short - acting molecules are a challenge. Yet, the risk - to - benefi t ratio must
be weighed when determining such fundamental characteristics of the fi nal dosage
form. For instance, a number of biopharmaceutical compounds are administered
subcutaneously, but this route of parenteral administration exhibits the highest
potential for immunogenic adverse events due to the presence of Langerhans cells
[14] . A compound ’ s immunogenic potential is related to a host of factors, both
TABLE 1 Factors That Determine Route of
Administration
Site of action
Therapeutic indication
Dosage
Potency/biological activity
Pharmacokinetic profi le
Absorption time from tissue vs. IV
Circulating half - life
Distribution and elimination kinetics
Toxicological profi le
Immunogenic potential
Patient population characteristics
Disease state
Pathophysiology
Age
Pharmacodynamic profi le
Onset and duration of action
Required clinical effect
Formulation considerations
Stability
Impurity profi le
FORMULATION ASSESSMENT 9
10 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
patient and treatment related; however, if an alternate, potentially safer route of
administration is available, it may be prudent to consider it. Other factors, such as
the frequency of dosing (especially into an immune organ such as the skin) and the
duration of treatment, can also dramatically increase the potential for immunogenic
reactions [14] . Many of the factors that contribute to the immunogenic potential of
biopharmaceuticals, such as impurities, degradation products, and native antigenic
epitopes, can be mitigated through altering the physicochemical properties of the
drug (e.g., pegylation [15, 16] , acylation [17, 18] , increased glycosylation to mask
epitopes [19] ) or changing the characteristics of the formulation [20, 21] . In reality,
the pharmaceutical industry has done a good job of recognizing the potential implications
of immunogenic reactions and readily embraced technologies that can either
mask or eliminate potential antigenic epitopes. However, additional research is
needed to further identify and remove immunogenic epitopes.
1.1.2.2 Pharmacokinetic Implications to Dosage Form Design
Biological agents are generally eliminated by metabolism into di - and tripeptides,
amino acids, and smaller components for subsequent absorption as nutrients or
clearance by the kidney, liver, or other routes. Renal elimination of peptides and
proteins occur primarily via three distinct mechanisms. The fi rst involves the glomerular
fi ltration of low - molecular - weight proteins followed by reabsorption into
endocytic vesicles in the proximal tubule and subsequent hydroysis into small peptide
fragments and amino acids [22] . Interleukin 11 (IL - 11) [23] , IL - 2 [24] , insulin [25] ,
and growth hormone [26] have been shown to be eliminated by this method. The
second involves hydrolysis of the compound at the brush border of the lumen and
subsequent reabsorption of the resulting metabolites [6] . This route of elimination
applies to small linear peptides such as angiotensin I and II, bradykinin, glucagons,
and leutinizing hormone releasing hormone (LHRH) [6, 27, 28] . The third route of
renal elimination involves peritubular extraction from postglomerular capillaries
and intracellular metabolism [6] . Hepatic elimination may also play a major role in
the metabolism of peptides and proteins; however, reticuloendothelial elimination is
by far the primary elimination route for large macromolecular compounds [29] .
Biopharmaceutical drug products are subject to the same principles of pharmacokinetics
and exposure/response correlations as conventional small molecules [6] .
However, these products are subject to numerous pitfalls due to their similarity to
nutrients and endogenous proteins and the evolutionary mechanisms to break them
down or prevent absorption. The types of pharmacokinetic - related problems that a
biotechnology drug development team may encounter range from lack of specifi city
and sensitivity of bioanalytical assays to low bioavailability and rapid drug elimination
from the system [6] . For example, most peptides have hormone activity and
usually short elimination half - lives which can be desirable for close regulation of
their endogenous levels and function. On the other hand, some proteins such as
albumin or antibodies have half - lives of several days and formulation strategies
must be designed to account for these extended elimination times [6] . For example,
the reported terminal half - life for SB209763, a humanized monoclonal antibody
against respiratory syncytial virus, was reported as 22 – 50 days [30] . Furthermore,
some peptide and protein products that persist in the bloodstream exhibit the
potential for idiosyncratic adverse affects as well as increased immunogenic poten
tial. Therefore, the indication and formulation strategy can prove crucial design
parameters simply based on clearance mechanisms.
1.1.2.3 Controlled - Release Delivery Systems
Given that the majority of biopharmaceutical products are indicated for chronic
conditions and may require repeated administrations, products may be amenable to
controlled - release drug delivery systems. Examples include Lupron Depot (leuprolide
acetate), which is delivered subcutaneously in microspheres [31] , and Viadur,
which is implanted subcutaneously [32] . Various peptide/protein controlled delivery
systems have been reviewed recently by Degim and Celebi and include biodegradable
and nondegradable microspheres, microcapsules, nanocapsules, injectable
implants, diffusion - controlled hydrogels and other hydrophilic systems, microemulsions
and multiple emulsions, and the use of iontophoresis or electroporation [33] .
These systems offer specifi c advantages over traditional delivery mechanisms when
the drug is highly potent and if prolonged administration greater than one week is
required [5, 33] . However, each of these systems has its own unique processing and
manufacturing hurdles that must be addressed on a case - by - case basis. These factors,
coupled with the diffi culties of maintaining product stability, limit the widespread
application of these technologies. However, the introduction of postapproval
extended - release formulations may also provide the innovator company extended
patent/commercial utility life and, as such, remains a viable option for postmarketing
development. A current example of this is observed in the development of a
long - acting release formulation of Amylin and Eli Lilly ’ s co - marketed Byetta
product.
1.1.3 ANALYTICAL METHOD DEVELOPMENT
The physical and chemical characterization of any pharmaceutical product is only
as reliable as the quality of the analytical methodologies utilized to assess it. Without
question, the role of analytical services to the overall drug product development
process is invaluable. Good analytical testing with proper controls could mean the
difference between a marketable product and one that is eliminated from development.
Analytical methodologies intended for characterization and/or assessment of
marketed pharmaceutical products must be relevant, validatable, and transferable
to manufacturing/quality assurance laboratories.
1.1.3.1 Traditional and Biophysical Analytical Methodologies
Typically, there are a handful of traditional analytical methodologies that are utilized
to assess the physical, chemical, and microbiological attributes of small -
molecule pharmaceutical products. While many of these testing paradigms can still
be utilized to assess biopharmaceuticals, these molecules require additional biophysical,
microbiological, and immunogenic characterization as well. In brief, analytical
methodologies should evaluate the purity and bioactivity of the product and
must also be suitable to assess potential contaminants from expression systems
as well as different isoforms and degradation products of the active. Biophysical
ANALYTICAL METHOD DEVELOPMENT 11
12 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
methodologies allow for assessment of the structural elements of the product with
respect to its activity. Such assessments include structural elements, such as the
folding of the molecule, and also encompass potential posttranslational modifi cations
and their impact on structure. A list of typical analytical parameters and
methodologies utilized to assess those parameters can be found in Table 2 .
The impact of a molecule ’ s biophysical characteristics on its clinical effi cacy
should be readily quantifi able. With respect to rational drug design, it is also
extremely important to minimize external factors that may infl uence the formation
of any adverse response. One such factor is the presence of degradation products
and drug - related impurities that may be responsible for an immune response. One
such industrial example is granulocyte - macrophage colony - stimulating factor [GM -
CSF, or Leukine (sargramostim), by Berlex Co.], which is produced as a recombinant
protein synthesized and purifi ed from a yeast culture, Saccharomyces cerevisiae . As
expected, the expression system has an impact on the fi nal product: sargramostim,
manufactured from S. cerevisiae , yields an O - glycosylated protein, while molgramostim
(Leucomax), synthesized using an E. coli expression system, is nonglycosylated
[34] . The E. coli – derived product exhibited a higher incidence of adverse reactions
in clinical trials and never made it to the market. With respect to the drug product,
the immunogenic reactions included [34, 35] :
TABLE 2 Analytical Methodologies and Their Utility for API and Drug Product
Characterization
Parameter
Assessed Methodologies Utility
Appearance Visual appearance,
colorimetric assays,
turbidity
Simple determination of physical
stability, i.e., are there particles in
solution, is the solution the correct
color/turbidity? Is the container
closure system seemingly intact?
Purity,
degradation
products and
related
substances
GPC/SEC - HPLC, RP -
HPLC, gel electrophoresis,
immunoassays, IEF, MS,
CD, CE
Gives a general idea of the relative
purity of the API and the drug
product. Are there impurities
related to the expression system?
Are there alternate API isoforms
present? Can degradation products
be distinguished from the active
component(s)?
Molecular
weight
determination
GPC/SEC - HPLC, gel
electrophoresis, multiangle
laser light scattering
(MALLS), laser diffraction
Is the product a single molecular
weight or polydisperse? Is the
molecular weight dependent on
posttranslational modifi cations?
Potency Biological activity (direct or
indirect)
Does the compound have reproducible
in vitro activity and can this be
correlated to in vivo?
pH Potentiometric assays Is the product pH labile or do pH
changes affect potency is such ways
that are not evident in other assays,
i.e., minimal degradation and/or
unfolding?
ANALYTICAL METHOD DEVELOPMENT 13
Parameter
Assessed Methodologies Utility
Primary
structural
elements
Protein sequencing, N - term
degradation (Edman
degradation), peptide
mapping, amino acid
composition, 2D - NMR
Verifi es primary amino acid sequence
and gives preliminary insight into
activity.
Secondary
structural
elements
CD, 2D - NMR, in silico
modeling from AA
sequence
Secondary structural elements result
from the primary sequence and help
defi ne the overall conformation
(3D folding) of the compound.
Tertiary
structural
elements
Disulfi de content/position,
CD
Determines correct folding and overall
integrity of the 3D product.
Qualitative determination for
denaturation potential. Also
correlates to immunogenic potential.
Agglomeration/
aggregation
Subvisual and visual Particle
size analysis,
immunogenicity
Indicator of physical instability. Also
gives an indication of immunogenic
potential.
Carbohydrate
analysis
RP - HPLC, gel
electrophoresis, AE -
HPLC, CE, MALDI - MS,
ES - MS, enzyme arrays
Ensures proper posttranslational
modifi cations and carbohydrate
content.
Water content
(lyophilized
products)
Karl Fischer, TGA, NIR Indicator of hydrolytic potential and
process effi ciency.
Immunogenic
potential
Surface plasmon resonance,
ELISA,
immunoprecipitation
Methodologies generally only give
positive/negative indicators of
immunogenic potential. In vitro
methodologies do not always
correlate to in vivo.
Sterility Membrane fi ltration Indicator of microbial contaminants
from manufacturing operations.
Bacterial
endotoxins
Limulus amebocyte lysate
(LAL)
Gives an idea of processing
contaminants and potentially host
organism contaminants.
Container
closure
integrity
Dye immersion, NIR,
microbial ingress/sterility
Demonstrates viability of container
closure system over the life of the
product.
Abbreviations : gel permeation chromatography (GPC), size exclusion chromatography (SEC), high -
performance, or high - pressure, liquid chromatography (HPLC), reverse phase (RP), isoelectric focusing
(IEF), mass spectrometry (MS), circular dichroism (CD), capillary electrophoresis (CE), nuclear magnetic
resonance (NMR), anion exchange (AE), matrix - assisted laser desorption ionization (MALDI),
electrospray ionization (ES), thermogravimetric analysis (TGA), near infrared (NIR), enzyme - linked
immunosorbent assay (ELISA)
TABLE 2 Continued
14 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
1. Formation of antibodies which bind and neutralize the GM - CSF
2. Formation of antibodies which bind but do not affect the effi cacy of
GM - CSF
3. Antibody formation against proteins not related to GM - CSF, but to proteins
from the expression system ( E. coli )
4. Antibodies formed against both product - and non - product - related proteins
5. No antibody formation
This example clearly illustrates not only the range of clinical manifestations with
respect to antibody formation to drug therapy but also how the choice of an expression
system can affect the fi nal product. In this example, the expression system was
responsible for the adverse events reported. This fi nding is certainly clinically relevant
considering the homologous product, sargramostim, has been on the U.S.
market for quite some time.
The above example also gives an indication of the relative importance of carbohydrate
analysis. Without question, protein glycosylation is the most complex of all
posttranslational modifi cations made in eukaryotic cells, the importance of which
cannot be underestimated. For many compounds, glycosylation can readily affect
protein solubility (as infl uenced by folding), protease resistance, immunogenicity,
and pharmacokinetic/pharmacodynamic profi les (i.e., clearance and effi cacy) [36] .
Typical analytical methodologies used to assess carbohydrate content are also listed
in Table 2 .
1.1.3.2 Stability - Indicating Methodologies
Analytical methodologies that are specifi c to the major analyte that are also capable
of separating and quantifying potential degradation products and impurities, while
simultaneously maintaining specifi city and accuracy, are deemed stability indicating.
Traditional stability - indicating high - performance liquid chromatography (HPLC)
methodologies for small molecules are developed and validated with relative ease.
Typically, the stability - indicating nature of an analytical method can be demonstrated
by subjecting the product to forced degradation in the presence of heat, acid,
alkali, light, or peroxide [37] . If degradation products are suffi ciently well resolved
from the active while maintaining specifi city and accuracy, the method is suitable.
In contrast to small molecules, there is no one “ gold standard ” analytical methodology
that can be utilized to determine the potential degradation products and impurities
in the milieu that may constitute a biopharmaceutical drug product. Furthermore,
a one - dimensional structure assessment (e.g., in terms of an absorption spectrum)
does not give any indication of the overall activity of the product, as is the case with
traditional small molecules. Thus, the stability assessment of biopharmaceuticals will
typically comprise a multitude of methodologies that when taken together give an
indication of the stability of the product. The overall goal is to assess the structural
elements of the compound as well as attempt to determine the relative quantities
of potential degradation products, as well as product isoforms and impurities, that
are inherent to the expression systems utilized for API manufacture. However, it is
still advised that bioactivity determinations are made at appropriate intervals
throughout the stability program, as discussed below. Furthermore, any biopharma
ceutical stability program should also minimally include an evaluation of the in vitro
immunogenicity profi le of the product with respect to time, temperature, and other
potential degradative conditions.
1.1.3.3 Method Validation and Transfer
Analytical method validation is the process by which scientists prove that the analytical
method is suitable for its intended use. Guidances available on validation
procedures for some traditional analytical methodologies [38] can be adapted to
nontraditional methodologies. The United States Pharmacopeia (USP) and National
Formulary (NF) do provide some guidance on designing and assessing biological
assays [39] , as does the U.S. FDA [40] . Essentially, validation determines the acceptable
working ranges of a method and the limitations of that method. At a minimum
the robustness, precision, and accuracy of quantitative methodologies should be
determined during support of API iteration and refi nement, while at the very least
the robustness of qualitative methodologies should be assessed. Of particular importance
for successful analytical method validation is ensuring that the proper standards
and system suitability compounds have been chosen and are representative
or analogous to the compound to be analyzed and traceable to a known origin
standard, such as the National Institute of Standards and Technology (NIST) or
USP/NF. If a reference standard from an “ offi cial ” source is not available, in - house
standards may be used provided they are of the highest purity that can be reasonably
obtained and are thoroughly characterized to ensure its identity, strength,
quality, purity, and potency.
Methods developed and validated during the product development phase are
routinely transferred to quality control or contract laboratories to facilitate release
and in - process testing of production batches. Ensuring that method transfer is executed
properly, with well - defi ned and reproducible system suitability and acceptance
criteria, is the responsibility of both laboratories. Experiments should consist
of all those parameters assessed during method validation and should include an
evaluation of laboratory - to - laboratory variation. This information will give an idea
of the reliability of the methodology and equipment used under the rigors of large -
scale manufacturing.
1.1.4 FORMULATION DEVELOPMENT
The previous sections have highlighted some of the limitations and diffi culties in
developing biotechnology - derived pharmaceuticals. Although there are major technological
limitations in working with these products, their synthesis and manufacturing
are signifi cantly more reproducible compared to naturally derived biologics.
Determining the most appropriate route of administration and subsequent formulation
is dependent on a number of factors, including the product ’ s indication, duration
of action, pharmacokinetic parameters, stability profi le, and toxicity. As
mentioned previously, biopharmaceuticals are typically delivered parenterally, and
thus we will focus on those studies required to successfully develop a parenteral
formulation of a biopharmaceutical agent. The goal of formulation development is
to design a dosage form that ensures the safety and effi cacy of the product through-
FORMULATION DEVELOPMENT 15
16 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
out its shelf life while simultaneously addressing the clinical needs of both the
patient and caregivers to ensure compliance. Formulation development is truly a
balancing act, attempting to emphasize the benefi ts of the therapy and patient compliance
while maximizing drug effi cacy and minimizing toxicity. As such, a number
of studies are required to properly design and develop a formulation, many of which
are discussed below.
1.1.4.1 Processing Materials and Equipment
An important factor in the quality and reproducibility of any formulation development
activity is the materials utilized for formulating and processing studies. In
addition, the choice of container closure systems for the API and the formulation
needs to be considered carefully to provide maximum product protection and
optimal stability. Variability between small - and larger scale development stages
may also be signifi cant depending on the API and materials involved during process
scale - up. It is important to conduct process development studies utilizing equipment
representative of what will be used for large - scale production, if possible. Implementing
this design approach will enable at least some limited dimensional analysis,
allowing for early identifi cation of critical design parameters, thereby facilitating
scale - up or permitting earlier attrition decisions and cost savings. Regardless, it is
important to consider the chemical composition and material properties of every
manufacturing component that may contact the drug product. For instance, processing
vessels may be made of glass, glass - lined steel, or bare steel, while stir paddles
used to ensure homogeneity made be manufactured of a number of different materials.
In short, any manufacturing unit that could potentially come into intimate
contact with either the formulation or the API should be demonstrated to be compatible
with the product, including sampling instruments, sample vials, analytical and
processing tubing, and so forth. Material incompatibility could result in something
as simple as unexplained analytical variability due to a loss of drug through adsorptive
mechanisms to something as serious as a loss of bioactivity or an increase in
immunogenic potential. Therefore, equipment design and materials would ideally
be consistent from formulation development through to scale - up and process validation;
however, this may not be readily feasible. As such, determining the chemical
and physical compatibility of each piece of processing equipment with the API is
critical to maintaining the physical and chemical attributes of the product. Furthermore,
such studies help eliminate potential sources of experimental variability and
give a better indicator as to the relative technological hurdles to successful product
development.
Material compatibility protocols must be clearly defi ned and require that analytical
methodologies be suitable for their intended use. Typically, product purity
methods and cleaning methodologies utilized to determine residual contaminating
product on processing equipment are used for compatibility studies as they are suf-
fi ciently sensitive and rugged to accurately determine product content in the presence
of a multitude of potential confounding factors. This is particularly important
when assessing potential metal, glass, and tubing compatibilities. Compatibility is a
function not only of the product ’ s intimate contact with surrounding materials but
also of the contact time and surface area with these equipment. As such, protocols
should be designed to incorporate expected real - world conditions the product will
see when in contact with the material. For instance, temperature, light, and mechanical
stimulation should mimic usage conditions, although study duration should
include time intervals that surpass expectations to estimate a potential worst case.
These factors should all be considered when examining potential process - related
stability.
1.1.4.2 Container Closure Systems
The ICH guideline for pharmaceutical development outlines requirements for container
closure systems for drugs and biologics [41] . The concept paper prepared for
this guidance specifi cally states that “ the choice of materials for primary packaging
should be justifi ed. The discussion should describe studies performed to demonstrate
the integrity of the container and closure. A possible interaction between
product and container or label should be considered ” [42] . In essence, this indicates
that the container closure system should maintain the integrity of the formulation
throughout the shelf life of the product. In order to maintain integrity, the container
closure system should be chosen to afford protection from degradation induced by
external sources, such as light and oxygen. In addition to the primary container, the
stability of the product should also be examined in the presence of IV administration
components if the product could be exposed to these conditions (see Section
1.1.5.6 ). Understanding the potential impact of product - to - container interactions is
integral to maintaining stability and ensuring a uniform dosage. For example, adsorption
of insulin and some small molecules has been demonstrated to readily occur
in polyvinyl chloride (PVC) bags and tubing when these drugs were present as
additives in intravenous (IV) admixtures [43] .
In addition to their use in large - volume parenterals and IV sets, thermoplastic
polymers have also recently found utility as packaging materials for ophthalmic
solutions and some small - volume parenterals [43] . However, there are many
potential issues with using these polymers as primary packaging components that
are not major concerns with traditional glass container closure systems, including
[44] :
1. Permeation of vapors and other molecules in either direction through the wall
of the plastic container
2. Leaching of constituents from the plastic into the product
3. Sorption (absorption and/or adsorption) or drug molecules or ions on the
plastic material
These concerns largely preclude the utility of thermoplastic polymers as the primary
choice of container closure system for protein and peptide therapeutics, although
the formulation scientist should be aware of the potential advantages of these
systems, such as the ease of manufacturability and their cost. These systems are also
fi nding greater utility in intranasal and pulmonary delivery systems.
Parenterally formulated biopharmaceuticals are typically packaged in glass containers
with rubber/synthetic elastomeric closures. Pharmaceutical glass is composed
primarily of silicon dioxide tetrahedron which is modifi ed with oxides such
as sodium, potassium, calcium, magnesium, aluminum, boron, and iron [45] . The USP
classifi es glass formulations as follows:
FORMULATION DEVELOPMENT 17
18 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
Type I, a borosilicate glass
Type II, a soda – lime treated glass
Type III, a soda – lime glass
NP, a soda – lime glass not suitable for containers for parenterals
The tendency of peptides to adsorb onto glass surfaces is well known and a major
concern in the pharmaceutical industry. This is especially important when the dose
of the active ingredient is relatively small and a signifi cant amount of drug is
adsorbed to these surfaces. In addition, the leaching of atoms or elements in the
glass ’ s silicate network into solution is also a potential issue. This is especially important
for terminally heat sterilized products where oxide additives included in the
silicate network are relatively free to migrate/leach, resulting in increased solution
pH, reaction catalysis, and so on [45] . As such, only type 1 treated glass is traditionally
used for parenterally administered formulations, where these alkaline - rich
phases in the glass have been eliminated, thus decreasing the potential for container
closure system interactions. Additional approaches, including surface treatment with
silicone (siliconization), have also been developed to minimize the interaction of
biotechnology products with free silanols (Si – OH) [46] .
Elastomeric closures are typically used for syringe and vial plungers and closures.
For vials, elastomers provide a soft and elastic material that can permit the entry of
a hypodermic needle without loss of the integrity [45] . For syringes, the closures not
only provide a permeation barrier but also allow for a soft gliding surface facilitating
plunger movement and drug delivery. Elastomeric polymers, however, are very
complex materials composed of multiple ingredients in addition to the basic polymers,
such as vulcanizing agents, accelerators, activators, antioxidants, fi llers, lubricating
agents, and pigments [45] . As leaching of these components into solution
is a potential issue, the compatibility of the drug formulation with the closures
must be studied early during the formulation development process. The choice and
type of elastomeric closure depends on the pH and buffer, if any preservatives
are present, the sterilization method, moisture vapor/gas protection, and active
compatibility [47] . In addition, the problem of the additives in rubber leaching
into the product can be reduced by the coating with specifi c polymers such as
Tefl on [48] .
Container closure systems required for implantable devices are further restricted
by the fact that they are required to be compatible with the formulation over the
intended shelf life and therapeutic application time as well as being biocompatible.
This means that the system not only must afford protection to and contain the formulation
but also cannot cause any potential adverse effects, such as allergy. Typically,
implantable systems are composed of biocompatible metals, such as titanium
or polymers such as polyethylene glycol or polylactic - co - glycolic acid.
1.1.4.3 Sterility Assurance
Maintaining the sterility of biopharmaceutical products is especially important due
to the relative potency and their innate potential for immunogenic reactions. Further,
the biochemical nature of these compounds enables them to serve as potential
nutrients for invading organisms. Methods for sterilizing small molecules include
heat terminal sterilization, terminal fi ltration coupled with aseptic processing techniques,
ultraviolet (UV) and gamma irradiation, ethylene oxide exposure (for containers
and packaging only), and electron beam irradiation. While terminal heat
sterilization is by far the most common sterilization technique, it normally cannot
readily be utilized for peptide or protein formulations due to the potential effects
of heat and pressure on the compound ’ s structure [48] . Furthermore, irradiation can
affect protein stability by cross - linking the sulfur - containing and aromatic residues,
resulting in protein aggregation [49] .
To overcome these issues, sterile fi ltration coupled with aseptic processing and
fi lling is the preferred manufacturing procedure for biopharmaceuticals. Garfi nkle
et al. refer to aseptic processing as “ those operations performed between the sterilization
of an object or preparation and the fi nal sealing of its package. These operations
are, by defi nition, carried out in the complete absence of microorganisms ” [50] .
This highlights the importance of manufacturing controls and bioburden monitoring
during aseptic processes. Newer technologies such as isolator technology have been
developed to reduce human intervention, thereby increasing the sterility assurance.
These technologies have the added benefi t of facilitating aseptic processing without
construction of large processing areas, sterile suites, or gowning areas [50] .
Even the most robust monitoring programs do not ensure the sterility of the fi nal
formulation. As such, aseptically processed formulations are traditionally fi ltered
through a retentive fi nal fi lter, which ensures sterility. Coupled with proper component
sterilization, traditionally by autoclaving, these processes ensure product sterility.
However, fi ltration is a complex unit operation that can adversely affect the drug
product through increased pressure, shear, or material incompatibility. Therefore,
fi ltration compatibility must be assessed thoroughly to demonstrate both product
compatibility, and suffi cient contaminant retention [51] . Parenteral Drug Association
(PDA) technical report 26 provides a thorough systematic approach to selecting
and validating the most appropriate fi lter for a sterilizing fi ltration application
[51] .
1.1.4.4 Excipient Selection
Pharmaceutical products are typically formulated to contain selected nonactive
ingredients (excipients) whose function is to promote product stability and enable
delivery of the active pharmaceutical ingredient(s) to the target site. These substances
include but are not limited to solubilizers, antioxidants, chelating agents,
buffers, tonicity contributors, antibacterial agents, antifungal agents, hydrolysis
inhibitors, bulking agents, and antifoaming agents [45] . The ICH states that “ the
excipients chosen, their concentration, and the characteristics that can infl uence the
drug product performance (e.g. stability, bioavailability) or manufacturability should
be discussed relative to the respective function of each excipient ” [42] . Excipients
must be nontoxic and compatible with the formulation while remaining stable
throughout the life of the product. Excipients require thorough evaluation and
optimization studies for compatibility with the other formulation constituents as
well as the container/closure system [52] . Furthermore, excipient purity may be
required to be greater than that listed in the pharmacopeial monograph if a specifi c
impurity is implicated in potential degradation reactions (e.g., presence of trace
metals) [48] .
FORMULATION DEVELOPMENT 19
20 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
One of the critical factors in excipient selection and concentration is the effect
on preferential hydration of the biopharmaceutical product [53, 54] . Preferential
hydration refers to the hydration layers on the outer surface of the protein and can
be utilized to thermodynamically explain both stability enhancement and denaturation.
Typical excipients used in protein formulations include albumin, amino acids,
carbohydrates, chelating and reducing agents, cyclodextrins, polyhydric alcohols,
polyethylene glycol, salts, and surfactants. Several of these excipients increase the
preferential hydration of the protein and thus enhance its stability. Cosolvents need
to be added in a concentration that will ensure their exclusion from the protein
surface and enhance stability [54] . A more comprehensive review of excipients utilized
for biopharmaceutical drug products is available elsewhere [48] .
Buffer Selection In addition to maintaining solution pH, buffers serve a multitude
of functions in pharmaceutical formulations, such as contributing toward overall
isotonicity, preferential hydration of proteins and peptides, and serving as bulking
agents in lyophilized formulations. The buffer system chosen is especially important
for peptide and proteins that have sensitive secondary, tertiary, and quaternary
structures, as the overall mechanisms contributing to conformational stabilization
are extremely complex [48] . Furthermore, a protein ’ s propensity for deamidation at
a particular pH can be signifi cant, as illustrated by Wakankar and Borchardt [55] .
This study illustrated stability concerns with peptides and proteins at physiological
pH in terms of asparagine (Asn) deamidation and aspartate (Asp) isomerization,
which can be a major issue with respect to circulating half - life and potential in vivo
degradation. This study and others also provide insight into predicting potential
degradative mechanisms based on primary and secondary structural elements allowing
for formulation design with these pathways in mind.
Selecting the appropriate buffer primarily depends on the desired pH range and
buffer capacity required for the individual formulation; however, other factors,
including concentration, effective range, chemical compatibility, and isotonicity
contribution, should be considered [56] . Some acceptable buffers include phosphate
(pH 6.2 – 8.2), acetate (pH 3.8 – 5.8), citrate (pH 2.1 – 6.2, p K 3.15, 4.8, and 6.4),
succinate (pH 3.2 – 6.6, p K 4.2 and 5.6), histidine (p K 1.8, 6.0, and 9.0), glycine
(pK 2.35 and 9.8), arginine (p K 2.18 and 9.1), triethanolamine (pH 7.0 – 9.0), tris -
hydroxymethylaminomethane (THAM, p K 8.1), and maleate buffer [48] . Additionally,
excipients utilized solely for tonicity adjustment, such as sodium chloride and
glycerin, may not only differ in ionic strength but also could afford some buffering
effects that should be considered [52] .
Preservatives In addition to those processing controls mentioned above (Section
3.1.4.3 ), the sterility of a product may be maintained through the addition of antimicrobial
preservatives. Preservation against microbial growth is an important
aspect of multidose parenteral preparations as well as other formulations that
require preservatives to minimize the risk of patient infection upon administration,
such as infusion products [52] . Aqueous liquid products are prone to microbial
contamination because water in combination with excipients derived from natural
sources (e.g., polypeptides, carbohydrates) and proteinaceous active ingredients
may serve as excellent media for the growth [57] . The major criteria for the selection
of an appropriate preservative include effi ciency against a wide spectrum of micro
organisms, stability (shelf life), toxicity, sensitizing effects, and compatibility with
other ingredients in the dosage form [57] . Typical antimicrobial preservatives include
m - cresol, phenol, parabens, thimerosal, sorbic acid, potassium sorbate, benzoic acid,
chlorocresol, and benzalkonium chloride. Cationic agents such as benzalkonium
chloride are typically not utilized for peptide and protein formulations because they
may be inactivated by other formulation components and their respective charges
may induce conformational changes and lead to physical instability of the API.
Further, excipients intended for other applications, such as chelating agents, may
exhibit some antimicrobial activity. For instance, the chelating agent ethylenediaminetetraacetic
acid (EDTA) may exhibit antimicrobial activity, as calcium is required
for bacterial growth.
Identifying an optimal antimicrobial preservative is based largely on the effectiveness
of that preservative at the concentration chosen. In short, it is not enough to
assess the compatibility of the preservative of choice with the API and formulation
and processing components. There also needs to be a determination of whether the
preservative concentration is suffi cient to kill certain standard test organisms. The
USP presents standard protocols for assessing the relative effi cacy of a preservative
in a formulation using the antimicrobial effectiveness test (AET) [58] . Briefl y, by
comparing the relative kill effi ciency of the formulation containing varying concentrations
of the preservative, the formulator can determine the minimal concentration
required for preservative effi cacy and design the formulation accordingly.
1.1.5 DRUG PRODUCT STABILITY
1.1.5.1 Defi ning Drug Product Storage Conditions
From a regulatory standpoint, the primary objective of formulation development is
to enable the delivery of a safe and effi cacious drug product to treat and/or mitigate
a disease state throughout its proposed shelf life. The effi cacy and in many cases the
safety of a product are directly related to the stability of the API, both neat and in
the proposed formulation under processing, storage, and shipping conditions as well
as during administration. As such, the concept of drug stability for biotechnology -
derived products does not change substantially from that of small molecules,
although the level of complexity increases commensurate with the increased complexity
of the APIs in question and the formulation systems utilized for their
delivery.
Stability study conditions for biotechnology - derived APIs and their respective
drug products are not substantially different from those studies conducted for small
molecules. Temperature and humidity conditions under which to conduct said
studies are outlined in ICH Q1A(R2), which incorporates ICH Q1F, stability study
conditions for zones III and IV climactic conditions [59] . Additional guidance specifi
c to conducting stability studies on biopharmaceutical drug products is given in
ICH Q5C [1] . However, the intention of ICH Q5C is not to outline alternate temperature
and humidity conditions to conduct primary stability studies; rather it
provides guidance with respect to the fact that the recommended storage conditions
and expiration dating for biopharmaceutical products will be different from product
to product and provides the necessary fl exibility in letting the applicant determine
DRUG PRODUCT STABILITY 21
22 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
the proper storage conditions for their respective product. Furthermore, this document
provides general guidance in directing applicants in the types of analytical
methodologies that may be used and direction on how to properly assess the stability
of these complex molecules [1] :
Assays for biological activity, where applicable, should be part of the pivotal stability
studies. Appropriate physicochemical, biochemical and immunochemical methods for
the analysis of the molecular entity and the quantitative detection of degradation
products should also be part of the stability program whenever purity and molecular
characteristics of the product permit use of these methodologies.
One recent approach to aid in defi ning the design space for protein and peptide
therapeutics has been to create empirical phase diagrams indicating the relative
stability of compounds based on altering conditions and assessing conformational
changes via a compilation of analytical techniques (Figure 1 ) [60 – 62] . These empirical
phase diagrams can be generated based on pH, temperature, salt concentration,
and so on, and, although seemingly laborious at fi rst glance, could provide invaluable
information in defi ning the extremes to which a compound may be subjected without
altering its conformation. For instance, if an empirical phase diagram determines
the safe temperature range for a compound is up to 35 ° C and an excursion occurs
to 33 ° C, this information would give the stability scientist a guideline as to the
appropriate course of action. Under the traditional testing paradigm of ICH Q1A,
where stability testing is limited to 25, 30, and 40 ° C, one may not know the compound
’ s upper transition temperature to induce conformational changes. If the
information is not already available, then additional excursion studies may need to
be conducted to assimilate this information and take the appropriate course of
action.
1.1.5.2 Mechanisms of Protein and Peptide Degradation
The inherent heterogeneity of peptide and protein drug substances results in their
relative sensitivity to processing, storage, and handling conditions as well as a mul-
FIGURE 1 Empirical phase diagram for ricin toxin A - chain generated using CD molar
ellipticity at 208 nm, ANS fl uorescence, and intrinsic Trp fl uorescence intensity data. Labels
indicate the state of the protein within the same region of color based on evaluation of a
compilation of data sets. (Reproduced with permission from ref. 62 .)
20
40
60
80
T
4 5 6 7 8 9
pH
titude of other factors. Most importantly, this heterogeneity results in a whole host
of potential degradative mechanisms, some of which are compiled in Table 3 and
include chemical instability pathways such as oxidation, hydrolysis of side chains
and potentially the peptide backbone, and deamidation of Asn and Gln side chains.
Also, physical instability manifesting in the form of protein unfolding, formation of
intermediate structures, aggregation, and adsorption to the surfaces of containers
and other equipment can be a major technical hurdle in developing any biopharmaceutical
and may or may not be related to chemical instability [63] . Further
complicating matters is that instability can potentially manifest in various ways and
may or may not be detectable by any one method. Taken together, however, the
compilation of methodologies utilized for stability assessment should give a good
approximation as to the degradative mechanisms of the compound in its respective
formulation. Further, bioactivity and immunogenicity assays should play integral
roles in assessing the relative stability of any biopharmaceutical compound. Briefl y
stated, the chemical and physical stability of products is extraordinarily diffi cult to
assess and will not be belabored here as good reviews on this topic are readily available
in the literature [63, 64] .
1.1.5.3 Photostability
In certain cases, exposure of pharmaceutical compounds to UV and visible light
could result in electronic excitation, termed vertical transition, that could ultimately
result in light - induced degradation. The ICH guideline Q1B [65] is a reference
on how to conduct photostability stress testing for pharmaceutical compounds.
In brief, compounds are exposed to an overall illumination of not less than 1.2
million lux hours and an integrated near - UV energy of not less than 200 Wh/m 2
[65] . These requirements are in addition to normal stability stress testing and
require the additional caveat that analytical methodologies are suitable to also
detect photolytic degradation products, as discussed above. A comprehensive discussion
of small - molecule photolytic degradative mechanisms is available for
further review [66] .
TABLE 3 Potential Degradative Mechanisms of Peptides and Proteins
Degradative Mechanism Site of Occurrence
Chemical
degradative
mechanisms
Oxidation Intrachain disulfi de linkages Met, Trp, Tyr
Peptide bond hydrolysis AA backbone
N - to - O migration Ser and Thr
. - to . - Carboxy migration Asp and Asn
Deamidation Asn and Gln
Acylation . - Amino and . - amino group
Esterifi cation/carboxylation Glu, Asp, and C-term
Physical
degradative
mechanisms
Unfolding Partial unfolding of tertiary structure
Aggregation Aggregation of subunits could result in
precipitation
Adsorption Adsorption to processing equipment and
container closure systems
Source : Modifi ed from Crommelin et al. [5] .
DRUG PRODUCT STABILITY 23
24 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
1.1.5.4 Mechanical Stress
Regulatory guidance on appropriate methods to evaluate the effect of shear stress
and process - handling stability studies is not available. However, these studies are
integral in determining the relative stability of the product with respect to mechanical
stresses introduced during development and manufacturing. Although not typically
recognized as a major degradative pathway for most small - molecule dosage
forms, the introduction of mechanical stress is recognized as a major challenge in
the formulation of semisolids and can potentially induce physical instability of biopharmaceuticals,
although the extent of this effect is currently unknown. For example,
processing shear may infl uence the protein ’ s outer hydration shell, altering the stabilizing
energy provided from preferential hydration and resulting in the exposure
of internal, nonpolar residues. This may facilitate aggregation if enough shear force
is provided. Alternately, the shear energy required to force unfolding has been
studied but has not been related to the fl uid dynamic shear experienced during
processing. Therefore, stress studies should include meticulous controls in the form
of temperature, light and humidity, and fl uid dynamic shear as a function of time.
Data from these studies could be incorporated into empirical phase diagrams, and/
or response surfaces, to help further defi ne the design space for the active and fi nished
drug product. Understanding the effects of stress introduced during the manufacturing
processing of biopharmaceutical products could facilitate the selection of
appropriate PAT tools and QbD incorporation in the development of these products.
Clearly, there is a considerable need for research in this area, and until the
extent of the possible effects are understood, this lack of knowledge poses an
unknown risk and prevents adequate risk assessment for biopharmaceutical development
activities consistent with ICH Q9.
1.1.5.5 Freeze – Thaw Considerations and Cryopreservation
The rapid or continuous freezing and thawing of protein products could contribute
signifi cantly to instability of the API. Such studies are typically designed to assess
the implications of potential transport and handling conditions. These conditions
include not only the manufacturing processing, storage, and shipment to warehouses
and pharmacies but also subsequent pharmacy storage and patient handling [52] .
Unpredictable and somewhat modest temperature fl uctuations could easily induce
degradation or conformational changes that may reduce bioactivity or expose antigenic
epitopes [5] . These effects could also be a result of altered preferential hydration
at the surface of the peptide or protein through salting - out effects induced by
rapid freezing, which could easily denature the product [67] .
1.1.5.6 Use Studies
Stability of biopharmaceutical compounds should also be determined under conditions
that mimic their normal usage. For instance, the stability of reconstituted
lyophilized products should be assessed with respect to time and temperature and,
if applicable, light and mechanical stimuli. Likewise, the stability of a compound
included in implantable devices and controlled - release microsphere formulations
should be determined over the course of its required use, under conditions which
mimic the heat, moisture, light, and enzymatic physiological conditions to which it
will be implanted. Such studies should also determine the release profi le of the
compound over these specifi ed conditions.
Drug products intended for IV administration are generally dosed as an initial
bolus followed by a slow infusion. Consequently, admixture studies of the compound
in potential IV fl uids, such as 0.9% (w/v) saline, 5% (w/v) dextrose, and Ringer ’ s
solution, should also be assessed to determine the relative stability of the compound
in this new environment. These studies are critical as the formulation dynamic that
protected and stabilized the compound has now been altered dramatically with
dilution. This environmental change could potentially impact the preferential hydration
of the compound as well as directly induce conformational changes based on
the diluent chosen and the compound ’ s potential degradative mechanism(s). Additional
contributing factors to instability in admixture solutions could be due to
changes in pH, mechanical mixing of the compound in the IV bag, adsorption of
the compound to the bag itself (which is typically polymeric), or IV sets used for
administration, as well as an increased potential for oxidative degradation. The suitability
of analytical methodologies should also be determined in the presence of
these additional analytes.
1.1.5.7 Container Closure Integrity and Microbiological Assessment
Ensuring that parenteral pharmaceuticals maintain their sterility over the course of
their shelf life is an integral part of any stability assessment [68] . Parenteral dosage
forms must be free from microbiological contamination, bacterial endotoxins, and
foreign particulate matter. Selection of the adequate sterile manufacturing process
has been briefl y discussed above. Determining the microbiological integrity of the
product over its shelf life also gives an indication of the relative quality of the container
closure system chosen for the formulation. Compendial sterility and endotoxin
testing are often used for this purpose; however, sampling is dependent on a
statistical evaluation of the batch size, unit fi ll volume, and method of product sterilization
[68] . Additionally, since these tests are destructive, it would be impossible
to test an entire stability batch to ensure viability of a container closure system.
Other nondestructive tests have been developed to determine the integrity of a
container ’ s closure system [69] . These tests could also serve as a surrogate indicator
of product manufacturing quality over time.
1.1.5.8 Data Interpretation and Assessment
Interpretation of primary stability data for determining expiration dating and
primary storage conditions has been outlined by ICH Q1E [70] . This guidance document
delineates broad methodologies for interpreting primary and accelerated stability
data and extrapolation of said data for determining expiry dating. Of course,
expiry dating cannot be made without reference to specifi cations for those primary
stability - indicating parameters assessed, which is discussed below. Traditionally, stability
assessments performed during preformulation will give an indication of the
potential storage conditions as well as allow for extrapolation of accelerated stability
studies to kinetic degradation rates. Typically this is done through Arrhenius
manipulations. However, as one would expect, these analyses are not readily
useful for biopharmaceutical products, as there is rarely a linear correlation between
QUALITY BY DESIGN AND SCALE-UP 25
26 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
temperature and the compound ’ s degradative rate. This is primarily due to the
complex and often competing degradative mechanisms as well as the potential for
so - called molten globule intermediate phases. In spite of these limitations, ICH Q5C
does provide relevant guidance in illustrating the fl exibility required for determining
storage conditions, as these products usually require a very narrow temperature
condition to maintain optimal stability. Further guidances may be needed to enhance
uniformity in testing methodology and enable the utilization of validated PAT
methodologies.
1.1.6 QUALITY BY DESIGN AND SCALE - UP
1.1.6.1 Unit Operations
Unit operations are defi ned as the individual basic steps in a process that when
linked together defi ne the process train and result in the fi nal product. In practical
terms, a unit operation is often defi ned as an individual step that is carried out on
one piece of equipment. Typical biopharmaceutical API unit operations may include
fermentation or bioreactor processes, cell separation through centrifugation or
microfi ltration, virus removal or inactivation, cell lysis and inclusion body precipitation,
product refolding, and purifi cation steps [71] . Conversely, those unit operations
for drug product manufacturing procedures would be similar to those seen in the
manufacture of a small molecule of comparable dosage form, namely mixing, fl uid
transfer, sterile fi ltration, dose fi lling, lyophilization, and so on. Of course, unit operations
will be dependent on the manufacturing process for the specifi c dosage form,
but careful preformulation and characterization studies will enable relatively
straightforward process design and ease subsequent scale - up activities. Modeling of
unit operations for both small and large molecules is a recognized gap in our ability
to achieve QbD. The application of accepted engineering methods to the problem
is the subject of active research.
1.1.6.2 Bioburden Considerations
Bioburden refers to the amount of microbial fl ora that can be detected on an item,
on a surface, or in a solution [68] . As mentioned previously, microbial contamination
and bioburden are especially important for biotechnology - derived parenteral products
since these products are typically capable of supporting microbial growth.
Special care should be taken to ensure not only that the fi nal packaged product
does not contain microbial contamination but also that manufacturing equipment
is also free from contamination. Monitoring bioburden and determining potential
levels of microbial contamination on equipment surfaces are particularly important
with respect to the material being evaluated.
In general, bioburden counts in parenteral solutions are obtained by conducting
the total aerobic counts and total yeast and mold counts as specifi ed in the USP
microbial limits test (61) or an equivalent test [72] . In addition, membrane fi ltration
of larger than specifi ed volumes may also be used to detect any microbial contamination
when sample results are expected to contain a negligible number of microbial
fl ora or in the presence of potential confounding factors, such as antimicrobial
preservatives [68, 72] . It is important to note that the presence of a high bioburden
count can present an endotoxin contamination problem, as whole microbial cells
and spores can be removed by sterilizing grade fi ltration (0.2 . m), while endotoxins
are not [68] . These issues also underscore the importance of cleaning methods and
their respective validation as well as assessing relevant product contamination on
manufacturing equipment.
1.1.6.3 Scale - Up and Process Changes
The FDA defi nes process validation as “ establishing documented evidence that
provides a high degree of assurance that a specifi c process will consistently produce
a product meeting its predetermined quality attributes ” [73] . While validation studies
are typically performed at full scale, in most cases scale - down or laboratory - scale
models were used to initially develop the manufacturing process. Consequently,
scale - down process precharacterization and characterization studies are considered
crucial to successful process validation for both API and drug product manufacturing
schemes [74] . Although they do require qualifi cation work and a signifi cant
commitment of time and resources, characterization studies provide signifi cant
insight into the critical process and control parameters for each unit operation as
well as improved success rates for process validation due to a better, more complete
understanding of the process [74] . In engineering terms, characterization studies
identify the critical parameters useful for dimensional analysis that enable successful
process scale - up.
While the above explanation attempts to simplify the scale - up process, it is not
meant to trivialize it. In fact, scale - up is probably the most diffi cult manufacturing
challenge for traditional small molecules, let alone biopharmaceuticals. Issues such
as homogeneous mixing, bulk product holding and transfer, and sterile fi ltration
could all be potentially compounded due to the increased scale and introduced
stress. However, a QbD approach to rational drug design should enable simplifi ed
process scale - up and validation. This is only true if experimental design approaches
have been utilized to identify the design space for the processes involved in the
production of the molecule. This is also where the greatest benefi t of developing
empirical phase diagrams early in development could materialize. Essentially, the
QbD approach identifi es the quality attributes of the product based on scientifi c
rationale as opposed to attempting to fi t the proverbial square peg into a round
hole through a trial - and - error approach. This rational design approach goes further
to identify the limiting factors of each unit operation and provides the means of
attempting to correlate how each unit operation affects the fi nal product quality
attributes.
In order to initiate a successful QbD program, the fi rst step is to identify those
process parameters that are essential to product quality and develop well - validated
analytical methodologies to monitor those parameters. In short, the process involves
identifi cation of the potential design space for production of the molecule and con-
fi rmation that design space through rational, deliberate experimentation. Ideally,
process monitoring should be done in real time to minimize production time and if
possible online; however, this may not always be the case or even necessary depending
upon the relative duration of the process to the test. Recognizing potential
quality metrics earlier in the development process could also potentially facilitate
QUALITY BY DESIGN AND SCALE-UP 27
28 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
greater fl exibility during product development and subsequent process characterization
[74] . Certainly, manufacturing site - specifi c differences could also potentially
introduce variability into processes. It is for this reason that site - specifi c personnel
training, process/technology transfer and validation, and stability assessments are
required to ensure product quality.
By defi nition, a process designed under the auspices of QbD should enable a
degree of process knowledge that allows for controlled process changes without
affecting the fi nal product or requiring regulatory approval. For immediate - and
controlled - release solid dosage products, SUPAC guidelines provide direction on
the studies to conduct to determine the impact of a process change. Although there
is some regulatory guidance available for biological products (e.g., “ Changes to an
Approved Application for Specifi ed Biotechnology and Specifi ed Synthetic Biological
Products ” or “ FDA Guidance Concerning Demonstration of Comparability of
Human Biological Products, Including Therapeutic Biotechnology - Derived Products
” ), process changes need to be evaluated on a case - by - case basis. The comparative
analysis of process changes should also be evaluated with respect to defi ned
product specifi cations. PAT will be invaluable in determining the potential impact
of process changes. While stability is often the main metric for small - molecule
drug product, bioactivity and immunogenicity will need to be added metrics for
biopharmaceuticals. Therefore, any process change should be approached subjectively
and care should be taken to validate the relative impact on the safety and
effi cacy of the product.
1.1.7 CONCLUDING REMARKS
Although the goals are the same, developing biotechnology molecules presents
challenges that are unique compared to the development of conventional small
molecules. The innate complexity of the molecular and macromolecular structures
requires three dimensionally viable stability assays and understanding. The complexity
of possible physiological responses and interactions requires an enhanced
understanding of the formulation and processing stresses to identify the minor but
critical changes that result in product unacceptability. A key to addressing these
challenges is the development of analytical techniques with the sensitivity and reliability
to detect and monitor such changes and to provide data to another gap -
closing activity — modeling unit operations. Also the need to develop meaningful
kinetic models is obvious to everyone involved in the development of both large
and small molecules. Linking this type of information to the major efforts in the
discovery arena is a necessary step to bringing the products of the future to
market.
The use of biotechnology products is increasing exponentially and many
opportunities exist to improve their development. The fi rst step may be defi ning
rational biotechnology - derived drug “ developability ” standards that can be assessed
during preclinical/early development testing. Such a tiered approach based upon
the potential risk, the confi dence in methodology, and benefi t has of course been a
proven strategy for small molecules, and a preliminary version applicable to biotechnology
drug products is likely possible today given the topics discussed in this
chapter.
ACKNOWLEDGMENTS
The authors would like to thank The School of Pharmacy and Pharmaceutical Sciences,
the Department of Industrial and Physical Pharmaceutics of Purdue University
and the National Institutes of General Medical Sciences (R01 - GM65448) for
their fi nancial support.
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32 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT
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33
1.2
REGULATORY CONSIDERATIONS IN
APPROVAL OF FOLLOW - ON PROTEIN
DRUG PRODUCTS
Erin Oliver, 1 Stephen M. Carl, 2 Kenneth R. Morris, 2
Gerald W. Becker, 3 and Gregory T. Knipp 1
1 Rutgers, The State University of New Jersey, Piscataway, New Jersey
2 Purdue University, West Lafayette, Indiana
3 SSCI, West Lafayette, Indiana
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
Contents
1.2.1 Introduction
1.2.1.1 Emergence of Biotechnology Industry
1.2.1.2 Challenges Facing “ Biogenerics ”
1.2.2 History of Biologics Regulation in United States
1.2.2.1 Early Biologics Regulation (1800s – 1990s)
1.2.2.2 Modern Biologics Regulation (1990s – Today)
1.2.3 Regulatory Classifi cation of Proteins
1.2.3.1 Defi nitions and Key Terminology
1.2.3.2 Application of Defi nitions to Proteins: Is It a Drug or a Biologic?
1.2.3.3 Regulatory Approval Path for Proteins
1.2.4 Regulation of Generic Drugs
1.2.4.1 History of Generic Drug Legislation in United States
1.2.4.2 Approval Process for Generic Drugs
1.2.4.3 Application of Generic Regulations to Biologics
1.2.5 Legal Arguments Related to Follow - On Proteins
1.2.5.1 Constitutionality of 505(b)(2) Process for Drugs
1.2.5.2 Constitutionality of 505(b)(2) Process for Follow - On Proteins
1.2.5.3 Applicability of 505(j)(1) or ANDA Process to Biogenerics
1.2.5.4 Current Rules Relating to Bioequivalence of Generic Drugs
1.2.5.5 Statutory Authority
1.2.6 Scientifi c Issues Related to Follow - On Proteins (Data Requirements)
1.2.6.1 “ Sameness ” as per Orphan Drug Regulations
1.2.6.2 “ Sameness ” as per Postapproval Change Guidances
34 REGULATORY CONSIDERATIONS IN APPROVAL
1.2.7 Proposed Regulatory Paradigm: Case Studies
1.2.7.1 Case Study 1: Fortical [Calcitonin - Salmon (rDNA Origin)]
1.2.7.2 Case Study 2: Omnitrope [Somatropin (rDNA Origin)]
1.2.7.3 Case Study 3: Generic Salmon Calcitonin
1.2.8 Summary and Conclusions
References
1.2.1 INTRODUCTION
The ongoing need to provide the U.S. population with cost - effective pharmacological
therapies has led to an emergent public health initiative in this country, namely
for generic versions of therapeutic proteins. Greater access to generic drugs was
made possible by the passage of the 1984 Drug Price Competition & Patent Term
Restoration Act, commonly referred to as Hatch – Waxman. Generics have historically
afforded considerable savings to the American consumer in need of prescription
medication. Ten years after the Hatch – Waxman amendments, the Congressional
Budget Offi ce estimated that purchasers saved a total of $ 8 – $ 10 billion on prescriptions
at retail pharmacies by substituting generic drugs for their brand - name counterparts
in 1994 [1] . To put those numbers in the context of today ’ s pharmaceutical
landscape, a recent report issued by the U.S. Department of Health and Human
Services estimates that generic drugs constitute 63% of the total prescription medicines
sold in the United States [2] . This same report suggests that generic drugs cost
approximately 11% of the total cost of branded pharmaceuticals (on a per - dose
basis).
At the same time, the development and use of therapeutic proteins have increased
dramatically, with more than 850 biotechnology drug products and vaccines currently
in trials [3] . Further, it is estimated that by the year 2010 nearly one - half of
all newly approved medicines will be of biological origin [4] . The industrial fi nancial
incentives for the pursuit of follow - on biologics (heretofore termed biogenerics) are
substantial with sales of biotechnology medicines in the United States rising 17%
to approximately $ 30 billion in 2005 and growing at an annual rate of about 20%
thereafter [3] .
Not unexpectedly, the U.S. Food and Drug Administration (FDA) is experiencing
mounting pressure to progress the cause of biogenerics. In a letter dated February
10, 2006, Senators Henry Waxman and Orrin Hatch (authors of the original “ generic ”
legislation) urged the FDA to develop and implement clear guidelines for the
approval of follow - on biological products for certain well - characterized proteins
like insulin and human growth hormone (HGH) [5] . Additionally, recent litigation
has compelled the FDA to take action on a pending drug application for a follow - on
protein (FOP) drug product [Omnitrope, somatropin (recombinant DNA, rDNA
origin)] [6] .
A signifi cant barrier to the emergence of “ biogenerics ” is the absence of a clear,
effi cient abbreviated pathway for approval. This hurdle is linked to signifi cant scientifi
c and legal issues in the United States in terms of how proteins are classifi ed
(drug vs. biologic) and subsequently regulated as well as how “ generics ” are tradi
HISTORY OF BIOLOGICS REGULATION IN UNITED STATES 35
tionally defi ned in terms of equivalence and substitutability. However, an examination
of the vast array of biologicals on the market today reveals that not all proteins
are created equal. This range of complexity may provide an opportunity for stepwise
progress on the regulatory front. This chapter presents the background to this multifaceted
issue and examines the key regulatory challenges facing biogenerics today.
An appropriate regulatory paradigm for the approval of FOPs is proposed and
supported though a discussion of recent case studies.
1.2.1.1 Emergence of Biotechnology Industry
The explosion of scientifi c advances over the last quarter century has spawned the
biotechnology industry and whole new classes of therapeutic agents for the treatment
and prevention of disease. In October of 1982, the FDA approved the fi rst
protein - based therapeutic created by DNA technology in the form of Humulin
(recombinant insulin). Developed by Eli Lilly & Co., with technical assistance from
Genentech, Humulin is indicated for the treatment of diabetes. At the time, the use
of recombinant technology was somewhat limited to the production of smaller,
nonglycosylated proteins such as insulin (51 amino acids) and HGH (191 amino
acids) using bacterial hosts. The seminal discovery by Columbia ’ s Richard Axel of
the process of cotransformation enabled complex protein production and glycosylation
and thus spurred the emergence of the modern biotechnology industry [7] .
The phenomenal growth observed in the biotechnology sector is notable in terms
of the extraordinary number and diversity of therapeutic peptides and proteins that
have been developed within a period of only about 20 years. Examples of therapeutic
proteins in current use include cytokines, clotting factors, vaccines, and monoclonal
antibodies, as illustrated in Table 1 [8] .
As presented in Table 2 , many of these “ early ” biotechnology products have
reached the end of their period of patent exclusivity [4 – 9] . Thus, it is appropriate to
now consider the next steps in the “ life cycle ” of these products as potential generic
drugs.
1.2.1.2 Challenges Facing “ Biogenerics ”
The diversity and complexity of biologic molecules that drive their utility as therapeutic
agents also contribute to the diffi culty in classifying them as pharmacological
entities, namely, whether they are drugs or biologics. This diffi culty in classifi cation
is of profound importance since there are fundamental differences in how the FDA
regulates drugs and biologics.
To appreciate the current challenges facing the pharmaceutical and biotechnology
industry, it is informative to review the historical background associated with
the classifi cation and regulation of biologics in the United States, particularly in the
context of the nation ’ s evolving drug regulation system.
1.2.2 HISTORY OF BIOLOGICS REGULATION IN UNITED STATES
Due to the scientifi c limitations of the early to mid - 1900s, signifi cant differences
existed between the approaches taken to manufacture and analyze biologics and
36 REGULATORY CONSIDERATIONS IN APPROVAL
TABLE 1 Examples of Therapeutic Peptide and Protein Molecules Currently Marketed
in United States
Peptides Antibiotics: bacitracin, bleomycin, gramicidine, capreomycin
Hormones: corticotropin, glucagon, gonadrolein HCl, leuprolide
acetate, histrelin acetate, oxytocin, secretin, goserelin acetate,
vassopressin
Others: polymixin B, eptifi batide, cyclosporine
Nonglycosylated
proteins
Interleukins: andresleukin (IL - 1), denileukin diftitox (fusion, protein -
IL - 2+ DT), anakinra (IL - 2)
Interferons: interferon alpha - n1, interferon alpha - n3, interferon alpha -
2a, peg interferon alfa - 2b, interferon alfacon - 1, Interferon alpha - 2b,
interferon beta - 1b, interferon gamma - 1b,
Enzymes/inhibitors: anistreplase, asparaginase, lactase, trypsin, alpha -
1 proteinase inhibitor, urokinase, deoxyribonuclease, fi brinolysin,
chymotrypsin, pancreatin, papain, urokinase
Growth factors/hormones: Filigrastim pegfi lgrastim, somatropin,
becaplermin, somatrem, menotropins
Antithrombotic agents: thrombin, fi brinogen, hirudin, hirulog, fi brin
Others: insulin, gelatin, prolactin, albumin (human), hemoglobin,
collagen
Glycosylated
proteins
Interferon beta - 1a
Antithrombotic agents: alteplase, drotrecogin alfa, antithrombin III
Antianemic: darbopoetin alfa, erythropoietin
Growth hormones: follitropin alpha, follitropin beta, chorionic
gonadotropin (Human)
Immuno globulins (IG): pertusssis IG, rabies IG, tetanus IG, hepatitis
B IG, varicella zoster IG, rho(D) IG, normal immune globulin,
lymphocyte anti - thymocyte, IB (equine)
Coagulation factors: factor VII antihemophilic factor, factor IX
(human, recombinant)
Factor VIII (others): etanercept (CSF), sargramostim (TNF)
Monoclonal
antiobodies
avciximab, alemtuzamub, basiliximab, gentuzumab, satumomab,
infl ixibam, palivizumab
drugs. This reality led to the creation of separate and distinct regulatory pathways
for drugs and biologics. As noted earlier, the developments in analytical chemistry
and improvements in process technologies have, in recent times, blurred the lines
between drug and biologic drug development. In the current era of pharmaceutical
development and standards harmonization, one might question the continued need
for two distinct pathways. Recognizing the shifting paradigm of drug development,
the history of biologics regulation is discussed below in two parts: early history and
present day.
1.2.2.1 Early Biologics Regulation (1800s – 1990s)
This country ’ s earliest experience with biologics dates back to the infectious scourges
of the late 1800s and early 1900s when epidemics of typhoid, yellow fever, smallpox,
diphtheria, and tuberculosis were being battled by new advances in immunology.
The discovery and development of vaccines and antitoxins led to the creation of a
HISTORY OF BIOLOGICS REGULATION IN UNITED STATES 37
TABLE 2 Patent Expiration Dates for U.S. Marketed Biologics
Brand Name Generic Name Indication Company
Patent
Expiry
Humulin Recombinant insulin Diabetes Eli Lilly Expired
Nutropin Somatropin Growth disorders Genentech Expired
Abbokinase Eudurase urokinase Ischaemic events Abbott Expired
Ceredase Alglucerase Gaucher disease Genzyme Expired
Cerezyme Imiglucerase Gaucher disease Genzyme Expired
Streptase Streptokinase Ischaemic events AstraZeneca Expired
Intron A IFN - . - 2b Hepatitis B and C Biogen/Roche Expired
Serostim Somatropin AIDS wasting Serono Expired
Humatrope Somatropin Growth disorders Eli Lilly Expired
Geref Sermorelin Growth hormone
defi ciency
Serono Expired
(2004)
Synagis Palivizumab Respiratory
syncytial virus
Abbott Expired
(2004)
Novolin Human insulin Diabetes Novo Nordisk 2005
Protropin Somatrem Growth hormone
defi ciency
Genentech 2005
TNKase Tenecteplase
TNK - tPA
Acute myocardial
infarction
Genentech 2005
Actimmmune IFN - . - 1b Chronic
granulomatous
disease;
malignant
osteoporosis
InterMune 2005, 2006,
2012
Activase,
Alteplase
tPA Acute myocardial
infarction
Genentech 2005, 2010
Proleukin IL - 2 HIV Chiron 2006, 2012
Epogen,
Procrit,
Eprex
Erythropoietin Anemia Amgen 2013
Neupogen Filgrastim (G - CSF) Anemia,
leukemia,
neutropenia
Amgen 2015
Note: Based on our search of available patent sites for only the reference product.
IFN - Interferon; tPA - Tissue Plasminogen Activator, IL - interleukin; HIV - Human Immunodefi ciency
Virus; G-CSF- Granulocyte-Colony Stimulating Factor; TNKase- Tenecteplase.
whole new “ biopharmaceutical ” industry. As demand increased, the pharmaceutical
manufacturers responded and in turn supplanted the government ’ s role in the public
supply of vaccines (per Vaccine Act of 1813) [10] . Unfortunately, the commercialization
of vaccines by smaller, less experienced, and likely less scrupulous manufacturers
led to problems. Similar to the history of drug regulation, early advances in
biologics regulation could be characterized as responsive rather than proactive.
Change often occurred following tragedy and the result of government ’ s attempt to
respond. Some of the key milestones of early biologics regulation are summarized
in Table 3 . The following years saw many administrative changes in terms of the
specifi c governmental agency responsible for regulating biologics, but with few
substantive changes to the regulations themselves.
38 REGULATORY CONSIDERATIONS IN APPROVAL
TABLE 3 Key Milestones in Early Biologics Regulation
1901 Ten children died in St. Louis from administration of tetanus - contaminated
diphtheria antitoxin. In this case, no safety testing had been performed prior to
use.
1902 Biologics Control Act (BCA) signed into law:
• Authorizing the regulation of commercial viruses, serums, toxins, and analogous
products
• Requiring the licensure of biologics manufacturers and establishments
• Providing governmental inspectional authority
• Making it illegal for the commercial distribution of product not manufactured
and labeled in accordance with the act
1906 Pure Food and Drug Act enacted (precursor of modern - day drug regulation). Lack
of mention of biologics as a class effectively represents fi rst distinction between
drug and biologic regulation.
1919 BCA amended:
• Required reporting of changes in equipment, manufacturing processes, personnel;
establishment of formal quality control procedures; and submission of samples
for regulatory inspection and approval for release
• Recognized potential that slight changes to manufacturing conditions (raw
materials, process, personnel, etc.) could have signifi cant and adverse effect on
product quality
• Required strict control of input (environment and manufacturing conditions)
rather than end - stage testing of quality attributes due to limitations in analytical
methodology to detect these effects
1937 Elixir sulfanilamide, containing the poisonous solvent diethylene glycol, kills 107,
many of whom are children.
1938 Food, Drug and Cosmetic Act (FDCA) enacted:
• Established concept of “ new drugs ” requiring proof of safety prior to marketing
• Required submission of an investigational new Drug (IND) application prior to
clinical use of an experimental drug in humans
• Required approval of a new drug application (NDA) prior to commercial sale of
drugs
• Granted federal government power of seizure of misbranded or adulterated
drugs
• Defi ned “ drugs ” comprehensively; not excluding potential of “ biologics ” to
function as drugs
1941 • Approximately 300 deaths and injuries result from distribution of sulfathiazole
tablets tainted with the sedative phenobarbital.
• Insulin Amendment passed to require FDA testing/certifi cation of purity and
potency.
1944 Public Health Service (PHS) Act enacted to consolidate and codify previous
biologics laws:
• Outlined licensing requirements for biologics — for both product (product
licensing application, or PLA) and establishment where the product was
manufactured (establishment licensing application, or ELA)
• Required submission of samples of each manufactured lot of all biologicals for
government testing and certifi cation prior to commercial release
• Required sponsors to own all of manufacturing facilities, effectively eliminating
multiparty or contract manufacturing
HISTORY OF BIOLOGICS REGULATION IN UNITED STATES 39
1.2.2.2 Modern Biologics Regulation (1990s – Today)
Whereas early biologics regulation was grounded by technical limitations, modern
biologics regulation is driven by tremendous advances in scientifi c knowledge.
Development of analytical tools and techniques has dramatically increased the
ability to characterize proteins and substantiate the structure, composition, and
function of the therapeutic molecule. These advances enable the detection of small
differences in molecular weight; elucidation of primary, secondary, and tertiary
protein structures; detection and quantifi cation of posttranslational modifi cations
(i.e., patterns of glycosylation); and improved understanding of structure – function
relationships and potential immunogenic responses. Simultaneously, developments
in the fi elds of pharmaceutical and biotechnological manufacturing have greatly
improved process effi ciency and control. This recent technological evolution has had
a direct impact on biologics regulation as refl ected below in several key events:
• In 1995, the FDA agreed to eliminate lot testing requirements for certain highly
characterized products once the company ’ s ability to consistently manufacture
product of acceptable quality was established.
• In 1996, the FDA and Congress dismantled the dual - licensing process, requiring
the submission of a single BLA (biologics license application), making the
content and format of a biologics application similar to that required for new
drug applications (NDAs).
• In 1996, the Center for Biologics Evaluation and Research (CBER) liberalized
its defi nition of “ legal manufacturer ” and eliminated many of the barriers to
cooperative, multiparty manufacturing arrangements.
• In 1997, Congress passed a noteworthy piece of legislation affecting modern
pharmaceutical regulation in the Food and Drug Modernization Act (FDAMA).
Among the many goals of the act was to harmonize the drug and biologic
approval processes.
In fact, current pharmaceutical/regulatory initiatives appear to extract the best
practices from biologic and drug approaches which can apply equally to both classes
of products:
• The Quality Systems Approach and GMPs for the 21st Century, two initiatives
being pursued by the FDA for drugs and devices, emphasize the utility of building
quality into the process, consistent with the strict control of “ input factors ”
seen in early biologic regulation.
• Initiatives such as Process Analytical Technologies build on the concept of
conventional drug product testing using increasingly sophisticated analytical
techniques to provide continuous process monitoring and fi nished - product
quality assurance of multiple pharmaceutical dosage forms.
• The current global initiative to harmonize electronic submission format and
content requirements effectively creates one standard data package for drugs
or biologics. Thus, the eNDA (electronic new drug application) or eBLA (electronic
biologics license application) will eventually be replaced by the eCTD
(electronic common technical document).
40 REGULATORY CONSIDERATIONS IN APPROVAL
1.2.3 REGULATORY CLASSIFICATION OF PROTEINS
Despite the blurring of lines between drugs and biologics, there remain two different
mechanisms to bring protein drug products to the U.S. marketplace. The choice of
approval framework is dependent on the protein ’ s classifi cation as a drug or biologic.
The history of this regulatory distinction is rooted in the technical differences
between small - molecule drugs and macromolecular biologics. Traditionally, drugs
were characterized as having well - defi ned chemistry. Conversely, biologics were
large, complex macromolecules whose active moiety defi ed characterization and
quantitation. By necessity, different means of assuring the safety and effi cacy of
these therapeutic products were required at the time. The modern - day consequence
is a legal system that distinguishes between proteins as drugs and proteins as biologics.
The distinction is based on statutory defi nitions as well as historical precedent
and has implications in terms of the approval pathways for original and follow - on
products.
1.2.3.1 Defi nitions and Key Terminology
Drugs are defi ned by the U.S. Food and Drug Act [FD & C Act, 21 U.S.C. 321(g)(1)] by
function as any article Federal Food, Drug and Cosmetic Act (a) intended for use
in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or
animals and (b) intended to affect the structure or function of the body [11] .
Biologics as a class may be regulated as drugs but are defi ned within the Public
Health Service Act [PHSA, 42 U.S.C. 262(a)] by category as “ a virus, therapeutic
serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic
product, or analogous product, or arsphenamine (or any other trivalent organic
arsenic compound), applicable to the prevention, treatment, or cure of diseases or
injuries of humans ” [12] .
A cursory examination of these defi nitions reveals that they are not mutually
exclusive, leading to confusion about how to appropriately and consistently
apply them. This point is illustrated when one reviews the history of how the FDA
has categorized and subsequently regulated these drugs and biologics as shown
below.
1.2.3.2 Application of Defi nitions to Proteins: Is It a Drug or a Biologic?
The answer to this fundamental question is not straightforward and has evolved
over time. Historically, some natural - source - derived proteins such as insulin, hyaluronidase,
menotropins, and Human Growth Hormone (HGH) have been regulated
as drugs. While other natural - source - proteins such as blood factors were regulated
as biologics. When recombinant proteins and monoclonal antibodies began development
in the 1970s – 1980s, these were regulated as follows:
1. By the Center for Drug Evaluation and Research (CDER) under the Food,
Drug and Cosmetic Act (FDCA) as drugs when they were hormones such as
insulin, HGH, and parathyroid hormone (PTH) derivatives
2. By the CBER under the PHSA as biologics when they were cytokines or blood
factors such as factor VIII for hemophilia
As other recombinant proteins and monoclonal antibodies came under development,
the CBER held primary responsibility for this review, with the CDER retaining
responsibility for hormones such as insulin and HGH. However, in 2003 all
therapeutic proteins were transferred from the CBER to the CDER. This reassignment
of review responsibility did not impact the legal classifi cation of these protein
products, such that the Center for Drug Evaluation and Research assumed responsibility
for the review and approval of biologics approved under Section 351 of the
PHSA.
The basic distribution of these therapeutic biologics to the respective FDA center
is refl ected in Table 4 ; however, many of the current complex biotechnology - derived
products do not fi t neatly into accepted defi nitions and require case - by - case classi-
fi cation [13] .
1.2.3.3 Regulatory Approval Path for Proteins
The relevance of the preceding discussion becomes important with the understanding
that therapeutic proteins classifi ed as drugs are governed under a different set
of laws than those classifi ed as biologics. Drugs are approved via submission of
NDAs under Section 505 of the FD & C Act, while biologics are supported by BLAs
under the PHSA. These two approval paths are similar in terms of application
content, that is, requirement of complete reports of clinical safety and effi cacy data
to support approval. However, only the drug regulation, that is, Section 505 of the
FD & C Act, has been amended to outline an abbreviated approval mechanism for
generic products.
1.2.4 REGULATION OF GENERIC DRUGS
1.2.4.1 History of Generic Drug Legislation in United States
In 1984, Congress responded to America ’ s need for safe, affordable medicines by
passing a pivotal piece of legislation, The Drug Price Competition and Patent Term
TABLE 4 FDA Center Regulatory Responsibility for Therapeutic Biological Products
CDER CBER
Monoclonal antibodies (in vivo use)
Proteins intended for therapeutic use:
Cytokines (e.g., interferons)
Enzymes (e.g., thrombolytics)
other novel proteins except those assigned to
CBER
Immunomodulators (nonvaccine, nonallergenic)
Growth factors, cytokines, some hormones and
monoclonal antibodies intended to mobilize,
stimulate, decrease, or otherwise alter the
production of hematopoietic cells in vivo
Cellular product, including products
composed of human, bacterial, or
animal cells
Vaccines
Allergenic extracts
Antitoxins, antivenoms, venoms
Blood, blood components, plasma -
derived products (e.g., albumin,
immunoglobulins, clotting factors,
fi brin sealants, proteinase inhibitors),
recombinant and transgenic versions
of plasma derivatives
REGULATION OF GENERIC DRUGS 41
42 REGULATORY CONSIDERATIONS IN APPROVAL
Restoration Act (Hatch – Waxman amendments). The intent of this act was to effectively
balance the need to encourage pharmaceutical innovation with the desire to
accelerate the availability of lower cost alternatives to approved drugs. The act also
sought to eliminate unnecessary or redundant clinical testing to protect patients
(reduce the number of patients in need receiving placebo in controlled clinical
trials) and conserve industry and agency resources. To accomplish the goal of faster
to market, cheaper alternatives, the amendments stipulated the following [14] :
• For Innovator Companies The act encouraged continued innovation, research,
and development activities by providing manufacturers with meaningful incentives
in the form of patent protection/restoration and marketing exclusivity,
thus allowing them to recoup some of their investments.
• For Generic Companies The act provided access to certain innovator information
without the threat of legal action via patent infringement suits (safe harbor
provisions), allowing generics the opportunity to prepare for market introduction
prior to the expiration of patent/exclusivity terms. This effectively limited
the period of innovator exclusivity to the statutory timelines.
1.2.4.2 Approval Process for Generic Drugs
The act served as a boon to the generic industry by paving the path to abbreviated
and accelerated drug approvals. From a legal perspective, the Hatch – Waxman
amendments modifi ed Section 505 of the FD & C Act to create two new abbreviated
approval pathways (see Table 5 ) [14] .
In essence, the abbreviated NDA (ANDA) and 505(b)(2) processes allow generic
manufacturers the ability to rely on what is already known about the drug and refer
to the agency ’ s fi nding of safety and effi cacy for the innovator. For an ANDA, the
generic product must meet certain criteria related to bioequivalence and product
sameness. However, a 505(b)(2) application often describes a drug with substantial
differences to the innovator (which would seem more closely related to FOPs).
1.2.4.3 Application of Generic Regulations to Biologics
A central question is “ Do biologics fall under the provisions of the Hatch – Waxman
Act? ” Since the Hatch – Waxman Act specifi cally amended the FD & C Act, biologics
TABLE 5 Description of NDA Approval Mechanisms
Traditional path 1. 505(b)(1) — Application that contains full reports of investigations
of safety and effectiveness to which sponsor has right of reference
(stand - alone NDA)
Abbreviated path 2. 505(b)(2) — Application that contains full reports of investigations
of safety and effectiveness, where the sponsor relies on studies
conducted by someone else to which the sponsor does not have
right of reference
Abbreviated path 3. 505(j)(1) — Abbreviated new drug application (ANDA) containing
information to show the product is a duplicate of an already
approved drug product
approved via a BLA under the PHSA are not covered by this legislation nor does
the PHSA have similar provisions for biogenerics. However, those few therapeutic
proteins approved via Section 505 of the FDCA as NDAs are covered by the
Hatch – Waxman amendments and thus are legally considered appropriate for fi ling
a 505(b)(2) or 505(j)(1) application. For simple, well - characterized peptides and
proteins regulated under Section 505 of the FD & C Act, mechanisms are already in
place to bring FOPs to the market. In fact, several FOPs have already been approved
by the FDA, including GlucaGen (glucagon recombinant for injection), Hylenex
(hyaluronidase recombinant human), Hydase and Amphadase (hyaluronidase), Fortical
(calcitonin salmon recombinant) Nasal Spray, and Omnitrope [somatropin
(rDNA origin)] [15] . Further details related to the latter two are presented in the
discussion of actual case studies.
1.2.5 LEGAL ARGUMENTS RELATED TO FOLLOW - ON PROTEINS
The legal arguments regarding the approval of biogenerics relate to several different
aspects of drug/biologics law.
1.2.5.1 Constitutionality of 505(b)(2) Process for Drugs
The agency ’ s authority to grant approval of drugs via the 505(b)(2) process has
previously been challenged by several companies. The nature of these challenges
has questioned the FDA ’ s right to use proprietary information of the innovator in
support of another company ’ s drug approval. Recall that the 505(b)(2) process
allows a company to use data for which it does not have right of reference (i.e.,
another company ’ s safety and effi cacy data) in support of its own application. The
FDA ’ s long - standing interpretation of the statute seems fi rm and well founded in
precedent since over 80 applications for drugs have been approved via the 505(b)(2)
route since its inception with indications ranging from cancer pain to Attention
Defi cit Disorder (ADD) [16] .
1.2.5.2 Constitutionality of 505(b)(2) Process for Follow - On Proteins
The constitutionality issues related to FOPs are similar to those mentioned above
for drugs, namely protection of proprietary information and intellectual property
rights. Some critics opine that issues unique to FOPs create additional legal hurdles.
For example, the rules pertaining to the disclosure of safety and effectiveness information
are different for biologics licensed under the PHSA and drugs approved
under the FDCA. When the rules were originally written (1974), it was thought that
safety and effectiveness for one biologic would not support the licensure of another.
So these data were deemed not to be protected trade secrets and could be publicly
disclosed immediately after issuance of the biologic ’ s license [see 21 CFR 601.51(e),
1974]. However, since this language applies strictly to the PHSA, it has no bearing
on discussions related to the 505(b)(2) process.
In other public challenges, opponents argue that the unique and complex nature
of biologics and the close relationship between their method of preparation and
clinical attributes require that the FDA use and disclose the manufacturing methods
LEGAL ARGUMENTS RELATED TO FOLLOW-ON PROTEINS 43
44 REGULATORY CONSIDERATIONS IN APPROVAL
and process information contained in an innovator ’ s application. Further, this use
and disclosure would violate Trade Secret and Constitutional Law (Fifth Amendment
“ taking clause ” ) [17, 18] .
The concept of “ the product is the process ” may have been applicable to early
biologics, but current capabilities allow the chemical, biologic, and functional comparison
of well - characterized protein drugs. The follow - on manufacturer need not
necessarily utilize the identical method of manufacture or proprietary technology
to reproduce a follow - on biologic with similar clinical safety and effi cacy. Additionally,
it is important to distinguish between the regulatory requirements for approval
of an actual generic protein (duplicate of innovator; see discussion below) and those
associated with a 505(b)(2), which requires a showing of similarity between two
products. Any differences between the two would need to be adequately supported
by bridging studies and appropriate clinical and/or nonclinical data.
The FDA has confi rmed this interpretation in its response to petitions fi led
regarding FOPs (both in general and targeted to specifi c applications). The FDA
has clearly said, “ the use of the 505(b)(2) pathway does not entail disclosure of trade
secret or confi dential commercial information, nor does it involve unauthorized
reliance on such data ” [18] .
1.2.5.3 Applicability of 505(j)(1) or ANDA Process to Biogenerics
Biogenerics per se, that is, protein drug products approved via 505(j)(1), would need
to demonstrate their bioequivalence to the innovator protein. However, due to their
complexity and heterogeneity, the classical biopharmaceutical principles upon which
the current ratings of therapeutic equivalence are based do not apply in their current
language to complex macromolecules. For example, due to the nature and complexity
of an immunogenic response, one concern would be if traditional bioequivalence
appropriately addresses the complex safety issues associated with biologics.
1.2.5.4 Current Rules Relating to Bioequivalence of Generic Drugs
The list of approved drug products with therapeutic equivalence (Orange Book)
was originally intended as an information source to states seeking formulary guidance
[19] . The list provides the FDA ’ s recommendations as to which generic prescription
drug products are acceptable substitutes for innovator drugs. The term
innovator is used to describe the reference listed drug, or RLD [21 CFR 314.94(a)(3)],
upon which an applicant (generic) relies in seeking approval of its ANDA. In layman
’ s terms the RLD describes the original NDA - approved drug and is often
referred to as the “ pioneer ” drug.
Under the Drug Price Competition and Patent Term Restoration Act of 1984,
manufacturers seeking approval to market a generic drug need to submit data to
the FDA demonstrating that their proposed drug product is bioequivalent to the
pioneer (innovator) drug product. A major premise underlying the 1984 law is that
bioequivalent drug products are therapeutically equivalent, will produce the same
clinical effect and safety profi le as the innovator product, and are therefore, interchangeable
[19] .
So how would FOPs be classifi ed using conventional defi nitions of bioequivalence?
To answer this question, it is necessary to review current legal defi nitions of
bioequivalence terms [19] :
• Two products are bioequivalent in “ the absence of a signifi cant difference in the
rate and extent to which the active ingredient or active moiety in pharmaceutical
equivalents or pharmaceutical alternatives becomes available at the site of drug
action when administered at the same molar dose under similar conditions in an
appropriately designed study ” [21 CFR 320.1(e)]. An appropriately designed
comparison could include (1) pharmacokinetic (PK) studies, (2) pharmacodynamic
(PD) studies, (3) comparative clinical trials, and/or (4) in vitro studies.
• Pharmaceutical equivalents are those drug products which are formulated to
contain the same amount of active ingredient in the same dosage form to meet
the same (compendial or other applicable) standards of quality.
• Pharmaceutical alternatives are drug products that contain the same therapeutic
moiety, or its precursor, but not necessarily in the same amount or dosage form.
Drug products are considered to be therapeutic equivalents only if they are
pharmaceutical equivalents and if they can be expected to have the same clinical
effect and safety profi le when administered to patients under the conditions
specifi ed in the labeling. Although pharmaceutical alternatives may ultimately
be proven bioequivalent, given their differences they are not automatically
presumed to be.
Given these defi nitions, FOPs would likely be considered pharmaceutical alternatives
if one presumes that pioneer and follow - on proteins are identical at a precursor
stage, prior to potential post - translational modifi cation. This presumption may also
be consistent with the similarity standard the agency applies to ascertain orphan
drug status (see discussion in Section 1.2.6 ). Follow - on proteins cannot be considered
to be therapeutic equivalents since they are not pharmaceutical equivalents
and cannot be expected to have the same clinical effect and safety profi le in the
absence of testing. This assertion is supported by the following:
• The potential impact of how posttranslational modifi cations, such as glycosylation,
can directly impact protein conformation and subsequently affect biological
activity, including the overall safety and effi cacy of the drug product.
• An underlying premise of bioequivalence assessments is a clearly defi ned pharmacokinetic/
pharmacodynamic relationship; however, the relation between
blood levels and effect is less clearly established for proteins [20] .
Consequently, within the current regulatory framework, FOPs are unique products
that may be “ similar ” but are not the same as innovator proteins, consistent with
their approval via a 505(b)(2) pathway. This interpretation is supported by the
FDA ’ s designation of Omnitrope as having a BX rating in the Orange Book. The
code BX in the Orange Book refers to drug products for which the data are insuf-
fi cient to determine therapeutic equivalence as compared to a therapeutic rating of
A indicative of interchangeability. This concept of similarity is also consistent with
the defi nitions proposed by the European Agency for the Evaluation of Medicinal
Products (EMEA) for generic versions of proteins [21] :
Bio - similar products: second and subsequent versions of biologics that are independently
developed and approved after a pioneer has developed an original version.
Bio - similar products may or may not be intended to be molecular copies of the innovator
’ s product; however, they rely on the same mechanism of action and therapeutic
indication.
LEGAL ARGUMENTS RELATED TO FOLLOW-ON PROTEINS 45
46 REGULATORY CONSIDERATIONS IN APPROVAL
1.2.5.5 Statutory Authority
Unlike the FDCA, which affords therapeutic protein drugs the legal pathway of
abbreviated drug approval for a FOP, the PHSA currently has no similar provisions.
Such a pathway for approval or licensure of FOP products under the PHSA would
require new legislation and recent congressional developments suggest that work is
underway to create this statutory pathway.
Legislation proposed on September 29, 2006, by U.S. Representative Henry
Waxman (D - CA) and Senator Charles Schumer (D - NY) seeks to amend the PHSA
to authorize the FDA to approve abbreviated applications for biologic products that
are “ comparable ” to previously approved (brand name) biologic products. Entitled
The Access to Life - Saving Medicine Act, this bill outlines a process by which the FDA
could determine, on a product - by - product basis, the studies necessary to demonstrate
comparability of a FOP product to a brand name product and assure its safety and
effectiveness. The act allows for an applicant to seek interchangeability with a brand
name product, recognizing that the extent of data to support such a designation must
be discussed with the FDA. To encourage the development of interchangeable products,
the bill would authorize tax incentives and periods of marketing exclusivity. The
bill would also seek to create an improved process to facilitate early resolution of
patent disputes which might otherwise delay competition [22] .
1.2.6 SCIENTIFIC ISSUES RELATED TO FOLLOW - ON PROTEINS
(DATA REQUIREMENTS)
The challenge of FOPs demonstrating similar quality, safety, and effi cacy to the
innovator product relates to the poor predictability of physicochemical characteristics
and biologic activity. For example, there are several different interferon - . and
erythropoietin . and . products currently on the market. These variants are characterized
by differences in sequence, glycosylation pattern, and in vitro measures of
specifi c activity; however, their clinical safety and effi cacy profi les are considered
similar [20] .
In contrast, different formulations of insulin and growth hormone containing the
same active ingredient exhibit signifi cant differences in bioavailability [20] . Additionally,
the inability to adequately predict immunogenic responses from in vitro
data or animal studies remains a concern.
The answer to the challenge is that generic manufacturers must go through a
similar process of in - depth characterization, including identifi cation of critical structural
elements of the product (structure/function) when developing a FOP. Although
the regulatory standards for demonstrating similarity are currently undefi ned, some
insight can be gleaned from consideration of FDA expectations in terms of granting
orphan drug status to similar proteins and assessing postapproval Chemistry, Manufacturing
and Controls (CMC) changes for innovator proteins.
1.2.6.1 “ Sameness ” as per Orphan Drugs Regulations
The Orphan Drug Act of 1983 was implemented in response to the government ’ s
concern that viable treatments for rare diseases were not being explored due to
excessive costs of drug development in comparison to the relatively small popula
tion of potential users (and sales). Orphan drugs are (a) those used to treat rare
diseases, defi ned by the act as affecting < 200,000 persons in the United States, or
(b) those drugs whose development costs would not be recovered through sales of
the drug. To encourage development, the government authorized incentives in the
form of marketing exclusivity (seven years), tax credits, protocol assistance, and
grants/contracts, with the fi rst being of primary importance to most drug sponsors.
Since exclusivity is awarded only to the fi rst designated product to obtain approval
for a given drug/indication, competition is fi erce. No approval would be given to a
subsequent sponsor ’ s application for the same product/indication unless it was
shown to be clinically superior (i.e., not the same). Thus, the agency needed to
develop criteria upon which it would make these determinations.
In 1992, the FDA ’ s orphan drug regulations fi rst established the conditions under
which the agency could determine product “ sameness ” of protein drugs and therefore
take action to block the approval of a second orphan drug product: “ two protein
drugs would be considered the same if the only differences in structure between
them were due to post - translational events, or infi delity of translation or transcription,
or were minor differences in amino acid sequence; other potentially important
differences, such as different glycosylation patterns or different tertiary structures,
would not cause the drugs to be considered different unless the differences were
shown to be clinically superior ” [23] . It should be noted that there may exist exceptions
to this rule that depend on the interpretation of each individual case. For
example, Eli Lilly & Co. successfully received orphan drug status in the late 1980s
for the naturally occurring HGH to compete with the previously marketed Met -
HGH, which only differed in the N - terminal methionine.
The support for clinical superiority could be based on evidence of greater effectiveness
and increased safety or represent a “ major contribution to patient care. ” In
short, orphan drug regulations utilize clinical data to demonstrate product differences.
Examples include [23] :
• 1996: Biogen ’ s Avonex (interferon . ) was considered to be clinically superior to
Berlex ’ s Betaseron based on improved safety (fewer site injection reactions).
• 1999: In a law suit involving generic paclitaxel, Baker Norton, challenged the
FDA ’ s sameness determinations based on active moiety alone, arguing that
factors such as formulation and labeling should be considered. The challenge
was unsuccessful.
• 2002: Serono ’ s Rebif (interferon .1a ) was awarded exclusivity based on the
clinical demonstration of improved effi cacy (reduced Multiple Sclerosis (MS)
exacerbations).
Therefore, it would appear that the orphan drug regulations provide some fl exibility
to the sponsor (generic) in establishing product sameness but also reaffi rm the
important role of clinical data in supporting product safety and effi cacy.
1.2.6.2 “ Sameness ” as per Postapproval Change Guidances
Guidelines for supporting postapproval changes to the chemistry, manufacturing,
and controls of approved products (SUPAC guidances) take a somewhat different
approach to establishing sameness. In essence, the SUPAC guidelines refl ect risk
SCIENTIFIC ISSUES RELATED TO FOLLOW-ON PROTEINS 47
48 REGULATORY CONSIDERATIONS IN APPROVAL
management practices in evaluating the potential of certain CMC changes to impact
the identity, strength, quality, purity, and potency of the product as they may relate
to overall safety and effi cacy.
A long - held contention within the biologics industry is that the product is the
process and, by extension, change is strongly discouraged. Without qualifi cation, this
rather dated thinking is inconsistent with the fl exibility required in managing change
throughout the life cycle of a product. Further, this thinking may serve to discourage
the implementation of advanced technologies designed to improve not only effi -
ciency but also product quality. Even current biologics regulations recognize
the need to accommodate change; 21 CFR 601.12 (for biologics) states that for
changes in the product, production process, quality controls, equipment, facilities,
and so on, an applicant must assess the effects of the change and demonstrate
through appropriate validation and/or other clinical and/or nonclinical laboratory
studies the lack of adverse effect of the change on the identity, strength, quality,
purity, or potency of the product as they may relate to the safety or effectiveness of
the product.
In fact, many of the challenges that generic manufacturers face in demonstrating
sameness of FOPs to reference listed drugs are similar to those encountered by
innovators in managing the dynamic CMC life cycle of a product. One of the tools
available to assess the potential impact of product differences is a comparability
protocol. The FDA described its expectations of the data requirements necessary
to support postapproval CMC changes to protein drug product and biologic products
in a Guidance to Industry on the use of comparability protocols for such products
issued in 2003 [24] . Underpinning the successful application of a comparability
protocol are extensive product development and characterization.
Initial Product Development Prior to undertaking any comparative analysis, a
manufacturer must perform two critical steps. First, the manufacturer needs to
conduct thorough process development and optimization of the therapeutic protein
product. Second, the sponsor (generic or innovator) needs to prospectively examine
the impact of changes to all critical processing parameters during the development
phase and determine the minimum data requirements necessary to assure the
absence of adverse impact to product quality, safety, or effi cacy. The current state
of technology provides us with better tools to more fully characterize the protein
drug substance and drug product at all stages of production.
Physicochemical Characterization and Process Development Some of the key steps
to process development and product characterization include:
• Production of a cell line/clone
• Identifi cation and characterization of critical raw materials (media, resins,
formulation excipients)
• Development of internal standards, in - process controls, product
specifi cations
• Conduct of extensive pilot - scale manufacturing development: fermentation and
downstream processing (separation and purifi cation)
• Performance of process scale - up and optimization studies
• Application of a comprehensive array of analytical techniques to fully characterize
the drug product at each stage of development. Table 6 provides examples
of methods to probe virtually every property of the protein and develop
a fi ngerprint of the molecule.
Other Testing Requirements The need for additional supportive studies beyond
physicochemical characterization will increase proportionately with the complexity
of the protein drug. The entire battery of tests may not be required for each FOP
but may include the following data, bioassay, preclinical (pharmacology/toxicology/
pharmacokinetic/pharmacodynamic), clinical safety and effi cacy, and immunogenicity.
The nature, number, and size of the trials should relate directly to the particular
drug/indication/patient population.
Bioassay A biological assay, or “ bioassay, ” is an analytical procedure capable of
measuring the biologic activity of a substance based on a specifi c functional, biologic
TABLE 6 Analytical Techniques for Physicochemical Characterization of Proteins
Parameter Test
Primary structure Amino acid sequencing, N - terminal Edman
sequencing, peptide mapping
Higher order structure CD, NMR, FTIR, Raman
Mass LC - ESI - MS, MALDI - TOF - MS
Size SDS - PAGE, DLS, SEC - MALLS
Hydrophobicity RP - HPLC
Binding Immunological binding
Sulfhydryl groups/disulfi de bridges Peptide mapping (under reducing and nonreducing
conditions)
Glycan analysis:
Monosaccharide analysis HPLC, MS
Sialic acid content HPLC
Molecular weight MALDI - MS, ESI - MS
Impurity profi le
Process - related impurities Immunoassay, HPLC, SDS - PAGE, MS, CD, capillary
gel electrophoresis, size exclusion chromatography • Cell substrate derived
• Cell culture derived
• Downstream derived
Product - related impurities
• Truncated forms
• Other modifi ed forms (i.e.,
deamidated, isomerized)
• Aggregates
Evaluation of stability HPLC
CD, Circular Dichroism; NMR, Nuclear Magnetic Resonance; FTIR, Fourier transform infrared spectroscopy;
LC - ESI - MS, Liquid chromatography electrospray ionisation mass spectrometry; MALDI -
TOF - MS, Matrix - assisted laser desorption ionization - time of fl ight - mass spectrometry; SDS - PAGE,
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis; DLS. Dynamic light scattering; SEC -
MALLS, Size exclusion chromatography - multi - angle laser light scattering; RP - HPLC, Reversed phase -
high performance liquid chromatography; HPLC, High performance liquid chromatography; MALDI - MS,
Matrix - assisted laser desorption ionization mass spectrometry; ESI - MS, Electrospray ionisation mass
spectrometry; MS, Mass spectrometry.
SCIENTIFIC ISSUES RELATED TO FOLLOW-ON PROTEINS 49
50 REGULATORY CONSIDERATIONS IN APPROVAL
response of the test system. Bioassays should be predictive of clinical effect and are
therefore used as a means of quantifying activity (in nonclinical manner) and ensuring
effi cacy throughout development. They are informative in equivalence studies
to the extent that a change affects a part of the molecule, which in turn impacts the
molecule ’ s biologic activity. Bioassays may be based on animal models, in vitro cell
lines, cell - based biochemical assays (i.e., kinase receptor activity), receptor binding
assays, or enzyme assays. The selection of an appropriate bioassay is driven in part
by the ability to demonstrate a correlation to clinical effect. An example of a predictive
bioassay is the measurement of the antiviral activity of interferon as a function
of its cytopathic effect on host cells [25] .
Nonclinical (Pharmtox, PK , PD ) In the context of FOPs, the original sponsor will
have demonstrated what the molecule per se does to the body; however, since the
formulation is likely different, nonclinical studies are useful in demonstrating a lack
of adverse impact due to dosage form, route of administration, excipient changes,
manufacturing contaminants, and supporting sameness of the active moiety.
Appropriate toxicology studies would include acute or subchronic testing in at
least one relevant small animal species. Pharmacokinetic studies are highly useful
in assessing the impact of changes in the manufacture of natural - source - and
recombinant - derived proteins. Standard approaches used in bioequivalence studies
[measurement of the area under the curve (AUC), Cmax, tmax ] can be used to make
direct comparisons of innovator and follow - on profi les. Pharmacodynamic studies
are similarly very informative. Direct comparison of innovator and follow - on products
can be made by evaluating appropriate surrogate markers of effi cacy (i.e.,
platelet aggregation following anticoagulation therapy).
Clinical ( PK , PD , Safety and Effi cacy) Human clinical studies can range in complexity
from standard - design PK studies to complicated, long - term effi cacy trials
evaluating one or more indications in multiple populations. Human PK studies are
used as the benchmark for establishing bioequivalence of conventional dosage
forms. For traditional pharmaceuticals for which reliance on systemic exposure may
not be suitable, PD or clinical safety and effi cacy may be performed to show
equivalence.
The appropriate clinical program is infl uenced by many factors, including the
degree of molecular complexity of the particular protein and the extent of physicochemical
characterization; the mode of action, indication(s), and use population(s);
the presence of established structure – activity relationships and validated bioassays;
and the results of preclinical testing.
As such, the nature and scope of each clinical support program need to be determined
on a case - by - case basis in consultation between the sponsor and the regulatory
agency.
Immunogenicity The observation of serious adverse events with the use of some
recombinant and natural source proteins [i.e., pure red cell aplasia (PRCA) detected
with erythropoietin use] has highlighted immunogenicity as a major issue for consideration
when assessing within and between manufacturer changes [8] . Although the
exact immunological mechanism responsible for the increased number of PRCA
cases is unknown, it appears to be linked to a formulation change associated with
Eprex, a European epoetin - . product. Replacement of the stabilizer human serum
albumin with polysorbate 80 and glycine correlated with a surge in PRCA reported
cases [4] . An immunogenic effect may have no clinical impact or it could have serious
clinical consequences as seen above. The immune response of the therapeutic protein
should be fully characterized using both immunoassays which detect antibodies that
bind to the drug as well as bioassays which detect neutralizing antibodies that might
block the protein ’ s desired biological effect. Ultimately, this testing needs to be performed
in humans, as animal testing is not truly predictive of human immune response.
Antibody detection techniques include enzyme - linked immunosorbent assay radioimmunoassay,
(ELISA), and surface plasmon resonance [8] .
Comparability Testing to Demonstrate “Sameness” Following the developmental
studies described above, comparative studies to directly evaluate pre - and postchange
materials to one another and assess the impact of any process changes may
be conducted. In a similar manner, comparative studies between pioneer drug and
the follow - on can be used to systematically evaluate the impact of any differences
between reference listed drug and proposed generic protein drug. When compiling
information into an analytical characterization database, the data should be directly
compared to the reference product and variation observed in multiple batches
of test product (generic) should be similar to that of the reference innovator
product.
The FDA ’ s expectations in this regard are apparent in their description of the
CMC data package supporting the comparability of Omnitrope to the innovator
protein Genotropin. The FDA asserted [18] :
Each biotechnology manufacturer, whether producing a new molecular entity or a
follow - on product must independently develop its own cell expression, fermentation,
isolation and purifi cation systems for the active ingredient in its product. Thus, the
manufacturing process for each active ingredient is unique to each manufacturer. Nevertheless,
as Sandoz has demonstrated in its Omnitrope application, for this relatively
simple recombinant protein, it is possible to determine that the end products of different
manufacturing processes are highly similar, without having to compare or otherwise
refer to the [proprietary] processes.
1.2.7 PROPOSED REGULATORY PARADIGM: CASE STUDIES
Based on the nature and complexity of therapeutic protein products, an approval
pathway for follow - ons may require moving away from the traditional generic paradigm
in place for small molecules and creating a biosimilar paradigm for complex
molecules. The proposed regulatory paradigm for the approval of FOP products
could be similar for protein drugs approved under Section 505 of the FDCA or
licensed as biologics under the PHSA and mirror the current 505(b)(2) process. This
pathway permits the sponsor and agency to determine exactly what studies are
necessary to support the proposed differences (see 21 CFR 314.54(a) [ “ a 505(b)(2)
application need contain only that information needed to support the modifi cation(s)
of the listed drug ” ]. Application of a 505(b)(2) paradigm removes the need to demonstrate
bioequivalence per se and potentially reduces innovator intellectual property
concerns that arise if a generic must “ duplicate ” the innovator. Guidance as to
how similar a “ biosimilar ” needs to be exists in the form of current regulations
related to orphan drugs and postapproval manufacturing changes.
PROPOSED REGULATORY PARADIGM: CASE STUDIES 51
52 REGULATORY CONSIDERATIONS IN APPROVAL
Several recent drug approvals illustrate how this regulatory framework may be
applied and are described in the sections to follow.
1.2.7.1 Case Study 1: Fortical [Calcitonin - Salmon ( r DNA origin)]
On August 17, 1995, the FDA approved Novartis ’ s NDA for Miacalcin (calcitonin -
salmon) Nasal Spray (Miacalcin NS) for the treatment of postmenopausal osteoporosis
in females greater than fi ve years postmenopause with low bone mass relative
to healthy premenopausal females. The active ingredient in Miacalcin NS is synthetic
salmon calcitonin. On March 6, 2003, Unigene submitted a new drug application
under Section 505(b)(2) for Fortical [calcitonin - salmon (rDNA origin)] Nasal
Spray which relied in part on data submitted in the Miacalcin NS NDA.
Comparability Program Fortical and Miacalcin NS differed in certain aspects,
such as the use of recombinant versus synthetic salmon calcitonin and the use of
different types and amounts of excipients. Given these differences, Unigene was
required to submit data to establish that the fi ndings of safety and effi cacy for Miacalcin
were relevant to Fortical (i.e., contain the same active ingredient and have
comparable bioavailability) and that the formulation differences did not impact
previous clinical profi le [26, 27] .
Comparability Results
Physicochemical Analysis Salmon calcitonin is a 32 - amino - acid, nonglycosylated
peptide hormone. It is structurally simple, possessing limited secondary structure
and a single disulfi de bond. The physicochemical characterization studies demonstrated
that the primary and secondary structure of Fortical ’ s recombinant salmon
calcitonin (sc) was identical to that of Miacalcin ’ s synthetic sc or naturally occurring
sc. Further, the tertiary structures of the three were indistinguishable.
Nonclinical PK / Tox The pharmacokinetic profi le of Fortical by different routes of
administration was compared to Miacalcin, demonstrating similarity in PK profi les
between the synthetic and recombinant peptides and toxicity results (28 - day rat
intranasal toxicity study) were acceptable, particularly in light of clinical safety
data.
Clinical PK / PD Calcitonin has a well - established mechanism of action; published
literature supports that salmon calcitonin, mediated through calcitonin receptors
located on osteoclasts, inhibits bone resorption, thereby increasing bone mineral
density. Since serum beta - CTx (C - telopeptides of type 1 collagen, corrected for
creatinine) is a recognized marker of bone resorption, the effect of administered
salmon calcitonin on serum beta - CTx is considered to be an adequate surrogate for
pharmacodynamic comparisons.
Fortical ’ s PD equivalence was shown in a double - blind, active - controlled, 24 -
week study in 134 postmenopausal women randomized to Fortical (200 IU per day)
or Miacalcin (200 IU per day). The primary outcome measure was change in serum
beta - CTx from baseline. The results fell within prespecifi ed PD equivalence limits
(. 0.08 to 0.06 ng/mL; equivalence margin of ± 0.2 ng.mL) and indicated Fortical was
not inferior to Miacalcin.
Fortical ’ s PK equivalence was assessed by comparing the relative bioavailability
of Fortical to Miacalcin in a multidose, crossover study of 47 healthy female volunteers.
Results indicated that Fortical was slightly more bioavailable than Miacalcin,
but given the demonstration of similar PD activity, this difference were not considered
to be clinically signifi cant.
Immunogenicity Archived samples from the 24 - week PD study were used to
compare the immunogenicity potential of both products. The results indicated there
was no difference in terms of total immune response and the response of neutralizing
antibodies between the two drugs.
Conclusion to Case Study 1 On August 12, 2005, the FDA approved Unigene ’ s
505(b)(2) application for Fortical for the same indication as Miacalcin NS [26, 27] .
In the FDA ’ s analysis no statistically and/or clinically signifi cant differences were
noted in any aspect of the comparability profi le, including clinical performance, and
Fortical was approved.
The basis of this comparison was strongly challenged in a citizen petition claiming
that (1) recombinant salmon calcitonin is not the same as the synthetic version
which could potentially cause differences in product effi cacy, safety, or both and
(2) only a long - term clinical study (actual bone fracture data) would provide adequate
support of sameness [28] . The FDA responded to this citizen petition by
asserting its decision that the comparability data presented above collectively constituted
suffi cient demonstration of sameness [27, 29] .
1.2.7.2 Case Study 2: Omnitrope [Somatropin ( r DNA origin)]
On August 24, 1995, the FDA approved NDA20 - 280 fi led by the Pharmacia &
Upjohn Company for Genotropin (somatropin) (rDNA origin) for injection. Since
that time, Genotropin has been marketed as a safe and effective therapy for growth
hormone defi ciency (GHD) in children and adults.
On July 30, 2003, Sandoz submitted a 505(b)(2) application for the approval of
its recombinant HGH product (recombinant somatropin) indicated for long - term
treatment of pediatric patients who have growth failure due to an inadequate secretion
of endogenous growth hormone and for long - term replacement therapy in
adults with GHD of either childhood or adult onset. This application relied in part
on data submitted in the Genotropin NDA.
Comparability Program As with the Fortical case study, Omnitrope and Genotropin
differed in certain aspects. As such, Sandoz was required to submit substantial
data to establish that Omnitrope was suffi ciently similar to Genotropin to warrant
reliance on FDA ’ s fi nding of safety and effectiveness for Genotropin to support the
approval of Omnitrope [18] .
Comparability Results
Physicochemical Analysis In terms of complexity, HGH is fairly simple and well -
characterized. Human growth hormone is a single - chain, 191 - amino - acid, nonglycosylated
protein with two intramolecular disulfi de bonds. Sandoz used a variety of
physicochemical tests and analytical methods to confi rm the primary, secondary, and
PROPOSED REGULATORY PARADIGM: CASE STUDIES 53
54 REGULATORY CONSIDERATIONS IN APPROVAL
tertiary structures, molecular weight, and impurity profi le. Characterization studies
performed to verify somatropin as the active ingredient in Omnitrope included
reverse - phase liquid chromatography/mass spetrometry (RP - HPLC/MS), DNA
sequencing, N - terminal and C - terminal sequencing, peptide mapping, circular
dichroism (CD) analysis, UV spectroscopy, one - dimensional nuclear magnetic
resonance spectroscopy (1D NMR), two - dimensional (2D) NMR, size exclusion
chromatography (SEC), isoelectric focusing (IEF), sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS – PAGE), and capillary zone electrophoresis.
Nonclinical PK / Tox Minimal toxicity data were needed on recombinant HGH
(rHGH) itself, since the clinical effects of HGH excess are well established and
understood and are extensively documented in published literature. Sandoz performed
toxicity studies to appropriately qualify impurities specifi c to Omnitrope,
that is, a subacute 14 - day rat study and a local (skin) tolerance study in rabbits.
Further, the bioactivity of Omnitrope was assessed using a validated weight gain
bioassay using a hypophysectomized (growth - hormone - defi cient) rats.
Clinical PK / PD HGH has a well - established mechanism of action. Omnitrope was
demonstrated to be pharmacokinetically and pharmacodynamically “ highly similar ”
to Genotropin. The dataset comprised a total of three PK/PD studies, including a
double - blind, randomized, two - way crossover study comparing Omnitrope and
Genotropin. Additionally, Sandoz conducted three sequential, multicenter phase 3
pivotal trials in 89 pediatric patients with GHD providing data in some patients for
up to 30 months. A fourth phase 3 trial ( n = 51, 24 months) was submitted as part
of its safety update. Collectively, these data in conjunction with the demonstrated
comparability to the reference listed product provide substantial evidence of Omnitrope
’ s safety and effectiveness.
Immunogenicity A signifi cant number of patients who were administered an
earlier version Omnitrope developed anti – growth hormone antibodies during the
fi rst and second phase 3 clinical trials. In response, Sandoz implemented changes to
the drug product to address this immunogenicity and evaluated the impact of these
changes clinically. Data from the 24 - month clinical study demonstrated that Omnitrope
has a low and acceptable level of immunogenicity (comparable to other
rHGH products) as none of the patients developed anti – growth hormone antibodies
during the duration of the study and only one patient developed anti – host cell
protein antibodies, which were of no detectable clinical consequence.
Conclusion to Case Study 2 This case provoked signifi cant challenges from interested
parties voiced via several citizen petitions [18] . Furthermore, the FDA ’ s delay
in approval prompted Sandoz to fi le suit to compel the FDA to rule on its application.
On April 10, 2006, the Washington, D.C., District Court ruled that the FDA
must meet its statutory obligations and take action on Sandoz ’ s outstanding NDA [6] .
On May 30, 2006, the FDA approved Omnitrope [somatropin (rDNA origin)] as a
“ follow - on protein product ” for use in the treatment of pediatric GHD. At the same
time, the FDA responded to the related citizen petitions and defended its position
that the data were adequate to demonstrate that Omnitorpe was suffi ciently similar
to Genotropin to enable reliance on the agency ’ s previous fi ndings of safety and
effi cacy for Genotropin. These data, in conjunction with the independent evidence
of safety and effi cacy provided by Sandoz, supported Omnitrope ’ s approval.
1.2.7.3 Case Study 3: Generic Salmon Calcitonin
On February 17, 2004, Nastech Pharmaceutical Company announced its fi ling of an
ANDA for a salmon calcitonin nasal spray drug product for the treatment of postmenopausal
osteoporosis. As with Fortical, Novartis ’ s Miacalcin was cited as the
reference listed drug; however, Nastech chose to submit an ANDA via the 505(j)(1)
route, rather than a 505(b)(2) application.
The distinction between the two regulatory routes has signifi cant implications for
FOPs. Whereas 505(b)(2) allows products to be “ suffi ciently similar, ” an ANDA
requires the applicant establish “ sameness ” of the active ingredients. The scope of
data necessary to demonstrate that the actives are the same is unclear. Additionally,
use of the ANDA route is appropriate for circumstances in which “ clinical studies
are not necessary to show safety and effectiveness. ” If clinical data are required as
proof of sameness, as in the previous example where clinical data were used to
demonstrate comparable immunogenicity, then the ANDA route may not represent
a viable regulatory path.
On July 10, 2006, Nastech was notifi ed by the FDA that its ANDA for
intranasal calcitonin salmon was not approvable at present based on concerns
relating to the potential for immunogenicity that might result from a possible
interaction between calcitonin salmon and chlorobutanol, the preservative in the
formulation.
Nastech has indicated it will continue to work with the agency to understand the
data requirements and regulatory options, but the fi nal resolution remains presently
unknown. This case study highlights the fact that demonstration of sameness of
therapeutic proteins is more complex than for other drugs and that true “ biogenerics
” may be hard to come by due to the complexity in establishing sameness versus
similarity.
1.2.8 SUMMARY AND CONCLUSIONS
This chapter provides an overview of the complex scientifi c, legal, and policy issues
facing the development of biogenerics today. Given the rising cost of health care
and prescription medications in this country and the pivotal and expanding role of
biologically derived products within the pharmaceutical landscape, these issues
present a challenge to industry, regulators, and legislators alike. Substantial progress
has already been made and the regulatory climate continues to evolve in response
to advancing science and technology. Recent FDA approvals provide insight into
the technical requirements for approval of well - characterized FOP products. They
also demonstrate the appropriate use of an abbreviated approval pathway, that is,
the 505(b)(2) pathway in place for drugs approved under the FDCA. Importantly,
recent legislative proposals seek to amend the PHSA to eliminate the current legal
barriers which prohibit abbreviated approval of protein biologics. This legislation
reaffi rms the need for the FDA to determine on a case - by - case basis the nature and
extent of supporting data required for a given product.
SUMMARY AND CONCLUSIONS 55
56 REGULATORY CONSIDERATIONS IN APPROVAL
REFERENCES
1. Congressional Budget Offi ce ( 1998 ), How Increased Competition from Generic Drugs
Has affected Prices and Returns in the Pharmaceutical Industry , Congressional Budge
Offi ce , Washington, DC .
2. Crawford , L. M. , Acting Commissioner of the Food and Drug Administration, in a Speech
to the Generic Pharmaceutical Association on February 26, 2005, available: http://www.
fda.gov/oc/speeches/2005/GPhA0301.html , accessed Apr. 23, 2005.
3. Comments of the Generic Pharmaceutical Association (GPhA) (Sept. 29, 2006), available:
http://www.gphaonline.org/AM/Template.cfm?Section=Media&Template=/CM/
HTMLDisplay.cfm&ContentID=2849 , accessed Jan. 23, 2007.
4. Schellekens , H. ( 2005 ), Follow - on biologics: Challenges of the “ next generation ” ,
Nephrol. Dial. Transplant . 20 ( Suppl. 4 ), iv31 – iv36 .
5. Congressional letter from Senators O. Hatch and H. Waxman to Andrew von Eschenbach,
Acting Commissioner of the Food and Drug Administration (Feb. 10, 2006), available:
http://www.henrywaxman.house.gov/news_letters_2006.htm , accessed Dec. 21, 2006.
6. Messplay , G. C. , and Heisey , C. ( 2006 ), Follow - on biologics: The evolving regulatory landscape
, Bioexec Int. , May, 42 – 45 .
7. Biotechnol. Law Rept. , 2003 , 22(5), 485 – 508 .
8. Comments from R. Williams, U.S. Pharmacopoeia (USP), to FDA Docket No. 2004N -
0355, Mar. 15, 2005 .
9. Herrera , S. ( 2004 ), Biogenerics standoff , Nat. Biotechnol. , 22 ( 11 ), 1343 – 1346 .
10. Scott , S. R. ( 2004 ) What is a biologic ?, Chapter 1 in Mathieu , M. , Ed., Biologics Development:
A Regulatory Overview , 3rd ed., Paraxel Intl. , Waltham, MA , pp. 1 – 16 .
11. Federal Food Drug and Cosmetic Act , available: http://www.fda.gov/opacom/laws/fdcact/
fdctoc.htm , accessed Apr. 21, 2005.
12. Public Health Service Act , available: http://www.fda.gov/opacom/laws/phsvcact/phsvcact.
htm , accessed Apr. 21, 2005.
13. U.S. Department of Health and Human Services, Food and Drug Administration Transfer
of Therapeutic Products to the Center for Drug Evaluation and Research , available:
http://www.fda.gov/cber/transfer/transfer.htm , accessed Apr. 23, 2005.
14. U.S. Department of Health and Human Services (DHHS) ( 1999 , Oct.), Food and Drug
Administration, Center for Drug Evaluation and Research , Guidance for Industry:
Applications covered by Section 505(b)(2), DHHS, Washington, DC.
15. U.S. Department of Health and Human Services, Food and Drug Administration, Omnitrope
(somatropin [rDNA origin]) questions and answers, available: http://www.fda.gov/
cder/drug/infopage/somatropin/qa.htm , accessed Dec. 21, 2006.
16. Letter of J. Woodcock. (CDER, FDA) to Docket Nos. 2001P - 0323/CP1, 2002P - 0447/CP1,
and 2003P - 0408/CP1 (Oct. 14, 2003 ).
17. Glidden , S. ( 2001 ), The generic industry going biologic , Biotechnol. Law Rept , 20 ( 2 ),
172 – 181 .
18. Letter from S. Galson (CDER, FDA) in response to Docket Nos. 2004P - 023 11CP1 and
SUP 1,2003P - 0 1 76lCP 1 and EMC 1, 2004P - 0171lCP1 and 2004N - 0355 (May 30, 2006 ).
19. U.S. Department of Health and Human Services, Food and Drug Administration, Center
for Drug Evaluation and Research, Offi ce of Pharmaceutical Science, Offi ce of Generic
Drugs , Electronic orange book: Approved drug products with therapeutic equivalence
evaluations, available: http://www.fda.gov/cder/ob/default.htm , accessed Apr. 21, 2005.
20. Schellekens , H. ( 2004 ), How similar do “ biosimilars ” need to be ? Nat. Biotechnol . 22 ( 11 ),
1357 – 1359 .
21. Webber , K. ( 2005 ), Relevant terminology. A presentation conducted at the Public Workshop
on the Development of Follow - On Protein Products, Sept. 14, 2004, available:
http://www.fda.gov/cder/meeting/followOn/followOnPresentations.htm , accessed Feb. 2,
2005.
22. Waxman , H. , Schumer , C. E. , and Clinton , H. R. (2006), Congress of the United States,
H.R. 6257, Access to Life - Saving Medicine Act, ” available: http://www.waxman.house.
gov/pdfs/bill_generic_biologics_9.29.06.pdf , accessed Sept. 29, 2006.
23. Mathieu , M. , and Evans , A. G. ( 2005 ), The FDA ’ s Orphan Drug Development Program ,
in Ed., New Drug Development: A Regulatory Overview , 7th ed., Paraxel Intl. , Waltham,
MA , pp. 307 – 317 .
24. U.S. Department of Health and Human Services (DHHS) ( 2003 , Sept.), Food and Drug
Administration, Center for Drug Evaluation and Research, Guidance for industry: Comparability
protocols — Protein drug products and biological products — Chemistry, manufacturing
and controls information , DHHS , Washington, DC .
25. Beatrice , M. ( 2002 ), Regulatory considerations in the development of protein Pharmaceuticals
, in Nail , S. , and Akers , M. , Eds., Development and Manufacture of Protein Pharmaceuticals,
Pharmaceutical Biotechnology , Vol. 14, Kluwer Academic/Plenum ,New York ,
pp. 405 – 457 .
26. Letter from R. Levy (Unigene) to FDA Docket No. 2004P - 0015 (Apr. 11, 2005 ).
27. Letter from S. Galson (CDER, FDA) in response to Docket No. 2004P - 0015/CP1 (Aug.
12, 2005 ).
28. Letter from N. Buc to FDA Docket No. 2004P - 0115/CP1 (Jan. 9, 2004 ).
29. FDA Week , 11(34), Aug. 26, 2005.
REFERENCES 57
59
1.3
RADIOPHARMACEUTICAL
MANUFACTURING
Brit S. Farstad 1 and Iv a n Pe n uelas 2
1 Institute for Energy Technology, Isotope Laboratories, Kjeller, Norway
2 University of Navarra, Pamplona, Spain
Contents
1.3.1 Introduction
1.3.1.1 Radiopharmacy
1.3.1.2 Characteristics of Radiopharmaceuticals
1.3.1.3 Ideal Characteristics of Radiopharmaceuticals
1.3.1.4 Radioactive Decay
1.3.1.5 Principles of Radiation Protection
1.3.1.6 Detection Devices for Clinical Nuclear Imaging
1.3.2 Product Development
1.3.2.1 Radionuclides
1.3.2.2 Carrier Molecules/Active Ingredients
1.3.2.3 Radiolabeling Techniques
1.3.2.4 Manufacturing Scale - Up
1.3.2.5 Automation
1.3.3 Manufacturing Aspects
1.3.3.1 Design of Manufacturing Sites
1.3.3.2 Design of Production Processes
1.3.3.3 Design of Production Equipment
1.3.3.4 Cleaning and Sanitation of Production Equipment
1.3.3.5 Environmental Control
1.3.3.6 Sterilization of Radiopharmaceuticals
1.3.3.7 Starting Materials
1.3.3.8 Labeling and Packaging
1.3.4 Product Manufacturing
1.3.4.1 Production of Radionuclides
1.3.4.2 Production of Radiopharmaceuticals
1.3.5 Quality Considerations
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
60 RADIOPHARMACEUTICAL MANUFACTURING
1.3.5.1 Documentation
1.3.5.2 Qualifi cation of Personnel
1.3.5.3 Quality Control
1.3.5.4 Validation and Control of Equipment and Procedures
1.3.5.5 Stability Aspects of Radiopharmaceuticals
1.3.6 Extemporaneous Preparation of Radiopharmaceuticals
References
Further Readings
1.3.1 INTRODUCTION
1.3.1.1 Radiopharmacy
Radiopharmacy is a patient - oriented science that includes the scientifi c knowledge
and professional judgment required to improve and promote health through assurance
of the safe and effi cacious use of radiopharmaceuticals. Radiopharmacy encompasses
studies related to the pharmaceutical, chemical, physical, biochemical, and
biological aspects of radiopharmaceuticals.
Radiopharmacy comprises a rational understanding of the design, preparation,
and quality control of radiopharmaceuticals, the relationship between
the physicochemical and biological properties of radiopharmaceuticals and
their clinical applications, as well as radiopharmaceutical chemistry and issues
related to the management, selection, storage, dispensing, and proper use of
radiopharmaceuticals.
1.3.1.2 Characteristics of Radiopharmaceuticals
A radiopharmaceutical is any medicinal product which, when ready for use, contains
one or more radionuclides (radioactive isotopes) included for a medicinal purpose.
This generic defi nition of radiopharmaceutical thus includes both diagnostic and
therapeutic radiopharmaceuticals.
A radiopharmaceutical can be as simple as a radioactive element such
as 133 Xe, a simple salt such as 131 INa, a small labeled molecule such as
l - ( S - [ 11 C]methyl)methionine, or a protein labeled with a radionuclide such as
99m Tc - labeled albumin or 90 Y - labeled monoclonal antibodies.
In clinical nuclear medicine, roughly 95% of radiopharmaceuticals are used with
diagnostic purposes. Radiopharmaceuticals are administered to the patients only
once, or a few times at most, in their lifetime. They contain minute amounts of active
ingredients, with a radionuclide somehow linked to or being the active ingredient
itself, with the main purpose of obtaining an image or a measure of their biodistribution.
Radiopharmaceuticals do not usually show any measurable pharmacodynamic
activity, as they are used in tracer quantities. Hence, there is no dose – response
relationship in this case and thus differs signifi cantly from conventional drugs.
Radiation is of course an inherent characteristic of all radiopharmaceuticals.
Hence, patients always receive an unavoidable radiation dose. In the case of therapeutic
radiopharmaceuticals, radiation is what produces the therapeutic effect.
The terms tracer, radiotracer , and radiodiagnostic agent , although long used as
equivalent to radiopharmaceutical, should be avoided. The preferred and correct
term is radiopharmaceutical , as the other names can be confusing or do not clearly
show the nature of these compounds as pharmaceuticals.
The composition of radiopharmaceuticals is not constant as it varies with time
as the radionuclide disintegrates. Very often, the half - life of the labeled molecule is
so short that it must be readily prepared just before its administration to the patient.
This implies in many cases the use of “ semimanufactured ” , such as radionuclide
generators, precursors, and cold kits that are also considered a medicinal product
according to directive 2001/83/EC.
1.3.1.3 Ideal Characteristics of Radiopharmaceuticals
Radiopharmaceuticals should have several specifi c characteristics that are a combination
of the properties of the radionuclide used as the label and of the fi nal radiopharmaceutical
molecule itself. The radiopharmaceutical should ideally be easily
produced (both the radionuclide and the unlabeled molecule) and readily available.
The half - life of the radionuclide should be adequate to the diagnostic or therapeutic
purpose for which it is designed. It has to be considered that radiopharmaceuticals
disappear from the organism by a combination of two different processes. The biological
half - life (showing the disappearance of a radiopharmaceutical from the body
due to biological processes such as metabolization, excretion, etc.) and the physical
half - life (due to the radioactive decay of the radionuclide). The combination of both
parameters gives the effective half - life :
T
TT
T T e
p b
p b
=
+
where T e is the effective half - life, T p the physical half - life, and T b the biological half -
life. Radiopharmaceuticals should have an effective half - life adequate to the use for
which they are intended. It should be short (hours) for diagnostic radiopharmaceuticals
(not longer than the time necessary to complete the study in question) and
longer for therapeutic radiopharmaceuticals (most often days) as the intended
effect should have a suffi cient duration.
The type of decay of the radiopharmaceutical should also be adequate for its
intended use. Diagnostic radiopharmaceuticals should decay by . emission, electron
capture, or positron emission, and never emit . or even . particles. On the contrary,
therapeutic radiopharmaceuticals should decay by . or . emission because the
intended effect is in fact radiation damage to specifi c cells.
Regarding the energy emission of diagnostic radiopharmaceuticals, the fi nally
produced . rays should be powerful enough to be detected from outside of the body
of the patient. The ideal energy for nuclear medicine equipment is around 150 keV.
. rays should be monochromatic and photon abundance should be high to decrease
the imaging time.
1.3.1.4 Radioactive Decay
Radionuclides are unstable nuclei that are stabilized upon radioactive decay. More
than 2000 unstable nuclides have been described so far, most of them radioactive.
INTRODUCTION 61
62 RADIOPHARMACEUTICAL MANUFACTURING
The stabilization process can proceed by several different processes, such as spontaneous
fi ssion, . - particle emission, . - particle emission, positron emission, . - ray
emission, or electron capture. In all decay processes the mass, energy, and charge of
radionuclides must be conserved, and many nuclides can decay by a combination
of any of the above - mentioned processes.
Fission is the process in which a nucleus breaks down into two fragments (thus
leading to two different new nuclides) with an emission of two or three neutrons
and a lot of energy. Spontaneous fi ssion is a rare process that can only occur in heavy
nuclei. Fission can also be produced by bombardment of certain nuclides with high -
energy particles (such as neutrons) and is in fact the nuclear process used for the
production of energy in nuclear energy plants by bombardment of highly enriched
uranium with neutrons.
The usual decay process of heavy nuclei is . - particle emission. An . particle is
a helium ion containing two protons and two neutrons. Alpha particles are heavy
particles that have a very short range in matter due to their mass, and radiopharmaceuticals
labeled with . emitters are used only with therapeutic purposes. Their
clinical use is very limited, and they are mainly used for research purposes or in
early phase clinical studies.
Radioactive nuclides that are neutron rich disintegrate by . decay. A . . particle
is originated by the conversion of a neutron into a proton, along with the emission
of an antineutrino to conserve energy in the decay process. Beta - emitting radionuclides
are also used in radiopharmaceuticals for therapeutic purposes.
Positron decay occurs in proton - rich nuclei. In this case, the positron (or .+ particle)
is originated by conversion of a proton into a neutron, along with the emission
of a neutrino to conserve the energy. Positrons are the antiparticle of electrons. In
a very fast process (10 . 12 s), emitted positrons collide with an electron of a nearby
atom and both particles disappear in a process called annihilation. The necessary
conservation of mass and energy accounts for the transformation of the mass of
both particles into energy, which is characteristically emitted in the form of two
511 - keV photons almost in opposite directions. Consequently, positron emitters are
used to label radiopharmaceuticals produced with diagnostic purposes by imaging.
Proton - rich nuclei can also decay by electron capture. In this process, an electron
from the innermost electron shell orbitals is captured into the nucleus and transforms
a proton into a neutron (and a neutrino is emitted for conservation of energy).
The vacancy created by the lost electron is fi lled by the transition of an electron
from a higher level orbital, and the energy difference between the intervening orbitals
is emitted as energy in the form of an X ray.
For any particular nucleus, several different energy states can be defi ned by
quantum mechanics. All the excited energy states above the ground state are referred
to as isomeric states and decay to the ground state by the so - called isomeric transition.
In . , positron, or electron - capture decay processes, the parent nucleus may
reach any of these isomeric states of the daughter nucleus. The energy difference
between the nuclear energy states can be emitted as . rays. A particular situation
for isomeric transition is that in which the excited state is long lived and is then
called the metastable state.
Radioactive Decay Equations, Magnitudes, and Units Radioactive decay is a
random process, being impossible to tell which particular atom from a group of atoms
will decay at a specifi c moment. It is then only possible to talk about the average
number of atoms that disintegrate during a certain period of time, giving the disintegration
rate ( . dN/dt ) of a particular radionuclide that is proportional to the total
number of radioactive atoms present at that time. This magnitude is usually called the
radioactivity (or mainly simply the activity) of a radionuclide and denoted by
A
dN
dt
N = . = .
where . is the decay constant and N the number of radioactive atoms. The previous
differential equation is mathematically solved leading to the exponential equation
N Ne t
t = .
0
.
where N t and N are the number of radioactive atoms present at time t = 0 and
t = t , respectively.
Radioactivity is expressed in becquerels (Bq), the Internationale System (SI) unit
for the magnitude A . One Becquerel is defi ned as one disintegration per second
(dps). Usual activities used in radiopharmacy are in the range of megabecquerels
or gigabequerels. There is (as usual) a non - SI unit called the curie (Ci). It was
initially defi ned in a trivial way as the disintegration rate of one gram of radium,
which was considered to be 3.7 . 10 10 dps. Thus the equivalence between the becquerel
and the curie is as follows:
1 27 10 1 37 11 Bq Ci Ci GBq = . = . .
The decay constant . is a specifi c characteristic of any single radionuclide, but being
related to probability, it is diffi cult to understand its meaning. Thus, a new magnitude
is defi ned: the half - life ( t 1/2 ), which is the time required to reduce the initial activity
of a radionuclide to one - half. In consequence, after one half - life the activity of a
radionuclide would be A /2, after two half - lives A /4, after three half lives A /8, and
so on.
The relationship between the decay constant . and the half - life t 1/2 can be derived
from the general radioactive decay equation
t1 2
2
/ = ln
.
An additional (and commonly misunderstood concept) is the mean life . ,which is
the average life of a certain group of radioactive atoms that is mathematically also
derived from the decay constant . as
.
.
= = = 1
2
1 44 1 2
1 2
t
t /
/ ln
.
1.3.1.5 Principles of Radiation Protection
Production, transportation, and use of radiopharmaceuticals, as radioactive products,
is governed by regulatory agencies dealing with radiation protection and
nuclear safety.
INTRODUCTION 63
64 RADIOPHARMACEUTICAL MANUFACTURING
In any case, and albeit the different regulation in different countries, as a general
principle only licensed personnel working in an authorized facility are authorized
to handle and use radiopharmaceuticals. Facilities and procedures are subject to
periodic inspection by offi cial radiation safety offi cers that control production and
handling of radioactive material, its transportation, proper use, as well as personnel
dosimetry and radioactive waste disposal.
The general principles of radiation protection are very simple:
Justifi cation. All procedures involving radioactive material must be justifi ed.
Optimization. The radiation exposure to any individual should be as low as reasonable
achievable. This principle is the widely known ALARA concept, an
acronym derived from as low as reasonable achievable.
Limitation. The radiation dose received by the personnel handling radioactive
material will never exceed the legally established dose limits. It has to be taken
into account that such limitations do not apply to patients receiving radiopharmaceuticals
as either diagnostic or therapeutic agents. But nuclear medicine
physicians, nuclear physicists, and radiopharmacists must ensure that the
amount of radiopharmaceutical administered to a patient is adapted to his or
her disease and optimized to obtained the intended result.
Operational Radiation Protection The fundamentals of operational radiation
protection (i.e., how to proceed when working with radioactive products) are based
on three factors: distance, time, and shielding. In any case, it is obvious that the
radiation hazard is increased with the activity of the radiation source, as can be
derived from the mathematical equation to calculate the exposure rate X given
by
X
A
d
= .
2
where A is the activity of the radiation source, . a constant that is characteristic of
every radionuclide, and d the distance to the source.
Distance should be increased as much as possible to decrease exposition and
exposure time should be reduced to a minimum. Adequate shielding (depending on
the radionuclide and its emission characteristics) should be used whenever possible
and handling of high activities should only be carried out by either automated
systems or proper manipulators.
1.3.1.6 Detection Devices for Clinical Nuclear Imaging
Diagnostic radiopharmaceuticals are mostly used for in vivo imaging of the biodistribution
of the radiopharmaceutical. Depending on whether . or positron emitters
are used, different devices are employed for clinical imaging. In any case, imaging
devices are based on detection of the high - energy photons coming from the body
of the patient upon administration and specifi c uptake of a radiopharmaceutical.
Advances in nuclear medicine imaging devices now permit in vivo noninvasive
imaging of such biodistribution and to obtain tomographic (i.e., three - dimensional)
images that can also give quantitative or semiquantitative information about the
amount of radiopharmaceutical and even its kinetics.
1.3.2 PRODUCT DEVELOPMENT
1.3.2.1 Radionuclides
When designing a radiopharmaceutical one should have in mind the potential
hazard the product may have to the patient. The goal must be to have maximum
amounts of photons with a minimum radiation exposure of the patient. For use in
therapy, . emitters and . emitters are particularly useful. For diagnostic purposes,
. emitters are most widely used. In general, those . emitters with a short physical
half - life and with a . energy between 100 and 300 keV are most widely used in
medical application, since these can easily be detected by standard . cameras.
However, positron emission tomography (PET) radiopharmaceuticals involve
short - lived radionuclides (positron emitters) giving a double set of photons at
511 keV each.
1.3.2.2 Carrier Molecules/Active Ingredients
The function of the carrier molecule is to carry the radioactivity to the target organ
and to make sure the radioactivity stays there. The uptake of radioactivity should
be as specifi c as possible in order to minimize irradiation of other organs and parts
of the body. This is particularly important when using radiopharmaceuticals for
therapy. But, also, for use in diagnostics, it is desirable that the radiopharmaceutical
is localized preferentially in the organ under study since the activity from nontarget
areas can obscure the structural details of the pictures of the target organ. It is
therefore important to know the specifi c uptake in an organ for a potential chemical
carrier and also the rate of leaking out of the organ/organ system. Thus, the target -
to - background activity ratio should be large. There are several approaches to develop
targeting radiopharmaceuticals. Radioimmunotargeting is one approach frequently
used for radiopharmaceuticals, where monoclonal antibodies (MAbs) or fractions
of MAbs are the carrier molecules for the radioactivity. These are binding specifi -
cally to receptors on cell surfaces in the target organs.
The target - binding surface of the cell has been well explored with a range of
tumor - associated and other antigens, identifi ed, and used for pathological tissue
characterizations.
The active analog approach in general, whereby a set of compounds is synthesized
so as to mimic features of a chosen natural compound, has been successful
[1] . The active analog approach includes the pharmacophore. The concept of the
pharmacophore is to look at features common to a set of drugs or compounds
binding to and acting on the same receptors.
1.3.2.3 Radiolabeling Techniques
When a labeled compound is to be prepared, the fi rst criterion to consider is whether
the label can be incorporated into the molecule to be labeled [2] . This may be
PRODUCT DEVELOPMENT 65
66 RADIOPHARMACEUTICAL MANUFACTURING
assessed from knowledge of the chemical properties of the two partners. Furthermore,
one needs to know the amount of each component to be added. This is particularly
important in tracer level chemistry and in 99m Tc chemistry.
In a radiolabeled compound, atoms, or groups of atoms of a molecule, are substituted
by similar or different radioactive atoms or groups of atoms. Saha [2] lists
six major methods employed in the preparation of labeled compounds for clinical
use: isotope exchange reactions, introduction of a foreign label, labeling with bifunctional
chelating agents, biosynthesis, recoil labeling, and excitation labeling. Among
these, three frequently used methods in radiopharmaceutical synthesis are briefl y
described below.
Isotope Exchange Reactions In isotope exchange reactions, isotopes of the same
elements having different mass numbers replace one or more atoms in a molecule.
Examples are labelling of iodide - containing material with iodine radioisotopes.
Since the radiolabeled and parent molecules are identical except for the isotope
effect, they are expected to have the same biological and chemical properties.
Introduction of a Foreign Label In this type of labelling, a radionuclide is incorporated
into a molecule primary by the formation of covalent or coordinated covalent
bonds. The tagging radionuclide is foreign to the molecule and does not label it
by exchange of one of its isotopes. Examples are 99m Tc – DTPA (Diethylenetriaminepentacetic
acid), 51 Cr - labeled red blood cells, and many iodinated proteins and
enzymes. In many compounds of this category, the chemical bond is formed by chelation.
In chelation, one atom donates a pair of electrons to the foreign acceptor atom,
which is usually a transition metal. Most of the 99m Tc - labeled compounds used in
nuclear medicine are formed by chelation.
Labeling by Bifunctional Chelating Agents In this approach, a bifunctional chelating
agent is conjugated to a macromolecule (e.g., protein) on one side and to a metal
ion by chelation on the other side. Examples of bifunctional chelating agents are
DTPA, metallothionein, diamide dimercaptide (N 2 S 2 ), and dithiosemi carbazone [2] .
There are two methods: the preformed radiometal – chelate method and the indirect
chelator — antibody method. Various antibodies are labelled by the latter, where
the bifunctional chelating agent is initially conjugated to a macromolecule, which is
then allowed to react with a metal ion, to form a metal – chelate – macromolecule
complex. Due to the presence of the chelating agent, the biological properties of
the labeled protein may be altered and must be assessed before clinical use.
1.3.2.4 Manufacturing Scale - Up
As the radiolabeled substances emerge from the laboratory to the clinics, there will
be a need for scaling up the batch size of the product. This can be done by increasing
either the total volume of the produced batches or the specifi c activity of the
product or both. When doing this, the following aspects should be considered:
The infl uence on the stability of the product itself due to possible radiolysis
The need for additional operator protection due to handling of increased amounts
of radioactivity
Product Stability The stability of a labeled compound is one of the major problems
in labeling chemistry. It must be stable both in vitro and in vivo. Many labeled
compounds are decomposed by radiation emitted by the radionuclides in them. This
kind of decomposition is called radiolysis. Radiation may also decompose the
solvent, producing free radicals that can break down the chemical bonds of the
labeled compounds (indirect radiolysis). In general, the risk of radiolysis increases
with higher specifi c activity of the product. In addition, the more energetic the radiation,
the greater is the radiolysis. Alpha emitters, leaving most of its energy close by
the molecules, and thus a high potential risk of radiolysis, give rise to major challenges
when scaling up is necessary.
Operator Radiation Protection Even for the largest commercial manufacturer of
radiopharmaceuticals, the batch volumes are small compared to nonradioactive
pharmaceuticals. So even a scaled - up production batch can be contained within a
limited space. When scaling up a radiopharmaceutical production, one always has
to assure that the radiation outside the contained work unit is acceptable for the
operator.
The production of a radiopharmaceutical will normally take place within a contained
box unit. Depending on the kind of radionuclides used and the amount of
radioactivity handled in the production process, the box units are shielded by lead
walls, typically 5 – 15 cm in thickness. When the box is used for production of radiopharmaceuticals
incorporating . - or . - emitting radionuclides, closed box units
without any lead coating are suffi cient. Working with these types of radionuclides
or with smaller amounts of . - emitting radioactivity, as in research scale, suitable
glove boxes can be used. When working with larger quantities of . - emitting radionuclides,
the material must be handled by either remote control equipment or
manipulator tongs incorporated in the wall.
1.3.2.5 Automation
Because of the unique operational and safety requirements of radiopharmaceutical
synthesis, the motivation for the development of automated systems is clear.
These unique constraints include short synthesis times and control from behind
bulky shielding structures that make both access to and visibility of radiochemical
processes and equipment diffi cult. The need for automated systems is particularly
expressed for PET radiopharmaceutical synthesis, with the short - lived
radionuclides emitting high - energy . photons at 511 keV. Automated synthesis
systems require no direct human participation. The short half - lives of the
PET radionuclides may require repeated synthesis during the day, thus being
a potential radiation burden for the operator when not using automated
systems.
Furthermore, radiopharmaceutical synthesis must be reliable and effi cient and
result in pharmaceutical - quality products. In addition, the processes must be well
documented and controlled. Automated systems may support all these challenges
and requirements.
One must keep in mind, though, that success in synthesis automation requires
fi rst and foremost innovative chemistry. PET radiosynthesis draws from a broad
chemistry knowledgebase rooted in synthetic organic chemistry [3] .
PRODUCT DEVELOPMENT 67
68 RADIOPHARMACEUTICAL MANUFACTURING
1.3.3 MANUFACTURING ASPECTS
1.3.3.1 Design of Manufacturing Sites
The manufacturing of radiopharmaceuticals is potentially hazardous. Both small -
and large - scale production must take place on premises designed, constructed, and
maintained to suit the operations to be carried out. Radiation protection regulations
stipulate that radionuclides must only be used in specially designed and approved
“ radioisotope laboratories. ” National regulations with regard to the design and classifi
cation of radioisotope laboratories must be fulfi lled. Such laboratories are normally
classifi ed according to the amount of the various radionuclides to be handled
at any time and the radiotoxicity grading given to each radionuclide. When planning
the layout of the laboratory, it is recommended to allocate separate working areas
or contained units for the various procedures to avoid possible cross - contamination
of radionuclides [4, 5] . Premises must be designed with two important aspects in
mind:
The product should not be contaminated by the operator.
The operator and the environment should be protected from contamination by
the radioactive product.
This is the basic principle of good radiopharmaceutical practice (GRPP).
One of the most important factors in planning a radioisotope laboratory is the
design of the ventilation system. Laboratories with medium and high grading must
be designed with the purpose of protecting the personnel from inhaling radioactive
gases or particles. The system should be designed to provide lower pressure at the
actual working area compared to the surrounding environment. Furthermore, the
system should have an appropriate number of air changes per hour and the replacement
air should be fi ltered. Air extracted from the area where radioactive products
are handled, though, should not be recirculated. Exhaust air to the environment
should be monitored for radioactivity, and it may be necessary to install active
charcoal fi lters to absorb radioactive gases and small particles [4] .
Aseptic production of radiopharmaceuticals, that is, when the products cannot
be terminally sterilized, will increase the requirements for the design and construction
of the premises. Contained workstations and clean - room technology will be
applied to a much higher degree. The general requirements for the design of such
premises are the same as for nonradioactive pharmaceuticals, including entry of staff
and the introduction of materials through air locks. The main difference is found in
the planning and design of the ventilation system. Laboratories for aseptic work
normally have a positive pressure relative to the surrounding areas. On the other
hand, in laboratories for work with radioactivity, it is good practice to have a negative
pressure to avoid the spread of radioactive material. In order to meet both
pharmaceutical and radiation protection requirements, it is necessary to balance
carefully the air pressures in the clean rooms, the air locks, and the surrounding
areas. From a pharmaceutical point of view a negative pressure in the area designated
for aseptic work can only be accepted in special cases. There are various ways
to meet the required balance between these apparently contradictory principles.
A frequently chosen solution is to use sealed production units or contained work
stations supplied with unidirectional airfl ow (UDAF) and with a lower pressure
compared to the aseptic laboratory. The laboratory itself may then have positive
pressure in relation to the surrounding premises.
Waste management is an important aspect when planning a radiopharmaceutical
manufacturing site. The key factor is to reduce the amount of radioactive waste to
a minimum. There should be a system for dividing the waste according to physical
half - life and radiotoxicity, both for solid and liquid waste. As an example, waste
containing . emitters is normally kept separately, when possible. National legislation
will vary considerably and infl uence the requirement that must be set for handling
of radioactive waste material.
1.3.3.2 Design of Production Processes
The design of a radiopharmaceutical production process depends very much upon
the kind of radiopharmaceutical to be made. Although most radiopharmaceuticals
are intended for parenteral use, also oral radiopharmaceuticals in different forms
are widely used. One must emphasize different factors when planning for production
of parenteral radiopharmaceuticals compared to oral radiopharmaceuticals.
Still, a common factor is the involvement of radioactive materials, and the radiation
protection of the personnel must always be an integral part of the design.
The production of a radiopharmaceutical will normally take place within a contained
box unit, consisting of either plastic walls or a combination of plastic and
stainless steel. The latter is more optimal for clean - room work. The box units may
be shielded by lead, either as large lead panels or as lead brick walls (see Figure 1 ).
Depending on the kind of radionuclides used and the amount of radioactivity
handled in the box, the walls are typically 5 – 15 cm in thickness. Shielded production
units like these are often called “ hotcells. ”
When the box is used for productions of radiopharmaceuticals incorporating . -
or . - emitting radionuclides, closed box units without any lead coating may be suffi -
cient. When handling radionuclides with mixed emitting properties, a possibility is
to concentrate the shielding to critical parts of the process. This can be done by use
of local shielding inside the production unit. However, for aseptic production, one
must keep in mind a potential disturbance of the airfl ow inside the box.
FIGURE 1 Shielding of box units (hot cells) with lead bricks. ( Photo courtesy of Institute
for Energy Technology .)
MANUFACTURING ASPECTS 69
70 RADIOPHARMACEUTICAL MANUFACTURING
Working with . and . radionuclides and also limited amounts of . - emitting
radioactivity, the boxes may be mounted with special protection gloves. When
working with larger quantities of . - emitting radionuclides, the material must be
handled by either remote control equipment or manipulator tongs incorporated in
the wall (see Figure 2 ).
The design of the elements and their assembly on the production unit should be
such that there are no radiation leaks at the interface.
When using lead bricks to construct the wall of the production unit, they should
have a special design. When they are stacked on top of each other, they should
interlock (see Figure 3 ). This is important to avoid cracks in the wall through which
radiation can escape.
Manipulator tongs are fi tted into the wall as part of a large tungsten sphere which
acts as a ball bearing and thereby allows more fl exibility for the movement of the
tong inside the box (see Figure 2 ). Lead glass windows, with good shielding properties,
are fi tted in the lead bricks to allow the operator to overlook the process.
When large lead panels are used, they should be reinforced with suitable steel
structures [International Organization for Standardization (ISO) 10648 - 1: 1997].
The surfaces of the lead shielding must be smooth and easy to clean. This can be
achieved by painting the surface of the wall.
FIGURE 2 Hot cells for manufacturing of larger quantities of . - emitting radionuclides.
( Photo courtesy of Institute for Energy Technology .)
FIGURE 3 Lead bricks are interlocked when they are stacked on top of each other. ( Photo
courtesy of Institute for Energy Technology .)
In general, the manufacturing of most radiopharmaceuticals consists of the
following:
Nuclear synthesis, synthesis of the radionuclide
Synthesis of the radiolabeled compound
Pharmaceutical formulation of the radiopharmaceutical
Nuclear Synthesis Except for radionuclides with ultrashort half - lives, like most
PET radionuclides, the production of these is normally performed well in advance
(see Section 1.3.4.1 ). Thus, the radionuclide is considered as a starting material and
must undergo controls as a starting material.
Synthesis of Radiolabeled Compound The complexity of a radiopharmaceutical
may differ greatly, with the radioactive element itself or simple salts as the less
complex. Very often, though, the radiolabel is part of a larger molecule, and thus a
radiolabeling procedure is required. This is part of the synthesis of the radiopharmaceuticals,
which also may involve chemical alteration of a precursor of the active
ingredient. Both labeling methods and synthesis may involve steps at elevated temperatures
or even cooling steps. Thus, equipment for heating or cooling must be part
of the production line. Furthermore, an important part of a synthesis is often the
purifi cation step, and equipment for this must be available. Typically, this is simple
chromatographic or ion exchange columns.
Planning of the process very much depends on the complexity of the process. In
general, keeping in mind the limited possibilities of direct handling of the materials,
it is important to keep the processes as simple as possible. For more complex processes,
automation may be the best solution, if available.
Pharmaceutical Formulation Even when the radiochemical part of a product is
simple, the radiopharmaceutical may be a complex solution. A pharmaceutical formulation
often contains additives in the form of buffers and preservatives: buffers
to keep the solution at a pH suitable for injection and preservatives to preserve the
integrity and effi ciency of the radiopharmaceutical.
Ideally, a solution for injection should be an isotonic solution with a neutral
(physiological) pH. However, the pH of a radiopharmaceutical is very important for
its stability, and for labelled compounds, the pH for optimal stability is not always
equivalent to physiological pH. For iodide solutions, the pH should be alkaline to
prevent loss of radioiodine. Reducing agents, such as thiosulfate, are often added to
radioiodide solutions to help this situation. A preservative can act as a stabilizer, an
antioxidant, or a bactericidal agent.
Some additives, like benzyl alcohol, are added for a double action. Benzyl alcohol
0.9% is widely used as a bactericide. In addition, benzyl alcohol reduces radiolysis
in radiopharmaceuticals and thus acts as a stabilizer.
1.3.3.3 Design of Production Equipment
The equipment used for manufacturing operation should be reserved exclusively
for radiopharmaceuticals [6] . Furthermore, two principles are of utmost importance
in the design of production equipment [4] :
MANUFACTURING ASPECTS 71
72 RADIOPHARMACEUTICAL MANUFACTURING
The equipment must be easy to repair after it has been installed in the production
unit.
The equipment must have a simple construction and be easy to assemble, so a
substitution can be done quickly when total renovation of the equipment is
necessary.
Glass is an important material in the construction of production equipment for
radiopharmaceuticals. This material will become discolored and brittle when affected
by radiation, and thus repair and/or change of parts of the equipment may be necessary.
Due to radioactive contamination of the equipment, repair and maintenance
can often be complicated, and time for decay must be included in the maintenance
period. To secure the continuous supply of products, it may be necessary to construct
two production lines in separate production units, where one is kept as a backup
facility.
Sometimes it will be necessary to substitute not only parts of a production line
but also the assembly of equipment as a whole. To facilitate this operation and
thereby reduce time and radiation exposure, it can be advantageous to build the
whole production line on a stainless steel support frame fi tted with simple connections
to electricity, water, and air supplies [4] . The complete withdrawal of a production
line from a box and the introduction of a new one can then be performed in a
very short time.
It is also important to keep in mind, when designing production equipment, that
all sense of touch is lost when fi ngers are replaced by remote handling tongs.
The design of the equipment must therefore be as simple as possible. On the
other hand, when using hot - cell units mounted with handling tongs, it may be favorable
to use more automated systems in the production line. Systems like these can
be run and controlled from steering panels outside the box unit.
Finally, equipment should be constructed so that surfaces that come in contact
with the product are not reactive, additive, or absorptive so as to alter the quality
of the radiopharmaceutical.
1.3.3.4 Cleaning and Sanitation of Production Equipment
Preparation equipment should be designed so it can be easily and thoroughly
cleaned. Procedures for cleaning, sanitation, and storage of production equipment
used in radiopharmaceutical production must be established. Special training is
necessary for personnel involved in this kind of work with regard to both clean -
room aspects and radiation protection aspects.
Before any equipment or materials used during production are removed from
the production unit, a check for radioactive contamination must be performed. After
removal, the equipment should be allowed to decay further in a special storage area
before it is cleaned and made ready for assembly again.
Glass equipment will normally be sterilized by dry - heat sterilization. Smaller
equipment, like plastic tubes and rubber stoppers, can be sterilized by autoclaving.
If available locally, also . irradiation may be a suitable method for sterilization
of equipment. One must keep in mind, though, that sterilization by irradiation may
change the composition of plastic and rubber materials. In addition, glass materials
may be discolored by . irradiation.
Production equipment that cannot be sterilized must be sanitized and disinfected
by an appropriate method. This can be done by use of biocides like alcohols (70%),
hydrogen peroxide, or formaldehyde - based chemicals or a combination of these.
These can either be used for surface disinfections by wiping or spraying or even
better by use of gas or dry fog systems for application of the disinfectants. The effect
of cleaning and sanitation should be monitored. Microbiological media contact
plates can be used to test critical surfaces, as inside the hot cells or glove boxes. The
test samples must then be handled and monitored as radioactive contaminated
units.
A system must be established for sanitation of all equipment before these are
transferred into clean areas.
1.3.3.5 Environmental Control
Workstations and their environment should be monitored with respect to radioactivity,
particulate, and microbiological quality. Active air sampling from production
units for radioactive products (hot cells or glove boxes) is subject to a safety consideration.
There is always a risk of bringing radioactive contaminated air outside
the workstation. To avoid the spread of radioactivity during the test, all possible
exhaust from the test equipment must be sampled and/or controlled.
A possible approach for testing of particulate and microbiological quality of air
inside the hot cells or glove boxes is to gain information about airborne particles
during simulated operations (without radioactivity).
The use of settle plates is common practice for monitoring of the microbiological
quality of air inside production units. These must then be placed as close a possible
to critical parts of the production process in order to show the real microbiological
burden to the product.
Warning systems must be installed to indicate failure in the fi ltered air supply to
the laboratory. Recording instruments should monitor the pressure difference
between areas where this difference is of importance.
1.3.3.6 Sterilization of Radiopharmaceuticals
Sterile radiopharmaceuticals may be divided into those which are manufactured
aseptically and those which are terminally sterilized. In general, it is advisable to
use a terminal sterilization whenever this is possible. Terminal sterilization is defi ned
as a process that subjects the combined product/container/closure system to a sterilization
process that results in a specifi ed assurance of sterility [7] . Since sterilization
of solutions normally means autoclaving (steam sterilization), one must assure that
the radiopharmaceutical product does not decompose when it is heated to temperatures
above 120 ° C. Many radiolabeled compounds are susceptible to decomposition
at higher temperatures. Proteins, such as albumin, are good examples of this. Others,
such as 18 F - fl uodeoxyglucose (FDG), can be autoclaved in some formulation but
not in others.
Furthermore, these processes take time, typically 20 – 30 min in total when heating
up to 121 ° C. For very short - lived product with a half - life of only a few minutes, this
is not an adequate method. On the other hand, these short - lived products are not
subject to any storage, and thus the risk of microbiological growth is more limited.
MANUFACTURING ASPECTS 73
74 RADIOPHARMACEUTICAL MANUFACTURING
Alternatively, a shorter cycle at a higher temperature might be used, assuming that
the temperature does not decompose the radiopharmaceutical.
If terminal sterilization is not possible, aseptic processing must be performed.
Aseptic processing is a process that combines presterilized materials and presterilized
equipments in a clean area.
Heating of radioactive solutions, particularly under elevated pressure (e.g., steam
sterilization), is also a matter of safety. In order to avoid any contaminated air to
escape if a container or a seal is broken, autoclaves used for radioactive solutions
should be placed inside negative - pressure sealed units. Autoclaves used for sterilizing
high - energy . - emitting radiopharmaceuticals should in addition be supplied with
proper lead shielding.
1.3.3.7 Starting Materials
As for manufacturing of other pharmaceuticals, a system should be established to
verify the quality of the starting materials used in manufacturing radiopharmaceuticals.
This system must assure that no material is used for production until it has
been released by a competent person [qualifi ed person (QP) or others given this
responsibility].
The starting materials as well as the packaging materials should be purchased
from qualifi ed vendors. It is recommended to use materials described in a pharmacopoeia,
whenever this is available. Supplier approval should include an evaluation
that provides adequate assurance that the material consistently meets
specifi cations.
Radionuclides involved in manufacturing radiopharmaceuticals must be considered
as starting materials. For very short - lived radionuclides, where batch analysis
is not possible, the validation of the production process of the radionuclide is of
utmost importance.
1.3.3.8 Labeling and Packaging
Packaging material should be purchased from qualifi ed vendors. Primary containers
and closures must be tested to verify that there are no interactions between the
radiopharmaceutical and packaging material during storage of the product.
Due to the risk of radiation exposure, it is accepted that most of the labeling of
the primary (direct) container is done prior to manufacturing. The empty vial can
be prelabeled with partial information prior to fi ltration and fi lling [6] . This procedure
should be designed so as to not compromise sterility or prevent visual inspection
of the fi lled vial. After fi lling of radioactive products, the primary containers
(vials) must be placed within a shielded container. These containers, which can be
made of lead or tungsten, vary in size and thickness depending on the amount of
radioactivity in the vial as well as the radiation properties of the radionuclide.
Radiopharmaceuticals containing . or . emitters may be placed in thin lead pots,
typically 2 – 4 mm in wall thickness. On the other hand, for vials containing regular
doses of high - energy . emitters, such as PET radionuclides, shielding with 3 – 5 cm
lead/tungsten may be needed.
Necessary information about the product must be given on the label of the lead
or tungsten container. Hence, there is no need to study the label on the direct con
PRODUCT MANUFACTURING 75
tainer. The name of the radiopharmaceutical, including the radionuclide, together
with the amount of radioactivity in the vial at a stated calibration time is part of the
necessary information. So is the expiry date of the product. Furthermore, the symbol
for radioactivity, designed as a black propeller, is obligatory on labels for radioactive
solutions.
When the products are intended for distribution and transport, the packaging
and labeling of the outer packages must be done according to the national regulation
of the country from which the shipments will depart, transfer, and arrive. The
outer packaging material must be properly tested in accordance with the type of
shipment, most frequently type A packages for radiopharmaceuticals. Furthermore,
the packages must be labeled with radionuclide data, such as type and amount of
radioactivity, along with the transport index (TI), which indicates the radiation from
the package at 1 m distance. While the information on the product itself (outside
the lead pot) is intended for the physicians, the information outside the package is
intended for the transport personnel.
1.3.4 PRODUCT MANUFACTURING
1.3.4.1 Production of Radionuclides
Radiopharmaceuticals are labeled with artifi cial radionuclides that are obtained by
bombardment of stable nuclei with subatomic particles or photons. Nuclear reactions
produced in such a way convert stable in unstable (radioactive nuclei). Several
kind of devices are used for such purposes, including nuclear reactors, particle accelerators,
and generators.
Various types of targets have been designed and used for both reactor and cyclotron
irradiation. In the design of targets, primary consideration is given to heat
deposition in the target by irradiation with neutrons in the reactors or charged
particles in the cyclotrons [2] . As the temperature can rise to 1000 ° C during irradiation
in both reactors and cyclotrons, the target needs proper cooling to avoid
burning. Most often, the targets are designed in the form of a foil to maximize the
heat dissipation. The target element should ideally be monoisotopic or an enriched
isotope to avoid extraneous nuclear reactions.
Nuclear Reactors Nuclear reactors are highly complex systems in which two kinds
of nuclear reactions are useful for the production of clinically useful radionuclides:
Neutrons produced by the fi ssion of heavy nuclides (such as 235 U or 239 Pu) are used
in a neutron capture (n, . ) reaction to produce an isotope of the same element
that is bombarded by the neutrons. Such reactions can be produced almost in all
elements with different probability. Examples of useful nuclear reactions are
130 Te(n, . ) 131 Te (which produces 131 I after emission of . particles with a half - life of
25 min), 50 Cr(n, . ) 51 Cr, 58 Fe(n, . ) 59 Fe, and 98 Mo(n, . ) 99 Mo. The second possibility for
the use of nuclear reactors is to use fi ssion reactions (n,f) in which a heavy nuclide
is broken down into two fragments. Many clinically relevant radionuclides can be
produced from thermal fi ssion of 235 U, such as 131 I, 117 Pd, 133 Xe, and 137 Cs. The isotopes
produced by this kind of fi ssion reaction must be separated and purifi ed by appropriate
chemical procedures, but since the chemical behavior of many different heavy
76 RADIOPHARMACEUTICAL MANUFACTURING
elements is similar, contamination can often become a problem in the isolation of
the radionuclide of interest.
As an example, and due to the particular interest of 99 Mo in radiopharmacy (as
it is the parent nuclide of 99m Tc in the 99 Mo – 99 mTc generator), the complex process
used to produce and purify 99 Mo is described below.
Molybdenum - 99 is produced by fi ssion of 236 U as follows:
235 1 236 99 135 1 2 U n U Mo Sn n + > > + +
After irradiation of the uranium target, it is dissolved in nitric acid and the fi nal
solution adsorbed on an alumina column that is washed with nitric acid to remove
uranium (and other fi ssion products). Molybdenum is fi nally eluted with ammonium
hydroxide and further purifi ed by absorption on an anion exchange column from
which ammonium molibdate is eluted with dilute hydrochloric acid after washing
the resin with concentrated HCl. The 99 Mo is obtained in no - carrier - added conditions,
and the most common contaminants can be 131 I and 103 Ru.
Particle Accelerators: Cyclotrons Both linear and circular particle accelerators
(cyclotrons) can be used, but the latter have many advantages and are mainly used
for the production of clinically relevant radionuclides.
A cyclotron is basically a cylinder - shaped high - vacuum chamber in which by
means of a magnetic fi eld and a radio - frequency system used to generate an alternating
electric fi eld, elemental particles can be accelerated to very high energies and
used as projectiles. The bombardment of stable elements loaded in a properly
designed target (either solid or fi lled with a liquid or a gas) induces different types
of nuclear reactions that fi nally lead to the production of radioactive elements.
Most cyclotrons accelerate negative particles (such as 2 H, 1 H, or even heavier
particles such as helium cations) that are stripped off the electrons in the stripping
foils that are used also to focus the beam on the target. As the energy of the incident
particle is increased, a much greater variety of nuclides can be produced.
When the nuclides produced have atomic numbers different from those of the
target elements, such preparations have no stable isotope of the intended element
and can be considered to be produced in no - carrier - added conditions.
The target material should ideally be monoisotopic to avoid the production of
extraneous radionuclides. However, in many cases this is not possible and only isotopically
enriched targets can be used, thus leading to the production of different
radionuclides. In this case appropriate methods must be used to separate the different
elements produced in the target.
An interesting concept that must always be taken into account in cyclotron -
produced radionuclides is the saturation activity characteristic of each target and
each nuclear reaction. The saturation activity is the activity of the radionuclide in
which the secular equilibrium is obtained between the activity produced in the
target and the disintegration of the radioisotope. The activity produced at a target
can be calculated by the equation
A A e t T = ..
S
/ A ( )( ) (ln ) 1 2 .
where A is the activity obtained for a radionuclide with a half - life of T after irradiation
of the target during a time of t at a current of . A microamperes. From the
PRODUCT MANUFACTURING 77
practical point of view, almost 97% of the saturation activity value is reached after
irradiation of the target for fi ve half - lives of the radionuclide. Longer irradiation
times do not produce signifi cant increases in the activity obtained. Methods to
obtain several cyclotron - produced radionuclides are described below.
Iodine - 123 can be produced either directly or indirectly in a cyclotron. Direct
reactions usually lead to 123 I contaminated with other iodine radioisotopes, such as
124 I or 125 I, due to side nuclear reactions. Using nuclear reactions such as 123 Te(p,n) 123 I,
122 Te(d,n) 123 I, or 124 (p,2n) 123 I produces 123 I that is obtained after dissolving the target
in hydrochloric acid by distillation into dilute NaOH.
In the indirect methods the radionuclide produced after bombardment of the
target is not 123 I, but a radionuclide that decays to 123 I with a short half - life. The most
widely used nuclear reactions produce 133 Xe (which decays to 123 I with a half - life of
2.1 h) by bombardment with high - energy 3 He or 4 He particles or 123 Cs (which decays
to 123 Xe with a half - life of 5.9 min, and then 123 Xe decays to 123 I) after irradiation of
124 Xe with high - energy protons. Complex processing and purifi cation processes must
be used to obtain 123 I in any of these cases, and adequate design and composition
of the target are critical to facilitate the process.
Thallium - 201 is obtained using an indirect reaction such as 203 Tl(p,3n) 201 Pb in
which 201 Pb decays to 201 Tl with a half - life of 9.4 h. Thallium - 201 can in this way be
obtained pure and free from other contaminants after several purifi cation steps and
letting the target product decay for 35 h.
Indium - 111 is produced by a direct nuclear reaction by irradiation of an 111 Cd
target with 15 - MeV protons. After irradiation the target is dissolved in HCl and
purifi ed in an anion exchange column.
Positron emission tomography has become a widely used diagnostic technique in
nuclear medicine. Ultrashort half - live radionuclides are used in these cases, and such
radionuclides are mostly obtained in small cyclotrons with high yields and short
irradiation times. The overall process will be described further in this chapter when
PET radiopharmaceuticals are described.
Generators A generator is constructed on the principle of the decay – growth relationship
between a parent radionuclide with longer half - life that produces by disintegration
a daughter radionuclide with shorter half - life. The parent and the
daughter radionuclide must have suffi ciently different chemical properties in order
to be separated. The daughter radionuclide is then used either directly or to label
different molecules to produce radiopharmaceutical molecules.
A typical radionuclide generator consists of a column fi lled with adsorbent material
in which the parent radionuclide is fi xed. The daughter radionuclide is eluted
from the column once it has grown as a result of the decay of the parent radionuclide.
The elution process consists of passing through the column a solvent that
specifi cally dissolves the daughter radionuclide leaving the parent radionuclide
adsorbed to the column matrix.
The main advantage of the generators is that they can serve as top - of - the - bench
sources of short - lived radionuclides in places located far from the site of a cyclotron
or nuclear reactor facilities.
A generator should ideally be simple to build, the parent radionuclide should
have a relatively long half - life, and the daughter radionuclide should be obtained
by a simple elution process with high yield and chemical and radiochemical purity.
The generator must be properly shielded to allow its transport and manipulation.
78 RADIOPHARMACEUTICAL MANUFACTURING
Several different generators are used in radiopharmaceutical procedures, but the
99 Mo/ 99m Tc is with great difference the most important generator of all of them and
will be described in detail later on in this chapter.
1.3.4.2 Production of Radiopharmaceuticals
More than 90% of the radiopharmaceuticals used in nuclear medicine are for diagnostic
use. PET radiopharmaceuticals, with their ultrashort half - lives, have become
a signifi cant part of this group of products. Hence PET investigation has been the
fastest growing imaging modality worldwide the last few years [8] .
Also for conventional radiopharmaceuticals used in diagnostic, it is favorable to
use products with short half - lives. Radionuclide generator systems are widely used
for supply of short - lived radionuclides/radiopharmaceuticals. Several generator
systems are available and routinely in use within nuclear medicine. Some of these
are listed in Table 1 .
Because of the short half - life, the coupling of the radionuclide to the carrier
molecule must be done immediately before the administration. Hence, there is a
need to have a constant supply of carrier molecules that can be labeled effi ciently
on site. For this purpose, several preparation kits have been developed.
Ready - for - use diagnostic radiopharmaceuticals which are intended for transport
over some distance typically include radionuclides with half - lives from 13 h and up.
Among these, products involving the radionuclide 131 I are used for both diagnostic
and therapeutic indications. This is based upon the mixed emitting properties of
the radionuclide, giving both . and . emission. The availability, price, and half - life
(8 days) of this radionuclide, together with the physical properties, have probably
made it the most commonly used radionuclide in radiotherapy. Although 131 I also is
frequently used for diagnostic purposes, the radiation characteristics of this radionuclide
are not really ideal for use in conventional scintigraphy (SPECT) due to
the high . energies. In addition, the . emission from this radionuclide gives the
patients an unnecessary radiation burden. Hence, other radionuclides are preferred
for use in diagnostic nuclear medicine.
The radioiodine 123 I, on the other hand, is very useful in nuclear medicine because
it has good radiation characteristics for scintigraphy, such as decay by electron
capture, a half - life of 13 h, and . emmision of 159 keV. However, the much shorter
half - life, together with the more complex radionuclide production, makes this radionuclide
less available and more expensive compared to 131 I.
There are several 131 I and 123 I radiopharmaceuticals on the market, for both oral
and parenteral administration. Ready - for - use radiopharmaceuticals that contain
TABLE 1 Several Radionuclide Generator Systems Useful in Nuclear Medicine
Parent Nuclide t1/2 Daughter Nuclide t1/2
68 Ge 280 days > 68 Ga 68 min
81 Rb 4.7 h > 81m Kr 13 s
99 Mo 66 h > 99m Tc 6 h
113 Sn 117 days > 113m In 100 min
188 W 69.4 days > 188 Re 17 h
PRODUCT MANUFACTURING 79
these radionuclides will normally be manufactured by radiopharmaceutical
companies and distributed to the marked according to a marketing authorization
(MA).
Although therapeutic application represents less than 10% of the nuclear medicine
investigations, therapeutic radiopharmaceuticals are a very important group
of radiopharmaceuticals. Hence, a brief description is outlined for production of
therapeutic radiopharmaceuticals following some other selected groups of
radiopharmaceuticals.
99 M o / 99m T c Generators The essential part of the most commonly available generator
system is a simple chromatography column to which the mother radionuclide is
absorbed on a suitable support material. The daughter radionuclide is a decay
product of the mother nuclide. Since it is the daughter nuclide that is used to label
the carrier molecules, it must be possible to separate this from the parent nuclide
by a chemical separation.
In a 99 Mo/ 99m Tc generator, the 99 Mo (molybdenum) is fi xed as molybdate to aluminum
oxide in the column. The daughter nuclide, 99m Tc (technetium), is eluted from
the column as pertechnetate when using saline solution. Molybdenum - 99 has a half -
life of 66 h, while 99m Tc has a half - life of 6 h. This is an ideal combination of half - lives,
giving a system where the daily supply of 99m Tc can easily be calculated from the
known amount of 99 Mo on the column. The half - life of 99m Tc, along with the radiation
characteristics of the nuclide, makes it excellent for use in nuclear medicine
imaging. After reconstitution of kits and formation of various radiopharmaceuticals,
this radionuclide is used in a major part of all nuclear medicine procedures.
Although the principle for the generators is similar, the design of 99 Mo/ 99m Tc
generators from different manufacturers can differ a lot. A drawing of a 99 Mo/ 99m Tc
generator is shown in Figure 4 . In general, the generator consists of a column with
adsorbent material where the radionuclide 99 Mo is applied. The column is combined
with a needle system necessary for the elution process. A sterile fi lter is fi tted on
the air inlet side of the needles to keep an aseptic system during elution. The saline
solution for elution may be supplied as a bulk solution suffi cient for several elutions
FIGURE 4 Typical radionuclide generator system ( ISOTEC, GE Healthcare, AS ).
1. Saline solution, volume: 5,10, or 15mL
2. Evacuated vial
3. Lead shield for eluate
4. Air filter (0.22 .m)
5. Special designed stainless steel needles
6. Glass column with Al2O3
7. Plastic container
8. Lead shield (min. 45-mm lead)
9. Laboratory shield (min. 50-mm lead)
80 RADIOPHARMACEUTICAL MANUFACTURING
or dispensed volumes suffi cient for a single elution. For both, vacuum is normally
used to run the elution of the column using sterile evacuated vials.
Finally, due to the relatively high radiation from 99 Mo, the system must be properly
shielded by either lead or a combination of lead and tungsten.
Whether the column is designed to contain liquid after and between elutions,
determine if this is a wet - column generator or a dry - column generator. When
liquid is retained at the column (wet generator), radiolysis of water on the column
may occur as a result . irradiation from 99 Mo. This may change the chemistry on
the column and thus reduce the yield when eluting the generator. Most commonly,
when manufacturing wet - column generators, oxidizing agents are added either to
the saline or to the column itself to avoid reduction of pertechnetate on the
column.
A radionuclide generator must be sterile and pyrogen free. Most commonly, the
generator is sterilized by autoclaving the entire column after the molybdate has
been bound to the aluminum oxide. Other critical procedures during the production
and the assembly of the generator must be performed under aseptic conditions.
Elution of the generator must also be carried out under aseptic conditions while
using only sterile accessories.
Other Generators Of the generators listed in Table 1 , two systems are of particular
interest in nuclear medicine today along with the 99 Mo/ 99m Tc generator, namely the
68 Ge/ 68 Ga generator and the 81 Rb/ 81m Kr generator.
68 G e / 68 G a Generator Germanium - 68 has a half - life of 271 days, and 68 Ga (gallium)
a half - life of 68 min. Gallium - 68 is a PET emitter, and this generator system is a
valuable source of a short - lived radionuclide in a radiopharmacy or nuclear medicine
department. However, the system is not as easy or effi cient as the 99 Mo/ 99m Tc
generator. On the other hand, the longer half - life of the mother nuclide allows use
of the system for several months.
This generator can be made up of aluminum loaded on a plastic or glass column.
Carrier - free 68 Ge in concentrated HCl is neutralized in ethylenediaminetetraacetic
acid (EDTA) solution and adsorbed to the column. Then 68 Ga is eluted from the
column with 0.005 M EDTA solution. Alternatively, 68 Ge is adsorbed on a stannous
dioxide column and 68 Ga is eluted with 1 N HCl [2] .
81 R b / 81m K r Generator Rubidium - 81 has a half - life of 4.6 h and decays to 81m Kr
(krypton) by electron capture. Krypton - 81m has a half - life of 13 s and decays by
isomeric transition emitting . rays of 190 keV. Being an inert gas 81m Kr is used for
lung ventilation study.
The parent 81 Rb is adsorbed on an ion exchange resin, and the daughter 81m Kr is
eluted with air. Because of the very short half - life of 13 s, the studies can be repeated
every few minutes, and no radiation safety precaution for trapping 81m Kr is needed
[2] .
Radiopharmaceutical Kits Radiopharmaceutical kits are nonradioactive ( “ cold ” )
products containing the sterile ingredients needed to prepare the fi nal radiopharmaceutical.
Immediately before administration to the patient, the radionuclide is
added. From the point of licensing, these semimanufactured products are defi ned
as radiopharmaceuticals, as they have no other application in medicine [2] .
PRODUCT MANUFACTURING 81
Most of these preparation kits have been developed for labeling of various substances
with 99m Tc. Labeling is normally a single - or two - step procedure consisting
of adding a solution of 99m Tc - pertechnetate to the preparation kit. The preparation
kit contains the ingredient necessary for labeling, such as the substance or ligand to
be labeled, a reducing agent, buffers for pH adjustments, and various stabilizers. The
reducing agent, very often a stannous salt, is added to bring the radionuclide into a
valence state with high reactivity.
Most preparation kits are lyophilized, and the reason for this is to extend the
shelf life of the products. Some preparation kits can in fact be stored for more than
one year. Since these products are not radioactive, conventional clean rooms and
clean - room technology can be applied for production of preparation kits. Most of
these products have to be produced aseptically, as they cannot be sterilized with
other methods. During lyophilization of the preparation kits used for 99m Tc labeling,
it is very important to remove all the oxygen from the kit vial. This is to ensure the
right valence of the tin salt. Normally, the vials are fi lled with an inert gas, such as
nitrogen, before the vials are closed completely. It is important, though, that the gas
is dried. Some manufacturer chose to not completely replace the removed oxygen,
giving a slightly negative pressure inside the kit vial. This may be favorable for the
kit - labeling procedure.
Therapeutic Radiopharmaceuticals Radiopharmaceuticals used for therapy
(radiotherapy) are designed such that, after administration, they act locally at a
target by either damaging or killing cells by irradiation. One of the attractions of
radionuclide therapy is the existence of radiation with quite different dimensions
of effectiveness, ranging from subcellular (Auger electrons) to hundreds of cell
diameters ( . particles). In between, . emitters have a tissue range equivalent to
only a few cell diameters [9] . Alpha emitters have a very high linear energy transfer
(LET), being very potent at short distances.
Table 2 lists a selection of radionuclides and radiopharmaceuticals used in
radiotheraphy.
TABLE 2 Selected Radionuclides and Radiopharmaceuticals Used for Radiotherapy in
Routine Use or as Part of Clinical Investigations
Radionuclide Mode of decay t1/2 Radiopharmaceuticals
131 I . . / . 8.04 days 131 I - NaI, 131 I - MIBG, 131 I - mAbs
90 Y . . 2.7 days 90 Y - colloid, 90 Y - DOTATOC, 90 Y - mAbs
186 Re . . / . 3.8 days 186 Re - sulfi de, 186 Re - HEDP
188 Re . . / . 17 h 188 Re - HEDP
177 Lu . . / . 6.6 days 177 Lu - DOTA - Tyr3 - octreotide
153 Sm . . / . 1.9 days 153 Sm - EDTMP
89 Sr . . 50.6 days 89 Sr - chloride
223 Ra . / . 11.4 days 223 Ra - chloride
211 At . 7.2 h 211 At - mAbs
213 Bi . 46 min 213 Bi - mAbs
166 Ho . . / . 26.8 days 166 Ho - colloid
169 Er . . 9.4 days 169 Er - citrate colloid
165 Dy . . / . 2.3 h 165 Dy - ferric hydroxide macroaggregate
32 P .. 14.3 days 32 P - ortho - phosphate
82 RADIOPHARMACEUTICAL MANUFACTURING
Pure . and . emitters are easy to shield, and thus production involving these can
be performed in sealed production units with no lead protection. One must keep in
mind, though, the potential hazard when inhaling some of these materials. Moreover,
many radionuclides used for radiotherapy have an additional . component.
Hence, local lead shielding may be necessary. If the . component is larger or represents
very high energy emission, a total lead shielded unit may be necessary. The
latter will be the case when manufacturing 131 I radiopharmaceuticals for therapy,
since 131 I is a radionuclide consisting of a high - energy . photon together with the .
component.
Radiopharmaceuticals for therapeutic use must have a high target - to - background
ratio. Targeted radiotherapy involves the use of molecular carrier such as a
receptor - avid compound or an antibody to deliver a radionuclide to cell
populations.
A challenge when performing radiolabeling of carrier molecules for targeted
radiotherapy is the potential risk of radiolysis due to the radiation characteristics
of the radionuclides involved. When increasing the specifi c activity, as part of the
scaling up, the risk of radiolytic decomposition of the labeled compound also
increases. This is particularly pronounced when using . emitters. The addition of
stabilizers in the form of scavengers can reduce this risk. Benzyl alcohol is an
example of a compound that acts as a scavenger by catching up with free radicals
in the solution.
Another approach is to use kit formulations also for this kind of product. Therapeutic
radiopharmaceuticals have been developed where the carrier molecule is
formulated in a lyophilized kit and supplied together with the radionuclide. An
example of this is the MAb ibritumomab tiuxetan formulated for labeling with the
. - emitting radionuclide 90 Y. Yttrium - 90 ibritumomab tiuxetan (Zevalin) is used in
the treatment of non - Hodgkin ’ s lymphoma (NHL).
The labeling is performed in a centralized radiopharmacy, hospital radiopharmacy,
or nuclear medicine department immediately before use.
Radioactive Sanitary Products Radioactive sanitary products could be considered
as radiopharmaceuticals according to the defi nition given in directive 2001/83/
EC, although there are signifi cant differences between radioactive sanitary products
and classical radiopharmaceuticals. The former can in fact be considered as
encapsulated radioactive sources, although with the use of microencapsulated sanitary
products (such as micrometer - sized glass or polymer beads loaded with a
radionuclide), the difference between both types is becoming more diffi cult to
establish.
In any case, radioactive sanitary products are delivered locally (and not systemically
or orally) for the local treatment of a disease. The idea is to give a high dose
of radiation to a specifi c part of the body by the implantation of the corresponding
sanitary product in the desired zone. The sanitary product must not be metabolized,
destroyed, or removed from the place it has been located during a suffi ciently long
time as to give the desired high radiation dose.
The most commonly used radioactive sanitary products are millimeter - sized
seeds or needles loaded with 103 P, 192 Ir, 90 Sr, or 125 I. Currently micrometer - sized or
even nanometer - sized beads loaded with 90 Y are being used for the treatment of
specifi c diseases.
PRODUCT MANUFACTURING 83
PET Radiopharmaceuticals PET radiopharmaceuticals are labeled with short -
lived positron - emitting radionuclides. Such radionuclides can either be produced in
a cyclotron or obtained from an appropriate radionuclide generator.
General Considerations The synthesis of PET radiopharmaceuticals has several
peculiarities substantially different from the procedures followed to prepare conventional
. - emitting radiopharmaceuticals. A very important issue that must be
considered is the specifi c activity. For all radiopharmaceuticals it is usually very high
and can be calculated from the formula
A
k
AT e
/
=
1 2
where A e is the specifi c activity, A the mass number of the radionuclide, and T 1/2 its
half - life. It is then clear that the achievable specifi c activity is higher for radionuclides
with shorter half - lives, as is the case for the most relevant PET radionuclides
( 18 F and 11 C). As an example, 18 F produced in no - carrier - added conditions can be
obtained with specifi c activities of almost 10 10 Ci/mmol, resulting in PET radiopharmaceuticals
with extremely high specifi c activities.
For PET radiopharmaceuticals we must always consider that synthesis processes
must be extremely fast. Consequently, synthesis schemes with as few steps as possible
must be used, and each of the steps must proceed with high effi ciency. The
incorporation of the radionuclide to the molecule should ideally be done in the fi nal
steps of the synthesis. In this way two objectives can be achieved: reduce the overall
synthesis time (thus increasing the yield) and reduce the number of side reactions
and secondary undesired products obtained during the synthesis.
The synthesis of PET radiopharmaceuticals is always carried out at very small
scale (only a few dozen micrograms of the radiopharmaceutical are obtained) and
each batch can sometimes only be used for a single patient or a few patients at most.
Consequently, there is always a big excess of the precursor in the reaction medium,
and proper purifi cation systems must be used to get rid of all the possible contaminants.
Such systems must also be very effi cient and fast, and the most usual is to
apply either semipreparative high - performance liquid chromatography (HPLC) or
solid - phase extraction - based procedures.
The position of the radionuclide in the molecule of interest is also critical as it
will affect the biological behavior of the radiopharmaceutical. Chemical reactions
must be designed to be stereospecifi c in many cases, as the production of a mixture
of different stereoisomers complicates the purifi cation of the fi nal radiopharmaceutical.
Synthesis procedures must also be easy to automate, as very elevated activities
are used for the synthesis of PET radiopharmaceuticals (several curies usually) and
appropriate radiation protection systems must be used.
PET Generators Table 3 summarizes the characteristics of some PET generators.
So far, the most widely used system has been the 82 Sr/ 82 Rb generator, although due
to the specifi c physical and chemical characteristics of the daughter radionuclide
and the half - life of the parent radionuclide, the 68 Ge/ 68 Ga generator is probably one
of the most interesting systems. Recent advances in gallium chemistry have permitted
the development of 68 Ga radiopharmaceuticals of clinical interest making
84 RADIOPHARMACEUTICAL MANUFACTURING
available PET studies at stand - alone PET centers without a cyclotron with other
compounds different from the classical 18 FDG.
PET Cyclotrons Cyclotrons used to produce positron emitters of clinical interest
(see Tables 4 and 5 ), mainly 18 F and 11 C, do not need to be very big. In fact, small
devices installed in hospital or academic institutions have long been used for such
purposes (see Figure 5 ). These devices are easy to operate and maintain, and even
with single - particle low - energy cyclotrons, it is possible to produce multicurie
amounts of 18 F and 11 C.
Some Positron Emitters of Clinical Interest Fluorine - 18 is undoubtedly the most
widely used positron - emitting radionuclide. This is mainly due to the wide use of
18 FDG, the PET radiopharmaceutical that has permitted PET to become an everyday
clinical tool. With the exception of 18 FDG and probably 18 FDOPA, the use of
other 18 F - labeled radiopharmaceuticals is very limited. However, the chemical and
physical characteristics of 18 F are excellent:
TABLE 4 Physical Characteristics of Some Positron
Emitters of Clinical Use
Isotope T1/2 (min) % E. + (keV)
11 C 20.4 99.7 960
13 N 9.9 99.8 1198
15 O 2.0 99.9 1732
18 F 109.6 96.7 634
TABLE 5 Nuclear Reactions for Production of Most
Widely Used Positron Emitters
11 C 13 N 15 O 18 F
14N(p,a)11C 16O(p,a)13 N 14 N(d,n) 15 O 18 O(p,n) 18 F
10 B(d,n) 11 C 13 C(p,n) 13 N 15 N(p,n) 15 O 20 Ne(d, . ) 18 F
11 B(d,2n) 11 C 12 C(d,n) 13 N 16 O( . ,pn) 18 F
11 B(p,n) 11 C 19 F(p,pn) 18 F
12 C(p,pn) 11 C
Note: Most common reactions used in small cyclotrons are bolded.
Different energies of the incident particle are needed for the different
nuclear reactions
TABLE 3 Selected of PET Generators
Generator Parent T1/2 Daughter T1/2
Fe/Mn 52 Fe 8.27 h 52m Mn 21.1 min
Zn/Cu 62 Zn 9.13 h 62 Cu 9.73 min
Ge/Ga 68 Ge 270.8 days 68 Ga 68.3 min
Sr/Rb 82 Sr 25.6 days 82 Rb 76.4 s
PRODUCT MANUFACTURING 85
It can easily be produced in very high quantities (up to 7 – 9 Ci per batch) even
in small cyclotrons with just a few hours irradiation time.
The mean positron emission energy of 18 F is just 0.64 MeV (the lowest of all
positron emitters with clinical use) and this has several important consequences:
The dose of radiation received by the patient will be lower and the
distance between disintegration of the radionuclide and the annihilation site
(after collision of the positron with an electron) is reduced, thus making PET
images with higher resolution possible.
The half - life of 18 F (109 min) is suffi ciently long to carry out complex synthesis
procedures, apply long PET imaging protocols, and carry out metabolite analysis.
Furthermore, it is possible to produce the radiopharmaceutical in a laboratory
and transport it to a distant site only equipped with an imaging device.
These kinds of “ satellite PET centers ” have boomed all around the world and
permitted the fast expansion of PET as an everyday clinical tool in certain
pathologies (mainly in oncological diseases).
Fluorine is not common in biological molecules, but many drugs contain this
atom. Fluorine and hydrogen have quite similar radii, and changing a hydrogen to
a fl uorine atom in a molecule does not usually generate substantial steric differences
between both molecules. Nonetheless, the electronegativity of fl uorine is usually
FIGURE 5 Small (less than 2 m in diameter) dual - beam negative ion cyclotron capable of
easily producing multicurie amounts of 18 F and 11 C. ( Photo courtesy of PET - CUN Center,
University of Navarra .)
86 RADIOPHARMACEUTICAL MANUFACTURING
going to change substantially the physicochemical properties of the molecule (reactivity,
hydrogen bonding, interactions with cognate receptors, metabolization, etc.).
It is not possible to assume that the biological behavior of a molecule and its fl uorinated
analog is going to be similar. On the contrary, it is advisable to fi nd substantial
differences in lipophilicity, biodistribution, protein binding, affi nity for receptors,
and so on. However, such modifi cations are in many cases very useful to permit the
use of a 18 F - fl uorinated analog as a PET radiopharmaceutical. In fact, that is the
case for the most widely use one: FDG. This compound, which accounts for probably
more than 90% of the PET studies performed in the world every day, is a glucose
analog that is taken up by the cells by GLUT transporters and metabolized just as
glucose at the very fi rst steps of glicolysis. But as a consequence of the change of
the C 2 OH group in natural glucose by a 18 F atom in FDG, the latter cannot be
isomerized (once phosphorilated) and suffers metabolic trapping being specifi cally
accumulated in tumoral cells.
Carbon - 11 has a very short half - life (just 20.4 min) but the chance to substitute
a carbon atom in any biological molecule by a positron - emitting 11 C is a very interesting
possibility. This has led to a substantial development of 11 C - labeled tracers.
The short half - life conditions everything and only PET centers equipped with a
cyclotron can have a clinical program with 11 C tracers. The production of the radiopharmaceutical
must in these cases be performed just before the imaging study and
is usually not started until the patient is already on the PET scanner.
The 12 C – 11 C substitution will produce chemically identical molecules and give the
chance to study many biological processes by this noninvasive methodology and can
also be used in new - drug research and development (R & D).
Synthesis of PET Radiopharmaceuticals Albeit the requirements for the synthesis
of PET radiopharmaceuticals previously described, the synthesis process could conceptually
be reduced to a very simple scheme, as shown in Figure 6 .
The concept is really simple, but there are considerable diffi culties in each of the
steps. In many cases it is diffi cult to synthesize a properly designed cold precursor
that will permit a simple direct reaction with few secondary products. No modifi ca-
FIGURE 6 General reaction scheme for synthesis of PET radiopharmaceuticals. The precursor
molecule (A) is designed with the adequate protecting groups ( ) and a reactive
leaving group ( . ). A reactive form of the radionuclide ( ) is covalently joined to the precursor
at the reaction site, while the leaving group is eliminated. An intermediate radioactive
product (B) is obtained that is hence deprotected (2) to produce the fi nal radiopharmaceutical
(C). A fast and effi cient purifi cation process of C is needed to get read of unreacted cold
precursor, radionuclide, and intermediate products.
PRODUCT MANUFACTURING 87
tions in the confi guration of the chiral centers should be produced during the overall
process and a simple purifi cation system able to purify the fi nal product in a very
short time should be found. Additionally, all the reactions should be very fast (just
several minutes at most) and be easy to automate to be performed in a computer -
controlled device placed in a shielded hot cell.
Production Process and Quality Control The production process includes the
following:
• Production of the radionuclide in the cyclotron and sending it to the PET
radiopharmaceutical laboratory
• Reaction of the radionuclide with an appropriate cold precursor, either in solution
or in solid phase
• Purifi cation of the radiopharmaceutical, usually by semipreparative radio
HPLC or solid - phase extraction
• Formulation of the fi nal product as an injectable solution (frequently including
phase change in a rotary evaporator) and the adjustment of tonicity and pH
• Sterile fi ltration or autoclaving
The quality control of the fi nal product must be carried out before release of the
batch (except for the sterility and the endotoxin tests for extremely short - lived
radionuclides). Consequently, all procedures must not only be very fast but also very
accurate, and in all cases it is very important to have a properly established quality
assurance system that might permit parametric release of the produced batches. The
quality control assays that must be carried out in the radiopharmaceutical includ
the following:
• Radionuclidic purity
• Radionuclidic identity
• Chemical purity
• Radiochemical purity
• Specifi c activity
• Residual solvents
• Visual inspection
• Tonicity
• pH
• Sterility
• Endotoxin
A PET radiopharmaceutical laboratory must include the cyclotron bunker (where
positron - emitting radionuclides are produced), the production laboratory, the
quality control laboratory, and several different ancillary areas.
In the production laboratory all synthesis and purifi cation processes are carried
out in remote - operated fully automated computer - controlled systems (synthesis
modules, see Figure 7 ) located in heavily shielded hot cells (see Figure 8 ). Dispensing
of individual doses is in many cases also carried out by automated systems.
88 RADIOPHARMACEUTICAL MANUFACTURING
1.3.5 QUALITY CONSIDERATIONS
1.3.5.1 Documentation
Good documentation constitutes an essential part of the quality assurance system.
As claimed in the European Community (EC) Guide to Good Manufacturing Practice
(GMP), Chapter 4: “ Clearly written documentation prevents errors from spoken
communications and permits tracing of batch history. ” In general, the requirements
for documentation related to manufacturing of pharmaceuticals, as set in the GMP
FIGURE 7 Automated synthesis module for PET radiopharmaceutical synthesis located in
a shielded hot cell. ( Photo courtesy of PET - CUN Center, University of Navarra .)
FIGURE 8 Production laboratory for PET radiopharmaceuticals. The 10 - cm lead shielded
hot cells contain computer - controlled automated synthesis modules. ( Photo courtesy of PET -
CUN Center, University of Navarra .)
regulations, are also valid for manufacturing of radiopharmaceuticals. A recent draft
proposal of EC GMP Annex 3, “ Manufacture of Radiopharmaceuticals, ” outlines
the following regarding this issue:
All documents related to the manufacture of radiopharmaceuticals should
be prepared, reviewed, approved, and distributed according to written
procedures.
Specifi cations should be established and documented for raw materials, labeling
and packaging materials, critical intermediates, the fi nished radiopharmaceutical,
and any other critical material.
Acceptance criteria should be established for the radiopharmaceutical, including
criteria for release and shelf life specifi cations.
Records of major equipment use, cleaning, sanitization or sterilization, and maintenance
should show the product, batch number, date and time, and signatures
of the persons involved.
Records should be retained for at least three years unless another time frame is
specifi ed in national requirements.
It is of utmost importance to have a system for implementing such documents.
Any new master document or a new version of such a document must be followed
by a training process for relevant operators. This training must be recorded as
well.
The recording of production data will make it necessary to bring batch documentation
into the radioisotope laboratory. Hence, it is important to have routines that
minimize the risk for radioactive contamination of the documents and to ensure
that any contaminated documents will not leave the controlled area. Today, the use
of computers instead of paper documents in the laboratory leaves most of the
paperwork outside the controlled area.
1.3.5.2 Qualifi cation of Personnel
As a general principle in GMP, there should be suffi cient qualifi ed personnel to
carry out all the tasks that are the responsibility of the manufacturer. Furthermore,
individual responsibilities should be clearly understood by the individuals and
recorded.
For personnel working with radiopharmaceuticals, training and qualifi cation
should cover general principles of GMP and radiation protection. This includes also
personnel in charge of cleaning premises and equipment used for this type of production.
All manufacturing operations should be carried out under the responsibility
of a QP with additional competence in radiation protection.
1.3.5.3 Quality Control
All quality control procedures that are applied to nonradioactive pharmaceuticals
are in principle applicable to radiopharmaceuticals. In addition, tests for radionuclidic
and radiochemical purity must be carried out. Furthermore, since radiopharmaceuticals
are short - lived products, methods used for quality control should
QUALITY CONSIDERATIONS 89
90 RADIOPHARMACEUTICAL MANUFACTURING
be fast and effective. Still, some radiopharmaceuticals with very short half - lives may
have to be distributed and used after assessment of batch documentation even
though all quality control tests have not been completed. It is acceptable, though,
for these products to be released in a two - stage process, before and after full analytical
testing. In this case there should be a written procedure detailing all production
and quality control data that should be considered before the batch is dispatched.
A procedure should also describe the measures to be taken by the QP if unsatisfactory
test results are obtained after dispatch (GMP, Annex 3).
The quality control tests fall in two categories: biological tests and physiochemical
tests. The biological tests establish the sterility and apyrogenicity, while the
physiochemical tests include radionuclidic, chemical, and radiochemical purity tests
along with determination of pH, osmotic pressure, and physical state of the sample
(for colloids).
For lyophilized preparation kits containing reducing agents, such as 99m Tc kits, a
test for moisture content can be necessary. Residual water in the freeze - dried pellet
may lead to oxidation of the reducing agent.
Radionuclidic Purity Radionuclidic purity is defi ned as the fraction of the total
radioactivity in the form of the desired radionuclide present in a radiopharmaceutical.
Radionuclide impurities may arise from impurities in the target material or from
fi ssion of heavy elements in the reactor [2] . In radionuclide generator systems, the
appearance of the parent nuclide in the daughter nuclide product is a radionuclidic
impurity. In a 99 Mo/ 99m Tc generator, 99 Mo may be found in the 99m Tc eluate due to
breakthrough of 99 Mo on the aluminum column. The presence of these extraneous
radionuclides increases the radiation dose to the patient and may also obscure the
scintigraphic image.
Radionuclidic purity is determined by measuring the characteristic radiations
emitted by individual radionuclides. Gamma emitters are distinguished from another
by identifi cation of their . energies on the spectra obtained from a NaI crystal or a
Ge (germanium) detector. This method is called . spectroscopy.
Pure . emitters are not as easy to check as the . emitters. However, they may be
checked for purity with a . spectrometer or a liquid scintillation counter.
Radiochemical Purity The radiochemical purity (RCP) of a radiopharmaceutical
is the fraction of the total radioactivity in the desired chemical form in the radiopharmaceutical.
Radiochemical impurities arise from decomposition due to the
action of solvent, change in temperature or pH, light, presence of oxidizing or reducing
agents, and radiolysis [2] . Examples of radiochemical purity are free 99m Tc -
pertechenetate and hydrolyzed 99m Tc in labeled 99m Tc radiopharmaceuticals. The
presence of radiochemical impurities in a radiopharmaceutical results in poor -
quality images due to the high background from the surrounding tissues and blood.
It also gives the patient unnecessary radiation doses.
A number of analytical methods are used to detect and determine the radiochemical
impurities in a given radiopharmaceutical. Most commonly used are
methods like paper (PC), thin - layer (TLC), and gel chromatography, paper and gel
electrophoresis, HPLC, and precipitation. A common principle for the different
methods is that they can chemically separate the different radiolabeled components
in the radiopharmaceutical. It may sometimes be necessary to perform more than
one test method, for instance, TLC and HPLC, to get a complete picture of the different
radiochemical impurities. Alternatively, one can use one chromatographic
method consisting of a constant stationary phase but varying the mobile phase
(solvent). An example is the radiochemical purity test of 99m Tc - methylenediphosphate
(MDP), a radiolabeled phosphate used in bone scintigraphy. When using two
TLC systems, one with sodium acetate as a solvent and one with methyl ethyl ketone
(MEK) as a solvent, the different 99m Tc compunds in the product can be determined.
A small aliquot of the radiopharmaceutical preparation is spotted on an instant
thin - layer chromatography (ITLC) strip. The strip is dipped into the chromatography
fl ask while keeping the spot above the solvent. During the chromatography
process, the different components of the sample distribute differently in the ITLC
strip, depending on the solubility and polarity of the components. In systems like
this, each component is characterized by an R f value, defi ned as the ratio of the distance
traveled by the component to the distance the solvent front has advanced
from the original point of application of the test material. The distribution of the
radioactive components on the strips can be monitored by use of an appropriate
device for measuring radioactivity and printed in a chromatogram. Figure 9 shows
typical chromatograms for 99m Tc - MDP in the TLC systems described above.
Chemical Purity The chemical purity of a radiopharmaceutical is the fraction of
the material in the desired chemical form. Chemical impurities may arise from the
breakdown of the material either before or after labeling. Chemical impurities may
also arise from the manufacturing process, such as aluminum in a 99m Tc eluate,
coming from the aluminum column on the generator. Residuals of solvent from the
radiopharmaceutical synthesis are also considered as chemical impurities. If the
chemical impurity is present before labeling, the result may be undesirable labeled
molecules. Furthermore, chemical impurities may cause a toxic effect. High -
performance liquid chromatography and gas chromatography (GC) are important
methods for determination of chemical impurities in a radiopharmaceutical.
FIGURE 9 Typical chromatograms for 99m Tc - MDP. The left strip and chromatogram are
obtained with ITLC - SG in sodium acetate. The right strip and chromatogram are obtained
in methyl ethyl ketone (MEK). When combining these, any free pertechnetate ( 99m TcO 4 . )
and/or hydrolyzed 99m Tc can be detected. Thus the fraction representing 99m Tc - MDP (RCP)
can be calculated.
L1 L2
TC-MDP + Hydr. Tc
TC-MDP + Tc04–
==> RCP =100%
3954
2966
1978
991
3
0.0
O F F
51.5 103.0 154.5 206.0
26054 1 1
19540
13027
6514
0
0.0
O
51.5 103.0 154.5 206.0
Counts
Counts
Distance (mm) Distance (mm)
QUALITY CONSIDERATIONS 91
92 RADIOPHARMACEUTICAL MANUFACTURING
Sterility and Pyrogen Testing Sterility indicates the absence of any viable bacteria
or microorganisms in a radiopharmaceutical preparation. Hence, sterility testing is
performed to prove that radiopharmaceuticals are essentially free of viable microorganism.
The test for microbial contamination of these products is best carried out
with fi lter methods. It is a great advantage to incubate only the fi lters instead of the
radioactive solutions.
The test is performed according to the Ph.Eur/USP monograph on Sterility tests
[13, 14] , but with an important modifi cation. Small batch sizes, typical for radiopharmaceuticals,
make it necessary to use smaller test volumes than required in the
monographs. Also the risk for radiation exposure supports this modifi cation.
All radiopharmaceuticals for human administration are required to be pyrogen
free. Also the tests for apyrogenicity must be modifi ed when applied for these products.
The classical rabbit test for pyrogens was never a convenient test for parenteral
radiopharmaceuticals. Practical problems due to radioactive rabbits and the need
for larger test volumes made this a diffi cult task. Today, the Limulus amebocyte test
(LAL) is the method of choice and has been accepted by the Ph. monographs for
many years. This test is normally done within an hour, compared to several days for
the rabbit test.
However, even the LAL test may be too time consuming for the very short lived
PET radiopharmaceuticals. Hence, less time consuming methods are in progress and
will probably improve this situation. Meanwhile, it is accepted that the test for apyrogenicity,
like the sterility test is for most radiopharmaceuticals, is fi nished after
release of the most short lived radiopharmaceuticals.
Bubble Point Testing of Filters Parenteral radiopharmaceuticals that are not terminally
sterilized must undergo a sterile fi ltration process as part of the aseptic
production procedure. Although the supplier certifi es the fi lters used, they must be
checked for integrity after use to assure that there has been no leakage during the
fi ltration. The integrity of the fi lter may be demonstrated by bubble point testing . In
this test, the fi lter is placed and monitored under controlled pressure. When the test
is done on wet fi lters, the pressure needed to push gas through the fi lter is defi ned
as the bubble point. A fi lter with given pore width has a corresponding bubble point
value. Most frequently, sterile fi ltration is performed by 0.22 - . m fi lters; hence the
bubble point is about 3 – 4 bars. However, the fi lter supplier should specify the bubble
point valid for a specifi c fi lter.
Since this is an in - process test, special caution must be given to radiation protection.
The test equipment should be placed within a closed and shielded unit and a
system should be in place to collect any radioactive spill from the test.
When the fi lter integrity test fails, the sterile fi ltration process must be rejected.
Visual Inspection of Finished Product As part of the quality control, all parenterals
will be subject to an inspection for the possible content of particles. Visual
inspection of radiopharmaceuticals is more complicated than for other pharmaceuticals,
as radiation protection guidelines strongly discourage any direct eye contact
with radioactive sources. Normally, the visual inspection of a radiopharmaceutical
is performed by placing the vial on a rotating station connected to a camera. The
station is properly shielded, and the operators can study the solution on a distant
screen.
1.3.5.4 Validation and Control of Equipment and Procedures
Preventive maintenance, calibration, and qualifi cation programs should be operated
to ensure that all facilities and equipment used in the manufacture of radiopharmaceuticals
are suitable and qualifi ed (GMP, Annex 3). Special emphasis should be put
on critical equipment for handling of radiopharmaceuticals, such as dose calibrators
that are used to check the accuracy of the dispensing of patient doses. Particular
programs are outlined for checking the dose calibrator, including constancy, accuracy,
linearity, and geometry. The general principles of validation outlined in the
GMP regulations are valid for radiopharmaceuticals as well as for other pharmaceuticals.
All validation activities should be planned and clearly defi ned and documented
in a validation master plan (VMP). Special emphasis should be given on
the validation of aseptic processes in the production of radiopharmaceuticals.
Studies, including media fi ll tests, must be performed and recorded to demonstrate
maintenance of sterility throughout the production process. This is particularly
important since most radiopharmaceuticals are dispatched and used before the
sterility test is fi nished.
1.3.5.5 Stability Aspects of Radiopharmaceuticals
As discussed already, radiopharmaceuticals are exposed to stability problems, particularly
when radiolabeled compounds are involved. Decomposition of labeled
compounds by radiolysis depends on the specifi c activity of the radioactive material,
the energy of the emitted radiation, and the half - life of the radionuclide. Particles,
such as . and . radiation, are more damaging than . rays, due to their short range
and local absorption in matter. The stability of a compound is time dependent on
exposure to light, change in temperature, and radiolysis. The longer a compound is
exposed to these conditions, the more it will tend to break down.
Stabilizers such as ascorbic acid and benzyl alcohol may be added to inhibit or
delay the decompostion. Many preparations are stored in the dark under refrigeration
to slow down the degradation of the material [2] . The expiry date of a radiopharmaceutical
is based upon data from stability studies designed to demonstrate
the described effects on the product after storage.
Hence, for most stability studies on radiolabeled compounds, the radiochemical
purity and pH are the most important physiochemical parameters to study. Moreover,
for parenteral radiopharmaceuticals, a stability study also has to demonstrate
the maintenance of sterility and apyrogenicity after storage.
1.3.6 EXTEMPORANEOUS PREPARATION OF
RADIOPHARMACEUTICALS
An extemporaneous preparation is defi ned as a product which is dispensed immediately
after preparation and not kept in stock [10] . Hence, many radiopharmaceuticals
could fall into this category due to their limited shelf life.
The use of extemporaneous preparation should be limited to situations where
there is no product with marketing authorization (MA) available. This could be
prepared based upon a prescription for a named patient (magistral preparation) or
a production based upon a formula and prepared on a regular basis. The latter is a
EXTEMPORANEOUS PREPARATION OF RADIOPHARMACEUTICALS 93
94 RADIOPHARMACEUTICAL MANUFACTURING
common situation for many radiopharmaceuticals. For radiopharmaceuticals with
short half - lives or rare indications, no sizable commercial market exists. Consequently,
no pharmaceutical company will be prepared to obtain a MA for a product
that will not yield a profi t due to these limitations. Still, there is a need from a
medical point of view to have such products available. For radiopharmaceuticals
incorporating radionuclides with a physical half - life of only a few minutes, only
local production is feasible. They are therefore prepared in hospital pharmacies or
laboratories and supplied for individual or small numbers of patients on a daily
basis.
The extemporaneous preparation of radiopharmaceuticals is regulated on a
national level, and hence this regulation may differ from country to country. The
Pharmaceutical Inspection Convention (PIC/S) has drafted a guide to good practices
for preparations of medicinal products in pharmacies [10] , valid for medicinal
products that do not have a MA, prepared extemporaneously or for stock. For
medicinal products prepared to a larger extent or for use in clinical trials, industrial
GMPs are applicable. Although the suggested guide outlines a general principle
according to GMP, different requirements are particularly evident when it comes to
documentation and quality control testing. There is also a discussion about the
grades of background environment needed for production, with a differentiation
between products with shelf lives less than or longer than 24 h [10] . While aseptic
manufacturing according to industrial GMP has to be performed in grade A with a
grade B background, this proposed guide opens for a relaxation to this. For an
aseptic preparation of a product with a shelf life of less than 24 h, using a biohazard
safety cabinet (BSC), the background environment may be grade D. Even for products
with a shelf life longer than 24 h, an extensively documented procedure may
allow grade C in background, as long as grade B clothing is worn. In general, the
referred draft guide is based much upon a risk related approach and is graduated,
depending on the size and type of prepared medicinal products.
As to documentation for extemporaneous prepared products, the proposed guide
set as a minimum requirement to specify the name, strength, and expiry date of the
product. If a product is prepared for a single patient (magistral production), it is
assumed that no end product testing will be required. For radiopharmaceuticals,
though, the activity in each dose must be measured before administration. Chemical
and microbiological quality control is not required for products that have a shelf
life of 24 h or less, provided that frequent process validation is performed. In addition,
chemical and microbiological information must be available to justify the shelf
life for the product.
For products that are prepared extemporaneously at a regular basis or even for
a limited stock, a product specifi c documentation (product fi le) is needed. This will
include specifi cations, instructions, and records but also a pharmaceutical assessment
of safety data, toxicity, biopharmaceutical aspects, stability, and product design.
The product fi le should also include a product review as soon as a product is used
repeatedly or over longer periods.
Furthermore, the drafted guide suggests that the level of end - product testing
for those products will depend on the associated risk connected to the scale of
operation, shelf life of the product, frequency of preparation, as well as type of
product (parenterals, orals) and type of facility where the product has been
prepared.
Independent of which regulation applies at a national level to extemporaneous
or magistral preparation of radiopharmaceuticals, the patients should be entitled to
expect that these products are prepared accurately, are suitable for use, and will
meet the expected standards for quality assurance. Pharmacists involved in this kind
of production must ensure that they and any other staff involved are competent to
undertake the tasks to be performed and that the requisite facilities and equipment
are available [11] . As for other radiopharmaceutical production, systems must be in
place to ensure the operator safety due to handling of radioactive materials. All
involved staff must have suffi cient training in radiation safety issues, in addition to
training in GMP.
REFERENCES
1. Britton , K. ( 1996 ), Radiopharmaceuticals for the future , Curr. Dir. Radiopharma. Res.
Dev . (Ed. by Stephen Mather ), viii. Developments in Nuclear medicine, Vol XXX. London,
UK.
2. Saha , G. B. ( 1998 ), Fundamentals of Nuclear Pharmacy , 4th ed., Springer , Heidelberg,
Germany .
3. Alexoff , D. L. Automation for the synthesis and application of PET radiopharmaceuticals,
BNL - 68614 Offi cinal File Copy.
4. Bremer , P. O. ( 1995 ), Aseptic production of radiopharmaceuticals , in Aseptic Pharmaceutical
Manufacturing , Vol. II, Application for the 1990s , Interpharm , Michael J. Groves and
Ram Murty , pp. 153 – 180 .
5. Nordic Council on Medicines . ( 1989 ), Radiopharmacy: Preparation and Control of Radiopharmaceuticals
in Hospitals , NLN Publications No. 26 , Uppsala, Sweden .
6. European Commision ( 2003 ), EU Guide to Good Manufacturing Practice , Annex 1 and
3, Brussels, Belgium, October 8.
7. Dabbah , R. ( 1995 ), Controlled environments in the pharmaceutical and medical products
industry: A global review from regulatory, compendial, and industrial perspectives , in
Aseptic Pharmaceutical Manufacturing, Vol. II, Application for the 1990s , Interpharm ,
Michael J. Groves and Ram Murty , pp. 11 – 40 .
8. Lee , M. C. , PET and PET/CT are the fastest growing imaging modalities worldwide, paper
presented at the 5th International Conference on Isotopes (5ICI), Brussels, Belgium, Apr.
25 – 29 , 2005 .
9. Zalutsky , M. R. , Pozzi , O. , and Vaidyanatha , G. , Targeted radiotherapy with alpha particle
emitting radionuclides, paper presented at the International Symposium on Trends in
Radiopharmaceuticals (ISTR -2005), Vienna, Austria, Nov. 14–18, 2005.
10. Pharmaceutical Inspection Convention (2006, Aug.), PIC/S guide to good practices
for preparation of medicinal products in pharmacies, PE 010 - 1 (Draft 2), Geneva ,
Switzerland .
11. Standards for good professional practice ( 2000 ), Pharm. J . 265 ( 7109 ), 233 .
12. Kowalsky , R. J. , and Falen , S. W. ( 2004 ), Radiopharmaceuticals in Nuclear Pharmacy and
Nuclear Medicine , American Pharmacists Association , Forrester Center, WV .
FURTHER READINGS
European Commision . ( 2006 ), EU Guide to Good Manufacturing Practice , Annex 3; draft
proposal, Brussels, Belgium, Apr. 12.
FURTHER READINGS 95
96 RADIOPHARMACEUTICAL MANUFACTURING
Rootwelt , K. ( 2005 ), Nukle . rmedisin , 2nd ed. Gyldendal Norsk Forlag AS , Oslo, Norway .
Schwochau , K. ( 2000 ), Technetium: Chemistry and Radiopharmaceutical Applications , VCH
Verlagsgesellschaft Mbh , Weinheim, Germany .
Welch , M. J. , and Redvanly , C. S. , Eds. ( 2002 ), Handbook of Radiopharmaceuticals , Wiley ,
Hoboken, NJ .
ASEPTIC PROCESSING
SECTION 2
99
2.1
STERILE PRODUCT
MANUFACTURING
James Agalloco 1 and James Akers 2
1 Agalloco & Associates, Belle Mead, New Jersey
2 Akers Kennedy & Associates, Kansas City, Missouri
Contents
2.1.1 Introduction
2.1.2 Process Selection and Control
2.1.2.1 Formulation and Compounding
2.1.2.2 Primary Packaging
2.1.2.3 Process Objectives
2.1.3 Facility Design
2.1.3.1 Warehousing
2.1.3.2 Preparation Area
2.1.3.3 Compounding Area
2.1.3.4 Aseptic Compound Area (If Present)
2.1.3.5 Aseptic Filling Rooms and Aseptic Processing Area
2.1.3.6 Capping and Crimping Sealing Areas
2.1.3.7 Sterilizer Unload (Cooldown) Rooms
2.1.3.8 Corridors
2.1.3.9 Aseptic Storage Rooms
2.1.3.10 Lyophilizer Loading and Unloading Rooms
2.1.3.11 Air Locks and Pass - Throughs
2.1.3.12 Gowning Rooms
2.1.3.13 Terminal Sterilization Area
2.1.3.14 Inspection, Labeling, and Packaging
2.1.4 Aseptic Processing Facility Alternatives
2.1.4.1 Expandability
2.1.5 Utility Requirements
2.1.5.1 Water for Injection
2.1.5.2 Clean (Pure) Steam
2.1.5.3 Process Gases
2.1.5.4 Other Utilities
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
100 STERILE PRODUCT MANUFACTURING
2.1.6 Sterilization and Depyrogenation
2.1.6.1 Steam Sterilization
2.1.6.2 Dry - Heat Sterilization and Depyrogenation
2.1.6.3 Gas and Vapor Sterilization
2.1.6.4 Radiation Sterilization
2.1.6.5 Sterilization by Filtration
2.1.7 Facility and System: Qualifi cation and Validation
2.1.8 Environmental Control and Monitoring
2.1.8.1 Sanitization and Disinfection
2.1.8.2 Monitoring
2.1.9 Production Activities
2.1.9.1 Material and Component Entry
2.1.9.2 Cleaning and Preparation
2.1.9.3 Compounding
2.1.9.4 Filling
2.1.9.5 Stoppering and Crimping
2.1.9.6 Lyophilization
2.1.10 Personnel
2.1.11 Aseptic Processing Control and Evaluation
2.1.11.1 In - Process Testing
2.1.11.2 End - Product Testing
2.1.11.3 Process Simulations
2.1.12 Terminal Sterilization
2.1.13 Conclusion
Appendix
References
Additional Readings
2.1.1 INTRODUCTION
The manufacture of sterile products is universally acknowledged to be the most
diffi cult of all pharmaceutical production activities to execute. When these products
are manufactured using aseptic processing, poorly controlled processes can expose
the patient to an unacceptable level of contamination. In rare instances contaminated
products can lead to microbial infection resulting from products intended to
hasten the patient ’ s recovery. The production of sterile products requires fastidious
design, operation, and maintenance of facilities and equipment. It also requires
attention to detail in process development and validation to ensure success. This
chapter will review the salient elements of sterile manufacturing necessary to
provide acceptable levels of risk regarding sterility assurance.
Commensurate with the criticality associated with sterile products, the global
regulatory community has established a substantial number of the basic requirements
that fi rms are expected to adhere to in the manufacture of sterile products.
The most extensive of these are those defi ned by the Food and Drug Administration
(FDA) in its 2004 Guideline on Sterile Drug Products Produced by Aseptic Processing
and the European Agency for the Evaluation of Medicinal Products (EMEA)
Annex 1 on Sterile Medicinal Products [1, 2] . Substantial additional information is
available from the International Organization for Standardization (ISO), the Parenteral
Drug Association (PDA), and the International Society for Pharmaceutical
Engineering (ISPE) (see Appendix ) [3] . The organizations have provided a level of
practical, experience - based detail not found in the regulatory documents, thereby
better defi ning practices that are both compliant with regulatory expectations and
based upon rational, evidence - based science and engineering.
Consideration of patient risk associated with pharmaceutical production emerged
largely from regulatory impetus, by which the regulatory community stated its
intended goal to structure its inspectional process using patient safety as a major
focus in determining where to allocate their inspectional and review resources.
Emanating from the International Conference on Harmonization (ICH) efforts to
produce a harmonized approach to pharmaceutical regulation, risk - based compliance
has been adopted in Europe, Japan, and the United States [4, 5] . Sterile products,
especially those made by aseptic processing, have been properly identifi ed as
a high priority by the global regulatory community. Several risk analysis approaches
have been developed that can help the practitioner review practices with the goal
of minimizing risk to the patient [6 – 8] .
2.1.2 PROCESS SELECTION AND DESIGN
The production of sterile products is profoundly impacted both by formulation and
the selection of primary packaging components. Design parameters for a facility and
selection of appropriate manufacturing technologies for the product require that
the formulation process and packaging components be chosen and evaluated in
advance.
2.1.2.1 Formulation and Compounding
The vast majority of parenteral formulations are solutions requiring a variety of
tankage, piping, and ancillary equipment for liquid mixing (or powder blending),
fi ltration, transfer, and related activities. Suspensions, ointments, and other similar
products, including the preparation of the solutions for lyophilized products, can be
manufactured in the same or very similar equipment. The scale of manufacturing
can vary substantially, with the largest batches being well in excess of 5000 L (typically
for large - volume parenteral production), down to less than 50 mL for radiopharmaceuticals
or biologicals customized for a particular patient.
The majority of this equipment is composed of 300 series austenitic stainless steel,
with tantalum or glass - lined vessels employed for preparation of formulations sensitive
to iron and other metal ions. The vessels can be equipped with external jackets
for heating and/or cooling and various types of agitators, depending upon the mixing
requirements of the individual formulation. In many facilities, a variety of tank sizes
are available for use. Larger facilities may have the high - capacity tanks permanently
installed and permanently connected to process utilities. Smaller vessels are generally
mobile and positioned in individual processing booths or rooms as needed.
PROCESS SELECTION AND DESIGN 101
102 STERILE PRODUCT MANUFACTURING
After sterilizing fi ltration (or sterilization by heat or other means), comparably sized
vessels are sometimes utilized to contain the product prior to and during the fi lling
process. These holding vessels are often steam sterilized along with the connecting
piping prior to use. There are a number of fi rms that fi ll directly from the compounding
vessel using in - line fi ltration eliminating the intermediate vessel. When this
approach is used, a small moist - heat - sterilized surge tank or reservoir tank may be
required, particularly with modern time – pressure fi lling systems. This practice may
reduce initial facility and equipment cost but places additional constraints
on operational fl exibility. The use of disposable equipment for compounding and
holding of sterile formulations is coming into greater use. This eliminates the cleaning
of vessels prior to reuse, but confi rmation of material compatibility is required.
Disposable equipment is often used with products manufactured in small to moderate
volumes, and while reducing initial equipment expenses disposable equipment
also results in contaminated waste, which cannot be recycled or reused and must be
treated appropriately.
Aseptic compounding as required for suspensions and other formulations in
which open - vessel processes are required mandate an ISO 5 environment providing
ideally > 400 air changes/hour in which these steps can be performed with minimal
opportunity for adventitious contamination. This could be accomplished using a
protective curtain and a unidirectional fl ow hood (UFH) or other more evolved
designs such as a restricted access barrier (RABs) system or an isolator (technologies
that provide a higher level of employee separation from the area in which
materials are handled can get by with lower air exchange rates). All activities requiring
opening of processing lines such as sampling or fi lter integrity testing should be
performed using similar protective measures. The preparation of sterile suspensions
requires a facility/equipment design capable of safe addition of sterile solids to a
liquid vehicle and is conventionally performed using a specifi cally designed processing
area to minimize contamination potential. Comparable and greater complexity
is generally required for creams, ointments, emulsions, and the increasingly common
liposome formulations.
Some sterile powder formulations (these are predominantly, but not exclusively,
antibiotics) may require sampling, mixing, milling, and subdivision activities similar
to those found in oral powder manufacturing. The facilities and equipment utilized
for these products is substantially different from that used for liquids, and the production
area bears little resemblance to that utilized for liquids. These materials are
received sterile and must be processed through sterilized equipment specifi cally
intended for powder handling in a fully aseptic environment with ISO 5 protection
over all open container activities.
2.1.2.2 Primary Packaging
The primary package for parenteral formulations provides protection to the sterile
materials throughout the shelf life. The components of the primary package are
every bit as important to contamination control and hence safety of the fi nished
product as the formulation itself, and their preparation must be given a comparable
level of consideration. The most commonly used container is glass; vials are still the
most common, although increasingly prefi lled syringes are chosen. Glass ampoules
are still seen. However, although convenient from a manufacturing perspective, the
diffi culty involved in opening ampoules while at the same time avoiding problems
with glass particulate or microbial contamination has reduced their popularity. The
use of plastic containers (as vials, ampoules, or syringes) is increasingly common
given their reduced weight and resistance to breakage. Blow - fi ll seal (BFS) and
form - fi ll seal (FFS) are utilized for the fi lling of numerous ophthalmic and other
noninjectable formulations in predominantly low - density polyethylene (LDPE)
containers. With the exception of ampoules and BFS/FFS, an elastomeric closure
system is also necessary to seal the containers. Some delivery systems (i.e., prefi lled
syringes, multichamber vials, and others may require more than one elastomeric
component to operate properly. In the case of vials, an aluminum crimp is applied
to secure the closure to the vial. Prefi lled syringes may require the preparation and
assembly of additional components such as needles, needle guards, stoppers, diaphragms,
or plungers, depending on the specifi cs of the design. Lyophilization is
required to ensure the stability of some formulations and requires the use of closures
that allow venting of the container during the freeze - drying process. Full
seating of the closure is accomplished within the lyophilizer using moving shelves
to seat the closure.
Glass is ordinarily washed prior to sterilization/depyrogenation to reduce contamination
with foreign material prior to fi lling. In aseptic fi ll processes, the glass is
then depyrogenated using dry heat. This can be accomplished using either a continuous
tunnel (common for larger volumes and high - speed lines) or a dry heat oven
(predominantly for small batches). The depyrogenation process serves to sterilize
the glass at the same time, and thus the glass components must be protected postprocessing.
This is generally accomplished by short - term storage in an ISO 5 environment
often accompanied by covering within a lidded tray. There are suppliers
that offer depyrogenated glass vials and partially assembled syringes in sealed packages
for fi lling at a customer ’ s site. In this instance, the supplier assumes responsibility
for the preparation, depyrogenation, and aseptic packaging. Glass ampoules are
available presealed and depyrogenated; the end user has merely to open, fi ll, and
reseal the syringe under appropriate conditions.
Plastic components (whether container or closure) can be sterilized using steam,
ethylene oxide, hydrogen peroxide, or ionizing radiation. The . irradiation is accomplished
off - site by a subcontractor with appropriate expertise as these methods are
considered the province of specialists because of the extreme health hazards directly
related to the sterilization method. Electron beam sterilization may also be done
by a contractor, although compact lower energy electron beam systems have been
introduced that allow sterilization in - house. Steam sterilization is ordinarily performed
in house, though many common components are becoming available presterilized
by the supplier. Preparation steps prior to sterilization vary with the
component and the methods used to produce the component. Rubber components
are washed to reduce particles, while this is less common with plastic materials.
Syringes vary substantially in design details and can be aseptically assembled
from individual components. However, increasingly, these are supplied as presterilized
partial assemblies in sealed containers.
The BFS and FFS are unique systems in that the fi nal container is formed as a
sterile container just prior to the aseptic fi lling step. The BFS requires careful control
over the endotoxin content of the LDPE (and other polymeric materials) beads
used to create the containers as well as the melting conditions utilized to form them.
PROCESS SELECTION AND DESIGN 103
104 STERILE PRODUCT MANUFACTURING
The FFS utilizes in - line sterilization/drying of the fi lm prior to shaping of the
containers.
2.1.2.3 Process Objectives
The production of parenteral products requires near absolute control over microorganisms.
Endotoxin contamination is a serious health concern, particularly among
neonates and infants and also requires a high level of control and validation. Additionally,
the control of foreign matter, including particles and fi bers of various types,
is also vitally important to end - user safety. Assuring appropriate control over these
potential contaminants requires careful attention to several factors: facility design,
equipment selection, sterilization procedures, cleaning regimens, management of
personnel, and the process details associated with compounding, fi lling, and sealing
of product containers. Each of these will be discussed in detail.
2.1.3 FACILITY DESIGN
To provide control of microbial, pyrogen, and particles controls over the production
environment are essential. The facility concerns encompass the entire building, but
the most relevant components are those in which production materials are exposed
to the environment.
2.1.3.1 Warehousing
Environmental protection of materials commences upon receipt where samples for
release are taken from the bulk containers. Protection of the bulk materials is
accomplished by the use of ISO 7 classifi ed environments for sampling. All samples
should be taken aseptically, which mandates unidirectional airfl ow and full operator
gowning. This practice is mandated by current good manufacturing practice (CGMP)
and assures that sampling does not introduce contaminants to the materials that
will be used in the production. Where central weighing/subdivision of active ingredients
and excipients are performed, similar protection is provided for identical
reasons. The expectation is that these measures reduce the potential for contamination
ingress into materials that have yet to receive any processing at the site. Materials
and components that are supplied sterile are received in this area, but samples
are often packaged separately by the supplier to eliminate the need for potentially
invasive sampling of the bulk containers. Where so - called delivery samples are used,
it is critical that these samples are known to be fully representative of the production
process. Additionally, where sterility or bioburden control of sampled materials
is critical, thought must be given to the methods used to reseal the containers to
ensure that moisture levels, bioburden levels, or in the case of sterile products sterility
assurance are not compromised.
2.1.3.2 Preparation Area
The materials utilized for production of sterile processes move toward the fi lling
area through a series of progressively cleaner environments. Typically, the fi rst step
is transfer into an ISO 8 [Class 100,000, European Union (EU) Grade D] environment
in which the presterilization preparation steps are performed. Wooden pallets
and corrugated materials should always be excluded from this zone (and any classifi
ed environment), and transfers of materials are performed in air locks designed
to reduce the potential for particle ingress and to a lesser extent microbial ingress.
Preparation areas provide protection to materials and components for a variety of
activities: component washing (glass, rubber, and other package components), cleaning
of equipment (product contact fi ll parts, process tools, etc.), and preassembly/
wrapping for sterilization. In some facilities, this area is also utilized to support
compounding operations in which case process utensils, small containers, and even
portable equipment will be cleaned and prepared for sterilization.
Careful attention must be given to material fl ow patterns for clean and dirty
equipment to prevent cross contamination. In larger facilities, the equipment wash
room may be a separate room proximate to the preparations area with defi ned fl ows
for materials and personnel. Ideally, materials should move through the facility in
a unidirectional fashion, with no cross over of any kind.
The preparations area typically includes storage areas where clean and wrapped
change parts, components, and vessels can be held until required for use in the fi ll
or compounding areas. (Just - in - time practices are desirable for all parenteral operations
to avoid extensive and extended storage of materials in the higher classifi ed
fi ll or compounding areas.) The preparations area is ordinarily located between the
warehouse and the fi lling/compounding areas and connected to each of those by
material/equipment air locks.
Preparation areas are supplied with high - effi ciency particulate air (HEPA) fi lters
(remote - mounted HEPAs are commonplace). The common design requirement is
more than 20 air changes per hour, turbulent airfl ow (see below), and temperature
and relative humidity controlled for personnel comfort. As in any clean room area
designed for total particulate control, the air returns should be low mounted.
Wall and ceiling surfaces should be smooth, easily cleaned, and tolerant of
localized high humidity. Floors should be typically monolithic with integral drains
to prevent standing water. Common utilities are water for injection, deionized
water, compressed air, and clean/plant steam. Clean - in - place (CIP) and sterilize -
in - place (SIP) connections may be present if the prep area supports compounding
as well.
Ordinarily, present within the preparation area are localized areas of ISO 5 unidirectional
airfl ow (Class 100) utilized to protect washed components prior to sterilization
and/or depyrogenation. These areas are not aseptic and should not be
subjected to the more rigorous microbial expectations of aseptic processing. They
are designed to reduce/eliminate the potential for particle contamination of
unwrapped washed materials. Operators accessing these protective zones wear
gloves at all times when handling materials.
Operators in the preparations area are typically garbed in low particle uniforms
(or suits) with shoe, hair, and beard covers. The use of latex or other gloves is
required when contacting washed components. Sterilized gowns and three - stage
gowning facilities are not required to enter or work in this ISO 8 environment.
Gowns are generally donned within a single - stage airlock, which is maintained at a
pressure slightly negative to the ISO 8 working environment. Separate personnel
entry/exit are not typically necessary for this lower classifi ed environment.
FACILITY DESIGN 105
106 STERILE PRODUCT MANUFACTURING
Equipment within the preparations area varies with the practices of the fi rm and
can include manual or ultrasonic wash/rinse sinks; single or double door automated
parts washers; batch or continuous glass washers; stopper washers for closure components;
CIP/SIP stations; equipment wrap areas (as described above); and staging
areas for incoming (prewash) components, dirty equipment, and cleaned components/
equipment. An adjacent classifi ed storage area(s) may be present in larger
facilities to accommodate the full variety of change parts and equipment that is not
in immediate use. Where the preparations area also supports compounding, it may
include additional equipment such as pH meters, fi lter integrity apparatus, and the
like in support of those operations. ( Note: Where compounding requires aseptic
conditions for rigorous control of bioburden, as is the case for unpreserved biologics
and other contamination - sensitive products, it is best to provide separate entry for
compounding. The moisture level and hence contamination potential in a typical
preparation area is unsuitable for entry into an aseptic compounding area).
Depending on the scale of the operation, the preparations area may include the
loading areas for both sterilizers and ovens. In high - throughput operations where
the use of tunnels for glass depyrogenation is more prevalent, glass washers and
tunnels for each fi lling line may be in separate ISO 8 rooms accessed from the
preparations area.
2.1.3.3 Compounding Area
The manufacture of parenteral solutions is ordinarily performed in ISO 7 (Class
10,000, EU Grade C) controlled environments in which localized ISO 5 unidirectional
fl ow hoods are utilized to provide greater environmental control during
material addition. These areas are designed to minimize the microbial, pyrogen, and
particle contributions to the formulation prior to sterilization. Depending upon the
scale of manufacture, this can range from small containers (up to 200 L) (disposable
containers are coming into use for these applications), to portable tanks (up to
600 L) to large fi xed vessel (10,000 L or more have been used) in which the ingredients
are formulated using mixing, heating, cooling, or other unit operations. Smaller
vessels are placed or rolled onto scales, while fi xed vessels are ordinarily mounted
on weigh cells. The vessels may be equipped for temperature and pressure measurement
instruments, as mandated by process requirements. Compounding areas often
include equipment for measuring mass and volume of liquid and solid materials
including, for example, graduated cylinders, and scales of various ranges, transfer
and metering pumps, homogenizers, prefi lters, and a variety of other liquid/powder
handling equipment. Liquid handling may be accomplished by single - use fl exible
hose, assemblies of sanitary fi ttings, or some combination thereof. A range of smaller
vessels to be used for the addition of formulation subcomponents or excipients to
the primary compounding tank may be required as well. Because parenteral formulations
can include aqueous and nonaqueous vehicles, suspensions, emulsions, and
other liquids, the capabilities of the compounding area may vary. Agitators can be
propeller, turbine, high shear, or anchor designs depending upon the requirements
of the products being manufactured, and it is not uncommon to fi nd examples of
each in larger facilities. It is preferable to perform as much of the process as possible
while the formulated liquid is nonsterile to ease sterilization requirements, although
precautions to prevent microbial and endotoxin contamination are important risk
abatement features.
FACILITY DESIGN 107
The formulation area is customarily a combination of open fl oor space, adjacent
to three - sided booths and individual processing rooms in which the ingredients are
handled and individual batches are produced. Walls and ceiling materials are selected
to be impervious to liquids and chemical spills and are easy to clean. Floors in these
areas are monolithic and should be sloped (at 1 – 3 : 100) to drains with appropriate
design elements and control procedures to eliminate backfl ow potential (regulatory
bans on drains in classifi ed areas are focused on protecting aseptic environments
and are inappropriate for nonsterile compounding areas). Pit scales should be
avoided in new installations; fl oor - mounted scales intended for cleaning underneath
the base are preferable.
Compounding areas are supplied with HEPA fi lters (ceiling - mounted terminal
HEPAs are more common, though central supply is possible in areas of low contamination
risk). The common design requirement is more than 50 – 60 air changes
per hour, turbulent airfl ow (see below), with temperature and relative humidity for
personnel comfort. Air returns may be at or near fl oor level, with localized extraction
provided as necessary to minimize dusting of powder materials. Where substantial
heat is generated from processing or sterilization, a ceiling or high wall return
may be more appropriate. Wall and ceiling surfaces are smooth, easy to clean, and
tolerant of localized high humidity. Floors are typically monolithic with integral
drains to prevent standing water. Common utilities are water for injection, deionized
water, nitrogen, compressed air, clean/plant steam, and heating and cooling media
for the fi xed and portable tanks. Water for injection use points are often equipped
with sanitizable heat exchangers for operator safety.
Cleaning of the fi xed vessels and portable tanks is accomplished using either
manual sequenced cleaning procedures or more commonly with a CIP system.
Cleaning of other items can be accomplished in a wash area accessed from the
compounding area or in a common wash room incorporating both fi lling and compounding
equipment. Sterilization of the nonsterile processing equipment and vessel
is often provided for as an option, even where it is not routinely required to control
product bioburden. Where production volumes or physical location dictate, the
compounding area may have a separate preparations area from that utilized to
support fi lling operations.
Personnel working in the compounding area typically wear a coverall (which may
be sterilized for contamination control as required), with head/beard covers, as well
as dust masks and sterile gloves. Additional personnel protective equipment may
be necessary for some of the materials being processed. A fresh gown should be
donned upon each entry into the compounding area. Separate gowning/degowning
rooms should be provided to minimize cross - contamination potential for personnel
working with different materials. As nonsterile compounding areas are often ISO
6 – 7 environments but are not aseptic, the more rigorous contamination controlling
designs required of aseptic gowning areas (see below) are somewhat reduced.
2.1.3.4 Aseptic Compounding Area (If Present)
Where products are fi lled using in - line fi ltration direct to the fi lling machine, an
aseptic compounding area may not be present. In those instances the fi nal sterilizing
fi lter will be located in the fi ll room.
Products that are held/processed in sterilized vessels prior to fi lling require an
aseptic compounding area. This is typically an ISO 7 in environment with localized
108 STERILE PRODUCT MANUFACTURING
ISO 5 unidirectional fl ow present where open - product containers or aseptic operations
are conducted. Some products may require larger ISO 5 suites with full HEPA
coverage rather than the more common ISO 5 clean booth design. Fixed vessels in
this area are cleaned and sterilized in situ, while portable vessels are typically relocated
to the wash area for cleaning. Sterilization of portable vessels may be accomplished
at an SIP station in the aseptic core, compounding, or preparations areas.
When accomplished outside the aseptic processing area, resterilization of the connecting
lines may be appropriate. Filters for sterilization of solutions from compounding
to holding vessels are typically located in this environment as well, with
sterilization by either SIP or sterilization in an autoclave. The use of integrated,
programmable logic controlled ( PLC) fi lter skids with automatic CIP/SIP and fi lter
integrity testing is frequently seen for contamination sensitive products.
Depending upon the formulations being produced, additional sterilized processing
equipment may be present in this area for use in the process. This can include
in - line homogenizers, static mixers, and colloid mills. Where sterile powders are
produced, the aseptic compounding processes can include blending, milling, and
subdivision equipment.
Aseptic compounding areas typically require a means to introduce sterile equipment,
tubing, and other items, so access to a sterilizer is desirable. The aseptic compounding
area may be contiguous to the aseptic fi lling suites. If it is not, separate
gowning areas must be provided for personnel as well as separate air locks/pass -
throughs (see below).
Personnel working in aseptic compounding wear full aseptic garb: sterile gown,
hood, face mask, goggles, foot covers, and gloves. Adaptations may be necessary
for potent/toxic compounds to assure operators are properly protected from
hazardous materials. Gowning areas are ordinarily shared with aseptic fi lling,
but where they are not shared a comparable design, albeit on a smaller scale,
is appropriate.
The facility design features match that of the aseptic fi lling room/aseptic processing
areas described in greater detail below. Utility services would mimic those utilized
in the nonsterile compounding area that is usually adjacent (next to or above)
to the aseptic compounding area. Temperature and humidity should be controlled
to similar levels as those required for aseptic fi lling. Since CIP/SIP systems tend to
generate heat and humidity, suffi cient capacity must be available to control temperatures
to approximately 18 – 20 ° C and < 50% relative humidity (RH).
2.1.3.5 Aseptic Filling Rooms and Aseptic Processing Area *
The fi lling of aseptic formulations (and many terminally sterilized products as well,
by reason of their lesser number) is performed in an ISO 5 (Class 100) environment,
which is accessed from an ISO 6/7 background environment in which personnel are
present. Some measure of physical separation is provided between the ISO 5 and
ISO 6/7 environments as a means of environmental protection as well as a reminder
to personnel to restrict their exposure to ISO 5.
* This section describes the conventional manned clean room; a later section in this chapter will address
alternative aseptic processing environmental control designs with somewhat different features and
control measures.
FACILITY DESIGN 109
In large operations an aseptic fi lling room is generally one of a multiple suite of
aseptic rooms which allow simultaneous production of multiple products. The fi lling
rooms are independent of each other; however, sharing the supporting rooms is
common. Sterilizer unload rooms, corridors, air locks, storage rooms, lyophilizer
loading rooms, and gowning rooms (each will be briefl y described as well) may all
be present, and their arrangement must suit production volumes. Where shared
common areas are required, the design should feature unidirectional materials fl ow
to prevent cross - contamination and to minimize the potential for mix - ups. In the
smallest facilities, only the gowning area might be separate from the fi ll room, and
all of the supportive activities could be inclusive in a single room (however, unloading
activities should not occur during fi lling operations). All of these aseptic processing
areas (APAs) are built to the same design standards: smooth, impervious ceilings,
walls and fl oors, fl ush - mounted windows, clean room door designs, coved corners,
fi nishes capable of withstanding the aggressive chemicals utilized for cleaning and
sanitization. Air returns throughout the APA are located at or near fl oor level.
Unidirectional airfl ow is provided over all exposed sterile materials, that is, fi ll zone,
sterilizer/oven/tunnel unload areas, and anywhere else sterile materials are exposed
to the environment. Air changes in these ISO 5 environments can approach 600 per
hour, though lesser values have proven successful. Air changes in the background
environment vary from 60 to 120 per hour.
The glass container fi ll rooms fi lling machines are connected to depyrogenating
tunnels and exit ports leading to capping stations. Batch handling of glass is discouraged
unless isolator systems are employed. In some operations, the in - feed and discharge
of containers/components may utilize trays, tubs, or bag systems for material
feed/discharge. Wherever possible, automation of component feeding should
be considered to reduce contamination risk. Supportive equipment present might
include carts, weigh stations, stoppering, crimping, sealing, and other fi ll system
related machinery depending upon requirements.
The product contact surfaces in this environment are typically removed for cleaning;
however, in some installations, the sterilization, transfer, and reinstallation of
the component feed hoppers present such diffi culty that these systems are decontaminated
in situ with a sporicidal agent, rather than removed after each use. These
units should still be removed for cleaning and sterilization on a validated periodic
basis to prevent the buildup of residues that might impact their in - situ decontamination
or create particle control problems. All other product contact surfaces should
be sterilized prior to each use. Nonsterilized items should not be allowed to enter
the ISO 5 portion of the fi ll zone, and sanitization is essential for all nonproduct
surfaces in the fi ll zone, as well as the surrounding background environment.
Discharge of sealed containers can be accomplished via a exit port or “ mouse
hole ” that allows for the passage of the containers from the APA to the surrounding
environment. Proper design of the mouse hole system ensures protection of the
classifi ed fi ll area from contamination fl owing against the fl ow of the containers. In
many instances the discharge is into a nonclassifi ed inspection area that may lead
directly to the secondary labeling/packaging area.
Personnel working in aseptic compounding wear full aseptic garb: sterile gown,
hood, face mask, goggles, foot covers, and gloves. Adaptations may be necessary for
potent/toxic compounds to assure operators are properly protected from hazardous
materials.
110 STERILE PRODUCT MANUFACTURING
2.1.3.6 Capping and Crimp Sealing Areas
The application of aluminum seals over rubber stoppers is essential to secure them
properly. In many older facilities this was accomplished outside the aseptic processing
area in an unclassifi ed environment. Current practice requires that air supplied
to this activity meet ISO 5 under static conditions. The protection of crimping has
resulted in a variety of designs to meet the requirement: Sterile crimps can be
applied with the aseptic core on the fi lling line; sterile crimps can be applied in a
separate crimping room accessible from the fi lling room. If the crimpling operation
is located within the APA, it should be in a separate room maintained at a negative
pressure differential relative to the fi lling environment. Crimping may alternatively
be performed in a classifi ed room accessed from a controlled but unclassifi ed environment.
In this case it is imperative to verify that the environmental controls satisfy
regulatory expectations for all relevant markets.
2.1.3.7 Sterilizer Unload (Cooldown) Rooms
Sterilizers/ovens are unloaded and items staged prior to transfer to the individual
fi ll rooms. ISO 5 air is provided over the discharge area of ovens (and autoclaves if
items are sterilized unwrapped) to provide protection until the items are ready for
transfer. The heat loads in this room may be such that special high - temperature
sprinkler heads may be necessary to avoid unintentional discharge when unloading
hot materials. This room may not be separate from the corridor used to connect the
fi ll rooms. It is ordinarily adjacent to any aseptic storage area.
2.1.3.8 Corridors
Corridors serve to interconnect the various rooms that comprise the APA. Fill
rooms, air locks, and gowning rooms are accessed from the corridor. They can also
be utilized for modest storage as well.
2.1.3.9 Aseptic Storage Rooms
In general, extensive use of in - process storage areas should be avoided. It is best to
operate the aseptic facility in a just - in - time mode in which components and
equipment are sterilized shortly before they are required for use in the fi lling or
compounding areas. Some limited storage is necessary for nonproduct contact
materials such as sanitizing agents, environmental supplies and equipment, and
other items.
2.1.3.10 Lyophilizer Loading and Unloading Rooms
The loading of lyophilizers is accomplished under ISO 5 environmental conditions
within the aseptic processing area. Several possible locations are possible: within
the aseptic fi ll room itself, in a separate room adjacent to the fi ll room, or in a separate
room remote from the fi ll room. There are pros and cons with each of these
selections which should be carefully considered in the facility design. There is a
universal expectation that fi lled containers of product should be maintained under
ISO 5 conditions during transfer and lyophilizer loading. Many modern facilities
incorporate automatic lyophilizer loading and unloading. Automation of loading,
unloading, and in the case of vials transfer to the crimping station greatly reduces
contamination risk and is highly recommended.
If manual transfer is unavoidable, location of the lyophilizer relatively close to
the fi lling line enables protected transfer to be accomplished rather easily. Remote
locations may require transfer of product in carts capable of providing ISO 5 quality
air. These carts will generally require battery power in order to run the necessary
air blowers and control systems. Alternatively, product trays could be placed in air -
tight carriers; this activity and the sealing of the carriers would have to be accomplished
under ISO 5 conditions. Locating the lyophilizer in the fi ll room may restrict
the ability to unload the dryer while the fi lling line is in use, particularly if the
lyophilizer is loaded and unloaded manually, which would increase the clean room
personnel load and potentially increase contamination risk.
The use of trays during lyophilization is less common, nevertheless, ring trays
with removable bottoms are sometimes used to transfer vials to/from the lyophilizer.
Where trays are used, they must be cleaned and sterilized prior to each batch. Large
lyophilization facilities will sometimes use an automated loading/unloading system
in which all shelves or a shelf at a time are processed. Regardless of the practice,
ISO 5 conditions are required for all areas of the facility in which partially stoppered
containers are transferred or handled. As previously mentioned, it may be possible
in some operations to transfer containers in a manner that they are not exposed to
the environment during transfer.
Upon completion of the drying process, the containers will ordinarily have their
stoppers fully seated on the container within the freeze dryer. The stoppered containers
are then passed through a sealing station in which aluminum crimps are
applied. This may be accomplished on the fi ll line, or using a separate crimping
machine. Precautions will need to be taken to ensure that only fully stoppered vials
are transferred to the crimping station. This can be accomplished by automatic
inspection systems of various designs. It is increasingly common for product transfer
to crimping and crimping itself to be done under unidirectional airfl ow. It should
be noted that a crimpling station will generally not meet ISO 5 particulate air quality
requirements when the crimper is operating since the generation of relatively high
levels of particulate is an inherent feature of this process.
2.1.3.11 Air Locks and Pass - Throughs
Air locks serve as transition points between one environment and another. Ordinarily,
they are designed to separate environments of different classifi cation: that is,
ISO 6 from ISO 7. When this is the case, they are designed to achieve the higher of
the two air quality levels in operation. If they are utilized for decontamination purposes
for materials/equipment that cannot be sterilized, but must be introduced into
the higher air quality environment, they may be fi tted with ultraviolet (UV) lights,
spray systems, vapor phase hydrogen peroxide generators, or other devices that may
be effectively utilized for decontamination of materials. Regardless of the design or
the decontamination method employed, the process should be validated to ensure
FACILITY DESIGN 111
112 STERILE PRODUCT MANUFACTURING
consistent effi cacy. The doors at each end can be automatically interlocked or
managed by standard operating procedure. In some instances a demarcation line is
used to delineate the extent to which individuals from one side should access the
air lock. It is good practice to carefully control and to minimize the time that any
operator spends accessing an air lock, therefore transfer of materials should be
carefully planned to minimize frequent and spontaneous access. Additionally, the
capacity of the air lock should be carefully considered relative to the actual production
requirements. Air locks that lack suffi cient capacity and that cannot provide
suffi cient air exchange will be less suited to the control of contamination into more
critical areas of the aseptic processing environment.
A smaller scale system with comparable capabilities is the pass - through. This
differs from the air lock primarily in dimension, as items are typically placed into the
pass - through by personnel, whereas the air lock is customary for pallet, portable
tanks, and larger items that are either rolled or mechanically lifted into position.
The operation of the pass - through can be either manual or automatic with similar
capabilities to that of the air lock described above. In general pass - throughs should
be supplied with HEPA fi lters and should be designed to meet the air quality level of
the higher air quality classifi cation room served. Pass - throughs should also be interlocked
and provide adequate facilities for decontamination of materials being
transferred.
Air locks and pass - throughs are bidirectional and can be used for movement in
either direction. When used as an exit route, the decontamination procedure can be
omitted. Where production volumes warrant separate entry and exit, air locks may
be necessary to maintain both adequate capacity and separation between clean and
used items. In an emergency, airlocks can serve as emergency exits for personnel, in
which case the interlocks can be overridden.
2.1.3.12 Gowning Rooms
The gowning area used for personnel entry/exit presents some unique problems.
Gowning facilities must be designed to the standards of the aseptic processing area,
yet personnel upon entry are certainly not gowned. Because ungowned staff will
release higher concentrations of contaminants into the environment, gown rooms
must be designed with suffi cient air exchange so that this contamination is effectively
and promptly removed. In general, the contamination load within a gowning
environment will require air exchange rates at the high end of recommended levels
for a given ISO 14644 air quality classifi cation. Gowning areas are separated into
well - defi ned zones where personnel can progress through the various stages of the
gowning process.
The most common approach in industry is a three - stage gowning area design in
which three linked rooms with increasing air quality levels are utilized to effi ciently
and safely affect clothing change. Staff should enter the fi rst state of the gowning
room wearing plant uniforms. No articles of outerwear worn outside the facility
should be worn to the gowning area. Therefore, a pregowning room equipped with
lockers is required so that operators can change into dedicated plant clothing prior
to moving to the gowning area. Generally, the pregowning locker area is not classi-
fi ed, although entry is controlled and temperature and humidity are maintained at
20 – 24 ° C and 50% ± 10%. The pregown area should have extensive hand - washing
facilities equipped with antibacterial soap, warm water, and brushes for cleaning
fi nger nails. Soap and water dispensing should be automatic and hands should be
air rather than towel dried. The pregown area should have typical clean room wall
and fl oor fi nishes along for frequent and rigorous cleaning and sanitization. The
pregown area is bidirectional as it is used as both an entry and exit point. Separate
pregown areas are required for female and male personnel. A typical complement
of garments for exit of the pregown area includes surgical scrubs or other nonparticulate
shedding plant uniform. Ideally, the uniform should have a high neck and
sleeves which extend to the lower wrist. Hair covers and beard covers are donned
in the pregown area.
Upon entry into the fi rst - stage gowning room, which is generally designed to an
ISO 7 air quality level, the operators often don a second hair cover, sterilized gloves,
and a sterilized surgical mask. In the second and third stages of the gowning area
room classifi cation is typically ISO 6 or ISO 6 followed by ISO 5 at the exit point.
Different fi rms have different gowning sequences. However, in every case the fl ow
of personnel and arrangement of gowning materials should be such that personnel
fl ow is in one direction. In the last of the three gowning stages, secondary protective
equipment can be donned, including sleeve covers and a second set of gloves. Some
fi rms will use tape to secure the gloves to the sleeves to prevent separation. A dry
glove decontamination point utilizing disinfectant foam is generally provided prior
to exiting the gowning area; this should be a hands - free operation. In some facilities
air showers, which provide a high - intensity blast of HEPA air for a predetermined
length of time, are employed after gowning is completed. Side - by - side gowning of
personnel should be avoided to preclude adventitious contamination. Similarly,
personnel exiting the aseptic area should use a separate degowning area. These
design practices are appropriate in all but the very smallest facilities where only a
single aseptic operator is present.
2.1.3.13 Terminal Sterilization Area
The terminal sterilization of fi nished product containers may be performed in the
same sterilizers utilized to supply the aseptic processing operations. The differing
process needs of terminal sterilization will sometimes dictate the use of sterilizers
specifi cally designed for terminal sterilization incorporating air - over pressure
systems, internal fans, and spray cooling. Where this is the case, the terminal sterilizer
is located proximate to the crimping/sealing areas. A double - door sterilizer
design is preferred with staging areas for fi lled containers to be sterilized and a
separate area for containers that have completed the process. Classifi cation of these
areas is not required as the containers are closed throughout the sterilization process.
The fl ooring materials in this area should be monolithic to allow for easy cleanup
in the event of container breakage.
2.1.3.14 Inspection, Labeling, and Packaging
These activities are performed on fi nished product containers in unclassifi ed environments.
The primary design requirements are straightforward: separation of products
to prevent mix - up, adequate lighting for the processes, and control over labeling
materials.
FACILITY DESIGN 113
114 STERILE PRODUCT MANUFACTURING
2.1.4 ASEPTIC PROCESSING FACILITY ALTERNATIVES
The successful production of parenteral drugs by aseptic processing requires an
environment in which microorganisms and particles are very well controlled. The
means to accomplish this has undergone substantial change over the last 50 years
(see Figure 1 ) with continuing refi nement. The earliest aseptic processing systems
used glove boxes with minimal (if any) airfl ow and manual disinfection in which
manual processes were performed. The availability of HEPA fi lters in the late 1950s
led to human - scale clean rooms in which processing equipment could be installed.
Aseptic processing changed radically once entire clean rooms became feasible.
As it had always been recognized that personnel were the dominant source of
contamination, the majority of designs utilized some measure of physical separation
between the operator and the critical zone (sterile fi eld) in which the aseptic
processing activities were performed. Separative devices (a term that is now
embodied in ISO 14644 - 7 Separative Enclosures) of different design and varying
capability have been successfully employed including fl exible curtains and fi xed
plastic shields with or without integrated gloves/sleeves [9] . In the most evolved
designs operation of the equipment is interlocked with the surrounding enclosure,
such that equipment stops running when the doors are opened. These latter designs
represented the pinnacle of clean room - based aseptic processing into the early
1990s.
Isolators represent a return to operator separation principles utilized during the
glove box era, albeit with substantial improvements in the form of rapid transfer
ports for material transfer, air - handling systems utilizing modern HEPA fi lters, and
reliable decontamination systems. The salient element of all isolator designs is the
completeness of separation between the internal and external environments. This
single feature affords vastly superior performance relative to manned clean rooms
in excluding personnel - derived contamination and has comparable advantages for
the containment of potent compounds. While initial adoption of the technology was
slowed by the novelty that isolators presented to users, much of the initial reluctance
has been overcome [10, 11] . Isolators for aseptic processing vary in complexity, size,
and amount of processing equipment. They can be utilized for processing ranging
Aseptic Processing Family Tree
Gloveboxes
Conventional
Cleanroom
Barrier
Systems
RABs
Closed
Isolators
Open
Isolators
BFS/FFS
FIGURE 1 Aseptic processing family tree.
from manual compounding of small batches to high - speed fi lling of fi nal product
containers. Depending upon the process requirements, isolators can be utilized for
containment of potent compounds (under negative pressure while still nonsterile)
during the compounding, aseptic operation (under positive pressure) for preparation
and transfer of components and aseptic containment (also under positive pressure)
for aseptic fi lling of the potent drug solution.
Firms that were intimidated by or unconvinced of the superiority of isolators
developed the restricted - access barrier (RAB) system as a potentially less complex
and less costly alternative [12] . The real - world utility of RABs systems is unknown;
there are still relatively few installations; thus, the experience base is still emerging.
Also unconfi rmed at this point are the actual validation and ongoing process control
requirements which make direct comparison of project time lines and overall costs
with isolators somewhat speculative.
There are specialized technologies such as BFS and FFS that are appropriate for
aseptic processing, but these are restricted to fi lling processes only. A number of
other new technologies are being developed for use in aseptic processing, including
vial isolators and closed vial fi lling [13 – 15] . All of these have the objective of reducing
contamination through reduction in human involvement or increased protection
of the container. Further advances in processing including gloveless isolator designs,
robotics, and others are already under active development to further improve the
safety of parenteral products.
2.1.4.1 Expandability
Large facilities often include design elements that facilitate later expansion of the
facility to add additional capacity. The most common of these is extension of an
aseptic corridor to additional fi lling suites; reservation of space for additional sterilizers;
and allocating space for additional or oversizing initial utility systems. Obviously,
these types of changes require careful design and must be properly managed
during execution to avoid impact on existing operations.
Isolation technology changes this dynamic signifi cantly by eliminating most of
the disruption on current activities, as fabrication of the isolator occurs off - site, and
installation can be minimally disruptive compared to what is required with a clean -
room design. Isolators are generally installed in ISO 8 space; therefore, it is possible
to build a rather large ISO 8 facility in which equipment can be moved, replaced,
or reconfi gured quite easily compared to conventional human - scale zoned aseptic
processing areas.
2.1.5 UTILITY REQUIREMENTS
Any utility in direct product contact is subject to formal qualifi cation through con-
fi rmation of the quality of the delivered material at each use point. Water - for -
injection (WFI) systems are considered the most critical of all, and the qualifi cation
period for WFI is the longest and may be as long as 3 months. The remaining product
contact utilities can be qualifi ed more rapidly. Nonproduct utilities requirements
can be satisfi ed by commissioning.
UTILITY REQUIREMENTS 115
116 STERILE PRODUCT MANUFACTURING
2.1.5.1 Water for Injection
The most important utility in sterile manufacturing is WFI. Not only is it a major
component in many formulations, it is also utilized as a fi nal rinse of process equipment,
product contact parts, utensils, and components. In some facilities it may be
the only grade of water available and is used for initial cleaning of items as well.
The WFI may be produced by either distillation (multiple effect or vapor compression)
or reverse osmosis (generally in conjunction with deionization) and is ordinarily
stored and recirculated at an elevated temperature greater than 70 ° C to prevent
microbial growth [16, 17] . Where cold water is required, it may be supplied by use
point heat exchangers or using a separate cold loop (usually without a storage
capability). Point - of - use cool water drops and reduced temperature circulation
loops are generally sterilized or high - temperature sanitized at defi ned and validated
intervals. The design details of the WFI system varies with the incoming water
quality, local utility costs, and operational demands. Very small operations may not
have a WFI system and will utilize larger (5 L or larger) packages of WFI for formulation
and cleaning.
Other grades of water may be present in parenteral facilities for use as initial
rinses and detergent cleaning. The water utilized for these purposes is generally of
relatively low bioburden and is often deionized, softened, ultra - fi ltered, or in some
instances prepared by distillation or reverse osmosis, resulting in chemical purity
similar to, if not identical to, WFI. Systems for the preparation of this water are
subject to qualifi cation, validation, and routine analysis to assure consistent quality.
2.1.5.2 Clean (Pure) Steam
Sterilizers and SIP systems in the facility are supplied with steam which upon condensation
meets WFI quality requirements (testing steam condensate for microbial
content is not fruitful). The steam can be produced directly from the water of suffi -
cient purity to meet the input requirements of the steam generator. Steam generators
are phase transition technologies that operate like a still, so it is no more
necessary to provide these devices with WFI feed water than it would be to double
distill WFI. (Production from WFI is certainly possible, but that is both expensive
and an unnecessary precaution.) Modest quantities of steam can be produced from
the fi rst effect of a multiple effect WFI still, however, with a resultant loss of WFI
output [18] .
2.1.5.3 Process Gases
Air or nitrogen used in product contact is often supplied in stainless steel piping
and ordinarily equipped with point - of - use fi lters; quite often an additional fi lter is
placed within the distribution loop or at the entry point into a room resulting in a
form of redundant fi ltration. Compressed air is typically provided by oil - free compressors
to minimize potential contaminants and is often treated with a drier to
obviate the possibility of condensation within the lines which could be a source of
contamination. Nitrogen is supplied as a bulk cryogenic liquid. Argon and carbon
dioxide have also been utilized as inerting gases, while propane or natural gas may
be needed for sealing of ampoules.
2.1.5.4 Other Utilities
The operation of a parenteral facility often entails other utilities for the operation
of the equipment. These include plant steam, jacket cooling water, and instrument
air.
2.1.6 STERILIZATION AND DEPYROGENATION
The preparation of the drug formulation, components, and equipment entails the
use of various sterilization/depyrogenation treatments to control bioburden, avoid
excessive pyrogens, and to sterilize. The selection of the specifi c process must always
fully consider the impact of the treatment on the items being sterilized/depyrogenated.
Sterilization and heat depyrogenation processes must balance the effect of
the treatment on the microorganism with the effect of that same treatment on the
materials being processed. The choice of one method over another is often based
upon achieving the desired sterilization/depyrogenation effect with minimal impact
on the items critical quality attributes.
2.1.6.1 Steam Sterilization
The method of choice in nearly every instance is moist heat due to its lethality,
simplicity, speed, and general ease of process development and validation. For the
majority of items, this is accomplished in a double - door steam sterilizer, which is
conventionally located between the preparations and aseptic processing (fi lling or
compounding) areas. Steam sterilizers are routinely utilized for items such as elastomeric
closures, process and vent fi lters, product contact parts, heat stabile environmental
monitoring equipment, tools and utensils, hoses, sample containers, and
other items unaffected by contact with saturated steam at commonly used sterilizing
temperature and pressure [19] . Similar items utilized in the nonsterile compounding
area would be processed in a similar manner. Regardless of their fi nal
destination or usage, items for steam sterilization should be protected from poststerilization
contamination by materials that are permeable to steam, air, or condensate
but impenetrable by microorganisms. The wrapping materials would be
maintained on the sterilized items until just prior to use. There are numerous
publications that provide additional details on steam sterilization procedures
[19 – 21] .
Sealed containers of aqueous solutions, suspensions, and other liquids can be
processed through steam sterilizers as well. These liquids might be used in formulation
or cleaning procedures, and sterilization in this manner may be more effi cient
and more reliable than sterilizing fi ltration. Larger volumes of aqueous liquids are
often sterilized in bulk using a jacketed and agitated pressure vessel (the vessel is
usually rated for full vacuum as well).
Steam SIP is a widely used practice for the sterilization of equipment prior to
the introduction of process materials and is the method of choice for holding tanks,
process transfer lines, lyophilizers, and other large items. Conceptually, it has many
similarities to sterilization in autoclaves but differs markedly due to the often
custom designs of process equipment requiring SIP. Systems must be designed with
careful consideration given to air removal and condensate draining, process sequenc-
STERILIZATION AND DEPYROGENATION 117
118 STERILE PRODUCT MANUFACTURING
ing, and poststerilization integrity to assure success [22] . Terminal sterilization of
fi nished product containers is addressed later in this chapter.
2.1.6.2 Dry - Heat Sterilization and Depyrogenation
The use of dry heat for depyrogenation (and sterilization) is almost universal for
glass containers. Temperatures of 250 ° C or higher are utilized to render the glass
endotoxin free. The depyrogenation is necessary because the washing of glass to
reduce particles can introduce unacceptable levels of gram - negative microorganisms
whose presence could result in pyrogen formation. The depyrogenation process
can assist in component surface treatment (siliconization is required for some formulations)
and will also render the glass sterile as well (depyrogenation temperature
conditions far exceed those needed for sterilization [23] ).
Sterilization by dry heat is only infrequently used, preference being given to the
use of steam (due to its higher speed) or dry - heat depyrogenation (affording an
added measure of safety using the same equipment). Where it is employed temperatures
in the range of 170 – 180 ° C are employed, and a batch oven is customarily
used.
Dry - heat processes are conducted in either batch ovens or continuous tunnels,
which are also installed between preparations and aseptic processing areas. Ovens
have lower capacity and are typically found in smaller facilities. They offer the ability
to handle items other than fi nal product containers and thus can replace autoclaves
in facilities where fi lling parts, feed hoppers, tools, and other items that must be
extremely dry. Ovens should be equipped with internal HEPA fi lters, recirculating
fans, heating/cooling coils, and a sophisticated control system [24] . Items prepared
for dry - heat treatment in ovens are inverted or covered to protect them after exiting
from the oven as there are no sealed protective systems suitable for the higher
temperatures necessary for dry - heat depyrogenation or sterilization. Oven discharge
is typically into a cool - down area (usually the same as that used for the sterilizer),
though in small facilities it might discharge directly into the fi ll room. Unless ovens
are used in conjunction with isolators, they require direct operator intervention to
transfer containers to the fi lling line and to charge the line with depyrogenated glass.
This constitutes a risky intervention which should be avoided. For this reason, batch
glass processing is rare in all but the lowest throughput facilities.
Dry - heat tunnels are typically utilized where the production volumes are higher
and allow for continuous supply of depyrogenated glass to the aseptic fi ll room.
Tunnels are operated at high temperatures ( > 300 ° C) to increase processing speed
and include a cooling zone that facilities discharge at or near room temperature.
Typically, heating of the glass to 300 ° C or more for 3 or more minutes will result in
much greater than the three - log endotoxin reduction required in current industry
standards. The air inside the tunnel is HEPA fi ltered, and newer designs allow for
dry - heat sterilization of the cooling zone as an added protective measure. Tunnels
must be positioned with some care as they ordinarily will terminate into a fi ll room.
A pressure differential between the cooling zone of the tunnel and the fi ll room is
critical for proper operation of the tunnel. The pressure differential must conform
to the requirements stipulated by the tunnel manufacturer. It is not necessary to
have a > 12.5 PA (particulate air) differential between the in - feed side of the heating
zone of the tunnel and the exit side of the cooling zone. It has been suggested by
some that, since the in - feed side of the tunnel is typically in ISO 7 or 8 space, a
greater differential is required; however, this is not true since the cooling zone is
ISO 5, and the heating zone is certain to be sterile and is also ISO 5 in terms of
particulate air quality. Their in - feed is often direct from a glass washer, which may
be remote from the main preparations area utilized for washing, wrapping, and
sterilizer loading.
2.1.6.3 Gas and Vapor Sterilization
The sterilization of materials using noncondensing gases (ethylene oxide, chlorine
dioxide, or ozone) or condensing vapors such as hydrogen peroxide is a supplementary
process intended for items that cannot be exposed to heat. The utilization of gas/
vapor designs is coming into increased use as a supportive technology for isolation
technology for presterilized items such as syringes and stoppers that must be introduced
into the isolators aseptic zone. Air locks using these agents can be utilized in
similar fashion for the supply of materials to manned clean rooms. Control over agent
concentration or injection mass, relative humidity, and temperature may be required
for these systems. There are different types of vapor processes available, and users
should generally follow the cycle development strategy suggested by the manufacturer
of the equipment they have chosen. Specifi c temperature and humidity ranges
may be required for some vapor processes to assure appropriate effi cacy [25, 26] .
2.1.6.4 Radiation Sterilization
The use of radiation within a parenteral facility would have been considered unthinkable
prior to the start of the twenty - fi rst century. While . irradiation is typically a
contracted service provided off - site, electron beam sterilization advances can make
the installation of an in - house (and generally an in - line) system a real possibility.
An in - line system would be utilized similarly to the gas/vapor systems described
above for treatment of external surfaces for entry into either a clean room or
isolator - based aseptic processing facility. The use of this same technology for terminal
sterilization is also possible [1] . Association for the Advancement of Medical
Instrumentation (AAMI)/ISO 11137 provides widely accepted guidance on the
development and validation of radiation sterilization processes.
2.1.6.5 Sterilization by Filtration
Filters are utilized to sterilize liquids and gases by passage through membranes that
retain microorganisms by a combination of sieve retention, impaction, and attractive
mechanisms [27] . In contrast with the other forms of sterilization that are destructive
of the microorganisms, fi lters rely on separation of the undesirable items (microorganisms
as well as nonviable particles) from the fl uid. Because fi ltration requires
passage of the fl uid from the “ dirty ” (upstream) side of fi lter to the clean (downstream)
side of the fi lter, the downstream piping and equipment must be both
“ clean ” and sterile prior to the start of the fi ltration process. This will ordinarily
require the use of SIP procedures or sterilization followed by aseptic assembly.
Sterilizing fi ltration of parenterals is a complex and often inadequately considered
subject, and numerous controls are required on the fi lter, fl uid, and sterilizing/
STERILIZATION AND DEPYROGENATION 119
120 STERILE PRODUCT MANUFACTURING
operating practices employed. PDA Technical Reports 26 and 40 can be instructive
in understanding the relevant concerns [28, 29] .
2.1.7 FACILITY AND SYSTEM: QUALIFICATION AND VALIDATION
Facilities for the manufacture of sterile products require the qualifi cation/validation
of the systems/equipment and procedures utilized for that production. Each system
described above and others with a direct/indirect impact on the quality of the products
being produced should be placed into operation using a defi ned set of practices.
The general approach is described below, and best practices include the development
of traceable documentation from project onset. The preferred approach begins
during a project ’ s conceptual design phase where provisions for meeting the CGMP
expectations and user requirement specifi cations establishing the technical basis for
the processes are fi rst defi ned. This is commonly followed by the validation master
planning exercise in which the user requirement specifi cations are used as a basis
for the development of acceptance criteria for process control studies. This effort
should be accompanied by an analysis of risk that considers product attributes,
target patient population, as well as technical and compliance requirements. Detailed
design follows in which the specifi cs of the various systems are refi ned. Construction
of the facility and fabrication of the process equipment follows and a variety of
controls are necessary during these activities to satisfy user requirements for compliance
of the various elements of the facility. Typically, factory acceptance testing
(FAT) will be done on all key process equipment, usually at the manufacturer ’ s
plant site; much of the information gathered during FAT can be referenced in the
qualifi cation activities to follow. Physical completion is followed by a well - defi ned
step termed commissioning in which construction and fabrication errors and omissions
are addressed. Site acceptance testing of installed process equipment may be
done in parallel with facility commissioning. Formal qualifi cation of the facility
ensues in which the installed systems and equipment are evaluated for their conformance
to the design expectations. The very last steps in this process are variously
termed performance qualifi cation. Detailed discussion of these subjects is not possible
within the constraints of this chapter, however the qualifi cation/validation of
equipment, systems, and processes has been extensively addressed in the literature
[30] .
2.1.8 ENVIRONMENTAL CONTROL AND MONITORING
Confi rmation of appropriate conditions for aseptic processing and its supportive
activities is required by regulation. In the highest air quality environment utilized
for aseptic processing, ISO 5, there is a general expectation that the air and surfaces
be largely free of microbial contamination and the number of particles be within
defi ned limits (less than 3500 particles greater than 0.5 . m/m 3 ). Proving the complete
absence of something is an impossible requirement, so the usual expectation is that
99+% of all samples taken from this most critical environment be free of detectable
microorganisms. The minimum monitoring expectations for these environments as
defi ned by the regulators are consistently attainable in nearly all instances,
especially those with lesser expectations. This is accomplished by proper design,
periodic facility disinfection, and measures to control the ingress of microorganisms
and particles for materials entering each environment from adjacent less clean areas
[31] .
2.1.8.1 Sanitization and Disinfection
Disinfection is customarily performed by gowned personnel during nonoperating
periods using such agents as phenolics, quaternary ammonium compounds, aldehydes,
and other nonsporicidal agents. The frequency of treatment varies with the
ability of the facility to maintain the desired conditions between disinfection. Sporicidal
agents such as dilute hydrogen peroxide or bleach are reserved for those
occasional periods when control over the spore population warrants and is often
employed after lengthy maintenance shutdowns or at the end of construction. Isolation
technology replaces the manual disinfection with reproducible decontamination
with a sporicidal agent and thus assures a superior level of environmental
control as compared to manned environments. The manual treatments fall short of
this level of control due to the uncertainties of the manual procedure and recontamination
of the environment as a consequence of the very personnel and activities
utilized to disinfect it. To mitigate these weaknesses, automatic sporicidal disinfection
of manned clean spaces has been developed by multiple vendors. Disinfection
of the less critical environments is accomplished in the same manner albeit on a less
frequent interval befi tting their higher allowable levels of microorganisms.
2.1.8.2 Monitoring
Aseptic environments are subject to a variety of monitoring systems including air,
surface, and personnel monitoring for viable microorganisms and for nonviable
particles. Environmental monitoring programs are often developed during the qualifi
cation of a new facility using a multiphase approach. Methods for the monitoring
and expectations for performance have been extensively discussed in the literature
and will only be addressed briefl y in the context of this chapter [1, 2, 31, 32] . In
general, the frequency and intensity of monitoring and concern for cleanliness
increases as the product progresses from preparation steps (typically in ISO 7/8
environments) to more important activities (nonsterile compounding in ISO 6) and
ultimately into the aseptic core (aseptic compounding and fi lling in ISO 5). Sampling
site and time selection should be a balance between the need to collect meaningful
data and avoidance of sampling interventions that could adversely (and inadvertently)
impact product quality. Microbiological sampling must always be done by
well - trained staff utilizing careful aseptic technique. This will both minimize risk to
the product and also improve the reliability of the data by reducing the likelihood
of false - positive results.
Air Sampling The relative cleanliness of air in the most critical environment is
assessed using passive sampling systems such as settle plates or estimated volumetrically
using active air samplers. Active air samplers should be designed to be isokinetic
in operation to avoid disruptions to unidirectional airfl ow. Considerable
variability has been reported among the several sampling methods employed for
ENVIRONMENTAL CONTROL AND MONITORING 121
122 STERILE PRODUCT MANUFACTURING
active air sampling, and there are also reports that active air sampling may have
advantages in terms of sensitivity. Passive sampling using settle plates can be a useful
adjunct in critical areas with limited access and where an active sampler might
interfere with airfl ow or entail a worrisome intervention risk. It must be recognized
that attempts to support the “ sterility ” of the cleanest aseptic environments (those
in ISO 5) by aggressive sampling may have exactly the opposite effect. Sampling
too frequently will increase process contamination risk by causing critical interventions
that are best avoided within these very clean environments. As personnel are
the greatest single source of microbial contamination and conduct the sampling,
sampling intensity should be carefully considered. There is no value to taking air
samples beyond those required to assess the relative cleanliness level within the
environment.
Surface Sampling Surfaces in the classifi ed environments are monitored using a
variety of methods but most commonly with contact plates (on smooth surfaces) or
swabs (for irregular surfaces). Surface sampling in aseptic environments (ISO 5/6)
is typically performed after the completion of the process to avoid the potential for
adventitious contamination of the production materials as a consequence of sampling
activities during the process. Fortunately, studies indicate that contamination
does not build up during typical processing operations in modern clean rooms.
Sampling with these materials may leave a trace of media or water on the sampled
surface, and cleaning of the surface immediately after sampling is commonplace.
Sampling of product contact surfaces (i.e., fi ll needles, feeder bowls, etc.) should only
be performed after completion of the process, and the results of this testing should
not be considered as an additional sterility test on the products. As in any form of
manual environmental sampling, the risk of contamination by samplers during the
processing of a sample makes the data less than completely reliable. Sampling of
surfaces such as walls and fl oors should not be overdone because with good attention
to aseptic technique they should be of little concern relative to actual process
risk. Sampling on these surfaces is probably most useful in assessing ongoing changes
in microfl ora and to confi rm the adequacy of the disinfection program.
Personnel Sampling The monitoring of personnel gown surfaces is an adaptation
of surface sampling in which samples are taken from surfaces on the operator. In
ISO 5 environments, this ordinarily entails the gloved hands and perhaps forearms.
As with any other sampling of a critical surface (the gloved hand is often in closest
proximity to sterile product contact surfaces and sterilized components), the sampling
should be performed at the conclusion of the aseptic activity. Sampling during
the midst of the process risks contamination of the product and should be avoided.
Sampling of other aseptic gown surfaces is ordinarily restricted to gowning certifi cation
or postmedia fi ll testing, where more aggressive sampling can sometimes be
informative. Whenever a gowned individual is sampled, the sample should be taken
in the background environment (not ISO 5), and the individual should immediately
exit and regown before continuing any further activity in the aseptic core area.
Sampling of personnel in less critical environments can be useful; however, meeting
regulatory expectations in these areas is ordinarily straightforward. Recommended
contamination levels often distinguish among the different room classifi cation levels
found within clean rooms. While this may seem reasonable, it is not completely
logical since operators often move frequently between these different levels of classifi
cation during the conduct of their work.
Total Particulate Monitoring Confi rming the ability of the facility ’ s heating, ventilation,
and air - conditioning (HVAC) system to maintain the appropriate conditions
throughout (to the extent practical) the classifi ed environments is most easily
accomplished using electronic total particle counters that can provide near immediate
feedback on conditions during production operations. Total particle samples can
be taken automatically, using permanently installed probes oriented into the unidirectional
airfl ow. As such, they can be positioned proximate to critical activities to
reaffi rm the continued quality of the air in the vicinity of the sterile materials and
surfaces. Manual total particulate air sampling can be a dangerous intervention and
therefore if required should be timed so as to minimize risk to product. Attempts
to correlate total particle counts with microbial counts have proven diffi cult. Correlations
are only meaningful when the source of foreign material is personnel since
people are the only source of airborne contamination within an aseptic processing
area. When personnel are the only source of particulate, the ratio between viable
and nonviable particles have been consistently found to be > 1000 : 1, which means
that in ISO 5 environments even relatively large total particulate count excursions
would typically contribute microbial contamination that fell far below the limit of
detection. Process equipment can and often does contribute airborne particulate
matter but not detectable levels of microbial contamination. Also, microbial sampling
is highly variable with respect to sensitivity, accuracy, precision, and limit of
detection making correlations, particularly in rooms of highest air quality. So, it
might seem logical to think that particle excursions are indicative of coincident
microbial excursions especially in the cleaner environments (ISO 5) where the
aseptic process takes place.
It is common practice for fi rms to interrupt their aseptic processes when atypical
total particulate excursions are observed so that the scientists and engineers can
determine the source of the foreign material. Monitoring frequency and expectations
in the less critical environments is always reduced relative to the critical aseptic
environments.
Where fi rms have introduced unidirectional air systems in preparations and
compounding areas for particle control, there is often the temptation to expect these
areas to meet the same microbial limits that these locations might attain in the
aseptic core. This temptation should be resisted to avoid unnecessary sampling and
deviations associated with expecting these environs to meet the conditions of aseptic
areas where sanitization frequency, background environment, and most importantly
personnel gowning are far superior to that found in the less clean locales [33] .
Housekeeping An important component of environmental control are the housekeeping
activities utilized to clean the facility external to the controlled environments.
Aseptic operations utilize a series of protective environments to protect the
sterile fi eld. Controls on the surrounding unclassifi ed areas are an important part
of the overall control scheme for sterile manufacturing. These unclassifi ed areas
support sterile operations in a variety of ways, and it is important to conduct activities
therein that assist in the environmental control. Routine housekeeping, periodic
sanitization, and even occasional environmental monitoring may be appropriate to
ENVIRONMENTAL CONTROL AND MONITORING 123
124 STERILE PRODUCT MANUFACTURING
assure that microbial and particle loads on items, equipment, and personnel entering
the classifi ed environments is appropriately controlled.
2.1.9 PRODUCTION ACTIVITIES
The preparation of sterile materials requires execution of a number of supportive
processes that together constitute the manufacturing process. They are intended to
control bioburden, reduce particle levels, remove contaminants, sterilize, and/or
depyrogenate. Nearly all of these activities occur within the controlled environments
and are subject to qualifi cation/validation.
2.1.9.1 Material and Component Entry
Prior to the start of any production activity, materials and components must be
transferred from a warehouse environment into a classifi ed environment. For most
items this will necessitate removal from boxes or cartons, transfer to a nonwooden
pallet, and passage through an air lock which serves as the transfer system between
the controlled and uncontrolled environments. Often components are contained
within plastic bags within a box or carton, and in some cases there are multiple bag
layers to facilitate disinfection and passage through air locks into different zones of
operation within the aseptic area. The fi rm may utilize an external disinfection
of the materials in conjunction with this transfer. The concern is for minimization
of particles and bioburden on these as yet unprocessed items in order to protect the
controlled environment.
Raw materials may be weighed in a weigh area in which they are transferred to
plastic bags and/or noncorrugate containers prior to the transfer. The weighing area
provides ISO 7 or better conditions, and may be a dedicated portion of the warehouse
proper; in a central weighing/dispensing area; or in a location contiguous to
the compounding area. Sterile ingredients are never opened anywhere other than
an aseptic environment and must be handled aseptically at all times including sampling
and processing of samples.
2.1.9.2 Cleaning and Preparation
Once the container component items have been introduced into the preparations
area, they must be readied for sterilization/depyrogenation. For many items this
consists of washing/rinsing processes designed to remove particles and reduce bioburden
and endotoxin levels. The application of silicone suspensions for glass or
closure materials is sometimes employed to provide lubrication allowing smoother
feeding of components or dispensing (elimination of product accumulation on vial).
Following the cleaning, items for sterilization are dried, wrapped, and staged/stored
for steam sterilization. Washed containers are either placed in trays or boxes for
depyrogenation in ovens or are directly loaded into dry - heat tunnels. It is common
practice to protect all washed items with ISO 5 air from the completion of washing,
through either wrapping or placement into a sterilizer or oven for passage into the
aseptic area. The intention is to avoid foreign matter that could result in contamination
of product.
It is increasingly common for components to be supplied by the vendor in a
ready - to - sterilize condition (washed and pretreated as necessary). Some items are
available in a ready - to - use confi guration with the supplier providing sterile and
pyrogen - free components. The use of supplier - prepared items eliminates the need
for preparation activities at the fi ll site and requires modifi cation of material in - feed
practices relative to on - site prepared items.
The process equipment (portable tanks, valves, fi ll needles, etc.) and consumable
materials (fi lters, hoses, gaskets, etc.) are prepared using a variety of methods. Portable
tanks are subjected to CIP (and perhaps SIP as well) in the preparation area.
Smaller items are disassembled (if necessary) and cleaned either manually or in a
cabinet washer. After cleaning they are wrapped and staged/stored prior to sterilization.
Tubing should not be reused; its preparation typically consists of fl ushing with
WFI followed by cutting to the required length. It is best to preassemble fi ll sets
with tubing, fi lters, and fi ll needles/pumps and then wrap them in preparation for
sterilization. This process obviates poststerilization assembly steps and therefore
mitigates contamination risk. These steps may be performed in ISO 5 environments
to reduce total particulate contamination on the items.
There are items that must be transferred into the aseptic processing area that
cannot be treated within a sterilizer/oven. These include portable tanks, electronic
equipment, and containers of sterile materials (ready - to - use items, sterile powders,
environmental monitoring media, etc.). Air locks, pass - throughs, and similar designs
are employed in which the exterior surfaces of the items are disinfected. The disinfection
process may be completed by personnel outside and/or inside the aseptic
area depending upon the specifi cs of the design.
At the completion of the cleaning process, the items should be free of contaminating
residues including traces of prior products, free of endotoxin, and well - controlled
in terms of total particulate and microbial levels. This level of control would be
appropriate regardless of whether the items, equipment, or components are to be
sterilized or not. Sterilization, other than by relatively high temperature dry heat, has
only a modest impact on endotoxin levels; cleaning provides the only means to
control endotoxin for materials and equipment that is sterilized by other means.
2.1.9.3 Compounding
Fixed equipment in the compounding area (nonaseptic or aseptic) is cleaned in
place. This eliminates traces of prior products, particles, and pyrogens. Sterilization
in place is required for the aseptic fi xed equipment and is sometimes employed for
the nonaseptic equipment as well as a bioburden control measure. Fixed transfer
lines must be cleaned and sterilized as well, and this is accomplished independently
or in conjunction with the vessels. The reuse of hoses and tubing is discouraged as
cleaning and extractables cannot be confi rmed beyond a single use.
The preparation of the product is performed within a classifi ed environment with
careful attention to the batch record, especially for time limits and appropriate
protection of materials during handling to guard against all forms of contamination.
This is proper for nonsterile compounding to minimize contamination prior to fi ltration/
sterilization and is required for aseptic compounding activities. Barrier designs
and other means of physically separating the worker from the product are recommended
as a minimum even in nonaseptic compounding. As compounding may
PRODUCTION ACTIVITIES 125
126 STERILE PRODUCT MANUFACTURING
expose the worker to a variety of potent/toxic materials, the use of personnel protective
equipment may be required. In extreme cases, the use of containment system
may be required to protect the compounding operator.
Where the compounding is nonaseptic, careful control over the environment,
materials, and equipment is still appropriate to reduce viable/nonviable levels and
to reduce the potential for endotoxin. Time limits should be imposed on manufacturing
operations for additional control over microorganisms and thus microbial
toxins.
Once the materials have been sterilized, interventions near either the formulation
or product contact surfaces/parts should be minimized. Direct handling of these
materials should only be done with sterilized tools or implements; nonsterile objects,
such as operator gloves, should never directly contact a sterilized surface. Sampling,
fi lter integrity testing, process connection, and other activities should all be designed
to eliminate the need for personnel exposure to sterile items.
Aseptic compounding is often a required activity for sterile products that cannot
be fi lter sterilized. The preparation of the sterile solids for use in these formulations
is outside the scope of this chapter, but it is often acknowledged as the most diffi cult
of all pharmaceutical processes to properly execute. Handling these materials at the
fi ll site is performed using ISO 5 environments, and the use of closed systems is
preferred [34] .
2.1.9.4 Filling
Aseptic fi lling is performed in ISO 5 environments, and a variety of approaches are
utilized with the technology choice largely dependent upon the facility design, batch
size, and package design. Older plants utilize manned clean rooms in which
aseptically gowned personnel operate the fi lling equipment: performing the setup,
supplying components, making any required adjustments, and conducting the environmental
monitoring. As human operators are directly or indirectly responsible
for essentially all microbial contamination, aseptic fi lling operations are increasingly
designed to minimize the potential for operator contamination to enter the critical
environment. Barriers of various sophistication and effectiveness are employed to
increase the protection afforded to sterile materials. The most evolved of the clean -
room designs are RAB systems in which personnel interventions are restricted to
defi ned locations. Many newer facilities utilize isolation technology in which the
fi lling environment is fully enclosed and personnel contamination is completely
avoided.
Filling designs for syringes and ampoules differ only with respect to the details
of component handling and closure design. However, it is wise not to underestimate
the infl uence of both component quality and component handling reliability on
contamination control in aseptic processing. Components that minimize the need
for intervention and equipment that is rather tolerant of component variability will
result in better contamination control performance. Aside from these distinctions,
the range of fi lling technologies previously described is also possible.
The fi lling of plastic containers is accomplished using two very different
approaches. Pre - formed containers can be sterilized in bulk, introduced into the
aseptic suite via air locks, oriented (unscrambled), and fi lled. Blow-fi ll - seal prepares
sterile bottles (most often LDPE) on line just prior to fi lling and sealing.
Filling of suspensions, emulsions, and other liquids may require slightly different
fi lling designs to assure uniformity of dose in each container. Ointments and creams
are sometimes fi lled at elevated temperatures to improve their fl ow properties
through the delivery and fi lling equipment. These are ordinarily fi lled into presterilized
plastic tubes that have largely replaced aluminum tubes for these formulations.
Powders are typically fi lled in vials using equipment specifi cally engineered for that
purpose.
An inerting gas (typically nitrogen, but other gases can be utilized) may be added
to the headspace of the container to protect formulations that are oxygen sensitive.
If the product is particularly sensitive to oxygen, purging may be done in the empty
container prior to fi lling and again immediately after fi lling. Products may also be
fi lled in an isolator under a nitrogen atmosphere if required. Products that require
inert gas purging will also generally require inert gas for pressurization of tanks to
provide motive force to drive the product through the fi lter(s) and into the fi lling
reservoir.
2.1.9.5 Stoppering and Crimping
If the product is not freeze dried, the primary closure or “ stopper ” is applied shortly
after completion of the fi lling process to better assure the sterility of the contents.
When the product is to be lyophilized, the stopper may be partially inserted after
fi lling and be fully seated after completion of the lyophilization cycle. Alternatively,
the container could be left open and a stopper applied after completion of the
drying.
Crimping is the act of securing the closure to the vial. It must be performed with
suffi cient uniform downward force to assure the container is properly secured. Too
little downward force results in inadequately secured closures, while excessive force
can result in container breakage. The force contributed by the crimp roller may be
controllable as well.
Applying the closure to syringes, ampoules, and other containers usually differs
in methodology from the approaches used for vials, but the objective is identical to
secure the container ’ s contents fully assuring the product ’ s critical quality attributes
(especially sterility) are maintained throughout its shelf life.
2.1.9.6 Lyophilization
Lyophilization (or freeze - drying) is a process utilized to convert a water - soluble
material fi lled into a container to a solid state by removal of the liquid while frozen.
The process requires the use of deep vacuums and careful control of temperatures.
By conducting the process under reduced pressure, the water in the container converts
from ice directly to vapor as heat is applied and is removed from the container
by the vacuum. The dissolved solids in the formulation cannot undergo this phase
change and remain in the container. At the completion of the cycle, the container
will be returned to near atmospheric pressure; stoppers are applied or fully seated
and crimped as described above. Lyophilization is particularly common with biological
materials whose stability in aqueous solution may be relatively poor. The time
period in solution and the temperature of the solution are kept at a specifi ed low
temperature to prevent product degradation [35] .
PRODUCTION ACTIVITIES 127
128 STERILE PRODUCT MANUFACTURING
As partially stoppered but unsealed containers must be transferred to the
lyophilizer from the fi ll line, various designs have been utilized to protect the containers
during this transit. Among the common alternatives utilized are the
following:
• Placement of the lyophilization in the wall of the fi ll room to allow for direct
loading
• Battery - operated unidirectional airfl ow carts to a remote lyophilizer
• ISO 5 – protected conveyors with single shelf loading
• Transfer utilizing isolator technology
The use of trays for supporting the containers during the transfer, loading, lyophilization,
and unloading steps was at one time common. The major problem with the
use of trays for this purpose was the heat/handling - related distortion of the tray
bottom that impacted the uniformity of the heating process in the freeze dryer. This
was overcome by the use of trays with bottoms that were removed after loading
and reinserted after completion of the drying. The current preference is for the
placement of the containers directly on the shelf eliminating the trays entirely. This
is accomplished by single height loading/unloading of the individual shelves with
various pusher designs.
The use of thermocouples to monitor product temperature inside selected
vials with the lyophilizer is still the prevalent practice. The utility of this data is
questionable and the current trend is to eliminate this “ requirement ” as soon as
possible to better assure sterility of the unsealed vials by eliminating placement of
the thermocouples.
The lyophilizer chamber and condenser should be cleaned with a CIP system
after each batch to prevent cross - contamination and, after cleaning, both should be
sterilized. If a slot door loading system is utilized, periodic opening of a full door in
the lyophilizer may be required to remove stoppers and glass that may have
fallen.
2.1.10 PERSONNEL
Aseptic processing in the pharmaceutical industry is almost entirely dependent
upon the profi ciency of the personnel assigned to this most critical of all activities.
The operators must be able to consistently aseptically transfer sterile equipment
and materials in a manner that avoids contamination of those materials [1] . This is
no mean feat given the contamination continuously released by personnel and the
prevailing need for personnel for execution of the process activities.
Personnel profi ciency in aseptic operations must be fi rmly established before
they are allowed to conduct critical aseptic process steps. Operators must master a
number of relevant skills in order to be declared competent. The usual progression
is from classroom training (CGMP, microbiology, sterilization, etc.) to relevant
practical exercises (aseptic media transfers, aseptic gowning rehearsals) and ultimately
to the core aseptic skills required (aseptic gowning certifi cation, aseptic
assembly/technique) using a growth medium. Through this approach the operator
gradually acquires the necessary skills to be a fully qualifi ed member of the production
staff. Training/qualifi cation of personnel is an ongoing requirement and must
be repeated periodically to assure the skills are maintained. Continuing evaluation
of operator qualifi cation is accomplished using written examinations, practical challenges,
documented observation, and participation in process simulation trials.
There is general acknowledgment of the risk associated with heavy reliance on
personnel for aseptic processing. This has fostered much of the innovative designs
for aseptic fi lling such as RABS and isolators where personnel are largely removed
from the critical environment. The future will undoubtedly witness aseptic technologies
where human interaction with sterile materials has been eliminated.
2.1.11 ASEPTIC PROCESSING CONTROL AND EVALUATION
The preparation of any pharmaceutical product requires controls over the production
operations to assure the end result is a product that meets the required quality
attributes. The methods utilized for this control are supported by formalized validation
studies in which proof of consistency is demonstrated by appropriately designed
experiments. The defi nition of appropriate operating parameters is the primary
objective of the development activities and is further confi rmed during scale - up to
commercial operations. The validation supports that the routine controls applied to
the process are appropriate to assure product quality [36] . This is typically accomplished
in formalized validation activities in which expanded sampling/testing of the
product materials is performed to substantiate their uniformity and suitability for
use [30] .
2.1.11.1 In - Process Testing
The sampling and testing of in - process materials during the course of the manufacturing
process can confi rm that essential conditions have been provided. This is
appropriate in preparation, compounding, and fi lling activities. Sampling in preparation
processes can confi rm the absence of particles, proper siliconization levels, and
cleanliness of equipment to assure that production items and equipment are suitable
for use. Samples for microbiological quality, must, as previously mentioned, always
be done by fully gowned staff under ISO 5 conditions using excellent aseptic techniques.
During compounding, in - process testing can confi rm proper pH, dissolution
of materials, bioburden, and potency prior to fi lling. Filling operations can be monitored
for fi ll volume (weight), headspace oxygen, and particles. These activities can
all be automated to reduce interventions. These are typical examples of in - process
controls utilized to assure acceptability of the process while it is underway. In the
event of an abnormal result, corrective measures could be applied before further
processing. The validation effort supports that these control measures are suffi cient
to assure product quality, when met during production operations. The sample
intervals, sizes, and locations for in - process testing are chosen to enhance the validation.
The tolerance limits are usually tightened relative to the release requirements
to further assure that no out - of - tolerance materials are produced.
ASEPTIC PROCESSING CONTROL AND EVALUATION 129
130 STERILE PRODUCT MANUFACTURING
2.1.11.2 End - Product Testing
Upon completion of the process, samples are taken to establish that the batch meets
the fi nal product specifi cations defi ned for release. Predefi ned sampling plans are
utilized to obtain representative samples of the entire batch, the prior validation
effort having assured through an expanded sampling effort that the process provides
a uniform product. End - product sampling often suffers from the inability to link an
anomalous result with a specifi c portion/segment of the batch. If the validation is
insuffi ciently rigorous, an out - of - specifi cation result will ordinarily result in rejection
of the batch and little opportunity to take effective corrective action.
The FDA has been supportive of the use of process analytical technologies
(PATs) as an improvement on end - product testing [37] . These are intended to act
as on - line indicators of critical product attributes enabling immediate corrective
action and preventing the production of off - specifi cation materials. This approach
is common in the continuous process industries where feedforward controls are
often employed. Their application to the more batch - oriented pharmaceutical/biotechnology
industry is an acknowledgment that this approach can assure product
quality more fully than a sampling - based approach. The PAT applications are still
relatively few in number, but their utility in lieu of traditional quality methods is
certainly promising.
The preceding relates solely to product quality attributes, based upon chemical
or physical requirements. Assurance of sterility, the most critical of all the quality
components for an aseptically fi lled sterile product relies on the following:
• The validation of the various sterilization processes for preparation of materials,
equipment, and formulations
• The design of the aseptic manufacturing process and facility
• The establishment and maintenance of a proper processing environment
• Most importantly, the profi ciency of the operating personnel directly involved
with the aseptic process
There is no direct means to evaluate the cumulative capability of these measures.
We infer success in aseptic processing through the evaluation of indirect measures
of performance: air pressure differentials, total particle counts, viable monitoring
results, and end - product sterility testing. The enormous challenge of aseptic processing
is that none of the in - process or end - product testing results can prove that the
attribute of sterility is attained with a high degree of certainty. Therefore, we rely
on validation and the demonstration of a validated state of control to infer the
adequacy of our contamination control efforts.
2.1.11.3 Process Simulations
An indirect means of assessing a facility ’ s aseptic processing performance is the
process simulation (or media fi ll) test [38] . This test substitutes a growth medium
for the product in the process from the point of sterilization through to closure of
the product container. The expectation is that successful handling of the growth
media through the operating steps provides assurance that product formulations
handled in a similar fashion would also be successful [39] . Process simulations
culminate in the incubation of the media - fi lled containers with success defi ned as a
limited number of contaminated units in a larger number of fi lled units. The result
is a contamination rate for the media fi ll, and not a direct indication of the level of
sterility assurance afforded to aseptically processed materials using the same procedures.
At the present time, the level of sterility provided to aseptically processed
materials cannot be measured. The FDA and EMEA have harmonized their expectations
relative to process simulation performance, but they have also asserted that
the goal in every process simulation is zero contamination [1, 2] . This formalized
expectation and recognition that patient safety should always be preeminent have
resulted in substantial improvements in aseptic processing technology over the last
20 years.
2.1.12 TERMINAL STERILIZATION
Terminal sterilization is a process by which product is sterilized in its fi nal container.
Terminal sterilization is the method of choice for products that are suffi ciently
stabile when subjected to a compatible lethal treatment. Because the process utilized
is expected to be lethal to the microorganisms present, is highly reproducible,
and generally readily validated, there is a clear preference for its use [1, 40, 41] .
The predominant method for terminal sterilization is moist heat, and a substantial
percentage of sterile products are processed in this manner. (Estimates range
from 5 to 15% of all sterile products are terminally sterilized.) The sterilization often
requires the attainment of a balance between sterility assurance and degradation of
the material ’ s essential properties [42] . The overkill sterilization method is preferred
for heat - resistant materials, and may be usable for terminal sterilization where the
formulation can tolerate substantial heat input. The bioburden/biological indicator
approach uses less heat input but requires increased control over the titer and
resistance of the bioburden organisms present.
The large - volume parenteral (LVP) industry sometimes uses dedicated nonaseptic
fi lling systems for its containers prior to subjecting them to terminal treatments.
These LVP systems may approach the aseptic designs described earlier, but they are
not supported by the same levels of environmental monitoring nor process simulation.
Application of terminal sterilization at small volume parenteral producers may
be done after the product is aseptically fi lled, although this practice is usual only
where the fi rm produces predominantly aseptically fi lled products and would not
have a fi lling system dedicated to terminally sterilized formulations. Product
that will be subject to terminal sterilization may be fi lled under clean conditions
with reduced environmental monitoring and control. However, control of total
particulate levels requires unidirectional airfl ow for critical fi lling or assembly
processes.
Terminal sterilization is most commonly accomplished by moist heat. Terminal
sterilization by other means is certainly possible, and a very limited number of parenteral
drugs are treated with dry heat or radiation after fi lling. There is growing
interest in the use of radiation, including low - energy E - beam, as a terminal treatment
suggesting more products will be processed in this manner.
Although there are numerous advantages to terminal sterilization, there can be
very good reasons for aseptically fi lling products that are stabile enough to be com-
TERMINAL STERILIZATION 131
132 STERILE PRODUCT MANUFACTURING
patible with a sterilization process. For example, multichamber containers that
cannot withstand terminal sterilization may provide a very important safety benefi t
to the patient by reducing aseptic admixture or reconstitution in the clinic. These
aseptic activities when conducted in clinics are generally not able to be done within
anything like the controls required in industrial aseptic processing. It is often benefi -
cial to discuss processing technology choices with regulatory authorities early in the
development of a new product.
2.1.13 CONCLUSION
The manufacture of parenteral drugs by aseptic processing has long been considered
a diffi cult technical challenge. These products require careful control and stringent
attention to detail to assure their safety. Aseptic processing done with discipline and
taking advantage of the numerous technical developments that have occurred over
the years results in sterile products that can be administered with complete confi -
dence. The wider adaptation of advanced aseptic processing will result in further
evolutionary improvements in aseptic processing. The industry is at the beginning
of an era in which human - scale aseptic processing will be completely replaced by
separative technologies and process automation. Additionally, improved in - process
controls are likely to be implemented making validation easier and easing the compliance
burden.
APPENDIX
Parenteral Drug Association, Bethesda, Maryland
TM 1: Validation of Steam Sterilization Cycles, 1978
TR 3: Validation of Dry Heat Processes used for Sterilization & Depyrogenation,
1981
TR 7: Depyrogenation, 1985
TR 11: Sterilization of Parenterals by Gamma Irradiation, 1988
TR 13: Fundamentals of an Environmental Monitoring Program, 2001
TR 22: Process Simulation Testing for Aseptically Filled Products, 1996
TR 26: Sterilizing Filtration of Liquids, 1998
TR 28: Process Simulation Testing for Sterile Bulk Pharmaceutical Chemicals,
2006
TR 34: Design & Validation of Isolator Systems for the Manufacture & Testing
of Health Care Products, 2001
TR 36: Current Practices in the validation of Aseptic Processing, 2002
TR 40: Sterilizing Filtration of Gases, 2005
International Society For Pharmaceutical Engineering, Tampa, Florida
Baseline Guide, Vol. 3: Sterile Manufacturing Facilities, 1999
Baseline Guide, Vol. 4: Water and Steam Systems, 2001
Baseline Guide, Vol. 5: Commissioning and Qualifi cation, 2001
REFERENCES
1. U.S. Food and Drug Administration (FDA) ( 2004 ), Guideline on sterile drug products
produced by aseptic processing, FDA, Washington, DC.
2. European Union (EU) (2006), Annex 1—Sterile medicinal products—draft revision.
3. International Organization for Standardization (ISO) , international standard 14644 1 - 3.
4. U.S. Food and Drug Administration (FDA) ( 2004 ), Pharmaceutical CGMPs for the
twenty - fi rst century —A risk-based approach, FDA, Washington, DC.
5. International Conference on Organization (ICH) ( 2005 ), Draft consensus guideline
quality risk management Q9, draft.
6. Whyte , W. , and Eaton , T. ( 2004 ), Microbiological contamination models for use in risk
assessment during pharmaceutical production , Eur J Parenteral Pharm Sci , 9 ( 1 ).
7. Whyte , W. , and Eaton , T. ( 2004 ), Microbial risk assessment in pharmaceutical cleanrooms ,
Eur J Parenteral Pharm Sci , 9 ( 1 ).
8. Agalloco , J. , and Akers , J. ( 2006 ), Simplifi ed risk analysis for aseptic processing: The
Akers - Agalloco method , Pharm Technol , 30 ( 7 ), 60 – 76 .
9. International Organization for Standardization (ISO) ( 2004 ), Cleanrooms and associated
controlled environments — Part 7: Separative devices (clean air hoods, gloveboxes, isolators
and mini - environments), ISO 14644 - 7 .
10. Agalloco , J. ( 2006 ), Thinking inside the box: The application of isolation technology for
aseptic processing , Pharm Technol ., p. S8 – 11 .
11. Lysford , J. , and Porter , M. ( 2003 ), Barrier isolators history and trends , Pharm Eng , 23 ( 2 ),
58 – 64 .
12. ISPE ( 2005 ), Restricted access barrier systems (RABS) for aseptic processing, ISPE defi -
nition, Aug. 16.
13. Wikol , M. ( 2004 ), GoreTM vial isolator, ISPE presentation, Feb. 12.
14. Py , D. ( 2004 ), Development challenges for intact sterile fi lling, PDA presentation,
Mar. 9.
15. Thilly , J. ( 2004 ), CVFL technology from lab scale to industry, PDA presentation, Mar. 8.
16. ISPE (2001), Water and Steam Systems Baseline® guide.
17. ISPE ( 1999 ), Sterile Manufacturing Facilities Baseline ® guide.
18. ISPE (2001), Water and Steam Systems Baseline® guide.
19. PDA ( 2006 ), Technical Monograph 1, Industrial moist heat sterilization in autoclaves,
draft 17.
20. Perkins , J. ( 1969 ), Principles and Methods of Sterilization in Health Sciences , Charles
Thomas , Springfi eld, IL .
21. Phillips , G. B. , and Morrissey , R. F. ( 1993 ), Sterilization Technology: A Practical Guide for
Manufacturers and Users of Health Care Products , Van Nostrand Reinhold , New York .
22. Agalloco , J. ( 1998 ), Sterilization in place technology and validation , in Agalloco , J. , and
Carleton , F. J. , Eds., Validation of Pharmaceutical Processes: Sterile Products , Marcel
Dekker , New York .
23. PDA ( 1981 ), Technical Report 3, Validation of dry heat processes used for sterilization
and depyrogenation.
24. Case , L. , and Heffernan , G. ( 1998 ), Dry heat sterilization and depyrogenation: Validation
and monitoring , in Agalloco , J. , and Carleton , F. J. , Eds., Validation of Pharmaceutical
Processes: Sterile Products , Marcel Dekker , New York .
25. Burgess , D. , and Reich , R. ( 1993 ), Industrial ethylene oxide sterilization , in Phillips , G. B. ,
and Morrissey , R. F. Eds., Sterilization Technology: A Practical Guide for Manufacturers
and Users of Health Care Products , Van Nostrand Reinhold , New York .
REFERENCES 133
134 STERILE PRODUCT MANUFACTURING
26. Sintim - Damao , K. ( 1993 ), Other gaseous sterilization methods , in Phillips , G. B. , and Morrissey
, R. F. Eds., Sterilization Technology: A Practical Guide for Manufacturers and Users
of Health Care Products , Van Nostrand Reinhold , New Youk .
27. Meltzer , T. , Agalloco , J. , et al. ( 2001 ), Filter integrity testing in liquid applications ; Revisited,
Part 1, Pharm Technol , 25 ( 10 ), and Part 2, Pharm Technol , 25 ( 11 ).
28. PDA (1998), Technical Report 26, Sterilizing fi ltration of liquids.
29. PDA (2005), Technical Report 40, Sterilizing fi ltration of gases.
30. Agalloco , J. , and Carleton , F. J. , Eds. ( 1998 ), Validation of Pharmaceutical Processes: Sterile
Products , Marcel Dekker , New York .
31. PDA (2001), Technical Report 13, Fundamentals of an environmental control program.
32. USP . 1116 . ( 2005 ), Microbiological control and monitoring environments used for the
manufacture of healthcare products , Pharm Forum , 31 ( 2 ), Mar. – Apr.
33. Agalloco , J. ( 1996 ), Qualifi cation and validation of environmental control systems , PDA
J Pharm Sci Technol , 50 ( 5 ), 280 – 289 .
34. PDA ( 2006 ), Technical Report 28, Process simulation testing for sterile bulk pharmaceutical
chemicals.
35. Trappler , E. ( 1998 ), Validation of lyophilization , in Agalloco , J. , and Carleton , F. J. , Eds.,
Validation of Pharmaceutical Processes: Sterile Products , Marcel Dekker , New York .
36. Chapman , K. G. ( 1984 ), The PAR approach to process validation , Pharm Technol , 8 ( 12 ),
22 – 36 .
37. Food and Drug Administration (FDA) ( 2004 ), PAT guidance for industry — A framework
for innovative pharmaceutical development, manufacturing, and quality assurance, FDA,
Washington, DC.
38. PDA ( 1998 ), Technical Report 22, Process simulation testing for aseptically fi lled
products.
39. Agalloco , J. , and Akers , J. ( 2006 ), Aseptic processing for dosage form manufacture: Organization
& validation , in Carleton , F. J. , and Agalloco , J. P. , Eds., Validation of Pharmaceutical
Processes: Sterile Products , Marcel Dekker , New York .
40. Food and Drug Administration (FDA) ( 1991 ), Use of aseptic processing and terminal
sterilization in the preparation of sterile pharmaceuticals, FR 56, 354 – 358 .
41. PIC/S41. ( 1999 ), Decision trees for the selection of sterilisation methods
(CPMP/QWP/054/98).
42. PDA ( 2006 ), Technical Monograph 1, Industrial moist heat sterilization in autoclaves,
draft 17.
ADDITIONAL READINGS
Akers , J. ( 2001 ), An overview of facilities for the control of microbial agents , in Block , S. S. ,
Ed., Disinfection, Sterilization and Preservation , 5th ed, Lippincott, Williams and Wilkins ,
Philadelphia , pp. 1123 – 1138 .
Akers , J. , and Agalloco , J. ( 1997 ), Sterility and sterility assurance , J Pharm Sci Technol 51 ,
72 – 77 .
Cole , J. C. ( 1990 ), Pharmaceutical Production Facilities — Design and Application , Ellis
Norwood , Chicester .
Institute of Environmental Science and Technology (IEST) ( 1995 ), Compendium of standards,
practices, and similar documents relating to contamination control, CC009/
IESCC009.2, IEST, Mt. Prospect, IL.
Ljungvist , B. , and Reinmueller , B. ( 1995 ), Ventilation and Airborne Contamination in Clean
Rooms , Pharmacia A/B , Stockholm .
Reinmuller , B. ( 2000 ), Microbiological risk assessment of airborne contaminants in clean
zones, Bulletin No. 52, Royal Institute of Technology/Building Services and Engineering,
Stockholm.
United States Pharmacopoeia/National Formulary ( 2006 ), 29, Chapter 1116, Microbial evaluation
of clean rooms, Rockville, Maryland, pp. 2969 – 2976 .
ADDITIONAL READINGS 135
FACILITY
SECTION 3
139
3.1
FROM PILOT PLANT TO
MANUFACTURING: EFFECT OF
SCALE - UP ON OPERATION OF
JACKETED REACTORS
B. Wayne Bequette
Rensselaer Polytechnic Institute, Troy, New York
Contents
3.1.1 Motivation
3.1.2 Background
3.1.2.1 Pharmaceutical Process Development
3.1.2.2 Batch Reactors
3.1.2.3 Reaction Calorimetry
3.1.3 Laboratory Vessels and Reaction Calorimeters
3.1.3.1 Material and Energy Balances
3.1.3.2 Estimating Fluid Properties and Heat Transfer Coeffi cients from Calorimeter
Data
3.1.3.3 Estimating Heat Flows
3.1.3.4 Relating Heat Flows and Conversion
3.1.3.5 Semibatch Reactions
3.1.3.6 Rapid Scale - Up Relationships
3.1.3.7 Strategy under a Cooling System Failure
3.1.4 Heat Transfer in Process Vessels
3.1.4.1 Heat Transfer Relationships
3.1.4.2 Effect of Reactor Type, Jacket Heat Transfer Fluid, and Reactor Fluid
Viscosity
3.1.4.3 Pilot - and Production - Scale Experiments
3.1.5 Dynamic Simulation Studies
3.1.6 Summary
References
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
140 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
3.1.1 MOTIVATION
There are many phases of process development between the discovery of an active
pharmaceutical ingredient and the design, construction, and operation of a manufacturing
process to produce a drug. A sequence of reactions and separations that
is successful at the bench scale may lead to a process that is unsafe, is diffi cult to
operate, or produces unsatisfactory product at the manufacturing scale. A manufacturing
process typically has a large sequence of steps, involving several different unit
operations (heat exchangers, reactors, separators, etc.), and a complete review of the
design and scale - up of these unit operations would constitute a chemical engineering
curriculum; thus, the focus of this chapter is the scale - up of jacketed batch
chemical reactors from the laboratory to the pilot plant and manufacturing. These
reaction vessels often serve many functions, including mixing, heating, cooling,
distillation, and crystallization.
Temperature control for laboratory reactors is typically easy because of high heat
transfer area – reactor volume ratios, which do not require large driving forces (temperature
differences) for heat transfer from the reactor to the jacket. Pilot - and
full - scale reactors, however, often have a limited heat transfer capability. A process
development engineer will usually have a choice of reactors when moving from the
laboratory to the pilot plant. Kinetic and heat of reaction parameters obtained from
the laboratory reactor, in conjunction with information on the heat transfer characteristics
of each pilot plant vessel, can be used to select the proper pilot plant
reactor.
Similarly, when moving from the pilot plant to manufacturing, a process engineer
will either choose an existing vessel or specify the design criteria for a new reactor.
A necessary condition for operation with a specifi ed reactor temperature profi le is
that the required jacket temperature is feasible. We have therefore chosen to focus
on heat transfer – related issues in scale - up. Clearly there are other scale - up issues,
such as mixing sensitive reactions. See Paul [1] for several examples of mixing scale -
up in the pharmaceutical industry.
In this chapter we discuss important issues as we move from laboratory to pilot
plant and manufacturing. A review of batch process operation and pharmaceutical
research is covered in Section 3.1.2 , followed by laboratory vessels and reaction
calorimetry in Section 3.1.3 . In Section 3.1.4 heat transfer in process vessels is presented,
including the effect of reactor type and heat transfer fl uid on the vessel heat
transfer capability. In Section 3.1.5 dynamic behavior based on simulation studies
is discussed.
3.1.2 BACKGROUND
3.1.2.1 Pharmaceutical Process Development
Anderson [2] presents a wide range of topics on pharmaceutical process development,
including a number of different problems related to process scale - up, such as
solvent and reagent selection, purifi cation, and limitations to various operations. He
notes that most reactors used for scale - up operations are selected for fl exibility in
running many different processes, especially for pilot plants and multiproduct manufacturing
plants.
BACKGROUND 141
Pisano [3] discusses the management of process development projects in the
pharmaceutical industry. Case studies are used to illustrate the effect of resource
allocation decisions at different stages of a project. While there has been a focus on
product development in the pharmaceutical industry, clearly process development
plays an important role in getting a product to market and lowering the long - term
product manufacturing costs.
3.1.2.2 Batch Reactors
Batch processes present challenging control problems due to the time - varying
nature of operation. Chylla and Haase [4] present a detailed example of a batch
reactor problem in the polymer products industry. This reactor has an overall heat
transfer coeffi cient that decreases from batch to batch due to fouling of the heat
transfer surface inside the reactor. Bonvin [5] discusses a number of important
topics in batch processing, including safety, product quality, and scale - up. He notes
that the frequent repetition of batch runs enables the results from previous runs to
be used to optimize the operation of subsequent ones.
LeLann et al. [6] discuss tendency modeling (using approximate stoichiometric
and kinetic models for a reaction) and the use of model predictive control (linear
and nonlinear) in batch reactor operation. Studies of a hybrid heating – cooling
system on a 16 - L pilot plant are presented.
Various aspects of the effect of process scale - up on the safety of batch reactors
have been discussed by Gygax [7] , who presents methods to assess thermal runaway.
Shukla and Pushpavanam [8] present parametric sensitivy and safety results for
three exothermic systems modeled using pseudohomogenous rate expressions from
the literature. Caygill et al. [9] identify the common factors that cause a reduction
in performance on scale - up. They present results of a survey of pharmaceutical and
fi ne chemicals companies indicating that problems with mixing and heat transfer
are commonly experienced with large - scale reactors.
3.1.2.3 Reaction Calorimetry
The microanalytical methods of differential thermal analysis, differential scanning
calorimetry, accelerating rate calorimetry, and thermomechanical analysis provide
important information about chemical kinetics and thermodynamics but do not
provide information about large - scale effects. Although a number of techniques are
available for kinetics and heat - of - reaction analysis, a major advantage to heat fl ow
calorimetry is that it better simulates the effects of real process conditions, such as
degree of mixing or heat transfer coeffi cients.
Regenass [10] reviews a number of uses for heat fl ow calorimetry, particularly
process development. The hydrolysis of acetic anhydride and the isomerization of
trimethyl phosphite are used to illustrate how the technique can be used for process
development. Kaarlsen and Villadsen [11, 12] provide reviews of isothermal reaction
calorimeters that have a sample volume of at least 0.1 L and are used to measure
the rate of evolution of heat at a constant reaction temperature. Bourne et al. [13]
show that the plant - scale heat transfer coeffi cient can be estimated rapidly and
accurately from a few runs in a heat fl ow calorimeter.
Landau et al. [14] use a heat fl ow calorimeter to investigate feasible pilot plant
operating conditions for the production of a pharmaceutical intermediate. They
142 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
determine kinetic and heat fl ow parameters using the calorimeter and further estimate
heat transfer parameters for a pilot - scale reactor. Simulation studies are used
to fi nd the required jacket temperature for desired batch reactor temperature pro-
fi les. Semibatch operation is shown to be safer than normal batch operation. Landau
[15] provides a detailed review of reaction calorimetry, including mathematical
expressions for energy balances, and a number of application examples.
3.1.3 LABORATORY VESSELS AND REACTION CALORIMETERS
As reviewed in Section 3.1.2.3 , reaction calorimeters can be used to better understand
and characterize scale - related process phenomena, such as mixing and heat
transfer. A heat fl ow calorimeter, the Mettler RC1e, is shown in Figure 1 . A
schematic of a similar calorimeter system is shown in Figure 2 [16] . A heat fl ow
calorimeter can be used to estimate:
• Physical parameters (heat capacity)
• Reaction rate constants
• Heat transfer coeffi cients (overall, U or, or fi lm, h i )
3.1.3.1 Material and Energy Balances
The overall energy balance for a process with no reaction has the form
Energy accumulation energy in heat transfer from jacket
energy i
=
+ n by calibration probe energy lost by ambient heat transfer .
FIGURE 1 Mettler RC1e heat fl ow calorimeter system ( www.mt.com ).
FIGURE 2 Schematic of HEL SIMULAR reaction calorimeter. From ref. 16 .
Additional heater
F3 F2 F1
Stirrer
Tamb
Qadd
n
Condenser
Tw,in
TR pHR pR
Tj,out
Tj,in
Tw,out and mw
Inert gas
venting
Circulation
thermostat,
heater, chiller
Liquid
surface
Oil jacket
Outlet valve Scale Scale Scale
which is shown mathematically as
( ) ( ) ( ) mc
dT
dt
UAT T q k T T p r j = . . + . . cal loss amb (1)
where ( mc p ) r
is the reactor thermal capacitance, T is the reactor temperature, T j is
the jacket temperature, U is the overall heat transfer coeffi cient, A is the area for
heat transfer, q cal is the heat fl ow from the calibration probe, and the fi nal term
accounts for heat loss from the reactor system. The thermal capacitance is composed
of the fl uid in the vessel as well as the inert components in contact with the fl uid,
including the vessel wall, agitator (stirrer), and sensors (e.g., thermocouple), as
shown in the equation
( ) mc V c m c p r p v pv = + . (2)
where V is the volume of liquid, . is the liquid density, c p is the liquid heat capacity,
m v is the mass of the vessel wall and other inerts, and c pv is the average heat capacity
of the vessel wall and inerts. The inert contributions and heat transfer to the ambient
can be found from extensive calibration studies. For small - scale reactors, such as
reaction calorimeters, the thermal mass of the inerts can be signifi cant. The thermal
capacitance ratio, sometimes called the Lewis number, is given as
.
. .
= =+
( ) mc
V c
m c
V c
p r
p
v pv
p
1 (3)
which can be on the order of 1.5 – 2 for a small - scale reactors and adiabatic calorimeters
but is often 1.05 – 1.10 for small pilot plant reactors and less than 1.02 for
manufacturing - scale reactors.
LABORATORY VESSELS AND REACTION CALORIMETERS 143
144 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
3.1.3.2 Estimating Fluid Properties and Heat Transfer Coeffi cients from
Calorimeter Data
In a heat fl ow calorimeter, a feedback controller is used to maintain a constant
desired reactor temperature by adjusting the jacket temperature. From (1), with a
constant calibration probe heat fl ow, at steady state ( dT / dt = 0), the overall heat
transfer coeffi cient can be found from
UA
q k T T
T Tj
=
. .
.
cal loss amb ( )
(4)
Also, the fl uid heat capacity can be found by ramping up the reactor temperature
and using
( )
( ) ( )
mc
UAT T q k T T
dT dt p r
j =
. . + . . cal loss amb
/
(5)
and solving for c p from (2), assuming that the reactor inert component contributions
are known from previous studies. An example calibration study is shown in Figure
3 , where a constant heat fl ow is applied from 35 to 42 min, enabling the heat transfer
coeffi cient to be estimated from the temperature difference using Equation (4) .
Then, the heat capacity is estimated from the temperature ramp applied between 5
and 20 min. It should be noted that the heat transfer coeffi cient and heat capacity
of the fl uid may vary with concentration and temperature. Typically, calibration
experiments are performed before and after the reaction; then the heat transfer
coeffi cient and heat capacity are assumed to vary linearly with conversion or batch
time. For polymerization reactions in particular, the viscosity can increase tremendously
with conversion, causing a substantial decrease in the heat transfer coeffi -
FIGURE 3 Example reaction calorimetry study without reaction. The overall heat transfer
coeffi cient area can be found during the steady - state temperature difference and known calibration
probe heat fl ow, between 35 and 42 min. The heat capacity can then be found from
the temperature ramp between 5 and 20 min.
RC1 Calibration profiles
Determine UA
Determine cp
Time, min Temperature, °C
28
26
24
22
20
20 30 40 50 60
18
0 10
reactor
jacket
cient. Reaction experiments can be run at several temperatures to fi nd the functional
relationship with temperature.
Since the heat transfer area as a function of liquid volume is known, the overall
heat transfer coeffi cient U can be calculated from (4). The overall heat transfer
coeffi cient is calculated as
1 1
U h
x
k i
g
g
= + (6)
where the jacket side resistance is negligible. The glass vessel heat transfer resistance
( x g / k g , thickness/thermal conductivity) can be used to fi nd the reactor fl uid heat
transfer coeffi cient ( h i ).
3.1.3.3 Estimating Heat Flows
The reaction heat fl ow can be found by rearranging (1), with the calibration heat
probe replaced by the reaction heat fl ow, to fi nd
q mc
dT
dt
UA T T k T T r p r j = + . + . ( ) ( ) ( ) loss amb (7)
The total heat released during the reaction can be found by integrating (7),
Q qdt r
tf
tot = .0 (8)
or, represented as a scaled (per - unit mass) total heat release,
Q
Q
V
Q
m tot
tot tot = =
.
(9)
The molar heat of reaction can be found from
.H
Q
n rxn
tot
rxn
=
.
(10)
where n rxn is the molar amount reacted.
As a “ fi rst - pass ” calculation, if it is assumed that the dominant heat transfer
resistance is on the reactor side, then the overall heat transfer coeffi cient ( U ) from
(4) can be used for scale - up.
3.1.3.4 Relating Heat Flows and Conversion
The reaction heat fl ows are directly related to the conversion of reactants [14] .
Consider a fi rst - order reaction of a limiting reactant, with the rate expression
dC
dt
kC = . (11)
LABORATORY VESSELS AND REACTION CALORIMETERS 145
146 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
where C is the molar concentration of the reactant. The heat fl ow is
q
dC
dt
H V kC H V r =( ) = . . . rxn rxn (12)
with an initial heat fl ow of
q kC H V r0 0 = . . rxn (13)
dividing (12) by (13), we fi nd the relationship between concentration and heat
fl ow:
C
C
q
q
r
r 0 0
= (14)
For an isothermal reaction, the solution to (11) is
C
C
e kt
0
= . (15)
so, the heat fl ow for an isothermal reaction is
q
q
e r
r
kt
0
= . (16)
Thus, the reaction rate constant k can be estimated from the reaction heat fl ow
without making any concentration measurements. Assuming an Arrhenius rate
expression
k Ae E RT = .
0
/ (17)
the rate constant at several temperatures can be used to estimate the frequency
factor ( A 0 ) and activation energy ( E ). (See ref. 14 for an example application.)
3.1.3.5 Semibatch Reactions
For extremely exothermic reactions it is necessary to slowly add the feed over time,
that is, operate in a semibatch fashion. The heat fl ow for a semibatch reaction can
be found from
q UAT T mc
dT
dt
m c T T r j p r f pf f = . + + . ( ) ( ) ( ) (18)
where mf is the mass fl ow rate of the feed stream. If the reactor temperature is
maintained constant, this reduces to
q UAT T mc T T r j f pf f = . + . ( ) ( ) (19)
For reactions with essentially instantaneous kinetics, the reaction rate is limited by
the feed addition rate. For other reactions, particularly if the reactor is operated at
too low of a temperature, a reactant concentration can “ build up, ” eventually reaching
an unsafe level that could lead to a rapid temperature rise and explosion. It is
important for these reactions to monitor the heat fl ow to confi rm that the reactant
concentration is not increasing to unacceptable levels.
3.1.3.6 Rapid Scale - Up Relationships
Lacking knowledge of the larger scale reactor, it is tempting to simply assume that
only the area for heat transfer varies upon scale - up. A natural parameter is the
cooling time , 1 defi ned as
.
. .
co= =
( ) mc
UA
V c
UA
p r p (20)
The heat transfer area varies with the square of the vessel diameter, and the volume
varies with the cube of the vessel diameter. Thus the area – volume ratio ( A / V ) varies
with volume as
A
V V
~
1
1/3
(21)
The inverse cooling time relationship for scale - up from volume V 1 to V 2 is
UA
V c
UA
V c
V
V p p . . . .
.
. .
.
. .
= .
. .
.
. .
( ) 2 1
1
2
1/3
(22)
The required reactor - jacket temperature difference on scale - up, with a constant
Lewis number, is
[ ] [ ] T T T T
V
V j j . = . ( ) 2 1
2
1
1/3
(23)
so the temperature difference can increase dramatically when a process is scaled up
several orders of magnitude. Reactor - jacket temperature difference constraints can
be particularly important for glass - lined vessels, where the limit is often 75 ° C.
3.1.3.7 Safety under a Cooling System Failure
In the event of a cooling system failure it can be assumed that the reactor operates
adiabatically. The adiabatic temperature rise can be found from
1 The notion of cooling time can be understood by writing (1) and assuming no calibration energy or heat
loss. Then (1) becomes . co ( dT / dt ) = . ( T . T j ). If a constant temperature difference T . T j is applied, it
will take . co time units for the reactor temperature to change by the temperature difference.
LABORATORY VESSELS AND REACTION CALORIMETERS 147
148 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
.T
Q
mcp r
ad
tot =
( )
(24)
and the fi nal temperature is
T T T final initial ad = +. (25)
As long as the fi nal temperature is less than some critical “ onset ” temperature where
a secondary decomposition reaction occurs, then the process can safely handle a
cooling system failure. If a batch reactor temperature cannot be assured to remain
less than the onset temperature after a cooling system failure, then a semibatch
operation should be used. As noted in Section 3.1.3.5 , it is necessary to assure that
reactant concentration is not increasing above an onset concentration where a
similar decomposition could occur with a cooling system failure.
3.1.4 HEAT TRANSFER IN PROCESS VESSELS
Based on initial heat fl ow calorimetry studies, a process development engineer must
choose the appropriate reactor vessels for pilot plant studies. A pilot plant typically
has vessels that range from 80 to 5000 L, some constructed of alloy and others that
are glass lined. In addition some vessels may have half - pipe coils for heat transfer,
while others have jackets with agitation nozzles. A process drawing for a typical
glass - lined vessel is shown in Figure 4 . In Sections 3.1.4.1 and 3.1.4.2 we review
fundamental heat transfer relationships in order to predict overall heat transfer
coeffi cients. In Section 3.1.4.3 we review experimental techniques to estimate heat
transfer coeffi cients in process vessels.
3.1.4.1 Heat Transfer Relationships
Reactor - Side Coeffi cient The reactor - side heat transfer coeffi cient is calculated
as
h a
k
D i
i
i
i i = Re Pr . . 0 67 0 33 (26)
where a is the agitation constant (0.33), k i is the fl uid thermal conductivity, Re i is
the Reynolds number, and Pr i is the Prandtl number,
Rei
ag i
i
D N
=
2 .
.
(27)
Pri
i pi
i
c
k
=
.
(28)
FIGURE 4 Typical 300 - or 500 - gal jacketed vessel ( www.pfaudler.com ).
SRW 3525 drive
Lubricated dry
mechanical seal
Drive nozzle
face
E
10.
5.
6.
3. 18.
12.
3. Legs (four)
45. Leg circle
54. O.D.
48. I.D.
13.
(3. Nozs.) (4. Nozs.)
A
B
C
D
F
Optional side
supports
Fin Battle w/RTD
Temperature Sansor
23. Span
Cryo-Lock CBT
2. Cplgs. (Two)
1/2. Cplg.
1/2. Cplg.
(4) 3/4. dia. holes
equally spaced on
a 10. BC
14 1/4. (6. Noz.)
4 1/4.
3. Noz.
1 1/2. Cplgs.
13 1/4.
1 1/2. agit.
nozs.
(Offset)
and N is the agitator rotation rate. It should be noted that the fi lm heat transfer
coeffi cient varies inversely with the viscosity, that is,
hi
i
~ .
1
0 33 .
(29)
Reactions where the viscosity increases substantially with conversion, such as some
polymerization reactions, can be particularly diffi cult to control upon scale - up.
Jacket - Side Coeffi cient Here the calculations are shown for a jacket equipped
with agitation nozzles that greatly increase the jacket fl uid velocity. The jacket “ swirl
velocity ” v j is calculated (iteratively) from the nonlinear algebraic relationship
[17]
m v v
fL
D
v
A n n j
e
j
f ( ) . =( ).. .
.. .
4
2
2
. (30)
HEAT TRANSFER IN PROCESS VESSELS 149
150 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
where mn the is the nozzle mass fl ow rate, v n is the nozzle velocity, the friction factor
is
f =
. 2 0 023
0 2
.
Re .
(31)
the jacket - side fi lm coeffi cient is
h
k
D j
j
e
j j = 0 027 0 8 0 33 . Re Pr . . (32)
and the Reynolds and Prandtl numbers are
Rej
e j j
j
D v
=
.
.
(33)
Prj
j pj
j
c
k
=
.
(34)
Overall Coeffi cient The overall heat transfer coeffi cient is found from the sum of
the resistances,
1 1 1
U h h
x
k
x
k
ff ff
i j
m
m
g
g
i j = + + + + + (35)
which includes reactor fi lm, jacket fi lm, vessel metal, vessel glass, and fouling factors
for both the reactor and jacket sides.
3.1.4.2 Effect of Reactor Type, Jacket Heat Transfer Fluid, and Reactor
Fluid Viscosity
Here we present examples of how the reactor type and heat transfer fl uid affect the
heat transfer coeffi cient. When the reactor fl uid has a low viscosity, the dominant
heat transfer resistance tends to be on the jacket side. When the reactor fl uid has a
high viscosity, however, the dominant resistance is typically on the reactor side.
Parameter values for the studies are presented in Figures 5 – 7 and are given in the
literature [18] .
The overall heat transfer coeffi cient is much higher for an alloy reactor/half - pipe
jacket than for a glass - lined carbon steel reactor/agitation nozzle jacket, as shown
in Figure 5 , where Syltherm is the heat transfer fl uid. Syltherm has a signifi cantly
lower heat transfer coeffi cient than an ethylene glycol mixture, as shown in Figure
6 , but is capable of operating over a wider range of temperatures. The reactor fl uid
viscosity has a tremendous effect on the overall heat transfer coeffi cient, as shown
in Figure 7 . This can be particularly important in polymerization reactions where
viscosity increases with conversion.
FIGURE 5 Overall heat transfer coeffi cient for 500 - gal reactors. Comparison of alloy half
pipe with glass - lined carbon steel (GLCS). Syltherm is the heat transfer fl uid. ( From ref. 18 ,
with permission .)
–50 0 50 100 150 200 250
20
30
40
50
60
70
80
Jacket temperature °C
Overall U, English units
Half pipe
Jacket w/nozzles
FIGURE 6 Overall heat transfer coeffi cient for 500 - gal GLCS reactor. Comparison of Syltherm
with Glycol. ( From ref. 18 , with permission .)
–50 0 50 100 150 200 250
15
20
25
30
35
40
45
50
55
60
Jacket temperature °C
Overall U, English units
Syltherm
Glycol
3.1.4.3 Pilot - and Production - Scale Experiments
The relationships shown in Section 3.1.3 are also pertinent to large - scale reactors.
By using different solvents and volumes of solvent, pilot and production reactor
heat transfer characteristics can be determined from a series of experiments. A
primary limitation, compared to reaction calorimeter characterization, is that a calibration
probe is rarely available. Thus, heat - up and cool - down studies, performed
HEAT TRANSFER IN PROCESS VESSELS 151
152 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
FIGURE 7 Overall heat transfer coeffi cient for 500 - gal GLCS reactor with glycol heat
transfer fl uid. Comparison of effect of reactor - side viscosity.
–20 0 20 40 60 80 100 120
10
15
20
25
30
35
40
45
50
55
60
Jacket temperature °C
Overall U, English units
500 gal. GLCS. 1 cp vs. 3000 cp
1 cP
3000 cP
by varying the jacket temperature and observing the changes in the reactor temperature
(for solvents with known heat capacity), are used to characterize the
reactor. The inverse cooling time,
UA
mc
dT dt
T T p r j ( )
=
.
/
(36)
can be estimated from the temperature data collected from a heat - up/cool - down
study. A characteristic example for a pilot - scale reactor is shown in Figure 8 . The
FIGURE 8 Temperature profi les (jacket inlet, jacket outlet, and reactor) for a pilot plant
reactor. ( From ref. 19 .)
Time, min
0 20 40 60 80 100 120 140 160 180
100
80
60
40
20
0
temperature, °C
FIGURE 9 Cooling time estimates based on data presented in Figure 8 . ( From ref. 19 .)
0 10
28
27
26
25
24
23
22
21
20
19
20 30
Jacket temperature, °C
mCp/UA, min
40 50 60 70 80 90
cool-down
heat-up
resulting cooling time estimates are shown in Figure 9 . Notice that the overall heat
transfer coeffi cient is clearly a function of the jacket temperature. The reduced heat
transfer at the lower jacket temperatures is due to the strong relationship between
viscosity and temperature for the 40% glycol solution used in the jacket. The discontinuity
in the cooling time estimate at around 45 ° C may be due to two factors.
One factor is the assumption of no heat loss from the vessel, which would tend to
lower the UA estimates during the heat - up phase. Another factor is the assumption
that the metal and glass inerts in the reactor are at the temperature of the reactor;
in practice it might be a better assumption that the reactor wall in particular is at a
temperature that is intermediate between the jacket and reactor temperatures.
The fl uid and inert thermal masses can be independently estimated by conducting
experiments with a number of different solvent amounts. From the cooling time
expression
( ) mc
UA
m c
UA
V c
UA
p r v pv p = +
.
(37)
writing this as a function of the reactor fl uid volume,
( ) mc
UA
m c
UA
c
UA
V p r v pv p = + .
.
(38)
and conducting experiments at a number of different fl uid volumes or, equivalently,
masses ( V . ),
( ) mc
UA
m c
UA
c
UA
V p r v pv p = + .. (39)
the linear regression can be used to fi nd the slope and intercept and thus estimate
the UA and m v c pv terms [19] . This approach is shown in Figure 10 for a jacket tem-
HEAT TRANSFER IN PROCESS VESSELS 153
154 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
FIGURE 10 Linear regression to estimate thermal mass and UA . ( From ref. 19 .)
200 250 300 350
Mass of water, kg
30
28
26
24
22
20
18
mCp/UA, min
perature of 60 ° C (based on a total of eight experiments at fi ve different reactor
fl uid volumes).
3.1.5 DYNAMIC SIMULATION STUDIES
Older pilot plant and manufacturing processes often used steam for heating and
water for cooling, with a switch - and - purge strategy between the two modes. Recent
process designs have two heat transfer fl uid systems (hot and cold heat transfer
fl uids) that are used for most of the heating and cooling needs. In addition, some
vessels may have nitrogen coolers for cryogenic operation.
A simplifi ed schematic for a jacket heat transfer service is shown in Figure 11
[18] . Here, two separate heat transfer fl uid headers are used, and the control valve
is on the outlet stream to reduce the temperature shocks that might occur if a single
FIGURE 11 Characteristic pilot plant vessel control strategy. Slave (secondary) controller
based on jacket outlet temperature is shown. The control valve is on the outlet stream to
minimize temperature gradients (when switching from hot to cold fl uids) that would be
imposed if the valve was on the inlet. ( From ref. 18 , with permission .)
TC1
TC2
From hot HT
fluid system
From cold HT
fluid system
To cold HT
fluid system
To hot HT
fluid system
control valve was on the inlet stream. Depending on the range of temperatures,
either ethylene glycol or a proprietary fl uid such as Syltherm is used. Depending
on whether heating or cooling is needed, either the hot or cold process control
valve is open. Similarly, on – off valves return fl uid to the appropriate distribution
system.
Although the heat transfer fl uid can be used over a wide range of temperatures,
the fi lm heat transfer coeffi cient is a strong function of temperature due to viscosity
effects. The “ cooling time ” of a large reactor operating at a low temperature can be
substantially longer than that of a small reactor operating at a high temperature
due to this strong temperature effect. Simulation studies can be used to:
• Understand the effect of heat transfer fl uid
• Understand possible performance limitations due to scale and operating
conditions
• Test the effect of specifi ed temperature gradient constraints
• Assist with controller design and selection of tuning parameters for system
start - up
Various levels of models can be used to describe the behavior of pilot - scale jacketed
batch reactors. For online reaction calorimetry and for rapid scale - up, a simple
model characterizing the heat transfer from the reactor to the jacket can be used.
Another level of modeling detail includes both the jacket and reactor dynamics.
Finally, the complete set of equations simultaneously describing the integrated
reactor/jacket and recirculating system dynamics can be used for feedback control
system design and simulation. The complete model can more accurately assess the
operability and safety of the pilot - scale system and can be used for more accurate
process scale - up.
In the simulation studies that follow, it is assumed that the reactor and jacket are
well mixed, resulting in differential equations for the material and energy balances
[18] . The reactor shell (including a glass lining, if used) and reactor internals (agitator
and baffl es) are at the same temperature as the reactor, so their “ thermal mass ”
is included in the reactor energy balance. Similarly, the jacket shell is at the jacket
temperature, with an associated thermal mass. The heat transfer area A is proportional
to the reactor liquid level (between volumes associated with the minimum
and maximum heat transfer area); also, the reactor shell thermal mass varies linearly
with the liquid level. Heat transfer coeffi cients are calculated using the relationships
presented in Section 3.1.4 ; see Garvin [20] or Dream [21] for detailed examples.
Parameters, viscosity in particular, are a function of temperature.
We focus on the effect of reactor size and material of construction on the expected
dynamic behavior of the reactors. Details on the model development and simulation
environment are presented elsewhere [18] . Figure 12 illustrates that a vessel can
have signifi cantly different dynamic behavior depending on whether it is being
heated or cooled (for illustrative purposes, the freezing point of water is neglected
in this simulation). The increase in reactor temperature results in a much faster
response than a decrease for two reasons: (i) the jacket heat transfer fl uid has a
much higher viscosity (resulting in a lower overall heat transfer coeffi cient) at low
temperatures and (ii) the fl uid fl ow rate/jacket temperature gain is proportional to
DYNAMIC SIMULATION STUDIES 155
156 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
FIGURE 12 Comparison of responses for ± 30 ° C reactor temperature setpoint changes at
t = 10 min; 500 - gal GLCS fi lled with water (1925 kg).
0 20 40 60 80 100 120 140 160 180 200
–40
–20
0
20
40
60
80
100
Time, min
Temperature, °C
FIGURE 13 Comparison of temperature responses for 30 ° C batch setpoint change; 500 - gal
GLCS, water (1925 L) vs. organic (1700 L).
0 20 40 60 80 100 120 140 160 180 200
20
30
40
50
60
70
80
90
100
Time, min
Temperature, °C
Water
Organic
the difference between the jacket temperature and make - up fl uid temperature,
which becomes small at low jacket temperatures. Notice that the initial response for
the temperature increase is constrained by the ramp limit of 5 ° C/min on the jacket
temperature. The temperature response of an organic solvent is much faster than
water because of the heat capacity difference, as shown in Figure 13 . The previous
plots were for simple heating/cooling applications (ref. 18 presents further studies
for cryogenic and semibatch systems).
3.1.6 SUMMARY
In this chapter we have presented an overview of scale - up considerations involved
as one moves from bench - scale reaction calorimetry to larger scale pilot plant and
production reactors. Our focus has been on heat transfer and single - phase processes,
addressing primarily the problem that the heat transfer area per unit reactor volume
decreases with scale. Clearly, there are many challenging problems associated with
multiphase vessels, with evaporation/distillation and crystallization as obvious
examples, but these topics are beyond the scope of this chapter.
REFERENCES
1. Paul , E. L. ( 1988 ), Design of reaction systems for specialty organic chemicals , Chem. Eng.
Sci. , 43 ( 8 ), 1773 – 1782 .
2. Anderson , N. G. ( 2000 ), Practical Process Research and Development , Academic , New
York .
3. Pisano , G. P. ( 1997 ), The Development Factory , Harvard Business School , Boston .
4. Chylla , R. W. , and Hasse , D. R. ( 1993 ), Temperature control of semi - batch polymerization
reactors , Comp. Chem. Eng. , 17 ( 3 ), 257 – 264 .
5. Bonvin , D. ( 1998 ), Optimal operation of batch reactors — A personal view , J. Proc. Cont. ,
8 ( 5 – 6 ), 355 – 368 .
6. LeLann , M. V. , Cabassud , M. , and Casamatta , G. ( 1999 ), Modeling, optimization and
control of batch chemical reactors in fi ne chemical production , Annu. Rev. Control , 23 ,
25 – 34 .
7. Gygax , R. W. ( 1990 , Feb.), Scale - up principles for assessing thermal runaway risks , Chem.
Eng. Prog. , 86 ( 2 ), 53 – 60 .
8. Shukla , P. K. , and Pushpavanam , S. ( 1994 ), Parametric sensitivity, runaway, and safety in
batch reactors: Experiments and models , Ind. Eng. Chem. Res. , 33 ( 12 ), 3202 – 3208 .
9. Caygill , G. , Zanfi r , M. , and Gavrildis , A. ( 2006 ), Scalable reactor design for pharmaceuticals
and fi ne chemicals production. 1: Potential scale - up obstacles , Org. Proc. Res. Dev. ,
10 ( 3 ), 539 – 552 .
10. Regenass , W. ( 1985 ), Calorimetric monitoring of industrial chemical processes , Thermochim.
Acta , 95 , 351 – 369 .
11. Kaarlsen, L. G. , and Villadsen, J. (1987), Isothermal reaction calorimeters—I. A literature
review , Chem. Eng. Sci. , 42 ( 5 ), 1153 – 1164 .
12. Kaarlsen , L. G. , and Villadsen , J. ( 1987 ), Isothermal reaction calorimeters — II. Data treatment
, Chem. Eng. Sci. , 42 ( 5 ), 1165 – 1173 .
13. Bourne , J. R. , Buerli , M. , and Regenass , W. ( 1981 ), Heat transfer and power measurements
in stirred tanks using heat fl ow calorimetry , Chem. Eng. Sci. , 36 , 347 – 354 .
14. Landau , R. N. , Blackmond , D. G. , and Tung , H. - H. ( 1994 ), Calorimetric investigation of an
exothermic reaction: Kinetic and heat fl ow modeling , Ind. Eng. Chem. Res. , 33 , 814 – 820 .
15. Landau, R. N. (1996), Expanding the role of reaction calorimetry , Thermochim. Acta , 289 ,
101 – 126 .
16. Obenndip , D. A. , and Sharratt , P. N. ( 2006 ), Towards an information - rich process development.
Part I: Interfacing experimentation with qualitatitive/semiquantitative modeling ,
Org. Proc. Res. Dev. , 10 ( 3 ), 430 – 440 .
REFERENCES 157
158 EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS
17. Bolliger , D. H. ( 1982 ), Assessing heat transfer in process - vessel jackets , Chem. Eng. , Sept.
20 , 95 – 100 .
18. Bequette , B. W. , Holihan , S. , and Bacher , S. ( 2004 ), Automation and control issues in the
design of a pharmaceutical pilot plant , Control Eng. Practice , 12 , 901 – 908 .
19. Zima , A. , Spencer , G. , and Bequette , B. W. ( 1996 ), Model development for batch reactor
calorimetry and control, Preprint, presented at the AIChE Annual Meeting, Chicago, IL,
Nov. 1996.
20. Garvin , J. ( 1999 ), Understand the thermal design of jacketed vessels , Chem. Eng. Prog. ,
95 ( 6 ), 61 – 68 .
21. Dream , R. F. ( 1999 ), Heat transfer in agitated jacketed vessels , Chem. Eng. , Jan., 90 – 96 .
159
3.2
PACKAGING AND LABELING
Maria In e s Rocha Miritello Santoro and Anil Kumar Singh
University of S a o Paulo, S a o Paulo, Brazil
Contents
3.2.1 Introduction
3.2.2 Packaging Materials
3.2.2.1 General Considerations
3.2.2.2 Glass as packaging material
3.2.2.3 Plastic as Packaging Material
3.2.2.4 Metal as Packaging Material
3.2.2.5 Applications: Some Examples
3.2.3 Quality Control of Packaging Material
3.2.3.1 General Considerations
3.2.3.2 Packaging Components
3.2.3.3 Inhalation Drug Products
3.2.3.4 Drug Products for Injection and Ophthalmic Drug Products
3.2.3.5 Liquid - Based Oral Products, Topical Drug Products, and Topical Delivery
Systems
3.2.3.6 Solid Oral Dosage Forms and Powders for Reconstitution
3.2.4 Importance of Proper Packaging and Labeling
3.2.5 Regulatory Aspects
3.2.5.1 General Considerations
3.2.5.2 Food, Drug and Cosmetic Act
3.2.5.3 New Drugs
3.2.5.4 Labeling Requisites
3.2.5.5 Prescription Drugs
3.2.5.6 Drug Information Leafl et
3.2.5.7 Other Regulatory Federal Laws
3.2.5.8 Fair Packaging and Labeling Act
3.2.5.9 United States Pharmacopeia Center for the Advancement of Patient
Safety
3.2.5.10 National Agency of Sanitary Vigilance (ANVISA, Brazil)
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
160 PACKAGING AND LABELING
3.2.5.11 International Committee on Harmonization (ICH)
3.2.5.12 European Union Regulatory Bodies
References
3.2.1 INTRODUCTION
The packaging of a pharmaceutical product fulfi ls a variety of roles, such as product
presentation, identifi cation, convenience, and protection until administration or use.
Selection of packaging requires a basic knowledge of packaging materials, the environmental
conditions to which the product will be exposed, and the characteristics
of the formulation. Several types of packaging are used to contain and protect the
pharmaceutical preparations, such as the primary packaging around the product and
secondary packaging such as a carton and subsequent transit cases [1] .
The principal objective of the modern pharmaceutical industry is to manufacture
pharmaceutical preparations presenting high quality, identity, purity, effectiveness,
and innocuity in order to guarantee the satisfaction and safety of patients. The
development of a new drug must involve the synthesis of a molecule, determination
of its pharmacological activity, industrial - scale production, and its commercialization
to guarantee quality of the fi nal product.
Packaging system development, including primary and secondary packaging
components, is of critical importance. The material should be selected based on the
characteristics of pharmaceutical product and dosage form. After the production
phase, packaging must be planned according to regulatory requirements and its
quality should be controlled according to the specifi cations.
Commercially, the packaging material is used as a barrier to protect the pharmaceutical
preparations against external factors that can degrade them and consequently
decrease their effectiveness and increase toxic effects.
Once the type of packaging material is decided based on such factors as size,
shape, capacity, and physicochemical properties, all these data, including quality
control tests, should be included in the specifi cation of the products in order to
assure the therapeutic effectiveness during its shelf life.
Several types of materials are in use in the preparation of containers and closure
systems: glass, plastics, metals, and combinations of these materials. However, care
should be taken in the selection of appropriate material. These materials should not
present any physical or chemical reactivity that could modify drug activity, quality,
purity, or physical characteristics of the drug and pharmaceutical preparations. Any
minor modifi cation in the pharmacopeial specifi cation is acceptable if it does not
present a threat to patient ’ s health.
The aim of this chapter is to discuss the importance of the packaging and labeling
of pharmaceutical preparations. The role of packaging and labeling in the pharmaceutical
industry has grown substantially over the past decade. The total packaging
operation is part of any drug development program. Pharmaceutical products generally
require a standard of packaging which is superior to that of most other products
in order to support and comply with their main requirements, such as effi cacy, integrity,
purity, safety, and stability.
PACKAGING MATERIALS 161
For these reasons packaging technology should be based on the understanding
of pharmaceutical products, characteristics of formulations, and dosage forms,
including the physical and chemical properties of the drug substance.
In the past, packaging concerns often arose only during the later steps of product
development. Today, packaging is integrated with the development step and is
among the earliest considerations of new pharmaceutical preparations being studied.
Labels of products can vary from the simple to the extremely complex. But, even
at the most basic level, product identifi cation should meet regulatory requirements.
More complex are the labels that make use of bar code technologies. New components
such as microchips, biosensors, and deoxyribonucleic acid (DNA) arrays are
making possible the development of new technologies leading to fi nished products
individually packed that require specialized packaging materials and design expertise.
The challenge now is to maintain low packaging cost, that is, always integrated
into the cost of the product itself.
Packaging in the post – World War II period benefi ted immensely from the commercialization
of plastics, which were little known or used in prior years. Since then,
the packaging industry has openly adopted plastics as a powerful new tool in the
development of new packaging forms and functions.
Quality control of a packaging component starts at the design stage. All aspects
of package development that may give rise to quality problems must be identifi ed
and minimized by good design. Identifying and correcting mistakes in packaging
will avoid product recall and rejection of pharmaceutical preparations [2, 3] .
3.2.2 PACKAGING MATERIALS
3.2.2.1 General Considerations
Packaging refers to all the operations, including fi lling and labeling, through which
a bulk product should pass to become a fi nished product. Usually, sterile fi lling is
not considered part of the packing process, although the bulk product is contained
in a primary container.
A packaging component means any single part of a container closure system.
Typical components are containers (e.g., ampules, vials, bottles), container liners
(e.g., tube liners), closures (e.g., screw caps, stoppers), closure liners, stopper overseals,
container inner seals, administration ports [e.g., on large - volume parenterals
(LVPs)], overwraps, administration accessories, and container labels [4] .
A primary packaging component is one that is or may be in direct contact with
the dosage form. A secondary packaging component is one that is not and will not
be in direct contact with the dosage form [4] .
A container closure system refers to the sum of packaging components that
together contain and protect the dosage form. This includes primary packaging
components and secondary packaging components, if the latter are intended to
provide additional protection to the drug product. A packaging system is equivalent
to a container closure system [4] .
The role of packaging material on the overall perceived and actual stability of
the dosage form is well established. Packaging plays an important role in quality
maintenance, and the resistance of packaging materials to moisture and light can
162 PACKAGING AND LABELING
signifi cantly affect the stability of drugs and their dosage forms. It is crucial that
stability testing of dosage forms in their fi nal packaging be performed. The primary
role of packaging, other than its esthetic one, is to protect the dosage forms from
moisture and oxygen present in the atmosphere, light, and other types of exposure,
especially if these factors affect the overall quality of the product on long - term
storage [5] .
The compliance packaging such as for fi xed - dose combination pills and unit
dosage form packaging is a therapy - related intervention and is designed to facilitate
medication regimens and so potentially improve adherence. Compliance packaging
can be defi ned as a prepackaged unit that provides one treatment cycle of the
medication, to both the pharmacist and the patient, in a ready - to - use package. This
innovation type of packaging is usually based on blister packaging that contain unit
therapeutic dose for one time use. The separate dosage units and separate days are
usually indicated on the dosage cards to help remind the patient when and how
much of the medication to take, for example, blister packed oral dosage forms with
drug information leafl ets and contraceptive pills [6, 7] .
The selection of packaging material for any pharmaceutical product is as important
as proper pharmaceutical dosage form. To guarantee the safe and adequate
delivery of drug product to the patient and improve patient compliance, the manufacturer
should consider the following factors:
1. Compatibility and safety concerns raised by the route of administration of the
drug product and the nature of the dosage form (e.g., solid or liquid based)
2. Kinds of protection the container closure system should provide to the dosage
form (e.g., photosensitive, hygroscopic, easily oxidized drug products)
3. Potential effect of any treatment or handling that may be unique to the drug
product in the packaging system
4. Patient compliance to the treatment and ease of drug administration
5. Safety, effi cacy, and quality of drug product throughout its shelf - life
The acquisition, handling, and quality control of primary and secondary packaging
materials and of printed materials should be accomplished in the same way as
that for the raw materials. The printed materials should be stocked in a reserved
place so the possibility of unauthorized access is avoided. The labels and other
rejected printed materials should be stored and transported with proper identifi cation
before being destroyed. There should be a destruction record of the printed
materials. Each batch of printed material and packaging material should receive a
specifi c reference number for identifi cation.
The identifi cation affi xed on the containers, on the equipment, in the facilities,
and on the product containers should be clear, without ambiguity, and in a format
approved by the company and contain the necessary data. Besides the text, differentiated
colors indicating its condition could be used (e.g., in quarantine, approved,
rejected, and cleaned).
The packing materials should attend to the specifi cations, giving emphasis to the
compatibility of the same with the pharmaceutical product that it contains. The
material should be examined with relation to visible physical and critical defects as
well as the required specifi cations.
PACKAGING MATERIALS 163
3.2.2.2 Glass as Packaging Material
A packaging system found acceptable for one drug product may not be appropriate
for another. Each application should contain enough information to show that each
proposed container closure system and its components are suitable for the intended
use.
Nonsterile Products
Solids Some topical drug products such as powders may be considered for marketing
in glass bottles with appropriate dispenser. These topical drug products may be
sterile and could be subject to microbial limits.
The most common glass - packed solid oral dosage forms are oral powders and
granules for reconstitution. A typical solid oral dosage form container closure system
is a glass bottle (although plastic bottles are also used) with a screw - on or snap - off
closure. A typical closure consists of a metal cap, often with a liner and frequently
with an inner seal.
The dry powders that are reconstituted in their marketed container need not be
sterile; however, the possibility of an interaction between the packaging components
and the reconstituting fl uid can’t be discarded. Although the contact time will be
relatively short when compared to the component/dosage form contact time for
liquid - based oral dosage forms, it should still be taken into consideration when the
compatibility and safety of the container closure system are being evaluated.
Powders for oral administration that are reconstituted in their market container,
however, have an additional possibility of interaction between the packaging components
and the reconstituting fl uid. Although the contact time will be relatively
short when compared to the component/dosage form contact time for liquid - based
oral dosage forms, it should still be taken into consideration when the compatibility
and safety of the container closure system are being evaluated.
Nonsolids For nonsterile products the preservative provides some protection, but
continual microbial challenge will diminish the effi cacy of the preservative, and
spoilage or disease transmission may occur [8] .
Antimicrobial preservatives such as phenylmercuric acetate are known to partition
into rubbers during storage, thus reducing the formulation concentration below
effective antimicrobial levels [9] . A complication of modern packaging is the need
for the application of security seals to protect against deliberate adulteration and
maintain consumer confi dence.
Sterile Products The sterile dosage forms share the common attributes that they
are generally solutions, emulsions, or suspensions and are all required to be sterile.
Injectable dosage forms represent one of the highest risk drug products (Table 1 ).
Any contaminants present (as a result of contact with a packaging component or
due to the packaging system ’ s failure to provide adequate protection) can be rapidly
and completely introduced into the patient ’ s general circulation. Injectable drug
products may be liquids in the form of solutions, emulsions, or suspensions or dry
solids that are to be combined with an appropriate vehicle to yield a solution or
suspension.
164 PACKAGING AND LABELING
Although ophthalmic drug products can be considered topical products, they
have been grouped here with injectables because they are required to be sterile and
the descriptive, suitability, and quality control information is typically the same as
that for an injectable drug product.
The potential effects of packaging component/dosage form interactions are
numerous. Hemolytic effects may result from a decrease in tonicity and pyrogenic
effects may result from the presence of impurities. The potency of the drug product
or concentration of the antimicrobial preservatives may decrease due to adsorption
or absorption.
A cosolvent system essential to the solubilization of a poorly soluble drug can
also serve as a potent extractant of plastic additives.
A disposable syringe may be made of plastic, glass, rubber, and metal components,
and such multicomponent construction provides a potential for interaction
that is greater than when a container consists of a single material.
Injectable drug products require protection from microbial contamination (loss
of sterility or added bioburden) and may also need to be protected from light or
exposure to gases (e.g., oxygen).
Performance of a syringe is usually addressed by establishing the force to initiate
and maintain plunger movement down the barrel and the capability of the syringe
to deliver the labeled amount of the drug product.
Solids For solids that must be dissolved or dispersed in an appropriate diluent
before being injected, the diluent may be in the same container closure system (e.
g., a two - part vial) or be part of the same market package (e.g., a kit containing a
vial of diluent).
Sterile powders or powders for injection may need to be protected from exposure
to water vapor. For elastomeric components, data showing that a component meets
the requirements of U.S. Pharmacopeia (USP) elastomeric closures for injections
will typically be considered suffi cient evidence of safety.
Nonsolids The package must prevent the entry of organisms; for example, packaging
of sterile products must be absolutely microorganism proof — hence the continued
use of glass ampules. Liquid injections are classifi ed as small - volume parenterals
(SVPs), if they have a solution volume of 100 mL or less, or as LVPs, if the solution
volume exceeds 100 mL [10] . Liquid - based injectables may need to be protected
from solvent loss.
An SVP may be packaged in a vial or an ampule. An LVP may be packaged in
a vial, a glass bottle or, in some cases, as a disposable syringe. Packaging material
for vials, and ampules are usually composed of type I or II glass. Stoppers and septa
in cartridges, and vials are typically composed of elastomeric materials.
Pharmaceuticals may interact with packaging and containers, resulting in the loss
of drug substances by adsorption onto and absorption into container components
and the incorporation of container components into pharmaceuticals. Diazepam in
intravenous fl uid containers and administration sets exhibited a loss during storage
due to adsorption onto glass [11, 12] .
Glass surfaces are also known to adsorb drug substances. Chloroquine solutions
in glass containers decreased in concentration owing to adsorption of the drug onto
the glass [13] .
PACKAGING MATERIALS 165
Rubber closures are also known to absorb materials, including drugs. Absorption
of preservatives such as chlorocresol into the rubber closures of injectable formulations
has been studied extensively [13] .
The water permeability of rubber closures used in injection vials is considered
an important parameter in assessing the closures, but quantitative prediction of
water permeability through rubber closures is diffi cult because the diffusion coeffi -
cient of water is dependent on relative humidity [14] .
Liquid - based oral drug products are usually dispensed in glass bottles (sometimes
in plastic), often with a screw cap with a liner, and possibly with a tamper - resistant
seal or an overcap that is welded onto the bottle. The same cap liners and inner
seals are sometimes used with solid oral dosage forms. A laminated material can be
used to overwrap glass bottles for extra safety.
The USP - grade glass packaging components are chemically resistant and can be
considered suffi cient evidence of safety and compatibility. In some cases (e.g., for
some chelating agents), a glass packaging component may need to meet additional
criteria to ensure the absence of signifi cant interactions between the packaging
component and the dosage form.
Several ophthalmic preparations are commercialized in glass containers. Although
the risk factors associated with ophthalmic preparations are generally considered
to be lower than for injectables, any potential for causing harm to the eyes demands
caution.
A large - volume intraocular solution (for irrigation) may be packaged in a polyolefi
n (polyethylene and/or polypropylene) container.
The liquid - based oral dosage forms may be marketed in multiple - unit bottles.
The dosage form may be used as is or admixed fi rst with a compatible diluent or
dispersant. Liquid - based oral drug products in glass container must meet the requirements
for USP containers. Glass containers are accepted as suffi cient evidence of
safety and compatibility. Performance is typically not a factor for liquid - based oral
drug products but should be considered while treating pressurized liquid - based oral
drug products (e.g., elixir spray).
Topical dosage forms such as unpressurized sprays, lotions, ointments, solutions,
and suspensions may be considered for marketing in glass bottles with appropriate
dispenser. Some topical drug products, especially ophthalmic, are sterile or may be
subject to microbial limits. In these cases, packaging material and handling should
be done as those for injectables.
3.2.2.3 Plastic as Packaging Material
For plastic components, data from USP biological reactivity tests will typically be
considered suffi cient evidence of safety. Whenever possible, extraction studies
should be performed using the drug product. If the extraction properties of the drug
product vehicle may reasonably be expected to differ from that of water (e.g., due
to high or low pH or to a solubilizing excipient), then drug product should be used
as the extracting medium. If the drug substance signifi cantly affects extraction characteristics,
it may be necessary to perform the extractions using the drug product
vehicle. If the total extract signifi cantly exceeds the amount obtained from water
extraction, then an extraction profi le should be obtained. It may be advisable
to obtain a quantitative extraction profi le of an elastomeric or plastic packaging
166 PACKAGING AND LABELING
component and to compare this periodically to the profi le from a new batch of the
packaging component. Extractables should be identifi ed whenever possible.
Nonsterile Products
Solids The most common solid oral dosage forms are capsules and tablets. A
typical solid oral dosage forms container closure system is a plastic, usually high -
density polyethylene (HDPE), bottle with a screw - on or snap - off closure and a
fl exible packaging system such as a pouch or a blister package. A typical closure
consists of a cap, often with a liner, frequently with an inner seal. If used, fi llers,
desiccants, and other absorbent materials are considered primary packaging
components.
A change in the selection of packing materials combined with a change in storage
conditions or conditions during administration of the drug products may provoke
stability problems.
Many studies have been conducted on predicting the role of packaging in moisture
adsorption by dosage forms. Adsorption of moisture by tablets contained in
polypropylene fi lms was successfully modeled from storage temperature and the
difference in water vapor pressure between the inside and outside of the packaging
[15] .
Chemical and physical degradation of packaged dosage forms caused by moisture
adsorption has been predicted from the moisture permeability of the packaging. For
example, strength changes of lactose – corn starch tablets in strip packaging [16] and
discoloration of sugar - coated tablets of ascorbic acid [17, 18] were predicted using
the moisture permeability coeffi cient of the packaging.
Typical fl exible forms of packaging containing solid oral dosage forms are the
blister package and the pouch. A blister package usually consists of a lidding material
and a forming fi lm. The lidding material is usually a laminate which includes a
barrier layer (e.g., aluminum foil) with a print primer on one side and a sealing agent
(e.g., a heat - sealing lacquer) on the other side.
The sealing agent contacts the dosage form and the forming fi lm. The forming
fi lm may be a single fi lm, a coated fi lm, or a laminate. A pouch typically consists of
fi lm or laminate which is sealed at the edges by heat or adhesive.
Solid oral dosage forms generally need to be protected from the potential adverse
effects of the following:
1. Water vapor (e.g., moisture may affect the decomposition rate of the active
drug substance or the dissolution rate of the dosage form)
2. Incident light (e.g., in case of photosensitive products)
3. Reactive gases (e.g., oxygen could provoke oxidative reactions)
Carefully selected packaging material may help protect drug products. For
example, a blister or pouch and use of secondary packing may be used to protect
pack photosensitive material, especially when a dark polymeric fi lm with a covering
lid made of aluminum is used for blister packing. Blister packaging using multilayer
HDPE material and selection of an adequate sealing technique may help prevent
moisture in the blister system. However, plastics and glass for packaging of solid
oral dosage forms and for powders for reconstitution should meet the requirements
PACKAGING MATERIALS 167
of the USP container test. Incorporating oxygen adsorbents such as iron powder
into packaging units can reduce the effect of oxygen. Protection from light can be
achieved using primary packaging (packaging that is in direct contact with the
dosage forms) and secondary packaging made of light - resistant materials. May be
involved in the photolytic degradation kinetics. The velocity of the photochemical
reaction may be affected not only by the light source, intensity, and wavelength of
the light but also by the size, shape, composition, and color of the container.
Great effort should be taken to stabilize a formulation in such a way that the
shelf life becomes independent of the storage conditions. The photostability of drugs
and excipients should be evaluated at the formulation development stage in order
to assess the effects of packaging on the stability of the fi nal product. Molsidomine
tablet preparations in inadequate primary containers (blister) without secondary
containers when exposed to irradiation may produce morpholine. These results
illustrate the importance of packaging for the stability of molsidomine [19] .
Three standard tests for water vapor permeation have been established by the
USP for use with solid oral dosage forms.
1. Polyethylene containers (USP . 661 . ) [10]
2. Single - unit containers and unit - dose containers for capsules and tablets (USP
. 671 . )
3. Multiple - unit containers for capsules and tablets (USP . 671 . ) [10]
The cotton and rayon used as fi llers in solid oral dosage form containers may not
meet pharmacopeial standards, but through appropriate tests and acceptance criteria
for identifi cation and moisture content, their adequacy should be shown. For
example, rayon has been found to be a potential source of dissolution problems for
gelatin capsules and gelatin - coated tablets.
Desiccants are often used to eliminate moisture in packaging when the moisture
resistance of the packaging is not suffi cient to prevent exposure. The utility of desiccants
has been assessed based on a sorption – desorption moisture transfer model
[20] .
Desiccants or other absorbent materials are primary packaging component. The
components should differ in shape and/or size from the tablets or capsules with
which they are packaged. Their composition should be provided and their inertness
should be proved through appropriate tests, and acceptance criteria should be
established.
A topical powder product may be marketed in a sifter - top container made of
fl exible plastic tubes or as part of a sterile dressing (e.g., antibacterial product). The
topical formulations in a collapsible tube can be constructed from low - density polyethylene
(LDPE), with or without a laminated material. Normally, there is no
product contact with the cap during storage. Thus usually there is no cap liner,
especially in collapsible polypropylene screw caps. Normally separate applicator
devices are made from LDPE. Product contact is possible if the applicator is part
of the closure, and therefore an applicator ’ s compatibility with the drug product
should be established, as appropriate (e.g., vaginal applicators).
Nonsolids Typical liquid - based oral dosage forms are elixirs, emulsions, extracts,
fl uid extracts, solutions, gels, syrups, spirits, tinctures, aromatic waters, and suspen
168 PACKAGING AND LABELING
sions. These products are usually nonsterile but typically need to be protected from
solvent loss, microbial contamination, and sometimes exposure to light or reactive
gases (e.g., oxygen).
The presence of a liquid phase implies a signifi cant potential for the transfer of
materials from a packaging component into the dosage form.
The higher viscosity of semisolid dosage forms and transdermal systems may
cause the rate of migration of leachable substances into these dosage forms to be
slower than for aqueous solutions. Due to extended contact, the amount of leachables
in these drug products may depend more on a leachable material ’ s affi nity for
the liquid/semisolid phase than on the rate of migration.
In addition to absorption onto and absorption into containers, transfer of
container components into pharmaceuticals may affect the perceived stability/
quality of drug dosage forms. Adsorption of volatile components from rubber
closures onto freeze - dried parenterals during both dosage form processing and
storage brought about haze formation upon reconstitution [21 – 23] . Leaching
of dioctyl phthalate, a plasticizer used especially in polyvingl chloride (PVC)
plastics, into intravenous solutions containing surfactants was observed [24, 25] .
Plastics contain additives to enhance polymer performance. PVC may contain
phthalate diester plasticizer, which can leach into infusion fl uids from packaging
[26] .
The liquid - based oral dosage forms may be marketed in multiple - unit bottles or
in unit - dose or single - use pouches or cups. The dosage form may be used as is or
admixed fi rst with a compatible diluent or dispersant. A liquid - based oral drug
pouch may be a single - layer plastic or a laminated material. The pouches may use
an overwrap, which is usually a laminated material.
For LDPE components, data from USP container tests are typically considered
suffi cient evidence of compatibility. The USP general chapters do not specifi cally
address safety for polyethylene (HDPE or LDPE), polypropylene (PP), or laminate
components.
In such cases, an appropriate reference to the indirect food additive regulations
[27] is typically considered suffi cient. This reference is considered valid for liquid -
based oral dosage forms which the patient will take only for a relatively short
time.
For liquid - based oral drug products which the patient will continue to take for
an extended period, that is, months or years, and is expected to extract greater
amounts of substances from plastic packaging components than from water (presence
of cosolvents), additional extractable information may be needed to address
safety issues.
Topical dosage forms such as creams, emulsions, gels, lotions, ointments, pastes,
and powders may be marketed in plastic materials. Topical dosage formulations are
for local (not systemic) effect and are generally applied to the skin or oral mucosal
surfaces. Some vaginal and rectal creams and nasal, otic, and ophthalmic solutions
may be considered for topical drug products.
A rigid bottle, a collapsible tube, or a fl exible pouch made of plastic material may
be used for liquid - based topical product. These preparations are marketed in a
single - or multiple - unit container. For example, dissolved drug (or any substance,
e.g., benzocaine) may diffuse in the suppository base and can, for instance, partition
into polyethylene linings of the suppository wrap.
PACKAGING MATERIALS 169
Topical delivery systems are self - contained, discrete dosage forms that are
designed to deliver drug for an extended period via intact skin or body surface, for
example, transdermal, ocular, and intrauterine.
These systems may be constructed of a plastic or polymeric material loaded with
active ingredients or a coated metal. Each of these systems is generally marketed
in a single - unit soft blister pack or a preformed tray with a preformed cover or
overwrap. The compatibility and safety for topical delivery systems are addressed
in the same manner as for topical drug products. Performance and quality control
should be addressed for the rate - controlling membrane.
Sterile Products
Nonsolids An SVP may be packaged in a disposable cartridge, a disposable syringe,
or a fl exible bag made of polymeric plastic. Flexible bags are typically constructed
with multilayered plastic (Table 2 ).
An LVP may be packaged in a vial, a fl exible bag, or, in some cases, a disposable
syringe. Packaging material for cartridges, syringes, vials, and ampules are usually
composed of polypropylene (Table 2 ).
Stoppers and septa in cartridges and syringes are typically composed of elastomeric
materials. An overwrap may be used with fl exible bags to retard solvent loss
and to protect the fl exible packaging system from rough handling.
Diazepam in intravenous fl uid containers and administration sets exhibited
a loss during storage due to adsorption onto and absorption into plastics
[11, 12] .
Absorption of clomethiazole edisylate and thiopental sodium into PVC infusion
bags was observed [28] .
The pH dependence of adsorption/absorption of acidic drug substances such as
warfarin and thiopental and basic drug substances such as chlorpromazine and diltiazem
indicates that only the un - ionized form of the drug substance is adsorbed
onto or absorbed into PVC infusion bags [29] .
The absorption was correlated to the octanol – water partition coeffi cients of the
drugs, suggesting that prediction of absorption from partition data is possible [30,
31] . Polymers such as Nylon - 6 (polycaprolactam) are known to adsorb drug substances
such as benzocaine [32] .
The ophthalmic drug products are usually solutions marketed in a LDPE bottle
with a dropper built into the neck. A few solution products use a glass container
due to stability concerns regarding plastic packaging components.
3.2.2.4 Metal as Packaging Material
Metal tubes constructed of a single material are the packaging material of choice
for topical dosage forms and may be tested readily for stability with a product. Tubes
with a coating, however, present additional problems. The inertness of coating material
must be established through adequate tests and guarantee that it completely
covers underlying material. The coating material must be resistant to creaking,
leaking, leaching, and solvent erosion. For example, frequently used aluminum tubes
have demonstrated reactivity with fatty alcohol emulsions, mercurial compounds,
and preparations with pH below 6.5 and above 8.0. Nonreactive, epoxy linings have
been found to make aluminum tubes resistant to attack [6] .
170 PACKAGING AND LABELING
TABLE 2 Parenteral Drug Administration Devices
Sterile Device Plastic Material
Containers for blood products Polyvinyl chloride
Disposable syringes Polycarbonate, polyethylene, polypropylene
Irrigating solution containers Polyethylene, polypropylene, polyvinyl chloride
Intravenous infusion fl uid containers Polyethylene, polypropylene, polyvinyl chloride
Administration sets Nylon (spike), polyvinyl chloride (tubing),
polymethylmethacrylate (needle adapter),
polypropylene (clamp)
Catheter Tefl on, polypropylene, thermoplastic elastomers
Source : From ref. 6 .
Some examples of plastic additives and parenteral drug administration devices used
as packaging materials for sterile products can be seen in Tables 1 and 2 .
Ophthalmic ointments are marketed in a metal tube with an ophthalmic tip.
Ophthalmic ointments that are reactive toward metal may be packaged in a tube
lined with an epoxy or vinyl plastic coating.
Metal containers, pressurized or not, may also be used for topical drug products.
Topical dosage forms include aerosols, emulsions, gels, powders, and solutions
and may be marketed in metallic fl asks, pressurized or not. Topical dosage formulations
are for local (not systemic) effect and are generally applied to the skin or
oral mucosal surfaces. Some vaginal and rectal creams and nasal and otic spray
drug products may be considered for marketing in metallic containers for topical
use.
A number of topical products marketed as a pressurized aerosol may be dispensed
in a metallic bottle with a screw cap. Topical dosage forms in aluminum tubes
usually include a liner. A tube liner is frequently a lacquer or shellac whose composition
should be stated. A metallic pressurized packaging system for a liquid -
TABLE 1 Plastic Additives
Type Purpose Examples
Lubricants Improve processability Stearic acid paraffi n waxes, polyethylene
(PE) waxes
Stabilizers Retard degradation Epoxy compounds, organotins, mixed
metals
Plasticizers Enhance fl exibility,
resiliency, melt fl ow
Phthalates
Antioxidants Prevent oxidative
degradation
Hindered phenolics (BHT), aromatic
amines, thioesters, phosphites
Antistatic agents Minimize surface static
charge
Quaternary ammonium compounds
Slip agents Minimize coeffi cient of
friction, especially
polyolefi ns
Dyes, pigments Color additives
Source : From ref. 6 .
PACKAGING MATERIALS 171
based topical product may deter solvent loss and may provide protection from light
when appropriate.
The droplet size of topical aerosol sprays does not need to be carefully controlled,
and the dose usually is not metered as in inhalers. The spray may be used to apply
the drug to the skin (topical aerosol) or mouth (lingual aerosol) and the functionality
of the sprayer should be addressed. The drug product has no contact with the
cap and short - term contact with the nozzle. A topical aerosol may be sterile or may
conform to acceptance criteria for microbial limits. However, the physical stability
of aerosols can lead to changes in total drug delivered per dose and total number
of doses that may be obtained from the container.
3.2.2.5 Applications: Some Examples
Many research papers in the scientifi c literature present studies showing the importance
of the effect of packaging materials on the stability of pharmaceutical and
cosmetic preparations:
1. Santoro and co - workers [33] presented results of the stability of oral rehydration
salts (ORSs) in different types of packaging materials. The objective of the
research was to give guidance on the adequate choice of packaging material presenting
the indispensable characteristics in order to protect ORS preparation. This
pharmaceutical preparation is essential to children living in developing countries
with tropical climate and its distribution is one of the programs of the World Health
Organization (WHO) [34] .
It has been proved in several research papers that water is the most important
factor in the component ’ s degradation of ORSs. To proceed with the study, the
pharmaceutical formulation was prepared by a pharmaceutical manufacture. The
batch was packed in six types of packaging material. After storage of samples for
36 weeks maintained at ambient temperature, at ambient temperature and 76%
relative humidity, and at 40 ° C with 80% relative humidity, analyses of water determination
were made at different intervals of time. Water determination was performed
by loss on drying at 50 ° C and Karl Fisher methods.
The studied ORS preparation contained anhydrous glucose (20 g), sodium chloride
(3.5 g), trisodium dehydrate citrate (2.9 g), and potassium chloride (1.5 g)
According to the results, the packaging material that better protected the ORS
preparation is the one constituted of polyester (18 g), aluminum (35%), and polyethylene
(50 g).
2. The effect of packaging materials on the stability of sunscreen emulsions was
also studied by Santoro and co - workers [35, 36] . The purpose of the research was
to study the stability of an emulsion containing UVA, UVB, and infrared sunscreens
after storage in different types of packaging materials (glass and plastic fl asks, plastic
and metallic tubes). The samples, emulsions containing benzophenone - 3 (B - 3), octyl
methoxycinnamate (OM), and Phycocorail , were stored at 10, 25, 35, and 45 ° C and
representative samples were analyzed after 2, 7, 30, 60, and 90 days. Stability studies
were conducted by analyzing samples at predetermined intervals by high -
performance liquid chromatography (HPLC) along with periodic rheological
measurements.
The proposed HPLC method enabled the separation and quantitative determination
of B - 3 and OM present in sunscreens. The method was successfully applied in
172 PACKAGING AND LABELING
the stability studies of the emulsions. The method is simple, precise, and accurate;
there was no interference from formulation components. The sample emulsions
stored at different temperatures presented similar rheological behavior, at least
during the period of the study (three months). Most of the samples showed a pseudoplastic
non - Newtonian thixotropic profi le. There were no signifi cant changes in
the physical and chemical stability of emulsions stored in different packaging material.
The studied glass and plastic packaging materials were found adequate for
storing referred solar protector emulsions.
3. Sarbach and co-workers [48] , studied the effect of plastics packaging materials
on parenteral pharmaceutical products. Compatibility studies of these containers
with different contents are required for drug registration. The authors demonstrated
the migration phenomena which occurred between a trilaminated fi lm and a parenteral
solution of metronidazole at 0.5%. The main migration products found in
the solution were e - caprolactam and a phthalic derivative. The authors also separated
several unidentifi ed compounds probably coming from the polyurethane
adhesive.
4. Molsidomine is sensitive to light and shows a fast decomposition in solutions
and in tablets. Thoma and co - workers [37] showed the importance of light - resistant
packaging material for photolabile pharmaceuticals. They irradiated molsidomine
preparations over a period of 72 h in a light cabinet according to storage at daylight
for about 4 – 6 weeks. Losses of 23 – 90% in tablets and 43 – 60% in solutions were
found. The photodegradation could be overcome by selection of suitable packaging
materials, colorants or vanillin. The degradation product morpholine after dansylation
was determined by HPLC and showed contents of 0.10 – 0.67 mg in tablets and
0.10 – 0.38 mg/mL in solution after irradiation.
These examples, among many others described in the scientifi c literature, illustrate
the importance of proper selection of packaging material for the stability and
effectiveness of pharmaceutical dosage forms.
3.2.3 QUALITY CONTROL OF PACKAGING MATERIAL
3.2.3.1 General Considerations
Several regulatory agencies as well as private agencies [Food and Drug Administration
(FDA), British Pharmacopoeia, WHO, USP] [4, 10, 34, 38] have issued guidelines
on the safety evaluation of materials and container closure systems. However,
the ultimate proof of the safety and suitability of a container closure system and
the packaging process is established by full shelf life stability studies. An important
step in such evaluations is characterization of the packaging materials and the
chemicals that can migrate or extract from container closure system components to
the drug product. This extractable material belongs to diverse chemical classes that
can migrate from polymeric materials, such as antioxidants, contaminants, lubricants,
monomers, plasticizers, and preservatives. Such basic information is critical to understanding
the biological safety and suitability of a container.
Establishing the safety of container closure systems is of key importance to the
medical and pharmaceutical industries (Table 3 ). It is no less important than the
contents themselves. The FDA ’ s document “ Guidance on Container Closure Systems
for Packaging Human Drugs and Biologics ” makes this point clear [4] .
The FDA ’ s guidance document requires the evaluation of four attributes to
establish suitability: protection, compatibility, safety, and performance/drug delivery.
The document also provides a structured approach to ranking packaging concerns
according to the route of drug administration and likelihood of packaging component
– dosage form interaction. A container closure system acceptable for one drug
product cannot be assumed to be appropriate for another. Each product should have
suffi cient information to establish that a container and its components are right for
their intended use [4] .
To establish suitability, all four attributes must be evaluated and be shown to
pose no concern to the drug product or to product performance. Suitability refers
to the tests used for the initial qualifi cation of the container closure system with
regard to its intended use. The guidance defi nes what tests must be done to evaluate
each of the attributes of suitability.
While the tests and methods described in Table 4 allow one to provide data that
the container closure system is suitable for its intended use, an application must also
describe the quality control (QC) measures that will be used to ensure consistency
in the packaging components. The principal considerations for the QC measures are
the physical characteristics and the chemical composition. By choosing two or three
of the tests done in the initial suitability study, a QC program can be established
that will ensure the consistency of the container closure system (Table 4 ).
Protection A container closure system should provide the dosage form with adequate
protection from factors (e.g., temperature, light) that can cause a degradation
in the quality of that dosage form over its shelf life. Common causes of such degradation
are exposure to light, loss of solvent, exposure to reactive gases (e.g., oxygen),
absorption of water vapor, and microbial contamination.
A container intended to provide protection from light or offered as a light - resistant
container must meet the requirements of the USP . 661 . light transmission test.
The procedure requires the use of a spectrophotometer, with the required sensitivity
TABLE 3 Examples of Packaging Concerns for Common Classes of Drug Products
Degree of Concern
Associated with
Route of
Administration
Likelihood of Packaging Component – Dosage Form Interaction
High Medium Low
Highest Inhalation aerosols and
solutions; injection;
injectable suspensions
Sterile powders
and powders
for injections
and inhalation
powders
High Ophthalmic solutions and
suspensions; transdermal
ointments and patches;
nasal aerosols and sprays
Low Topical solutions and
suspensions; topical and
lingual aerosols; oral
solutions and suspensions
Topical powders;
oral powders
Oral tablets and
oral (hard and
soft; gelatin)
capsules
QUALITY CONTROL OF PACKAGING MATERIAL 173
174 PACKAGING AND LABELING
and accuracy, adapted for measuring the amount of light transmitted by the plastic
material used for the container.
The ability of a container closure system to protect against moisture can be
ascertained by performing the USP . 661 . water vapor permeation test. The USP
sets limits on the amount of moisture that can penetrate based upon the size and
composition of the plastic components [HDPE, LDPE, or polyethylene terephthalate
(PET)].
Evaluating the integrity of the container can be done in several ways. Two of the
most common tests are dye penetration and microbial ingress. Container closure
systems stored in a dye solution and exposed to pressure and vacuum cycles are
examined for dye leakage into the container. The microbial ingress is similar in
fashion but determines the microbial contamination of the contents when soaked
in a media contaminated with bacteria. Other quantitative tests that can be run are
vacuum/pressure decay, helium mass spectrometry, and gas detection.
Compatibility Packaging components that are compatible with a dosage form will
not interact suffi ciently to cause unacceptable changes in the quality of either the
dosage form or the packaging component. A leachability study designed to evaluate
the amount and/or nature of any chemical migrating from the plastic material to
the drug product should be considered. The study should evaluate substances that
migrate into the drug product vehicle for the length of shelf life. The drug product
should be evaluated at regular intervals, such as at one, three, or six months or one
or two years, until the length of the shelf - life claim has been met.
Analytical techniques such as liquid chromatography/mass spectrometry (LC/
MS) to evaluate nonvolatile organics, gas chromatography/mass spectrometry (GC/
MS) to evaluate semivolatile organics, and inductively coupled plasma (ICP) spectroscopy
to detect and quantitate inorganic elements should be a part of this
study.
Unknown impurities and degradation products can be identifi ed using liquid or
gas chromatography along with MS. Information or substances identifi ed from
extractable chemical evaluation can be used to help prepare standards specifi c for
the plastic container being studied during leachability studies. Development and
validation of the selective analytical method should be thoroughly studied before
its application in the detection of leachable chemicals in active drug substance and
drug product.
Organoleptic and chemical changes such as precipitates, discoloration, strange
odor, and pH modifi cation are signs of degradation of drug product. Changes in the
physical characteristics of the container, such as brittleness, should be evaluated
using thermal analysis and hardness testing. An infrared spectroscopic scan can fi ngerprint
the materials and also provide proof of identity. Spectrophotometry and
LC with ultraviolet detection can be used for the analysis of drug product stored at
different stress conditions. These tests can be used for the quality control of drug
product as well as for conducting stability studies on different products stored in
the same container material.
Safety Packaging components should be constructed of materials that will not
leach harmful or undesirable amounts of substances to which a patient will be
exposed when being treated with the drug product. This consideration is especially
important for those packaging components which may be in direct contact with the
dosage form, but it is also applicable to any component from which substances may
migrate into the dosage form (e.g., an ink or adhesive). Determining the safety of
a packaging component is not a simple process, and a standardized approach has
not been established. However, an extraction study should be one of the fi rst
considerations.
A good knowledge regarding possible extractable material could help analysts
develop specifi c and selective methods to identify extractables from container
closure components under various control extraction study conditions.
Precise information on the synthesis of the polymer and descriptions of the
monomers used in the polymerization, the solvents used in the synthesis, and the
special additives that have been added during material production as well as knowledge
of degradation products that may be released into the drug product are also
important.
Some potential extractable chemicals from packaging materials are water soluble,
while others are soluble only in nonpolar environments. The USP includes physicochemical
tests for plastics based on water extracts, while water, alcohol, and hexane
extracts are required for polyethylene containers under controlled temperature and
time parameters (70 ° C for 24 h for water and alcohol and 50 ° C for 24 h for hexane).
The USP physicochemical tests for extractables should be a part of all suitability
programs, regardless of the criticality of the drug dosage form. USP elastomeric
closures for injections should also be a part of the extractables study to establish
safety. These USP tests, which have evolved over many years, are relevant, sensitive,
rapid, and inexpensive. They help establish material safety.
TABLE 4 Properties of Suitability Concerns and Interactions
Attributes Concerns and Interactions Proposed Methods
Protection Exposure to light, moisture,
microbial ingress, and
oxidation from presence
of oxygen
USP . 661 . light transmission and water
vapor permeation, container integrity
(microbial ingress, dye penetration,
helium leak)
Compatibility Leachable induced
degradation, absorption
or adsorption of drug,
precipitation, change in
pH, discoloration,
brittleness of packaging
materials
Leachability study (migration of chemicals
into drug product) using LC/MS, GC/MS,
ICP/AA, pH, appearance of drug and
container, thermal analysis (DSC, TGA),
and infrared (IR)
Safety No leached harmful or
undesirable amounts of
substances to expose
patients treated with drug
Extraction study (USP physicochemical
tests – plastics), USP elastomeric closures
for injections, toxicological evaluation,
USP biological reactivity and complies
with CFR, additives and purity
Performance Container closure system
functionality, drug
delivery
Functionality (improved patient compliance
or use), delivery (transfer dose in right
amount or rate)
Abbreviations : DSC, differential scanning calorimetry; ICP, Inductively coupled plasma spectrometer;
AA, Atomic absorption.
Source : From ref. 39 .
QUALITY CONTROL OF PACKAGING MATERIAL 175
176 PACKAGING AND LABELING
The safety of material can be guaranteed by using appropriate analytical methods
and instrumentation to identify and quantitate extracted chemicals. Liquid and gas
chromatography and MS are powerful analytical tools that can separate and quantitate
volatile and nonvolatile chemicals along with useful structural information.
The mass spectrum or fragmentation pattern acquired for each molecule makes
these excellent and effective tools for identifying unknown impurities or degradation
products.
Toxicological evaluation of identifi ed and unidentifi ed impurities from a container
can help improve the safety index of drug products. The toxicological evaluation
should take into consideration container closure system properties, drug
product formulation, dosage form, route of administration, and dose regimen. A
close correlation between chemical and toxicological information can provide better
control on safety and compatibility of containers and closures.
Performance The fourth attribute of the suitability of the container closure system,
performance and drug delivery, refers to its ability to function in the manner for
which it was designed. There are two major considerations when evaluating performance.
The fi rst consideration is functionality that may improve patient compliance,
[e.g., a two - chamber vial or intravenous (IV) bag], or improve ease of use (e.g., a
cap that contains a counter, a prefi lled syringe). The second consideration is drug
delivery, which is the ability of the packaging system to deliver the right amount or
rate (e.g., a prefi lled syringe, a transdermal patch, a metered tube, a dropper or spray
bottle, a dry - powder inhaler, and a metered - dose inhaler).
3.2.3.2 Packaging Components
Quality control refers to the tests typically used and accepted to establish that, after
the application is approved, the components and the container closure system continue
to possess the characteristics established in the suitability studies.
To ensure consistency, protection, compatibility, safety, and performance of the
packaging components, it is necessary to defi ne QC measures that will be used to
ensure consistency in the packaging components. These controls are intended to
limit unintended postapproval variations in the manufacturing procedures or materials
of construction for a packaging component and to prevent adverse effects on
the quality of a dosage form.
The USP tests and studies for establishing suitability and QC of container closure
system and for associated component materials are summarized in Table 5 .
Hydrolysis and oxidation are the two main routes of degradation for the majority
of drugs. To gain more information, the drug could be subjected to a range of temperature
and relative humidity conditions. In addition, photostability studies could
be conducted by exposure to artifi cial or natural light conditions. Elevated temperature,
humidity, and light stress the drug and induce rapid degradation. Harmonized
guidelines are available for new drug substances and products and may provide
useful information to characterize degradation processes and selection of appropriate
packaging material.
The primary packaging must physically protect the product from the mechanical
stresses of warehousing, handling, and distribution. Mechanical stress may take a
TABLE 5 U.S. Pharmacopeia General Tests and Assays
Chapter Topic
. 1 . Injections
. 51 . Antimicrobial preservatives — effectiveness
. 61 . Microbial limit tests
. 71 . Sterility tests
. 87 . Biological reactivity tests, in vitro
. 88 . Biological reactivity tests, in vivo
. 161 . Transfusion and infusion assemblies
. 381 . Elastomeric closures for injections, biological test procedures, physicochemical
test procedures
. 601 . Aerosols
. 661 . Containers: light transmission; chemical resistance — glass containers; biological
tests — plastics and other polymers; physicochemical tests — plastics;
containers for ophthalmics — plastics; polyethylene containers; polyethylene
terephthalate bottles and polyethylene; terephthalate G bottles; single - unit
containers and unit - dose containers for nonsterile; solid and liquid dosage
forms; customized patient medication packages
. 671 . Containers — permeation: multiple - unit containers for capsules and tablets;
single - unit containers and unit - dose containers for capsules and tablets
. 691 . Cotton (or the monograph for purifi ed rayon USP)
. 771 . Ophthalmic ointments
. 1041 . Biologics
. 1151 . Pharmaceutical dosage forms
Source : From ref. 10 .
variety of forms, from impact through vibration in transit and compression forces
on stacking.
The demands for mechanical protection will vary with product type: Glass
ampules will require greater protection than plastic eye drop bottles, for example.
Other protection is required from environmental factors such as moisture, temperature
changes, light, gases, and biological agents such as microorganisms and, importantly,
humans.
The global market for medicinal products requires that the products are stable
over a wide range of temperatures ranging from subzero in the polar region, 15 ° C
in temperate zones, up to 32 ° C in the tropics. Along with this temperature variation,
relative humidity can vary from below 50% to up to 90%, a feature that the packaging
should be able to resist if necessary. The majority of packaging materials (including
plastics) are to some degree permeable to moisture and the type of closure
employed, such as screw fi ttings, may also permit ingress of moisture. The susceptibility
of the product to moisture and its hygroscopicity will have to be considered
and may require packaging with a desiccant or the use of specialized strip packs
using low - permeability materials such as foil.
Temperature fl uctuations can lead to condensation of moisture on the product
and, with liquids, formation of a condensate layer on top of the product. This latter
problem is well known and can lead to microbiological spoilage as the condensate
is preservative free.
QUALITY CONTROL OF PACKAGING MATERIAL 177
178 PACKAGING AND LABELING
If the product is sensitive to photolysis, then opaque materials may be required.
Most secondary packaging materials (e.g., cartons) do not transmit light, but in
some cases specialized primary packaging designed to limit light transmission is
employed.
The package must also prevent the entry of organisms; for example, packaging
of sterile products must be microorganism proof — hence the continued use of glass
ampules. For nonsterile products the preservative provides some protection, but
continual microbial challenge will diminish the effi cacy of the preservative, and
spoilage or disease transmission may occur.
The packaging material must not interact with the product either to adsorb substances
from the product or to leach chemicals into the product. Plastics contain
additives to enhance polymer performance. PVC may contain phthalate diester
plasticizer, which can leach into infusion fl uids from packaging. Antimicrobial preservatives
such as phenylmercuric acetate are known to partition into rubbers and
plastics during storage, thus reducing the formulation concentration below effective
antimicrobial levels.
A complication of modern packaging is the need for the application of security
seals to protect against deliberate adulteration and maintain consumer confi dence.
The active products used must also be stability tested in the proposed packaging
material.
The FDA guidance for industry suggests considering consistency in physical and
chemical composition. Using a few simple tests, the quality of components and ultimately
the container closure system can be monitored.
Physical Characteristics The physical characteristics of interest include dimensional
criteria (e.g., shape, neck fi nish, wall thickness, design tolerances), physical
parameters critical to the consistent manufacture of a packaging component (e.g.,
unit weight), and performance characteristics (e.g., metering valve delivery volume
or the ease of movement of syringe plungers). Unintended variations in dimensional
parameters, if undetected, may affect package permeability, drug delivery performance,
or the adequacy of the seal between the container and the closure. Variation
in any physical parameter is considered important if it can affect the quality of a
dosage form.
Physical considerations such as water vapor transmission to evaluate seal integrity,
thermal analysis such as DSC to monitor melting point and glass transitions of
plastics, and IR scanning to prove identity should be part of an ongoing quality
control monitoring program.
Chemical Composition The chemical composition of the materials of construction
may affect the safety of a packaging component. New materials may result in new
substances being extracted into the dosage form or a change in the amount of known
extractables. The chemical composition may also affect the compatibility, functional
characteristics, or protective properties of packaging components by changing rheological
or other physical properties (e.g., elasticity, resistance to solvents, or gas
permeability).
The chemical composition should also be evaluated by performing the simple but
informative USP physicochemical tests using water, drug product vehicle, and
alcohol extractions of plastic components. Specifi cations should be set for nonvola
tile residue (total extractables) during the initial suitability tests and then used to
monitor the level of polar and nonpolar extractables as part of a quality control
plan.
A change in the composition of packaging raw material or a change in formulation
is considered a change in the specifi cations. Due care must be taken to guarantee
the safety, compatibility, and performance of a new dosage form in a new
packaging system.
The use of stability studies for monitoring the consistency of a container closure
system in terms of compatibility with the dosage form and the degree of protection
provided to the dosage form is essential. Except for inhalation drug products, for
which batch - to - batch monitoring of the extraction profi le for the polymeric and
elastomeric components is routine, no general policy concerning the monitoring of
a packaging system and components with regard to safety is available.
Secondary packaging components are not intended to make contact with the
dosage form. Examples are cartons, which are generally constructed of paper or
plastic, and overwraps, fabricated from a single layer of plastic or from a laminate
made of metal foil, plastic, and/or paper. In special cases, secondary packaging components
provide some additional measure of protection to the drug product. In such
cases it could be considered a potential source of contamination and the safety of
the raw materials should be taken into consideration.
3.2.3.3 Inhalation Drug Products
Inhalation drug products include inhalation aerosols (metered - dose inhalers); inhalation
solutions, suspensions, and sprays (administered via nebulizers); inhalation
powders (dry - powder inhalers); and nasal sprays. The carboxymethylcellulose
(CMC) and preclinical considerations for inhalation drug products are unique in
that these drug products are intended for respiratory tract compromised patients.
This is refl ected in the level of concern given to the nature of the packaging components
that may come in contact with the dosage form or the patient (Table 4 ).
In October 1998, the FDA issued guidance for industry regarding container
closure systems such as metered - dose inhaler (MDI) and dry - powder Inhaler (DPI)
drug products.
3.2.3.4 Drug Products for Injection and Ophthalmic Drug Products
Injectable dosage forms are sterile and represent one of the highest risk drug products.
Injectable drug products may be liquids in the form of solutions, emulsions,
and suspensions or dry solids that are to be combined with an appropriate vehicle
to yield a solution or suspension.
Cartridges, syringes, vials, and ampules are usually composed of type I or II glass
or polypropylene frequently used to deliver SVP and LVPs. Flexible bags are typically
constructed with multilayered plastic. Stoppers and septa in cartridges, syringes,
and vials are typically composed of elastomeric materials. An overwrap may be used
with fl exible bags to retard solvent loss and to protect the fl exible packaging system
from rough handling.
Injectable drug products require protection from microbial contamination (loss
of sterility or added bioburden) and may also need to be protected from light or
QUALITY CONTROL OF PACKAGING MATERIAL 179
180 PACKAGING AND LABELING
exposure to gases (e.g., oxygen). Liquid - based injectables may need to be protected
from solvent loss, while sterile powders or powders for injection may need to be
protected from exposure to water vapor.
For elastomeric components, data showing that a component meets the requirements
of USP elastomeric closures for injections should typically be performed to
assure safety. For plastic components, USP biological reactivity tests are recommended
to assure evidence of safety. Whenever possible, the extraction studies
described in USP should be performed using the drug product. Extractables should
be identifi ed whenever possible. For a glass packaging component, data from USP
“ Containers: Chemical resistance — Glass containers ” will typically be considered
suffi cient evidence of safety and compatibility. In some cases (e.g., for some chelating
agents), a glass packaging component may need to meet additional criteria to
ensure the absence of signifi cant interactions between the packaging component
and the dosage form.
The performance of a syringe is usually addressed by establishing the force to
initiate and maintain plunger movement down the barrel and the capability of the
syringe to deliver the labeled amount of the drug product.
Ophthalmic drug products are usually solutions marketed in a LDPE bottle with
a dropper built into the neck or ointments marketed in a metal tube lined with an
epoxy or vinyl plastic coating with an ophthalmic tip.
Since ophthalmic drug products are applied to the eye, compatibility and safety
concerns should also address the container closure system ’ s potential to form substances
which irritate the eye or introduce particulate matter into the product (USP
. 771 . , ophthalmic ointments).
3.2.3.5 Liquid - Based Oral Products, Topical Drug Products, and
Topical Delivery Systems
The presence of a liquid phase implies a signifi cant potential for the transfer of
materials from a packaging component into the dosage form.
Liquid - Based Oral Drug Products Typical liquid - based oral dosage forms are
elixirs, emulsions, extracts, fl uid extracts, solutions, gels, syrups, spirits, tinctures,
aromatic waters, and suspensions. These products are usually nonsterile but may be
monitored for changes in bioburden or for the presence of specifi c microbes.
A liquid - based oral drug product typically needs to be protected from solvent
loss, microbial contamination, and sometimes exposure to light or reactive gases
(e.g., oxygen). For glass components, data showing that a component meets the
requirements of USP “ Containers: Glass containers ” are accepted as suffi cient evidence
of safety and compatibility. For LDPE components, data from USP container
tests are typically considered suffi cient evidence of compatibility.
The USP general chapters do not specifi cally address safety for polyethylene
(HDPE or LDPE), PP, or laminate components. A patient ’ s exposure to substances
extracted from a plastic packaging component (e.g., HDPE, LDPE, PP, laminated
components) into a liquid - based oral dosage form is expected to be comparable to
a patient ’ s exposure to the same substances through the use of the same material
when used to package food [27] .
Topical Drug Products Topical dosage forms include aerosols, creams, emulsions,
gels, lotions, ointments, pastes, powders, solutions, and suspensions. These dosage
forms are generally intended for local (not systemic) effect and are generally applied
to the skin or oral mucosal surfaces. Topical products also include some nasal and
otic preparations as well as some ophthalmic drug products. Some topical drug
products are sterile and should be subject to microbial limits.
A rigid bottle or jar is usually made of glass or polypropylene with a screw cap.
The same cap liners and inner seals are sometimes used as with solid oral dosage
forms. A collapsible tube is usually constructed from metal or is metal lined from
LDPE or from a laminated material.
Topical Delivery Systems Topical delivery systems are self - contained, discrete
dosage forms that are designed to deliver drug via intact skin or body surface,
namely transdermal, ocular, and intrauterine.
Each of these systems is generally marketed in a single - unit soft blister pack or
a preformed tray with a preformed cover or overwrap. Compatibility and safety for
topical delivery systems are addressed in the same manner as for topical drug products.
Performance and quality control should be addressed for the rate - controlling
membrane. Appropriate microbial limits should be established and justifi ed for each
delivery system.
3.2.3.6 Solid Oral Dosage Forms and Powders for Reconstitution
The most common solid oral dosage forms are capsules and tablets. A typical container
closure system is a plastic (usually HDPE) or a glass bottle with a screw - on
or snap - off closure and a fl exible packaging system, such as a pouch or a blister
package. If used, fi llers, desiccants, and other absorbent materials are considered
primary packaging components.
Solid oral dosage forms generally need to be protected from the potential adverse
effects of water vapor, light, and reactive gases. For example, the presence of moisture
may affect the decomposition rate of the active drug substance or the dissolution
rate of a dosage form. The potential adverse effects of water vapor can be
determined with leak testing on a fl exible package system (pouch or blister package).
Three standard tests for water vapor permeation have been established by the USP,
namely polyethylene containers (USP . 661 . ), single - unit containers and unit - dose
containers for capsules and tablets (USP . 671 . ), and multiple - unit containers for
capsules and tablets (USP . 671 . ).
3.2.4 IMPORTANCE OF PROPER PACKAGING AND LABELING
The Poison Prevention Packaging Act ( www.cpsc.gov/businfo/pppa.html) requires
special packaging of most human oral prescription drugs, oral controlled drugs,
certain normal prescription drugs, certain dietary supplements, and many over - the -
counter (OTC) drug preparations in order to protect the public from personal injury
or illness from misuse of these preparations.
IMPORTANCE OF PROPER PACKAGING AND LABELING 181
182 PACKAGING AND LABELING
In many countries there are very strict regulations for packaging of many drug
substances. Nevertheless, special packaging is not required for drugs dispensed
within a hospital setting for inpatient administration. Manufacturers and packagers
of bulk - packaged prescription drugs do not have to use special packaging if the drug
will be repackaged by the pharmacist.
Various types of child - resistant packages are covered in ASTM International
standard D - 3475. Medication errors linked to poor labeling and packaging can be
controlled through the use of error potential analysis. The recognition that a drug
name, label, or package may constitute a hazard to safety typically occurs after the
drug has been approved for use and is being marketed. Calls for change almost
always result from accumulating reports of serious injuries associated with the use
of a drug.
Numerous reports of medication errors are being reported, some of which have
resulted in patient injury or death. In a number of these reports, a medication was
mistakenly administered either because the drug container (bag, ampule, prefi lled
syringe and bottle) was similar in appearance to the intended medication ’ s container
or because the packages had similar labeling. Obviously, the severity of such errors
depends largely on the medication administered.
The problem of medical errors associated with the naming, labeling, and packaging
of pharmaceuticals is being very much discussed. Sound - alike and look - alike
drug names and packages can lead pharmacists and nurses to unintended interchanges
of drugs that can result in patient injury or death. Simplicity, standardization,
differentiation, lack of duplication, and unambiguous communication are
human factors that are relevant to the medication use process. These factors have
often been ignored in drug naming, labeling, and packaging.
The process for naming a marketable drug is always lengthy and complex and
involves submission of a new entity and patent application, generic naming, brand
naming, FDA — or other corresponding organization all over the world — review, and
fi nal approval. Drug companies seek the fastest possible approval and may believe
that the incremental benefi t of human factor evaluation is small. Very often, the
drug companies are resistant to changing, for example, brand names. Although a
variety of private - sector organizations in many countries have called for reforms in
drug naming, labeling, and packaging standards, the problem remains.
Drug names, labels, and packages are not selected and designed in accordance with
human factor principles. FDA standards or other corresponding organizations in
other countries do not require application of these principles, the drug industry has
struggled with change, and private - sector initiatives have had only limited success.
A number of factors can contribute to the mistaking of one medication for
another. Failure to read the package label is one cause. Another if a medication is
stored in the wrong location or if clinicians select the medication based solely on
the appearance of its package. Also, confusion can occur between medications with
names that look alike or sound alike or between premixed medications packaged
in similar - looking containers. Another potential source for confusion with premixed
medications is the presence of different concentrations of the same medication in
a particular location (e.g., a package with 100 mg/mL concentration of a drug could
be mistaken for one with 10 mg/mL concentration).
Daily, physicians, nurses, and pharmacists base medical decisions on the information
provided by a drug product ’ s labeling and packaging. Unfortunately, poor
labeling and packaging have been linked all too often to medication errors. To help
practitioners avoid errors, drug manufacturers should present information in a clear
manner that can be grasped quickly and easily.
To determine what presentation is most clear, manufacturers should invite and
consider the input of physicians, nurses, and pharmacists, because they work with
these products every day and are more likely than label and package designers to
discover potential problems. Such input provides the basis for failure and effect
analysis (FMEA), also known as error potential analysis or error prevention analysis.
FMEA is a systematic process that can predict how and where systems might
fail. Using FMEA, health care practitioners examine a product ’ s packaging or labeling
in order to identify the ways in which it might fail. A number of steps to reduce
confusion and improve the readability of a drug product ’ s label have already been
determined through the use of FMEA.
The fi rst step is to reduce label clutter. Only essential information, such as the
brand and generic names, strength or concentration, and warnings, should appear
prominently on the front label. Numerous deaths have been prevented through the
addition of a warning to concentrated vials of injectable potassium chloride, for
example. Another step includes the use of typeface to enhance distinctive portions
of look - alike drug names on look - alike packaging.
Medication errors are also associated with poor product packaging design. Unfortunately,
medication errors linked to poor labeling and packaging are sometimes
used in the health care environment to justify the damage. Participation of an expertise
from health care practitioners, during labeling and packaging design phase,
might have prevented several errors.
Whether for established drugs or new entities going through the approval process,
the principles of safe practice in naming, labeling, and packaging are the same and
must be very well controlled. Safety experts may differ about specifi c details, but
there is little disagreement about the fundamental principles that should be incorporated
into the drug approval process.
Based on reports of errors associated with packaging and labeling, many recommendations
have been proposed. Some of them are:
1. Avoid storing medications with similar packaging in the same location or in
close proximity.
2. Follow the American Society of Health System Pharmacists (ASHP) guidelines
or other legislation of a specifi c country for preventing hospital medication
errors [40, 41] . The ASHP ’ s recommendations include the following:
Fully document all medication prescription and deliveries and instruct staff
that discrepancy or misunderstanding about prescription or patient
information should be verifi ed with the prescribing physician. Staff
members should be told that all caregivers (regardless of level) have the
duty to question the prescribing physician (regardless of the physician ’ s
relative position in the hospital hierarchy) if they have concerns about
a drug, dose, or patient.
Periodically train staffs in practices that will help avoid medication errors.
Ensure that the medication storage and distribution to hospital locations
outside the pharmacy are supervised by hospital pharmacy staff only.
IMPORTANCE OF PROPER PACKAGING AND LABELING 183
184 PACKAGING AND LABELING
Nonpharmacists should not be allowed to enter the pharmacy if it is
closed.
3. Perform failure mode and effects analysis. This is a technique used to identify
all medication errors that could occur, determine how they occur, and estimate
what their consequences would be. Steps then should be taken to prevent
errors from occurring, when possible, and to minimize the effects of any errors
that do occur.
4. Report any information relating to medication errors to the Medication Errors
Reporting Program operated by USP convention [10] and the Institute for
Safe Medication Practice (ISMP) or other corresponding institutions in the
different countries. The program shares information on medication errors with
health care professionals to prevent similar errors from recurring.
5. Hospitals should report incidents in which a device caused or helped cause a
medication error.
6. Urge suppliers to provide clear and unique labels and packages for their
various individual medications.
Some other considerations relating to standards for drug names, labeling standards,
and packaging standards are as follows:
1. Standard for Drug Names. The most critical issue in drug name selection is that
one name should not be easily confused with another. This applies to both generic and
brand names. A name must neither sound like that of another drug nor look like
another drug name when it is written out by hand. From the industry ’ s standpoint, the
challenge is to fi nd a name that is easy to recollect and appropriate for the connotation
desired, do not lead astray (safe), and not already a trade name.
Nowadays, increasing sophisticated and effective methods are available for determining
the likelihood of confusion by sound or sight.
2. Labeling Standards. To minimize the possibility of error, labels should be easy
to read and avoid nonessential material. The name of the drug, and not the name
of the manufacturer, should be the most prominent feature and should be in at least
12 - point type. The use of color is very controversial; some believe that all colors
should be prohibited to force personnel to read the labels.
In the 1990s, a Washington State legislator proposed that every drug product
entering the state must have a color - coded label. There was concern on the part of
many that the state legislature would turn this idea into law. The prospect of having
to color - code all the drugs entering a single state galvanized a response by industry,
regulators, practitioners, and safe experts who agreed to revise pharmaceutical
labeling. A Committee to Reduce Medication Errors was formed to study the
problem. The effort eventually satisfi ed the color coders and the proposed legislation
was dropped.
The committee made several recommendations for standardizing and simplifying
labels:
1. Eliminate unnecessary words from the label, such as “ sterile, ” “ nonpyrogenic, ”
and “ may be habit forming. ”
2. Allow some abbreviations such as “ HCl ” and “ Inj. ”
3. Make label information consistent.
4. Require that vials containing medication that must be diluted bear the words
“ Concentrated, must be diluted ” in a box on the label, that the vial have a
black fl ip - top with those words on it, and that the ampules carry a black
band.
3. Packaging Standards. While there is no evidence that trademark colors and
logos on boxes pose a problem, the use of color on bottle tops and labels creates
many diffi culties. There are dozens of drugs whose names are quite different but
whose packages look alike. This creates the potential for error when people “ see ”
what they expect to see on the label.
Standards need to be set for color on both caps and labels. Some believe that
prohibiting all color would be safest — in effect, taking away a cue that could divert
someone from reading the label.
3.2.5 REGULATORY ASPECTS
3.2.5.1 General Considerations
Once the fi nished dosage form is made, the product should be packed into the
primary container and labeled. Additional packaging and labeling are also included.
Because of the many products and labeling materials, personnel in this area must
be alert to prevent mix - ups. Controls and in - process checks should be carried out
throughout the packaging/labeling operation to ensure proper labeling.
Some examples of good manufacturing practices (GMP) requirements specifi c
to packaging and labeling in different countries are as follows:
In the United States the requirements should be written procedures designed to
assure that correct labels, labeling, and packaging materials are used for drug
products; such written procedures should be followed. These procedures
should incorporate features such as prevention of mix - ups and cross -
contamination by physical or spatial separation from operations on other drug
products.
In Canada, packaging operations are performed according to comprehensive and
detailed written operating procedures or specifi cations, which include identi-
fi cation of equipment and packaging lines used to package the drug, adequate
separation, and, if necessary, the dedication of packaging lines packaging different
drugs and disposal procedures for unused printed packaging materials.
Packaging orders are individually numbered.
In the European Union, the requirements should be formally authorized in the
“ packaging instructions ” for each product containing pack size and type. They
are normally included in process controls with instructions for sampling and
acceptance limits [42] .
3.2.5.2 Food, Drug and Cosmetic Act
About 100 years after its foundation, the Congress of the United States recognized
that subjects related to safety and public health could not exclusively be state
dependent and measures should be taken to protect the population in vital areas.
Therefore, the federal government became interested in regulating products for
consumption.
REGULATORY ASPECTS 185
186 PACKAGING AND LABELING
In 1906, the Congress approved the Wiley Law to avoid the production, sale, or
transport of food, medications, and alcoholic beverages that were inadequate or
falsifi ed, poisonous, or harmful. It was the fi rst food and medication regulation
adopted in interstate commerce. The Congress was given power to regulate commerce
between foreign nations and several U.S. states.
In 1912 a civil code law was enacted prohibiting any false affi rmation of curing
or therapeutic effect on medication labels. The current law was enacted on June 27,
1938, and regulates food, medications, medical devices, and diagnostic and cosmetic
products. The law of 1938 stopped regulating the trade of alcoholic beverages. This
law stated, among other recommendations, the following:
1. The label of each medication had to give the name of each active component
and the quantity of some specifi c substances, active or not.
2. Cosmetics had to be inoffensive and be properly labeled and packaged.
The 1938 law states that the label of a medication should contain adequate information
regarding its use. However, in practice, it became evident that some pharmaceuticals
and medications had to be administered by or under the orientation of
a medical practitioner, due to the inability of a layman to diagnose a disease, choose
an effective treatment, and recognize the cure or the symptoms. Several products
were thus classifi ed, but “ the prescription concept of a medication ” was introduced
only after Alteration in the Law of Durham - Humphrey ’ s in 1951. Since then, a label
had to carry the warring “ Caution, the Federal Law prohibits dispensation without
medical prescription. ” The use of these medications had to be restricted to prescription
by a practitioner and the packing or printed material inside had to contain
adequate information so that the practitioner could prescribe them safely.
Alterations in 1962 of the 1938 Law constituted an attempt to establish rigid
controls on the research, production, divulging, promotion, sale, and use of medications
as well as to assure its quality, effi ciency, and effectiveness [43] .
3.2.5.3 New Drugs
Before starting clinical trials in humans, an authorization should be obtained from
the FDA. This is known as a clinical trial authorization request for a new medication
(AEM), on which it is necessary to establish the following:
1. The name that best describes the medication, including the chemical name
and the structure of any new molecule
2. A complete list of medication components.
3. A quantitative composition of the medication.
4. The name and address of the vendor and an acquired description of the new
drug
5. The methods, facilities, and controls used for the production, processing, and
packing of a new medication
6. All available results available from preclinical and clinical trials
7. Copies of medication labels and the informative material that will be supplied
to the researchers
8. A description of the scientifi c training and the appropriate experience considered
by the proponent to qualify a researcher as an adequate expert to
investigate the medication
9. The names and “ curricula vitae ” of all researchers
10. An investigation layout planned for test accomplishment in humans
Solicitations for release of new medications are generally very extensive,
sometimes thousands of pages. The information has to be enough to justify the
affi rmations contained in the label of the proposed medication with respect to
effectiveness, dosage, and safety. The exact composition of the content on the
medication label is usually decided by consensus between the proponent and the
FDA.
The requisites for solicitation of new medications, whether by prescription or not,
are identical. The instructions contained in the medication labels for use without
prescription should demonstrate that the medication can be used safely without
medical supervision.
Once the medications are perfected, the publicity related to them has to be routinely
presented to the FDA.
The rules of 1985 also changed the requisites regarding addendums that are
necessary when alterations are proposed in the medication or in its labeling, for
example.
In regulations promulgated by the FDA on February 12, 1972, a clinic should be
called upon regarding the effectiveness of a medication. After that the information
may be included in the label or in the drug informative leafl et with eligible sentence
and defi ned by dark lines that contour it [43] .
Other dispositions contained in the alterations to the 1962 law are as follows:
1. Immediate registration with the FDA before starting the production, repacking,
or relabeling of medications and later annual registration, with inspections
to be made at least once every two years.
2. Supportive inspections in the factory, particularly where prescription medications
are produced.
3. The procedures used by the manufacturers should be in conformity with the
good manufacturing practices, which permits the government to better inspect
of all the operations.
4. The common name should be presented on the label.
5. The publicity of a prescription medication should present a brief summary
mentioning the secondary effects, the contraindications, and the medication
effectiveness.
6. All antibiotics are subject for certifi cation procedures.
3.2.5.4 Labeling Requisites
According to a 1962 law, the main requisites for labeling are as described below.
The labeling of over - the - counter medications is regulated by the Food, Drug and
Cosmetic Act, which states:
REGULATORY ASPECTS 187
188 PACKAGING AND LABELING
A medication should be considered falsifi ed unless the label contains: 1. Indications of
adequate use and 2. Adequate warnings regarding the pathological indications in those
it should not be used or not for children use, when its use can be dangerous for health,
of dosages, methods or interval of administration, or unsafe application, of mode and
in necessary form for patients ’ protection.
“ Indications of use ” were defi ned in the regulations as information with which even
a layman can use the medication safely and for the purpose to which it is
designated.
The label of an over - the - counter medication must refer to the active substances,
but it is not necessary to indicate its relative quantity, except where the ingredient
leads to habituation. In this case the warning “ Can lead to habituation ” should
appear on the label.
A drug can be considered falsifi ed if it does not provide, besides indications of
adequate use, warnings against its use in some pathological conditions (or for children)
in which the medication can constitute a health risk. Regulations have suggested
warnings that can be used for most well - known dangerous substances.
3.2.5.5 Prescription Drugs
Specifi c requisites for labeling of ethical medications or of prescription medications
are also found in the Food, Drug and Cosmetic Act. These need not to contain
“ adequate indications of use ” ; however, they must contain indications for the
practitioner, inside or outside the package in which the medication is going to be
dispensed, with adequate information for its use. This information may in -
clude indications, effects, dosages, route of administration, methods, frequency
and duration of administration, important dangers, contraindications, secondary
effects, and cautions “ according to which the practitioners can prescribe the medication
assuredly and for the desirable effects, including those for which it is
proclaimed. ”
Regarding all medications, the act requires that the label present a precise affi rmation
on the weight of the content, measure or counting, as well as the name and
manufacturer ’ s address, packer or distributor.
The label of a prescription medication destined for oral administration has to
contain the quantity or proportion of each active substance.
If the medication is for parenteral administration, the quantity or proportion of
all the excipients have also to be mentioned on the label, except for those that are
added to adjust pH or make it isotonic, in which case only the name and its effect
are needed. However, if the vehicle for injection is water, this does not need to be
mentioned.
If the medication is not to be administered by any of the routes mentioned above,
for example, a pomade or a suppository, all excipients must to be mentioned, except
for perfuming agents. Perfumes can be designated as such without the need to
mention the specifi c components.
Coloring agents can be assigned without being specifi ed individually, unless this
is required in a separate section for regulation of coloring agents, and inoffensive
substances added exclusively for individual identifi cation of each product need not
be mentioned.
The only warning that is necessary, “ Attention: the Law prohibits the dispensing
without prescription, ” should be on the label of a prescription medication or in its
secondary packing if the label is too little to contain it.
3.2.5.6 Drug Information Leafl et
The inclusion of a drug information leafl et is not compulsory whatever the medication.
However, all medications, whether prescription or of over the counter, have to
contain a label with adequate indications for use. If the medication label does not have
enough space to contain all the information, the drug information leafl et has to be
included with necessary information. The drug information leafl et and labels containing
indication information must include the date when the text was last revised.
To satisfy the act, the drug information leafl et usually included in the prescription
medication packaging should contain “ adequate information on usage, including
indications, effects, dosages, methods, route, frequency and duration of administration.
Any important dangers, contraindications, secondary effects and cautions,
based on which the practitioner can prescribe the medication safely and for desirable
effects, including those for which a clam is made. ” To present the information
in a uniform manner, the FDA issued labeling policies describing its format and the
order and headings for the drug information leafl et description, action, indication,
contraindications, alerts, cautions, adverse reactions, dosage and administration,
overdose (when applicable), and as it is supplied.
The drug information leafl et can contain the following optional information:
Animal pharmacology and toxicology
Clinical studies
References
Other specifi c cautions on medication have to appear in a visible manner at the
beginning of the drug information leafl et so that practitioners, pharmacists, and
patients can easily see them.
According to GMP, an inspector should be cautious with several aspects of drug
production, including the following:
1. Product containers and other components have to be tested and be considered
adequate for their intended use only if they are not reactive, departure byproducts,
or even have absorption capacity; so that they do not affect the safety,
identity, potency, quality, or purity of the medication or its components.
2. Packing and labeling operations should be adequately controlled to (1) guarantee
that only those medications that own quality standards and attain established
specifi cations in their production and control be distributed, (2) avoid
mix - ups during the fi lling operations, packing, and labeling, (3) assure that the
labels and labeling used are correct for the medication, and (4) identify the
fi nished product with a batch or a control number that allows determination
of the batch production and control history.
Application of the federal law on food, drug, and cosmetics is the FDA ’ s responsibility,
which is a subdivision of the Department of Health and Human Services.
REGULATORY ASPECTS 189
190 PACKAGING AND LABELING
The institution is managed by a Commissaries and is subdivided into several departments:
Food safety and applied nutrition (CFSAN), Drug evaluation and research
(CDER), Biologics evaluation and research (CBER), Devices and radiological
health (CDRH), Veterinary medicine (CVM), Toxicological research (NCTR),
Regulatory affairs (ORA) and the offi ce of the commissioner (OC) [4, 44] .
3.2.5.7 Other Regulatory Federal Laws
There are other federal laws with which a pharmacist should be familiar. Perhaps
the most important are laws on packing and labeling, operations that are regulated
by the FDA and the Federal Communications Commission (FCC). The law on
packing and labeling is targeted mostly to protecting the consumer. In the case of
liquid the ingredients should be on the visible part of the package. The law presents
specifi c requisites concerning the location and size of the type. Violation of this law
can lead to apprehension by the FDA or a withdrawal order from the FCC.
Many times a pharmacist involved in developing a product is called upon in the
publicity of the medication. For this, he or she must understand the politics of the
regulatory agency involved. The FCC, according to the Federal Law of Commerce,
has jurisdiction over the announcement and promotion of all consumables, including
medications and cosmetics.
This law extends to all publicity and has to do with practices of fraudulent publicity
and with promotion that is understood to be false and fraudulent. In general, the
FCC controls the publicity of nonprescription drugs and cosmetics with respect to
false or fraudulent affi rmations, and the FDA is responsible for labeling of medications
and for all publicity related to prescription medications. The principal objective
of this is to avoid unnecessary duplication of procedures while enforcing the law.
The agencies work closely together and the FCC relies strongly on the FDA due to
its scientifi c knowledge. Any government has the right to approve laws for its citizens
’ protection. This right constitutes the base on which laws regulate the drug
substance, the drug product, and its production, distribution, and sale. It is common
that these laws exist at a district level, state level, and national level and deal with
falsifi cation and adulteration, fraudulent publicity, and maintenance of appropriated
sanitary conditions.
Most U.S. states specify the purity requisites, labeling, and applicable packaging
of a medication that are generally defi ned in identical language in federal law.
Almost all states, prohibit the commercialization of a new medication until an
authorization request for commercialization of a new medication has been submitted
to the FDA and has been approved. Medication labeling requisites in each
state are established, just as the local laws are defi ned, taking into consideration
arguments and information, such as name and place of activity (production), content
quantity, drug name, name of ingredients, quantity or proportion of some ingredients,
usage indications, warning regarding dependence, caution against deterioration
(degradation), warning about situations in which the use can be dangerous, and
special requisites for labeling of offi cial drugs [43] .
3.2.5.8 Fair Packaging and Labeling Act [44]
The FDA through Fair Packaging and Labeling Act regulates the labels on many
consumer products, including health products. Title 15: Commerce and Trade
Chapter 39: Fair Packaging and Labeling Program [44]
Section 1451. Congressional Delegation of Policy Informed consumers are essential
to the fair and effi cient functioning of a free market economy. Packages and
their labels should enable consumers to obtain accurate information as to the quantity
of the contents and should facilitate value comparisons. Therefore, it is hereby
declared to be the policy of the Congress to assist consumers and manufacturers in
reaching these goals in the marketing of consumer goods [44] .
Section 1452. Unfair and Deceptive Packaging and Labeling: Scope of
Prohibition
(a) Nonconforming Labels It shall be unlawful for any person engaged in the
packaging or labeling of any consumer commodity (as defi ned in this chapter) for
distribution in commerce, or for any person (other than a common carrier for hire,
a contract carrier for hire, or a freight forwarder for hire) engaged in the distribution
in commerce of any packaged or labeled consumer commodity, to distribute or
to cause to be distributed in commerce any such commodity if such commodity is
contained in a package, or if there is affi xed to that commodity a label, which does
not conform to the provisions of this chapter and of regulations promulgated under
the authority of this chapter.
(b) Exemptions The prohibition contained in subsection (a) of this section shall
not apply to persons engaged in business as wholesale or retail distributors of consumer
commodities except to the extent that such persons (1) are engaged in the
packaging or labeling of such commodities, or (2) prescribe or specify by any means
the manner in which such commodities are packaged or labeled.
Section 1453. Requirements of Labeling; Placement, Form, and Contents of
Statement of Quantity; Supplemental Statement of Quantity
(a) Contents of Label No person subject to the prohibition contained in section
1452 of this title shall distribute or cause to be distributed in commerce any packaged
consumer commodity unless in conformity with regulations which shall be
established by the promulgating authority pursuant to section 1455 of this title
which shall provide that:
• (1) The commodity shall bear a label specifying the identity of the commodity
and the name and place of business of the manufacturer, packer, or
distributor;
• (2) The net quantity of contents (in terms of weight or mass, measure, or
numerical count) shall be separately and accurately stated in a uniform location
upon the principal display panel of that label, using the most appropriate units
of both the customary inch/pound system of measure, as provided in paragraph
(3) of this subsection, and, except as provided in paragraph (3)(A)(ii) or paragraph
(6) of this subsection, the SI metric system;
• (3) The separate label statement of net quantity of contents appearing upon or
affi xed to any package:
• (A)
• (i) if on a package labeled in terms of weight, shall be expressed in pounds,
with any remainder in terms of ounces or common or decimal fractions of
REGULATORY ASPECTS 191
192 PACKAGING AND LABELING
the pound; or in the case of liquid measure, in the largest whole unit (quart,
quarts and pint, or pints, as appropriate) with any remainder in terms of
fl uid ounces or common or decimal fractions of the pint or quart;
• (ii) if on a random package, may be expressed in terms of pounds and
decimal fractions of the pound carried out to not more than three decimal
places and is not required to, but may, include a statement in terms of the
SI metric system carried out to not more than three decimal places;
• (iii) if on a package labeled in terms of linear measure, shall be expressed
in terms of the largest whole unit (yards, yards and feet, or feet, as appropriate)
with any remainder in terms of inches or common or decimal fractions
of the foot or yard;
• (iv) if on a package labeled in terms of measure of area, shall be expressed
in terms of the largest whole square unit (square yards, square yards and
square feet, or square feet, as appropriate) with any remainder in terms of
square inches or common or decimal fractions of the square foot or square
yard;
• (B) shall appear in conspicuous and easily legible type in distinct contrast (by
topography, layout, color, embossing, or molding) with other matter on the
package;
• (C) shall contain letters or numerals in a type size which shall be
• (i) established in relationship to the area of the principal display panel of
the package, and
• (ii) uniform for all packages of substantially the same size; and
• (D) shall be so placed that the lines of printed matter included in that statement
are generally parallel to the base on which the package rests as it is
designed to be displayed; and
• (4) The label of any package of a consumer commodity which bears a representation
as to the number of servings of such commodity contained in such
package shall bear a statement of the net quantity (in terms of weight or mass,
measure, or numerical count) of each such serving.
• (5) For purposes of paragraph (3)(A)(ii) of this subsection the term “ random
package ” means a package which is one of a lot, shipment, or delivery of packages
of the same consumer commodity with varying weights or masses, that is,
packages with no fi xed weight or mass pattern.
• (6) The requirement of paragraph (2) that the statement of net quantity of
contents include a statement in terms of the SI metric system shall not apply
to foods that are packaged at the retail store level.
(b) Supplemental Statements No person subject to the prohibition contained in
section 1452 of this title shall distribute or cause to be distributed in commerce any
packaged consumer commodity if any qualifying words or phrases appear in conjunction
with the separate statement of the net quantity of contents required by
subsection (a) of this section, but nothing in this subsection or in paragraph (2) of
subsection (a) of this section shall prohibit supplemental statements, at other places
on the package, describing in nondeceptive terms the net quantity of contents: Provided
, That such supplemental statements of net quantity of contents shall not
include any term qualifying a unit of weight or mass, measure, or count that tends
to exaggerate the amount of the commodity contained in the package.
Section 1454. Rules and Regulations
(a) Promulgating Authority The authority to promulgate regulations under this
chapter is vested in (A) the Secretary of Health and Human Services (referred to
hereinafter as the “ Secretary ” ) with respect to any consumer commodity which is
a food, drug, device, or cosmetic, as each such term is defi ned by section 321 of title
21; and (B) the Federal Trade Commission (referred to hereinafter as the “ Commission
” ) with respect to any other consumer commodity.
(b) Exemption of Commodities from Regulations If the promulgating authority
specifi ed in this section fi nds that, because of the nature, form, or quantity of a particular
consumer commodity, or for other good and suffi cient reasons, full compliance
with all the requirements otherwise applicable under section 1453 of this title
is impracticable or is not necessary for the adequate protection of consumers, the
Secretary or the Commission (whichever the case may be) shall promulgate regulations
exempting such commodity from those requirements to the extent and under
such conditions as the promulgating authority determines to be consistent with
section 1451 of this title:
(c) Scope of Additional Regulations Whenever the promulgating authority determines
that regulations containing prohibitions or requirements other than those
prescribed by section 1453 of this title are necessary to prevent the deception of
consumers or to facilitate value comparisons as to any consumer commodity, such
authority shall promulgate with respect to that commodity regulations effective
to:
• (1) establish and defi ne standards for characterization of the size of a package
enclosing any consumer commodity, which may be used to supplement the label
statement of net quantity of contents of packages containing such commodity,
but this paragraph shall not be construed as authorizing any limitation on the
size, shape, weight or mass, dimensions, or number of packages which may be
used to enclose any commodity;
• (2) regulate the placement upon any package containing any commodity, or
upon any label affi xed to such commodity, of any printed matter stating or
representing by implication that such commodity is offered for retail sale at a
price lower than the ordinary and customary retail sale price or that a retail
sale price advantage is accorded to purchasers thereof by reason of the size of
that package or the quantity of its contents;
• (3) require that the label on each package of a consumer commodity (other
than one which is a food within the meaning of section 321(f) of title 21) bear
(A) the common or usual name of such consumer commodity, if any, and
(B) in case such consumer commodity consists of two or more ingredients, the
common or usual name of each such ingredient listed in order of decreasing
predominance, but nothing in this paragraph shall be deemed to require that
any trade secret be divulged; or
REGULATORY ASPECTS 193
194 PACKAGING AND LABELING
• (4) prevent the nonfunctional - slack - fi ll of packages containing consumer commodities.
For purposes of paragraph (4) of this subsection, a package shall be
deemed to be nonfunctionally slack - fi lled if it is fi lled to substantially less than
its capacity for reasons other than (A) protection of the contents of such
package or (B) the requirements of machines used for enclosing the contents
in such package.
(d) Development by Manufacturers, Packers, and Distributors of Voluntary Product
Standards Whenever the Secretary of Commerce determines that there is undue
proliferation of the weights or masses, measures, or quantities in which any consumer
commodity or reasonably comparable consumer commodities are being distributed
in packages for sale at retail and such undue proliferation impairs the
reasonable ability of consumers to make value comparisons with respect to such
consumer commodity or commodities, he shall request manufacturers, packers, and
distributors of the commodity or commodities to participate in the development of
a voluntary product standard for such commodity or commodities under the procedures
for the development of voluntary products standards established by the
Secretary pursuant to section 272 of this title. Such procedures shall provide adequate
manufacturer, packer, distributor, and consumer representation.
(e) Report and Recommendations to Congress upon Industry Failure to Develop or
Abide by Voluntary Product Standards If (1) after one year after the date on which
the Secretary of Commerce fi rst makes the request of manufacturers, packers, and
distributors to participate in the development of a voluntary product standard as
provided in subsection (d) of this section, he determines that such a standard will
not be published pursuant to the provisions of such subsection (d), or (2) if such a
standard is published and the Secretary of Commerce determines that it has not
been observed, he shall promptly report such determination to the Congress with
a statement of the efforts that have been made under the voluntary standards
program and his recommendation as to whether Congress should enact legislation
providing regulatory authority to deal with the situation in question.
Section 1455. Procedures for Promulgation of Regulations
(a) Hearings by Secretary of Health and Human Services Regulations promulgated
by the Secretary under section 1453 or 1454 of this title shall be promulgated,
and shall be subject to judicial review, pursuant to the provisions of subsections (e),
(f), and (g) of section 371 of title 21. Hearings authorized or required for the promulgation
of any such regulations by the Secretary shall be conducted by the Secretary
or by such offi cer or employees of the Department of Health and Human
Services as he may designate for that purpose.
(b) Judicial Review; Hearings by Federal Trade Commission Regulations promulgated
by the Commission under section 1453 or 1454 of this title shall be promulgated,
and shall be subject to judicial review, by proceedings taken in conformity
with the provisions of subsections (e), (f), and (g) of section 371 of title 21 in the
same manner, and with the same effect, as if such proceedings were taken by the
Secretary pursuant to subsection (a) of this section. Hearings authorized or required
for the promulgation of any such regulations by the Commission shall be conducted
by the Commission or by such offi cer or employee of the Commission as the Commission
may designate for that purpose.
(c) Cooperation with Other Departments and Agencies In carrying into effect the
provisions of this chapter, the Secretary and the Commission are authorized to
cooperate with any department or agency of the United States, with any State, Commonwealth,
or possession of the United States, and with any department, agency, or
political subdivision of any such State, Commonwealth, or possession.
(d) Returnable or Reusable Glass Containers for Beverages No regulation adopted
under this chapter shall preclude the continued use of returnable or reusable glass
containers for beverages in inventory or with the trade as of the effective date of
this Act, nor shall any regulation under this chapter preclude the orderly disposal
of packages in inventory or with the trade as of the effective date of such
regulation.
3.2.5.9 United States Pharmacopeia Center for the Advancement of Patient
Safety [45]
For nearly 33 years, the USP has been reporting programs for health care professionals
to share experiences and observations about the quality and safe use of
medications. This year, the USP Center for the Advancement of Patient Safety
publishes its sixth annual report to the nation on medication errors reported to
MEDMARX (Table 6 ). It was observed that drug product packaging/labeling is one
of the main courses of medication errors in hospitals.
3.2.5.10 National Agency of Sanitary Vigilance ( ANVISA , Brazil)
ANVISA is a federal organization linked to Brazil ’ s Health Ministry, which has the
incumbency of looking after medication quality and other health products aimed at
patients ’ safety. Several documents regarding GMP and quality control are easily
accessed. The agency is also responsible for establishing enforcing the rules and can
take corrective measures and punish the offenders [46] .
Product stability and compatibility with the conditioning material are distinct,
separate, and complementary concepts which should be applied to the pharmaceutical
product before being made available for health care.
TABLE 6 Selected Causes of Error Related to Equipment, Product Packaging/Labeling,
and Communication in ICUs
Cause of Error N (Nonharmful + Harmful) Percent Harmful
Label (the facility ’ s) design 1,236 6,9
Similar packaging/labeling
Packaging/container design
Label (manufacturer ’ s) design
Brand/generic names look - alike
Source : MEDMARX Data Report: A Chartbook of 2000 – 2004 Findings from Intensive Care Units
(ICUs) and Radiological Services.
REGULATORY ASPECTS 195
196 PACKAGING AND LABELING
In the compatibility test between formulation and the conditioning material,
several options of conditioning materials are evaluated to determine the most adequate
for the product.
The environmental conditions and periodicity analyses can be the same as those
mentioned for the stability studies for the formulation. In this phase, the possible
interactions between the product and the conditioning material which is in direct
contact with the medication are verifi ed. Phenomena such as absorption, migration,
corrosion, and others that compromise integrity can be observed. Considering that
these types of tests are generally destructive, it is necessary to defi ne the number of
samples to be tested.
In ANVISA ’ s documents, different types of tests are established that should be
carried out with different types of available materials and employed for conditioning
medications and cosmetics (cellulose packagings, metallic, plastic, pressurized,
etc.) [46] .
3.2.5.11 International Committee on Harmonization ( ICH )
In the document “ Good Manufacturing Practice Guide for Active Pharmaceutical
Ingredients (APIs) ” of the ICH Harmonized Tripartite Guideline, the following
instructions are given for packaging and identifi cation labeling of APIs and intermediates
[47] .
General
• There should be written procedures describing the receipt, identifi cation, quarantine,
sampling, examination and/or testing and release, and handling of packaging
and labeling materials.
• Packaging and labeling materials should conform to established specifi cations.
Those that do not comply with such specifi cations should be rejected to prevent
their use in operations for which they are unsuitable.
• Records should be maintained for each shipment of labels and packaging
materials showing receipt, examination, or testing, and whether accepted or
reject.
Packaging Materials
• Containers should provide adequate protection against deterioration or contamination
of the intermediate or API that may occur during transportation
and recommended storage.
• Containers should be clean and, where indicated by the nature of the intermediate
or API, sanitized to ensure that they are suitable for their intended use.
These containers should not be reactive, addictive, or absorptive so that the
quality of the intermediate or API complies with the specifi cations.
• If containers are reused, they should be cleaned in accordance with documented
procedures and all previous labels should be removed or defaced.
Label Issuance and Control
• Access to the label storage areas should be limited to authorized personnel.
• Procedures should be used to reconcile the quantities of labels issued, used,
and returned and to evaluate discrepancies found between the number of containers
labeled and the number of labels issued. Such discrepancies should be
investigated and the investigation should be approved by the quality unit(s).
• All excess labels bearing batch numbers or other batch - related printing should
be destroyed. Returned labels should be maintained and stored in a manner
that prevents mix - ups and provides proper identifi cation.
• Obsolete and outdated labels should be destroyed.
• Printing devices used to print labels for packaging operations should be controlled
to ensure that all imprinting conforms to the print specifi ed in the batch
production record.
• Printed labels issued for a batch should be carefully examined for proper identity
and conformity to specifi cations in the master production record. The
results of this examination should be documented.
• A printed label representative of those used should be included in the batch
production record.
Packaging and Labeling Operations
• There should be documented procedures designed to ensure that correct packaging
materials and labels are used.
• Labeling operations should be designed to prevent mix - ups. There should be
physical or spatial separation from operations involving other intermediates or
APIs.
• Labels used on containers of intermediates or APIs should indicate the name
or identifying code, the batch number of the product, and storage conditions,
when such information is critical to assure the quality of intermediate API.
• If the intermediate or API is intended to be transferred outside the control of
the manufacturer ’ s material management system, the name and address of the
manufacturer, quantity of contents and special transport conditions, and any
special legal requirements should also be included on the label. For intermediates
or APIs with an expiry date, the expiry date should be indicated on the
label and certifi cate of analysis. For intermediates or APIs with a retest date,
the retest date should be indicated on the label and/or certifi cate of analysis.
• Packaging and labeling facilities should be inspected immediately before use
to ensure that all materials not needed for the next packaging operation have
been removed. This examination should be documented in the batch production
records, the facility log, or other documentation system.
• Packaged and labeled intermediates or APIs should be examined to ensure that
containers and packages in the batch have the correct label. This examination
should be part of the packaging operation. Results of these examinations should
be recorded in the batch production or control records.
REGULATORY ASPECTS 197
198 PACKAGING AND LABELING
• Intermediate or API containers that are transported outside of the manufacturer
’ s control should be sealed in a manner such that, if the seal is breached
or missing, the recipient will be alerted to the possibility that the contents may
have been altered.
3.2.5.12 European Union Regulatory Bodies
European regulatory requirements say little to date about container closure integrity
of parenteral or sterile pharmaceutical products. Regulations provide for
package integrity verifi cation of parenteral vials to be supported by the performance
of sterility tests as part of the stability program. More specifi c information is
described in the European Union (EU) 1998 “ Rules Governing Medical Products
in the European Union, Pharmaceutical Legislation. ” These GMP regulations
require that the sealing or closure process be validated. Packages sealed by fusion
(e.g., ampules) should be 100% integrity tested. Other packages should be sampled
and checked appropriately. Packages sealed under vacuum should be checked for
the presence of vacuum. While not as detailed as the FDA guidances, it is evident
that the EU rules also require the verifi cation of parenteral product package seal
integrity. It is important to note that the EU rules specifi cally require 100% product
testing for fusion - sealed packages, sampling and testing of all other packages, and
vacuum verifi cation for packages sealed under partial pressure [42] .
The vacuum/pressure decay test is performed by placing the package in a tightly
closed test chamber, a pressure or vacuum is applied inside the chamber, and then
the rate of pressure/vacuum change in the chamber over time is monitored. The rate
or extent of change is compared to that previously exhibited by a control, nonleaking
package. Signifi cantly greater change for a test package is indicative of a leak.
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density polyethylene containers on some hospital - manufactured eye drop formulations.
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fl uid containers and administration sets , Am. J. Hosp. Pharm. , 37 , 496 – 500 .
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surfaces and silicone - coated surfaces as models of surfaces of containers , Chem. Pharm.
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sorption of pharmaceutical preparation under the shelf condition , Yakugaku Zusshi ,
98 , 986 – 996 .
16. Nakabayashi , K. , Tuchida , T. , and Mima , H. ( 1980 ), Stability of packaged solid dosage
forms. I. Shelf - life prediction of packaged tablets liable to moisture damage , Chem.
Pharm. Bull. , 28 , 1090 – 1098 .
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forms. II. Shelf - life prediction for packaged sugar - coated tablets liable to moisture and
heat damage , Chem. Pharm. Bull. , 28 , 1099 – 1106 .
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forms. III. Kinetic studies by differential analysis on the deterioration of sugar - coated
tablets under the infl uence of moisture and heat , Chem. Pharm. Bull. , 28 , 1107 – 1111 .
19. Tonnesen , H. H. ( 1996 ), Photostability of Drugs and Drug Formulations , CRC Press ,
London .
20. Kontny , M. J. , Koppenol , S. , and Graham , E. T. ( 1992 ), Use of the sorption – desorption
moisture transfer model to assess the utility of a desiccant in a solid product , Int. J. Pharm. ,
84 , 261 – 271 .
21. Pikal , M. J. , and Lang , J. E. ( 1978 ), Rubber closures as a source of haze in freeze dried
parenterals: Test methodology for closure evaluation , J. Parenteral drug Assoc. , 32 ,
162 – 173 .
22. Jaehnke , R. W. O. , Kreuter , J. , and Ross , G. ( 1990 ), Interaction of rubber closures with
powders for parenteral administration , J. Parenteral sci. Tech. , 44 , 282 – 288 .
23. Jaehnke , R. W. O. , Kreuter , J. , and Ross , G. ( 1991 ), Content/container interactions: The
phenomenon of haze formation on reconstitution of solids for parenteral use , Int. J.
Pharm. , 77 , 4755 .
24. Moorhatch , P. , and Chiou , W. L. ( 1974 ), Interactions between drugs and plastic intravenous
fl uid bags. II: Leaching of chemicals from bags containing various solvent media ,
Am. J. Hosp. Pharm. , 31 , 149 – 152 .
25. Venkataramanan , R. , Burckart , G. J. , Ptachcinski , R. J. , Blaha , R. , Logue , L. W. , Bahnson ,
A. C. , and Brady , G. J. E. ( 1986 ), Leaching of diethylhexyl phthalate from polyvinyl
chloride bags into intravenous cyclosporine solution , Am. J. Hosp. Pharm. , 43 , 2800 –
2802 .
26. Boruchoff , S. A. ( 1987 ), Hypotension and cardiac arrest in rats after infusion of mono
(2ethylhexyl) phthalate (MEHP), a contaminant of stored blood , N. Engl. J. Med. , 316 ,
1218 – 1219 .
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27. U.S. Food and Drug Administration , Code of Federal Regulations (CFR) — Title 21, Food
and drugs, Chapters 174 – 186, available: http://www.access.gpo.gov/nara/cfr/index.html ,
accessed Mar. 11, 2005.
28. Kowaluk , E. A. , Roberts , M. S. , Blackburn , H. D. , and Polack , A. E. ( 1981 ), Interactions
between drugs and polyvinyl chloride infusion bags , Am. J. Hosp. Pharm. , 38 , 1308 – 1314 .
29. Illum , L. , and Bundgaard , H. ( 1982 ), Sorption of drugs by plastic infusion bags , Int. J.
Pharm. , 10 , 339 – 351 .
30. Illum , L. , Bundgaard , H. , and Davis , S. S. ( 1983 ), A constant partition model for examining
the sorption of drugs by plastic infusion bags , Int. J. Pharm. , 17 , 183 – 192 .
31. Atkinson , H. C. , and Duffull , S. B. ( 1990 ), Prediction of drug loss from PVC infusion bags ,
J. Pharm. Pharmacol. , 43 , 374 – 376 .
32. Richardson , N. E. , and Meakin , B. J. ( 1974 ), The sorption of benzocaine from aqueous
solution by nylon 6 powder , J. Pharm. Phamacol. , 26 , 166 – 174 .
33. Santoro , M. I. R. M. , Kedor - Hackmann , E. R. M. , and Moudatsos , K. M. ( 1993 ), Estabilidade
de sais de reidrata c a o oral em diferentes tipos de embalagem . Bol. Sanit. Panam. ,
115 , 310 – 315 .
34. World Health Organization (WHO) ( 2003 ), The International Pharmacopoeia, Tests and
General Requirements for Dosage Forms: Quality Specifi cations for Pharmaceutical Substances
and Tablets , 3rd ed., Vol. 5, WHO , Geneva.
35. Santoro , M. I. R. M. , Oliveira , D. A. G. C. , Kedor - Hackmann , E. R. M. , and Singh , A. K.
( 2004 ), Quantifying benzophenone - 3 and octyl methoxycinnamate in sunscreen emulsions
, Cosm. & Toil. , 119 , 77 – 82 .
36. Santoro , M. I. R. M. , Oliveira , D. A. G. C. , Kedor - Hackmann , E. R. , and Singh , A. K. ( 2005 ),
The effect of packaging materials on the stability of sunscreen emulsions , Int. J. Pharm. ,
13 , 197 – 203 .
37. Thoma , K. , and Kerker , R. ( 1992 ), Photoinstability of drugs. 6. Investigations on the photosansibility
of molsidomine , Pharm. Ind. , 54 , 630 – 638 .
38. British Pharmacopoeia ( 2002 ), Her Majesty ’ s Stationary Offi ce, London, pp A144, 135 –
136, 196, 671 – 673, 778 – 780, 976 – 978, 1145 – 1146.
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Med. Packaging News , 3 , 76 – 78 .
40. ASHP Council on Professional Affairs ( 1993 ), ASHP Guidelines on preventing medication
errors in hospital , Am. J. Hosp. Pharm. , 50 , 305 – 314 .
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errors in hospital , Am. J. Hosp. Pharm. , 58 , 3033 – 3041 .
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43. Lachman , L. , Lieberman , H. A. , and Kanig , J. L. ( 2001 ), Teoria e pr a tica na ind u stria
farmac e utica , Funda c a o Calouste Gulbenkian , Lisboa .
44. U.S. Food and Drug Administration, Fair Packaging and Labeling Act . Title 15 —
Commerce and Trade, Chapter 39 — Fair Packaging and Labeling Program, available:
http://www.fda.gov/opacom/laws/fplact.htm accessed Mar. 11, 2005.
45. Santell , J. P. , Hicks , R. W. , and Cousins , D. D. ( 2005 ), MEDMARX Data Report: A Chartbook
of 2000 – 2004 Findings from Intensive Care Units and Radiological Services , USP
Center for Advancement of Patient Safety , Rockville, MD .
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Produtos Cosm e ticos , ANVISA , Bras i lia .
47. International Organization on Harmonisation (2000), ICH harmonized tripartite guideline:
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http://www.ICH.org , accessed June 23, 2005.
48. Sarbach , C. , Yagoubi , N. , Sauzieres , J. , Renaux , C. , Ferrier , D. , and Postaire , E. ( 1996 ),
Migration of impurities from a multi-layer plastics container into a parenteral infusion
solution , Int. J. Pharm. , 140 , 169 – 174 .
201
3.3
CLEAN - FACILITY DESIGN,
CONSTRUCTION, AND
MAINTENANCE ISSUES
Raymond K. Schneider
Clemson University, Clemson, South Carolina
Contents
3.3.1 Introduction
3.3.2 Planning for Project Success
3.3.2.1 Needs Assessment
3.3.2.2 Front - End Planning
3.3.2.3 Preliminary Design
3.3.2.4 Procurement
3.3.2.5 Construction
3.3.2.6 Start - Up and Validation
3.3.2.7 Summary
3.3.3 Design Options
3.3.3.1 Clean - Facility Scope
3.3.3.2 Design Parameters
3.3.3.3 Architectural Design Issues
3.3.3.4 Materials of Construction
3.3.3.5 HVAC System
3.3.3.6 Clean - Room Testing
3.3.3.7 Utilities
3.3.4 Construction Phase: Clean Build Protocol
3.3.4.1 General
3.3.4.2 Level I Clean Construction
3.3.4.3 Level II Clean Construction
3.3.5 Maintenance
Appendix A: Guidelines for Construction Personnel and Work Tools in a Clean
Room
Appendix B: Cleaning the Clean Room
Bibliography
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
202 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
3.3.1 INTRODUCTION
While there are discrete steps in the design and construction of a pharmaceutical
manufacturing plant project, those projects deemed successful incorporate certain
practices that promote fl ow of the construction process toward completion on time
and within budget. Proper front - end planning is not completed until it results in
appropriate values for design parameters, “ buy - in ” at all levels of management, and
clear direction for the design phase. Engineering the clean room in accordance with
recognized industry practice would produce construction documents that facilitate
clear procurement and construction planning as well as a focused, effi cient, construction
effort. A full return on the energy expended through the construction phase
cannot be realized without a well - executed start - up and validation process that
provides baseline data for effective ongoing operation and maintenance.
The steps in the clean - room construction project include:
Needs assessment
Front - end planning
Preliminary design
Construction document development
Procurement
Construction
Start - up and validation
One of the truisms of the construction industry is that the greatest impact on the
cost of a facility can be made at the earliest stages of the process. The construction
process can be likened to a snowball rolling down a snow - covered hill. It grows and
gains momentum, seemingly taking on a life of its own, until it can only be brought
under control with a major effort. So too with manufacturing plant projects. Careful
work during the fi rst three stages will ensure that the project begins on a well -
directed course and moves to a successful conclusion.
Sometimes the special nature of pharmaceutical manufacturing plant projects
clouds the fact that building such a plant is in fact a construction project. The facility
engineering team of a small to medium company may be tempted to turn away from
such projects due to the projects ’ perceived uniqueness and leave the key decision
making to others. In fact, it is the construction experience of that team that is most
required to keep the project costs under control. The way to accomplish this is for
the team to be involved in the process from its earliest stages.
Let us review the steps in such a project and identify what should occur at each
step and the potential for trouble.
3.3.2 PLANNING FOR PROJECT SUCCESS
3.3.2.1 Needs Assessment
It is during this early stage that a requirement for a clean manufacturing facility is
perceived. The need for the facility may be precipitated by a new product, an
improved product, an improved manufacturing methodology, new or more stringent
regulation requirements, or perhaps a change in marketing strategy.
At this point a study should be undertaken to determine the benefi ts to be realized
by the new facility as well as the costs to be incurred. Costs arise from not only
construction but also ongoing operation and maintenance. These costs are affected
by the plant location and the availability of a trained or trainable workforce. Does
the day - to - day operation of the facility generally require that special attire be worn?
Are special procedures, possibly more time consuming than those presently used,
required? It is important that this study is complete and accurate in order to prevent
any unrealistic expectations on the part of management and plant operations and
to permit advanced planning for revised procedures once the facility is in use.
The study should describe the goals of the project, its impact on present operations,
budget restraints, tentative schedule, and path forward. It will serve as the
basis for front - end planning and will provide the standard against which the success
of the program is measured.
3.3.2.2 Front - End Planning
While the needs assessment study may be conducted by a limited number of people,
the front - end planning process should be open to all. Plant facilities people will be
bearing the brunt of the responsibility for bringing the facility online, on schedule,
and within budget. Process people are responsible for ensuring that the facility will
adequately house the process equipment and that the facility incorporates suffi cient
space, utilities, process fl ow considerations, and provision for fl ow of people and
material to support the goals of the building program. Human resources people
have to staff the facility, either out of the present employee pool or from the general
local labor market. They must know the requirements of potential employees as
well as the conditions under which they will be working.
Procurement people will be purchasing furnishings and process equipment for
the plant as well as overseeing the contracts let to the design and construction professionals.
Operations people should have input regarding design parameters such
as temperature, humidity, lighting, vibration, cleanliness class, and energy needs.
Materials handling people should participate in order to understand the requirements
for storing and transporting raw materials as well as retrieving, storing, and
shipping fi nished goods from the plant.
An integral part of the front - end planning team should be the design professionals
charged with developing the plant design based on client input in such a way as
to satisfy as many requirements developed in needs assessment as possible. This
team may be assembled internally but frequently is drawn from specialty builders,
architectural and engineering (A & E) fi rms, and design/build fi rms active in the
pharmaceutical industry. The team of design professionals should have pharmaceutical
experience on facilities comparable in size and complexity to that being planned
as well as extensive experience in construction projects of all types. The design team
may offer design only, design/build, procurement, construction management, or
combinations of these services. This design team should be considered a resource
during the front - end planning phase. It is the wise client who takes advantage of
the experience of the design team, permitting them a large role as facilitators of the
planning sessions.
PLANNING FOR PROJECT SUCCESS 203
204 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
An appropriate design team will demonstrate expertise in contamination control
philosophies, space planning, code compliance, and mechanical and electrical design
and will be familiar with materials of construction currently being used in pharmaceutical
projects. It is frequently helpful to include a member of the construction
team in the front - end planning effort to advise on constructibility of the facility
being planned. Unrealistic construction schedules will be avoided and fi eld rework
will be minimized if appropriate attention is paid to the construction phase early in
the planning process.
3.3.2.3 Preliminary Design
Front - end planning typically utilizes the expertise of client process people to convey
the requirements of the pharmaceutical facility to the design team. With this information
in hand the design team begins the facility design incorporating process
needs, code requirements, safety issues, material and personnel fl ow, work - in - process
storage, utility needs, and so on, into a fi rst - cut approach.
Client representatives have an opportunity to review the effort and begin fi ne
tuning the design to incorporate late - breaking process changes. The preliminary
design is a target that helps both the design team and the client solidify design goals.
Change is inexpensive, and therefore encouraged, at this stage and buy - in by all
concerned is a major objective of this phase of the design effort.
A budget based on the agreed - upon preliminary design should be developed to
make sure that the overall project is on course. This will minimize surprises further
along in the design/build process. Ideally the design will be “ cast in stone ” at the
end of the preliminary phase. This permits the production work on the design documents
to proceed unhindered. The more unknowns left at the end of the preliminary
phase, the more diffi cult it will be to complete design documents in a timely
fashion.
Construction Document Development The construction documents should convey
the intent of the design team and client to the construction team. A good set of
construction documents should result in a tight spread of construction bids as there
should be little room for varying interpretation on the part of the potential construction
contractors. The drawings should have suffi cient notes to convey the design
intent without creating a cluttered appearance. The written specifi cations should be
as brief as possible consistent with clarity.
Complicated documents create the impression that a project may be more
involved, and therefore more costly, than it should be. Cautious contractors may
unnecessarily infl ate their bid to cover perceived contingencies. Specifi cations that
are too wordy may be diffi cult to follow and similarly result in higher prices as
bidders make sure all bases are covered. No one likes surprises.
The development of construction documents should be a straightforward process
with little involvement by the client except to monitor the process and ensure that
the original design intent is followed. While changes will always occur during this
phase ( “ cast in stone ” is a euphemism for “ let ’ s keep the changes under control ” ),
they are certainly less costly at this point than during the construction phase. It is
desirable to minimize such changes. A continuous sequence of changes suggests that
the preliminary design phase was not entered into seriously. It demonstrates a lack
of preparedness on the part of the client and a lack of ability to communicate and
draw out the client ’ s needs on the part of the design team. A sense of clarity of
purpose slips away with ongoing change and the possibility for errors in construction
documents, which eventually surface as costly construction changes, increases.
3.3.2.4 Procurement
A detailed scope of work describing the materials and services required is a vital
part of the procurement process. There is no purpose to keeping the project bidders
in the dark regarding what is required of them. The role of the procurement function
is to obtain maximum value, that is, the best quality and schedule at the lowest
price. The clearer the scope of work and construction documents, the better will be
the chance of this happening. A low price is not a good value if the schedule slips
by several months as a result. A marginal plant that does not maintain design conditions
or meet production goals is a poor value even if it was delivered within
schedule.
The procurement process should qualify potential bidders by ensuring that similar
pharmaceutical projects have been delivered on time, within budget, and on schedule.
References should be checked. It is expected that references offered by a
potential bidder would have good things to say about that bidder, but this is not a
certainty and pointed questioning about personnel, schedule, quality, change orders,
follow - up, and so on, can help develop a warm feeling or an uncertain feeling about
potential bidders. If bids are in fact quite close, it is the quality of references that
might suggest a particular bidder be given preference.
There are a number of ways in which the project can be procured. Use of in - house
engineering and construction expertise may work in special situations or on smaller
projects. Typically problems arise when facilities departments, stretched to their
limit with ongoing plant requirements, must lower the priority of the new facility to
meet other commitments. Schedules may stretch out unacceptably.
A number of specialty contractors have proven over the years to be adept at
installing small turnkey facilities of limited complexity in a timely and economical
fashion. If extensive engineering is required, if local code compliance becomes an
issue, if complex process requirements must be met, or if the client requirements
exceed the experience of the supplier there could be cause for concern.
Design/build is a popular approach in that it suggests a single source of responsibility
for all phases of the project. Frequently fi rms billing themselves as “ design/
build ” are strong in either design or build, but not both. The strong design fi rm can
put the essentials on paper but the fi nal price and schedule may suffer. The strong
construction fi rm may lack the expertise to create an appropriate manufacturing
environment, particularly where clean - room expertise is required. The project may
be outstanding in all respects except performance. A good review of references is
essential before selecting a design/build fi rm.
Construction management has been increasingly used on larger projects. A good
construction management fi rm will work closely with the client - selected design
company to review constructability and adequacy of construction documents. It will
assist to qualify bidders, maintain schedule, track costs, administer and oversee, and
generally ensure that a team incorporating the strongest skills is assembled to complete
the project. Pharmaceutical experience is essential.
PLANNING FOR PROJECT SUCCESS 205
206 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
3.3.2.5 Construction
The construction process should proceed smoothly if the remarks presented above
are followed. Cost can increase during this phase if changes must be implemented.
While change is inevitable, a construction change procedure negotiated during the
bidding phase and in place during construction will keep such change from getting
out of control.
The requirement for “ building clean ” has arisen in recent years as more stringent
clean rooms have become more popular. Imposing a clean construction protocol on
contractors can lengthen the schedule and increase cost. The protocol should be
developed during the construction document phase and be an integral part of the
bid documents. Once the decision is made to work clean, protocols developed
should be followed by everyone on the jobsite associated with the clean areas. A
poorly conceived and enforced protocol will be a costly and futile exercise. The
tendency to build clean on every new or retrofi t project should be carefully evaluated
and a practical protocol should be developed consistent with the needs of the
project.
Client end users should be encouraged to observe construction as it progresses.
They will be more intelligent about how their facility was built and therefore more
attuned to maintaining the facility once it is completed and in operation. While
suggestions should be welcomed as construction progresses, it is important that a
chain of command be enforced. Any questions or suggestions or concerns should
not be expressed to workers on the site but rather through project management
channels. In this way good ideas can be implemented and bad ideas shelved without
impacting the construction effort in a negative manner. Note the one exception to
this practice is in regard to safety. Everyone on the site has safety responsibility.
Any unsafe acts should be questioned and supervisors consulted immediately.
3.3.2.6 Start - Up and Validation
Subcontractors on the jobsite should be responsible for start - up as well as installation
of equipment. Equipment manufacturers typically have personnel available to
ensure appropriate start - up procedures are followed. If several trades are involved
in the installation of a particular piece of equipment, then one trade should be
assigned, by contract, as having coordinating responsibility for that piece of equipment.
This will minimize “ fi nger pointing ” when equipment does not start or operate
properly. This can be a sensitive issue and a construction manager can set the tone
for cooperation in this area.
An independent contractor responsible to the construction manager or owner
should do testing and balancing (TAB) of mechanical systems. All start - up should
be complete and initial valve or damper settings made (and recorded) by the subcontractor
before testing and balancing begins. The TAB contractor should not have
to repair equipment or troubleshoot inoperative equipment but rather only adjust
and verify performance of equipment.
A separate contractor should certify clean - room areas. This might be the TAB
contractor if that fi rm is suitably qualifi ed. There should be no question of equipment
being operative at this stage of the project since start - up and testing and
balancing are complete. Certifi cation is the verifi cation of facility compliance
DESIGN OPTIONS 207
with clean - room specifi cations. If the facility design is well conceived and the construction
team has installed a quality project, any certifi cation test failure will most
likely be corrected through fairly minor adjustments. Failure of the clean room to
pass certifi cation tests might require redesign but more frequently requires some
equipment adjustment or perhaps a fi lter repair and then a retest. It is important
that a clear understanding of responsibility be communicated before problems are
encountered. Failure to plan for potential problems could result in extending the
schedule and incurring unforeseen costs at a crucial point in the project.
3.3.2.7 Summary
Recognizing the step - by - step process involved in even the smallest pharmaceutical
project can help focus attention in a manner that will result in a successful project.
The formal schedule of a well - conceived project will include needs assessment,
front - end planning, and preliminary design. It is important that project progress is
measured against such a schedule and not just by the visual impact caused by bricks
and mortar being installed.
3.3.3 DESIGN OPTIONS
3.3.3.1 Clean - Facility Scope
The purpose of this section is to identify design and construction options for those
parts of a pharmaceutical facility intended to house process equipment. These suggestions
are intended to assure that the facilities, when used as designed, will meet
the requirements of current good manufacturing practices (cGMPs). Air cleanliness
within the facility may range from International Organization for Standardization
(ISO) 5 (Class 100) through ISO 8 (Class 100,000). In addition, areas may be considered
clean or labeled as “ controlled environment ” without having a cleanliness
class assigned to the space. Note that throughout this chapter cleanliness class will
be described using the designation presented in the new ISO 14644 (e.g., ISO 5, ISO
8) and parenthetically as presented in the currently obsolete (but widely understood
and quoted) U.S. Federal Standard 209 (e.g., Class 100, Class 100,000).
A cleanliness classifi cation in accordance with the latest revision of ISO 14644 is
generally inadequate by itself to describe a facility used for pharmaceutical processes.
The presence of viable particles (living organisms) within the particle count
achieved by applying methods described in the standard may affect the product
within the facility. A measure of both viable and nonviable particles is required to
provide suffi cient information upon which to base a decision regarding the suitability
of the clean room for its intended purpose.
The options presented herein are intended to provide facilities that will effectively
restrict both viable and nonviable particles from entering the clean areas,
minimize contamination introduced by the facility itself, and continuously remove
contaminants generated during normal operations.
Measurement of total particle count in the clean room is described in ISO 14644.
This count may be composed of viable, nonviable, or nonviable host particles with
a viable traveler. There is no generally accepted relationship between total particle
208 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
count and viable particle count. While maintaining appropriate particle counts is
important in clean - room design and operation, a protocol designed to identify viable
particles should be inherent in the certifi cation/validation testing of a pharmaceutical
clean room.
No facility design can compensate for excessive contamination generated within
it. In addition to effective facility design, the user must also institute a routine maintenance
program as well as maintain personnel and operational disciplines that limit
particles both entering and being generated within the facility.
While this section identifi es options for contamination control in facility design,
any such options must be implemented in accordance with all appropriate government
and regulatory building and safety codes. The design guideline is nonspecifi c
as regards biological or chemical materials that may be used within the facility but
generally addresses bulk pharmaceutical chemical plants (BPCs), secondary manufacturing
chemical plants, bulk biopharmaceutical plants, and plants used for fi ll and
fi nish operations. Good practice as well as any regulations governing biological and
pharmaceutical processes conducted within the facility must be adhered to as
required and could modify some of the suggestions contained herein.
3.3.3.2 Design Parameters
The design of the facility is based upon specifi cation of certain design parameters.
These in turn are used to calculate building system equipment capacities and aid in
the selection of the appropriate types of equipment that are required. Design
parameters that may be critical are discussed below.
Cleanliness Classifi cation The classifi cation of the clean areas is determined by
the using organization consistent with the level of nonviable and viable particulate
contamination acceptable to the process conducted within the facility. This may be
governed by regulatory agencies, client organizations, or company protocols. Target
goals are set for nonviable particle count in accordance with the ISO. Viable particle
target goals should be stated in colony - forming units (CFU) per square centimeter.
In accordance with ISO 14644, particle goals will typically be identifi ed for “ at rest ”
and “ operational ” modes.
In the absence of other guidance governing the cleanliness classifi cation and
acceptable levels of microbial contamination of the clean room, the values presented
in Table 1 may be used. The room grades presented are from most critical
(A) to least critical (E). The defi nition of criticality is left to the clean - room user
organization.
Other Design Parameters Facility design parameters that support the process
within the clean room should be established by the user organization. Parameters
such as temperature, humidity, lighting requirements, sound level, and/or vibration
may be process driven or comfort driven and therefore are selected to accommodate
specifi c process or comfort requirements as determined by the end user.
Local Control Under some circumstances, cleanliness requirements can be
achieved through the use of localized controls such as clean tents, glove boxes,
minienvironments, or isolators. These provide unidirectional fi ltered airfl ow within
DESIGN OPTIONS 209
a limited area. They may be located within a facility that provides the necessary
temperature and humidity conditions or they may be provided with integral environmental
control equipment designed to maintain necessary conditions.
Air Change Rate The airfl ow pattern and air change rate in a clean room largely
determines the class of cleanliness that can be maintained during a given operation.
Non - unidirectional fl ow clean rooms rely on air dilution as well as a general ceiling -
to - fl oor airfl ow pattern to continuously remove contaminants generated within the
room. Unidirectional fl ow is more effective in continuously sweeping particles from
the air due to the piston effect created by the uniform air velocity. The desired air
change rate is determined based on the cleanliness class of the room and the density
of operations expected in the room. An air change rate of 10 – 25 per hour is common
for a large, low - density ISO 8 (Class 100,000) clean room. ISO 7 (Class 10,000) clean
rooms typically require 40 – 60 air changes per hour. In unidirectional fl ow clean
rooms, the air change rate is generally not used as the measure of airfl ow but rather
the average clean - room air velocity is the specifi ed criterion. The average velocity
in a typical ISO 5 (Class 100) clean room will be 70 – 90 ft/min. A tolerance of plus
or minus 20% of design airfl ow is usually acceptable in the clean room. The foregoing
values have been found to be appropriate in many facilities. Generally air change
rate or air velocity is not a part of regulations. It is left to the user to demonstrate
that the selected design parameter is appropriate for the products being manufactured.
An exception to this may be in the case of fi lling operations where a unidirectional
fl ow velocity of 90 ± 20 ft/min may be required.
Pressurization A pressure differential should be maintained between adjacent
areas, with the cleaner area having the higher pressure. This will minimize infi ltration
of external contamination through leaks and during the opening and closing of
personnel doors. A minimum overpressure between clean areas of 5 Pa [0.02 in. of
TABLE 1 An Example of Cleanliness Classifi cation Goals
Room
Grade
Cleanliness
Class a
Particle Counts e Microbial Contamination
At Rest Operational
Air
Sample
Settle
Plates
Contact
Plates
Glove
Print
(cfu/m 3 ) (cfu/4 h) b (cfu/plate) c (cfu/glove) d
A f M3.5 (100) 3,500 3,500 < 1 < 1 < 1 < 1
B g M3.5 (100) 3,500 35,000 10 5 5 5
C M5.5 (10000) 350,000 3,500,000 100 50 25 —
D M6.5 (100000) 3,500,000 N/A 200 100 50 —
E Uncontrolled N/A N/A N/A N/A N/A N/A
a In accordance with U.S. Federal Standard 209E.
b 90 - mm - diameter settling plate. These are average values and individual plates may have < 4 h of exposure.
c 55 - mm contact plates.
d Five - fi ngered glove.
e Maximum particle counts per cubic meter > 0.5 . m.
f Unidirectional airfl ow at 90 ft/min.
g Non - unidirectional airfl ow.
210 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
water column (in. WC)] is recommended. The pressure between a clean area and
an adjacent unclean area should be 12 – 14 Pa (0.05 in. WC). Where several clean
rooms of varying levels of cleanliness are joined as one complex, a positive - pressure
hierarchy of cleanliness levels should be maintained, including air locks and gowning
rooms. Note that for certain processes and products it may be desirable to have a
negative pressure relative to the surrounding ambient in one or more rooms when
containment is a major concern. A “ room within a room ” may have to be designed
to achieve this negative pressure yet still meet the needs of clean operation.
Temperature Control Where occupant comfort is the main concern, a temperature
of 68 – 70 ° F ± 2 ° F will usually provide a comfortable environment for people wearing
a typical lab coat. Where a full “ bunny suit ” or protective attire is to be worn, room
temperature as low as 66 ° F may be required. If the temperature is to be controlled
in response to process concerns, the value and tolerance should be specifi ed early
in the design phase to ensure that system selection is appropriate and that budgeting
is accurate. Note that a tight tolerance (e.g., ± 1 ° F or less) will typically be more
costly to maintain than a less stringent tolerance.
Humidity Control The humidity requirement for comfort is in the range of 30 –
60% relative humidity (RH). If process concerns suggest another value, it should
be specifi ed as soon as possible in the design process. Biopharmaceutical materials
sensitive to humidity variations or excessively high or low values may require stringent
controls.
3.3.3.3 Architectural Design Issues
Facility Layout The facility layout should support the process contained within
the clean room. While a rectangular shape is easiest to accommodate, other shapes
may be incorporated into the facility as long as appropriate attention is paid to
airfl ow patterns. The facility should be able to accommodate movement of equipment,
material, and personnel into and out of the clean room. The layout of the
clean suite should facilitate maintaining cleanliness class, pressure differentials, and
temperature/humidity conditions by isolating critical spaces and by excluding nonclean
operations. See Figure 1 . The potential for cross - contamination is addressed
as both an architectural and a mechanical issue. Generally, in a facility where multiple
products are to be processed, each product has a dedicated space, isolated
physically from adjacent spaces, and each has its own air conditioning system, independent
of adjacent systems.
Air Locks or Anteroom This is a room between the clean room and an unrated
or less clean area surrounding the clean room or between two rooms of differing
cleanliness class. The purpose of the room is to maintain pressurization differentials
between spaces of different cleanliness class while still permitting movement
between the spaces. An air lock can serve as a gowning area. Certain air locks may
be designated as an equipment or material air lock and provide a space to remove
packaging material and/or to clean equipment or materials before they are introduced
into the clean room. Interlocks are recommended for air lock door sets to
prevent opening of both doors simultaneously. The air lock is intended to separate
the clean from the unclean areas.
DESIGN OPTIONS 211
Prior to equipment or raw materials being introduced into the clean room, they
should be prepared. This may mean removing an outer package wrap or perhaps
surface cleaning of the object. Material handling equipment used within the clean
room should be dedicated to the clean room. Physical barriers may be integrated
into the material air lock design to prevent material handling equipment from
leaving the clean room or outside equipment from passing into the clean room.
Windows Windows are recommended in interior clean - room walls to facilitate
supervision and for safety, unless prohibited by the facility protocol for visual security
reasons. Windows in exterior building walls adjacent to a clean space are problematic.
Windows can be a source of leakage and can result in contaminants entering
the space. Windows should be placed to permit viewing of operations in order to
minimize the need for non - clean - room personnel to enter the clean room. Windows
should be impact - resistant glass or acrylic, fully glazed, installed in a manner that
eliminates or minimizes a ledge within the clean space. Double glazing is frequently
used to provide a fl ush surface on both sides of the wall containing the window.
Windows may be included if there is a public relations requirement for visitors to
view the operations. Speaking diaphragms or fl ush, wall - mounted, intercom systems
are recommended near all windows to facilitate communication with occupants of
the clean room.
Pass - Through A pass - through air lock should be provided for the transfer of
product or materials from uncontrolled areas into the clean room or between areas
of different cleanliness class (Figure 2 ). The pass - through may include a speaking
diaphragm, intercom, or telephone for communication when items are transferred
and interlocks to prevent both doors from being opened at the same time. A cart -
size pass - through installed at fl oor level can be used to simplify the movement
of carts between clean areas. Stainless steel is typically the material of choice
(Figure 3 ).
FIGURE 1 Sample clean - room lay - out.
Bench
Emergency exit
Main clean room
Material
air lock
Window wall
(Eg. 4' wide x 3' high x no. of windows)
Gowning
room
Personal
Locker
Area
Air lock
Clean-room
entrance
Clean-room
exit
Pass-thru
window
Clean
Garment
Storage
Soiled
Garment
Disposal
Pass-thru
window
212 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
Gowning Room Gowning rooms should be designed to support the garment protocol
established for the facility. A typical gowning room may have a wall - or fl oor -
mounted coat rack for clean garment storage (Figure 4 ); a bench specifi cally designed
for clean - room use (Figure 5 ); a full - length mirror installed near the door for
gowning self - inspection; storage for new packaged garments; and bins for disposal
of soiled garments.
Personal lockers and coat racks for the storage of notebooks, coats, and personal
items should be located outside the gowning room or in an anteroom separate from
FIGURE 2 Stainless steel pass - through with interlock designed to permit safe passage of
small items between spaces of differing cleanliness. ( Courtesy of Terra Universal. )
FIGURE 3 Cart pass - through enabling larger amounts and sizes of items to be transported.
Note that the cart shown is not to be taken from the clean room. Typically a physical barrier
is incorporated into the cart pass - through design. ( Courtesy of Terra Universal. )
DESIGN OPTIONS 213
the clean gowning area. Restroom facilities may also be located outside the gowning
room or in an anteroom adjacent to the clean gowning area. A common gowning
room design has two areas divided by a bench. The “ unclean ” area is used to remove
and store outer garments. Stepping over the bench as the clean - room footwear is
being put on ensures that the “ clean ” side of the gowning room will remain that
way. Final donning of the clean - room garb is then accomplished.
FIGURE 4 Furnishings in the gowning room are typically of a nonshedding material such
as the stainless steel designs shown. The gown rack will generally have a ceiling - mounted
HEPA fi lter above it to continually bathe the garments in clean air. ( Courtesy of Terra
Universal. )
FIGURE 5 The stainless steel clean benches have has a perforated seat to permit airfl ow
from ceiling to fl oor essentially unobstructed. ( Courtesy of Terra Universal. )
214 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
Male and female gowning rooms may be required depending on the make - up of
the work force and the type of garments being used.
Siting A clean room that serves as an element of a larger process line should be
integrated into the line to permit movement of personnel and materials in and out
of the room. A free - standing clean room may be located in any convenient site;
however, certain conditions adjacent to the facility may degrade its performance.
Vibration sources inside or near a clean room will encourage particle release within
the room and under severe conditions may cause leaks in fi lters and ductwork.
Heavy equipment, including the heating, ventilation, and air conditioning (HVAC)
system components, pumps, house and vacuum system, ought to be vibration isolated.
Location of a clean room directly adjacent to heavy equipment or loading
docks that see heavy truck traffi c and other sources of vibration, shock, and noise
may be problematic. The outdoor air intake for the clean - room makeup air must be
carefully located to prevent overloading of fi lters or entrance of contaminating gases
that the fi lter will not remove. Clean - room air intakes should not be located near
loading docks, traffi c lanes, or other areas where vehicles may drive through or idle.
These intakes should not be located near the exhaust locations of other processing
facilities. Use of gas - phase fi ltration may be required if the quality of make - up air
is not acceptable.
3.3.3.4 Materials of Construction
Walls Generally wall material selection should be based on the operations and
material handling equipment to be used within the space. The walls should be
strong enough to withstand repeated impact of carts or other equipment without
deterioration. The materials should also be selected with the sanitizing protocol
in mind. Chemicals, high - pressure wash, and steam can cause reduced wall life if
proper materials are not selected. Seamless walls, to the extent possible, are
desirable.
Basic steel stud construction with gypsum board paneling can be used in biopharmaceutical
clean rooms when appropriately coated with a nonshedding fi nish.
Modular wall systems utilizing coated steel or aluminum panel construction are
growing in popularity due to the ability to easily retrofi t a lab or production space
at a later date with minimal disruption and construction debris. Stainless steel may
be appropriate but costly. Modular systems have been developed that address the
concerns of the biopharmaceutical clean - room user relative to surface fi nish integrity
and smooth surfaces. The joint between adjacent modular panels is commonly
treated with a gunnable sealant to provide a smooth, cleanable joint that will not
hold contaminants.
Concrete masonry unit (CMU) construction is widely used (Figure 6 ). It can
prevent buildup of contaminants when fi nished with an epoxy or other smooth,
chemical - resistant coating. Where retrofi t is not a regular practice, the strength of
concrete block and its long life recommend it.
Rounded, easy - to - clean corners and smooth transitions between architectural
features such as windows and walls (Figure 7 ) should be featured in all wall system
designs, whether modular or “ stick built. ”
DESIGN OPTIONS 215
Wall Finishes Inexpensive latex wall paints will deteriorate over time and are unacceptable
in clean rooms. Acceptable wall fi nishes include epoxy paint, polyurethane,
or baked enamel of a semigloss or gloss type. These may be applied in the factory to
metal wall system panels. Field application of epoxy to gypsum board or CMU should
be done to ensure a smooth, nonporous, monolithic surface that will not provide a
breeding site for organisms. Exposed outside corners in high traffi c areas as well as
on lower wall surfaces may have stainless steel facings or guards to prevent impact
FIGURE 6 A CMU wall treated with block fi ller and epoxy fi nish to provide a smooth,
cleanable wall surface. ( Courtesy of Niagara Walls. )
FIGURE 7 A window detail that provides a smooth, easy - to - clean surface on both wall
faces. ( Courtesy of Portafab. )
216 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
damage to the wall. This is particularly true when gypsum board construction is used.
Corner and wall guards should extend from the fl oor to at least the 4 - ft height. Traditionally
the clean room has been white throughout as an indication of the clean nature
of the facility and to identify it as a special work space. Other colors may be used in
the clean room to provide an interesting environment as long as the materials of construction
do not contribute particles to the air stream and will withstand the sanitizing
agents and procedures used in the facility (Figure 8 ).
Doors Entry should be through air locks to maintain clean - room pressure differentials.
Emergency exit doors should incorporate a panic - bar mechanism (or a
similar emergency opening device) with alarms for exit only. Emergency exit doors
must be secured in a manner that prevents entry from the outside yet permits exiting
from within. All doors should include essentially air - tight seals. Neoprene seals are
generally acceptable. Brush - type door seals are not recommended. Foam rubber
door seals are not recommended as these have been found to quickly deteriorate
and shed particles. All personnel doors and swinging equipment doors should
include self - closing mechanisms. Manual and automatic sliding doors may be useful
when space is an issue or to facilitate movement between spaces of similar cleanliness
class for personnel whose hands are otherwise engaged. As the mechanism of
such doors can generate particles, a design specifi cally intended for clean - room
application should be selected.
Ceilings The ceiling fi nish should be similar to that used on the walls. The requirements
for sanitizing typically address the ceiling as well as the walls and ceiling
material and fi nish selection should refl ect this. Suspended ceilings using an inverted -
T grid and lay - in panels may have a place in that part of the clean - room suite not
subjected to the rigors of frequent sanitizing and where the possibility of trapped
FIGURE 8 A modular wall system has been installed in a manner that provides a smooth
surface for cleaning. The fi t of the components and the method of sealing are important when
a modular wall is selected. ( Courtesy of Portafab. )
DESIGN OPTIONS 217
spaces to support organism growth is not considered an issue (Figure 9 ). When suspended
panel ceilings are used, the panels must be securely clipped or sealed in
place to prevent movement due to air pressure changes.
Modular wall systems designed for biopharmaceutical applications frequently
have a “ walk - on ” ceiling designed using materials and fi nish similar to the wall. A
rounded, easy - to - clean intersection between ceiling and walls should be a feature
of the clean - room ceiling design, whether modular or stick built. Monolithic (seamless)
ceilings can be installed using inverted - T grid supports and gypsum panels
(Figure 10 ). This design permits incorporation of fi ltration and lighting into what is
essentially a monolithic ceiling.
FIGURE 9 A suspended ceiling utilizing lay - in panels and lay - in lighting troffers. A variety
of cleanable materials can be used for the panels. The lay - in lights should be of
a design that will provide appropriate service based on the cleaning protocol to be used.
( Courtesy of CleanTek. )
FIGURE 10 An area of HEPA fi lters is installed above a process machining. Tear drop
lighting is used to permit maximum fi lter coverage. A monolithic ceiling construction of
gypsum panels suspended from a framework. The panels are fi nished with an epoxy coating
compatible with cleaning/sterilization procedures. ( Courtesy of CleanTek. )
218 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
Floors Commonly used fl oor fi nishes for biopharmaceutical clean rooms include
sheet vinyl installed using heat - welded or chemically fused seams to provide a seamless
surface. Troweled epoxy and epoxy paint (Figure 11 ) have also found wide use.
Compatibility of the fl oor material with solvents, chemicals, and cleaning agents to
be used in the room must be considered. A minimum 4 - in. cove at the junction of
fl oor and walls is recommended to facilitate cleaning. Some modular wall systems
have a recess or offset that permits sheet vinyl to be installed in a manner that
creates a seamless junction between fl oor and wall. When a stick - built approach is
used, care should be taken to design cleanable intersections of walls and fl oors
(Figure 12 ).
3.3.3.5 HVAC System
Air Side The clean - room HVAC system must be designed to maintain the required
particulate cleanliness, temperature, humidity, and positive pressure at the expected
outside environmental extremes and during the expected worst - case use operations.
Rapid recovery from upset conditions such as door openings and contaminant -
generating events is also a consideration. The high cost of conditioning outside air
suggests that as much air as possible be recirculated. Recirculated air should be
high - effi ciency particulate air (HEPA) fi ltered in those spaces requiring a cleanliness
classifi cation in accordance with ISO 14644. Air that may be hazardous to
health, even after HEPA fi ltration, should be exhausted after appropriate treatment.
The required quantity of make - up air is calculated based on process exhaust plus
air leakage from the clean room. A rate of two air changes per hour for clean room
pressurization may be used in the absence of a more detailed calculation of air
FIGURE 11 The process area is subjected to substantial chemical action due to the sterilizing
protocol. It has a troweled epoxy, easy - to - clean fi nish. ( Courtesy of Dex - O - Tex. )
DESIGN OPTIONS 219
leakage. Make - up air should be drawn from the outdoors, conditioned, and fi ltered
as necessary before being introduced into the clean - room recirculation air stream.
Care should be taken to ensure that make - up air intakes are not drawing in contaminated
air.
The potential for cross - contamination is an issue that should be addressed. A
fl exible manufacturing facility is one in which a variety of products can be manufactured
simultaneously. If the facility has a single air - handling system, the likelihood
of materials from one space intruding into an adjacent space is high. For this
reason each fi lling or compounding operation, or operation where noncompatible
product can be expected to be picked up by the air stream, should be served by its
own air - handling system (Figure 13 ). Isolated systems will minimize the possibility
of cross - contamination. This can be a costly option and should not be undertaken
FIGURE 12 The wall system used in the facility incorporates a monolithic sheet vinyl fl ooring
junction between fl oor and wall face. Note the coving run up the wall around the edges
to provide a smooth surface for cleaning. ( Courtesy of Portafab. )
FIGURE 13 The air handler has several stages of fi ltration combined with heating, cooling,
humidifi cation, and dehumidifi cation capability. ( Courtesy of Air Enterprises. )
220 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
lightly. The current use of the plant and the anticipated future use should be assessed
before a blanket decision that may lead to costly duplicated systems is made.
Filtration The fi ltration system for a biopharmaceutical clean room typically consists
of several stages of fi lters. Prefi lters are selected, sized, and installed to maximize
the life of the fi nal HEPA fi lters. With proper selection of prefi lters, the fi nal
HEPA fi lters should not require replacement within the life of the fi lter media and
seal materials, a period of several years (perhaps as long as 10 – 15 years). Make - up
air is commonly fi ltered by a low - effi ciency [30% as set by the American Society of
Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE)] prefi lter followed
by an intermediate - (60% ASHRAE) or high - effi ciency (95% ASHRAE)
fi nal fi lter (Figure 14 ). A screen should be included at the make - up air inlet to keep
out pests and large debris. The make - up air is then directed to the recirculating air
handler which also may have a low - effi ciency prefi lter, although prefi ltration of
recirculated clean - room air is often omitted because of its high cleanliness level
even after having passed through the clean room. The air is then directed through
HEPA fi lters into the clean room. HEPA fi lters must be a minimum of 99.97% effi -
cient on 0.3 - . m particles in accordance with military standard Mil - F - 51068 or the
Institute of Environmental Science and Technology IEST - RP - CC001. Note that the
fi ltration system for an unrated “ controlled area ” is the same, except that the HEPA
fi lter stage may be omitted. Refer to Figure 15 .
Filter Location HEPA fi lters may be installed in a facility either within an air
handler or at the inlet to a plenum above the clean room or in the clean room ceiling.
High - velocity HEPA fi lters, that is, fi lters with a face velocity up to 500 ft/min, are
frequently installed in air handlers serving Class 100,000 clean rooms and are also
used in make - up air handlers. Where hazardous materials may be trapped by the
fi lters a “ bag - in – bag - out ” fi lter arrangement, such as that depicted in Figure 16 , may
FIGURE 14 Non - unidirectional clean - room with lay - in HEPA fi lter modules.
Make-up air unit
Outside
air
95% Prefilter
Cooling coil
Reheat coil
Humidifier
Fan
Prefilter
Cooling coil
Fan
Air handler
30% Prefilter
Clean room
HEPA filter modules
Preheat coil
Air handler
Prefilter
Cooling coil
Fan
DESIGN OPTIONS 221
be employed. Figure 17 shows a schematic arrangement with HEPA fi lters installed
in the air handler. During the design phase care should be taken to provide access
to both the upstream and downstream face of these fi lters to permit periodic challenging
and leak testing.
To provide HEPA fi ltered air over a limited area within a larger controlled space,
a ceiling - mounted pressure plenum may be used. This plenum has an air distribution
means at its lower face that permits air to be introduced in a unidirectional manner
over the critical process area. Refer to Figure 18 .
HEPA fi lters are installed at the upper face of the pressure plenum and the
plenum is pressurized with fi ltered air. The ceiling - mounted HEPA fi lters have a
face velocity up to 100 – 120 ft/min. This is somewhat higher than the HEPA fi lters
serving the rest of the clean room. The fi lters are commonly supplied with air by a
FIGURE 15 Several panel - type fi lters commonly used as prefi lters in air handlers. Second
from left is a high - dust - loading fi lter available in ASHRAE effi ciency as high as 95% frequently
used in make - up air handlers. If HEPA fi ltration of the make - up air is required, the
high - velocity duct - mounted HEPA fi lter third from the left is appropriate. It can tolerate face
velocities up to 500 fpm, compared to the standard HEPA, which is usually designed for face
velocity on the order of 90 – 100 fpm. A standard HEPA designed for bio - pharma facility
ceiling installation is shown at right. ( Courtesy of of CamFil. )
FIGURE 16 The “ bag - in – bag - out ” fi lter unit contains a HEPA fi lter and permits personnel
to change the fi lter without coming into contact with possibly hazardous materials that may
have been fi ltered from the air. ( Courtesy of Flanders Filters Inc. )
222 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
duct distribution network consisting of rectangular or round trunk ducts and fl exible
or rigid round branch ducts. Full coverage, typical for ISO 5 (Class 100) clean rooms,
or partial coverage, for higher class (less stringent) clean rooms, can be accomplished
using 2 . 4 - ft lay - in HEPA fi lter modules installed in the ceiling.
3.3.3.6 Clean - Room Testing
ISO 14644 describes methodology and instrumentation for particle counting in the
clean room. The tests described there are the basis for assigning a cleanliness rating
FIGURE 17 Non - unidirectional clean - room with air handler mounted HEPA fi lters.
Make-up air unit
Outside
air
95% Prefilter
Cooling coil
Reheat coil
Humidifier
Fan
Prefilter
Cooling coil
Fan
Air handler
30% Prefilter
Clean room
Preheat coil
Air handler
Prefilter
Cooling coil
Fan
Ceiling diffuser
HEPA filter
HEPA filter
FIGURE 18 Non - unidirectional clean - room with critical area unidirectional fl ow
plenum.
Make-up air unit
Outside
air
95% prefilter
Cooling coil
Reheat coil
Humidifier
Fan
Prefilter
Cooling coil
Fan
Air handler
30% Prefilter Preheat coil
Air handler
Prefilter
Cooling coil
Fan
Unidirectional airflow over
critical process machine/surface
Standard velocity
HEPA filters
Pressure plenum
Air distributor
Clean room
High-velocity HEPA filters
to the facility. IEST - RP - CC006 similarly provides a procedure for particle counting
but goes beyond that to a full series of tests that can be conducted to determine the
effectiveness of the clean - room design and operability. The determination of which
tests should be run is up to the clean - room end user. As a minimum, particle counting,
room pressurization, and fi lter leakage tests should be run. Other tests dealing
with airfl ow patterns, temperature, humidity, lighting, and sound levels are available.
The array of tests selected is determined by the owner based on the effect the
various design parameters will have on the product. The data obtained in acceptance
tests become baseline data against which future testing is compared to determine
if clean - room performance is changing over time. Ongoing periodic monitoring of
the facility will ensure that clean - room performance degradation is identifi ed as it
occurs. Pass – fail criteria are not part of the ISO standards but are to be developed
on a case - by - case basis by the end user of the facility. These standards become part
of the operational protocol of the facility.
The clean - room testing described here is part of the commissioning or validation
process wherein all equipment in the facility is run, tested, and observed to ensure
it is working as designed.
3.3.3.7 Utilities
Biopharmaceutical clean - rooms typically house process equipment requiring utilities
such as pure water, electricity, vacuum, and clean compressed air. The source of
these utilities is usually outside the clean room. During the design phase a utility
matrix is developed, in conjunction with end users and equipment manufacturers,
identifying all equipment and the utilities required. This is the basis for determining
the capacity of the utility systems as well as the point - of - use location of specifi c
utilities.
When bringing the utilities to the point of use, care should be taken to ensure
that the clean room is not compromised. A clean construction protocol should be
implemented and wall, ceiling, and fl oor penetrations, if needed, should be fl ashed
and sealed in such a manner as to prevent contaminants from entering the clean
room. Such entry points should also be smoothly sealed to ensure that there are no
crevices to harbor organisms. Drains should be avoided in the clean room wherever
possible. When this is not possible, the drains should be covered when not in use
with a means specifi cally designed for biopharmaceutical clean - room application.
Such means are tight, smooth, cleanable, and corrosion resistant.
In small facilities an individual pipeline may be run from outside the facility to
the point of use. In large facilities a utility chase (Figure 19 ) that enables major
utility lines to be brought to the vicinity of process tools may be provided. Final
hook - up between the chase and point of use then becomes a relatively simple,
minimally intrusive procedure. The utility chase concept is also benefi cial in facilities
that undergo frequent retrofi t or upgrade.
3.3.4 CONSTRUCTION PHASE: CLEAN BUILD PROTOCOL
Ongoing experience has demonstrated that an aggressive clean construction protocol
program is generally not required for biopharmaceutical facilities that do not
CONSTRUCTION PHASE: CLEAN BUILD PROTOCOL 223
224 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
carry a cleanliness rating. Where cleanliness classifi cations less stringent than Class
10,000 are used, standard construction techniques followed by careful cleanup and
wipe - down within the clean space have proven quite acceptable. Cleanliness levels
of Class 1000 or Class 10,000 are achievable shortly after startup and maintainable
thereafter. For cleanliness levels of Class 100 a somewhat more restrictive protocol
is required. Once a facility is up and running, any intrusion into clean areas for retrofi
t work should be done in conjunction with some level of clean build protocol
in place, dependent on the rating of the facility and the degree of disruption
encountered during the retrofi t project.
The levels of clean construction described herein can provide a practical means
of meeting operational cleanliness goals in a cost - effective fashion. Each project,
whether new construction or retrofi t of an existing process, should have as part of
it an evaluation of the required elements of the build clean protocol to be employed.
The information provided below is broad and can act as a template for the protocol
put in place for a specifi c project.
A key to successful clean construction is the appointment of an individual as a
clean - room monitor who is well versed in the clean - room construction protocol.
That person is charged with maintaining a clean environment and monitoring the
activities of all personnel within the clean area during the construction phase and
is concerned with maintaining budget and schedule goals. The clean - room monitor
should have the confi dence to make “ real - world ” decisions supporting the “ spirit ”
of the protocol as well as the “ letter. ”
FIGURE 19 The utility chase is located between two clean rooms. Major utility lines are
installed within the chase and hook - up lines for local pieces of process equipment are connected
through the clean - room walls. A major benefi t of this arrangement is that installers
need not be fully garbed in clean - room attire to access the utility lines. ( Courtesy of
CleanTek. )
3.3.4.1 General
All clean - facility construction, while employing standard construction techniques,
should be accomplished in a manner that does not create excessive particulate
contamination. A temporary lay - down area within the building adjacent to the clean
area should be set aside for storage of clean construction components. All tools used
for clean construction should be in an “ as - new ” condition and be cleaned and
inspected prior to use. The pass – fail criteria for tool and material inspection is “ no
visible dirt. ”
Cleanup within the clean area at the end of each shift should consist of broom
cleaning and vacuum cleaning the fl oor with a clean vacuum, that is, a vacuum with
a HEPA fi lter (99.97% effi cient on 0.3 - . m particles).
Clean - facility construction materials should be left in an outer shipping wrap
until moved to the temporary lay - down area, where they should then be unwrapped
and wiped down before being moved into the clean space.
Adherence to these guidelines will make fi nal clean - up faster and acceptable
start - up and certifi cation/validation more certain. While a goal of clean construction
is rapid start - up and certifi cation/validation, a long - range goal is the maintenance
of the facility cleanliness without intrusion, over an extended period of time, of
contaminants deposited during construction due to a poor protocol or improper
implementation of the protocol.
Appendix A and Appendix B offer a template for working in a clean environment
as well as clean - room cleaning procedures. Procedures should be modifi ed
with caution to suit a particular project.
3.3.4.2 Level I Clean Construction
Level I clean construction is used for all areas with a cleanliness rating of Class 1000
(ISO 6) or higher (less stringent), including those spaces within which clean processes
are conducted in minienvironments/isolators and those unrated areas identi-
fi ed as being “ controlled environments. ”
Standard construction techniques are used until the clean - room envelope is
completed, HEPA fi lters with protective fi lm in place are installed and air handlers
are ready to start. The clean envelope consists of clean - room walls, ceiling, and fl oor.
Prior to starting the air handlers, a thorough clean - up of the space within the clean
envelope is accomplished as described in Appendix B, 2A – 2J. Following clean - up
and start - up of the clean - space air - handling system, particle counts should quickly
drop to well within operational requirements.
Once the clean room is operational, as described above, additional construction
related to process equipment installation and facility modifi cation within the clean
room can be done in compliance with the Guidelines in Appendix A.
3.3.4.3 Level II Clean Construction
This is used for construction of those areas rated at Class 100 (ISO 5) employing a
100% HEPA fi lter ceiling. Generally standard construction techniques should be
used. The clean - room envelope includes walls, ceiling, fl oor, return ductwork, supply
fans, and supply ductwork.
CONSTRUCTION PHASE: CLEAN BUILD PROTOCOL 225
226 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
All ductwork sections should be cleaned and sealed with plastic wrap at the time
of fabrication until just prior to installation or start - up to prevent contaminants from
accumulating inside air - handling passageways. The sections of ductwork should be
unsealed only as required for installation. Open ends of ducts and fans should
remain sealed until connecting duct is about to be installed. A fi nal isopropyl alcohol
(IPA) wipe - down of all interior duct sections and fan surfaces should be done
immediately prior to installation.
When general construction of the clean room is completed, steps 2A – 2J of
Appendix B describing coarse cleaning can be implemented. Following coarse
cleaning the protective fi lm can be removed from the HEPA fi lters and the air -
handling system started.
Successful completion of the cleaning process described above will indicate that
installation of process equipment may begin. Note that procedures described in
Appendix A should be followed. After installation of all process equipment or when
the clean room is to be prepared for certifi cation, steps 2K – 2O of Appendix B for
fi nal wipe - down can be followed.
A black - and - white felt rub - down test is performed to demonstrate adequate
cleanliness of the interior clean - envelope surfaces. This test consists of both black -
and - white felt being wiped over any surface for 1 m linear distance with a fi rm hand
pressure. No residue should be visible on the cloth. Each cloth should be 60 cm
square black or white static - free natural fi ber felt folded with cut edges inside to a
25 - cm square. The cut edges should be sealed with an approved latex sealant.
3.3.5 MAINTENANCE
To maximize the life and effectiveness of the facility, it must be maintainable. The
facility should be designed to permit ongoing day - to - day preventive maintenance
of the mechanical systems and, should a failure occur, permit needed repairs to be
made in an expeditious manner. Perhaps of equal importance is the janitorial maintenance
required to keep the facility suitable for pharmaceutical manufacturing.
Proper janitorial maintenance begins with the design of the facility and evolves into
an operational protocol, personnel training, and effective implementation.
In the design phase it is important to provide suffi cient access to mechanical and
process equipment to enable preventive maintenance procedures to be carried out
with minimum effort. Typically manufacturer ’ s installation instructions offer guidelines
as to how much space should be left open around equipment to permit removal
of critical components. One driver of construction cost is fl oor space. Making a space
as small as possible to house an operation presumably will result in fi rst - cost savings.
If the space does not provide suffi cient access for lubrication, fi lter changes, belt
adjustments, and the like, there is a strong possibility that this preventive maintenance
will be ignored. A predictable result is shortened equipment life and the disappearance
of any fi rst - cost savings that may have been realized. If there is a major
equipment failure that requires replacement of an inaccessible component, the cost
associated with knocking down a wall to gain equipment access will very likely
negate fi rst - cost savings.
Storage of maintenance items should be identifi ed early in the design process.
Spare - parts storage, janitorial supply storage, janitors ’ sink closets, repair work shops,
and storage space for consumable maintenance items (e.g. air fi lters) will require fl oor
space in the facility design. Frequently tools are dedicated to the clean facility or are
required specifi cally for unique process equipment and must also have a storage area.
Accommodation of these items is an important part of the planning process.
A requirement of a clean facility is that the cleaning materials should be specifi -
cally intended for use in a “ clean ” operation, should be kept in good ( “ like new ” )
repair, and should not be used in other, nonclean, areas of the facility. Using general
cleaning materials manned by the “ house ” janitorial staff will invariable introduce
more contamination into the clean portions of the facility than it removes.
A central housekeeping vacuum is very useful in keeping contamination under
control. While “ wet - and - dry ” versions of the central vacuum are available, the
manner in which each is to be used should be carefully reviewed to ensure that it
is in keeping with the sanitary requirements of the facility. A common housekeeping
procedure addresses spills with local clean - up and uses a dry - type central vacuum
for dry particulate contaminants.
APPENDIX A: GUIDELINES FOR CONSTRUCTION PERSONNEL AND
WORK TOOLS IN A CLEAN ROOM
1.0 General requirements
A. Makeup will not be allowed inside the clean room.
B. Smoking will not be allowed in or around the clean room.
C. Tobacco chewing will not be allowed in the clean room.
D. Paper or paper by - products will not be allowed in the clean room except
clean - room approved paper and pens.
E. Prints or papers will be allowed only if totally laminated in plastic and
cleaned with isopropyl alcohol prior to entry.
F. Lead pencils will not be allowed in the clean room. Ball point pens only.
G. Clean - room garments, to include shoe covers, coveralls, and head cover,
will be worn within the clean room.
H. Clean - room garments will not be unfastened or unzipped while inside the
clean room.
I. No writing will be allowed on the clean - room garments.
J. Food and drink will not be allowed in the clean room.
K. Combing of hair will not be allowed in the clean room or gowning area.
L. Stepping on chairs, work benches, test equipment or process equipment is
not allowed.
M. Damaged garments (rips, worn booties, torn gloves) will be replaced immediately.
Do not wait for a convenient time. DO IT NOW!
N. Tool pouches are not allowed in a clean room.
O. All work areas and adjacent areas will be vacuumed after completion of
work and prior to leaving the clean room.
P. ALWAYS wash hands before entering the clean room to remove residues
from food, smoke, and/or other sources.
2.0 Personnel
A. All personnel working inside a clean room will be required to follow all
dress codes associated with the particular clean space.
APPENDIX A 227
228 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
B. Street clothes or company uniforms will be allowed as standard undergarments
provided they are well maintained and clean. No such garments will
be allowed that are soiled with grease, dirt, or any detectable stains.
C. Any garments producing excessive fi bers (such as fuzzy sweaters) will not
be allowed as an undergarment.
D. Standard safety shoes (or other specifi ed footware) will be required. Shoe
covers must be worn.
E. Bare feet, socks, and stockings are not allowed inside booties.
F. Coats, lunches, and private items will not be allowed inside the clean
room.
3.0 Gowning procedure
A. Each individual is responsible for knowing and using the correct method
of gowning prior to entering the clean room. (See Figure 20 .)
1. Clean shoes prior to entering the gowning room.
2. The order of dress should be as follows:
a. Shoe covers
b. Hairnet/beard cover (required after fi nal cleaning)
c. Hood
d. Coveralls
e. Face cover (required after fi nal cleaning)
f. Gloves (required after fi nal cleaning)
B. Ensure that hoods are tucked inside neck opening of coveralls and pants
legs are tucked and snapped inside booties. Garments are to be snapped
FIGURE 20 Clean - room garments are intended to keep contaminants from entering the
clean room. In a critical environment the “ bunny suit ” shown may be required. In a less critical
environment a lab coat may suffi ce. The clean - room construction protocol should identify
the type of garment that will be required at various stages of construction and for process
equipment installation. ( Courtesy of Terra Universal. )
closed at the neck, wrist and ankle opening and sleeves tucked inside
gloves.
C. All head hair must be covered at all times.
D. Do not allow garments to touch the fl oor while dressing or undressing.
E. Avoid leaning on walls, lockers, or other personnel at all times. DO NOT
place feet on benches.
F. The order of undress should be as follows:
1. Gloves
2. Coveralls
3. Face cover and hood
4. Shoe covers
G. If you will be reentering the clean room, unsoiled garments may be hung
for reuse; gloves are not to be reused.
4.0 Work tools, parts, and equipment
A. All tools and equipment used in a clean room should be in like - new
condition.
B. All parts will be removed from their shipping container prior to cleaning
and introduction into the clean room. NO PAPER PRODUCTS will be
allowed inside the clean room.
C. All tools, parts, and equipment will be properly cleaned prior to entering
the clean room. Minimum cleaning should be a total wipe - down with isopropyl
alcohol, using certifi ed clean - room wipes, to assure that the last wipe
does not leave visible residue on the wipe. Parts should be blown off
outside the clean room using fi ltered nitrogen when available.
D. All parts and equipment should be sent through the equipment wipe - down
area (material air lock) and not carried through the gowning area.
5.0 Working in a Clean Room
A. A major concern when working in a clean room is the generation of particles
of the size that cannot be seen and spreading these particles throughout
the clean room. Every possible precaution must be taken to contain
these contaminants and protect the clean - room environment. Everything
that is done as a standard operation must be analyzed to determine if it
will adversely affect the cleanroom. If you have any concerns, ask the clean -
room monitor before you damage the environment and incur unnecessary
clean - up cost.
B. All procedures must be reviewed with the clean - room monitor to ensure
compliance with clean - room operation practices. All procedures that can
generate particles should be done outside the clean - room whenever possible.
In the listing below all prohibited procedures are subject to review
by the clean - room monitor. The intent is to get the job done; however, some
preplanning with the clean - room monitor can result in a positive result and
a clean facility.
1. Drilling:
a. All power drills will be wrapped to encapsulate any contamination
generated during operation.
b. Drills may be operated in sealed enclosures equipped with an exhaust
vacuum.
APPENDIX A 229
230 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
c. Surface to be drilled will be tented or vacuumed to prevent the
spread of contamination.
2. Grinding: NO GRINDING will be allowed in the clean room.
3. Welding: NO WELDING will be allowed inside the clean room.
4. Soldering: May be allowed after total review.
5. Painting: NO PAINTING will be allowed after the start - up of the clean
room.
6. Sanding/fi ling: Will be allowed only within a properly tented space.
7. Cutting: Will be allowed with clean - room approved vacuums removing
particles created.
APPENDIX B: CLEANING THE CLEANROOM
1.0 Final cleaning: During this cleaning phase, the clean - room proper should be
prepared for certifi cation to the appropriate cleanliness level. Extreme care
must be exercised by all those involved in this procedure to minimize the
potential for contamination.
2.0 Procedure
A. Secure the entrance to the clean space “ envelope, ” the gowning area, and
the entrance from the gowning room to the clean room with locks or
limited access via card keys. Post a restriction notice: YOU ARE ENTERING
A CONTROLLED, PARTICULATE - FREE ENVIRONMENT,
CONTACT ____________ FOR PERMISSION TO ENTER.
B. Perform two coarse cleanings of the clean room. Each cleaning should
include the following:
1. Wipe - down of the HEPA fi lter grid, all lights, walls, fl oors, windows, and
all exposed interior surfaces. This will include any outlet boxes or fl oor/
wall recesses.
2. Wipe - down should be by clean potable water and mild nonphosphate
detergent using clean, lint - free cloths approved by the clean - room
monitor.
3. A second wash - down should commence using clean potable water in
the same manner.
4. Floors should be scrubbed and polished using a fl oor - polishing machine.
No wax is to be used.
C. Partitions and fl oors should be washed to maintain a dust - free condition.
D. Access into the clean room should be restricted to discourage infi ltration
of outside particulates.
E. Tacky mats 3 ft by 6 ft should be installed inside the entrance of the gowning
area as well as at the entrance to the clean room.
F. Foot covers should be worn by all personnel entering the clean room after
the second coarse cleaning is complete.
G. Caulking crew should be assigned and commence work after the second
coarse cleaning is completed. They should complete all caulking as required
by specifi cation.
H. Simultaneously with the caulking procedure, air handlers and associated
ducts and plenums should be checked for cleanliness.
I. The HEPA fi lter protective fi lm should be removed. Air - handling equipment
should be activated and the clean space pressurized to maintain a
positive static pressure.
J. From this point forward, clean - room garments and head covers should be
worn by all personnel entering the clean space.
K. Commence with the fi rst of two fi nal wipe - downs. Nonshedding clean -
room wipes (saturated with isopropyl alcohol) or tacky wipes should be
used. All exposed surfaces should be wiped.
L. A particle counter should be installed in the clean space and samples taken
at several control points over the next 48 hours. A steady decrease in the
particle count over time should be achieved.
M. If particle counts stabilize at a level above that desired, a search for fi lter
leakage will be required.
N. If the search for fi lter leakage fails to fi nd a leak, the entire area should be
recleaned as described for the fi nal wipe - down.
O. Once the air standards are achieved, fi nal air balance can begin followed
by clean - room certifi cation testing.
BIBLIOGRAPHY
U . S . Food and Drug Administration, Washington, DC
21 CFR Part 210, Current good manufacturing practice in manufacturing, processing, packing,
or holding of drugs.
21 CFR Part 211, Current good manufacturing practice for fi nished pharmaceuticals.
Institute of Environmental Sciences and Technology, Rolling Meadows, IL
IEST - RP - CC001.4: HEPA and ULPA Filters , Nov. 7, 2005 .
IEST - RP - CC002.2: Unidirectional Flow Clean - Air Devices , Jan. 19, 1999 .
IEST - RP - CC003.3: Garments Systems Considerations for Cleanrooms and Other Controlled
Environments , Aug. 11, 2003 .
IEST - RP - CC004.3: Evaluating Wiping Materials Used in Cleanrooms and Other Controlled
Environments , Aug. 23, 2004 .
IEST - RP - CC005.3: Gloves and Finger Cots Used in Cleanrooms and Other Controlled
Environments , May 1, 2003 .
IEST - RP - CC006.3: Testing Cleanrooms , Aug. 30, 2004 .
IEST - RP - CC008 - 84: Gas - Phase Adsorber Cells , Nov. 1, 1984 .
IEST - RP - CC012.1: Considerations in Cleanroom Design , Mar. 1, 1998 .
IEST - RP - CC013 - 86 - T: Equipment Calibration or Validation Procedures , Nov. 1, 1986 .
IEST - RP - CC016.2: The Rate of Deposition of Nonvolatile Residue in Cleanrooms , Nov. 15,
2002 .
IEST - RP - CC018.3: Cleanroom Housekeeping: Operating and Monitoring Procedures , Jan. 1,
2002 .
IEST - RP - CC019.1: Qualifi cations for Organizations Engaged in the Testing and Certifi cation
of Cleanrooms and Clean - Air Devices , Jan. 23, 2006 .
IEST - RP - CC023.2: Microorganisms in Cleanrooms , Jan. 31, 2006 .
BIBLIOGRAPHY 231
232 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES
IEST - RP - CC026.2: Cleanroom Operations , July 21, 2004 .
IEST - RP - CC027.1: Personnel Practices and Procedures in Cleanrooms and Controlled Environments
, Apr. 1, 1999 .
IEST - RP - CC028.1: Minienvironments , Sept. 1, 2002 .
IEST - RP - CC034.2: Hepa and ULPA Filter Leak Tests , June 23, 2005 .
IEST - STD - CC1246D: Product Cleanliness Levels and Contamination Control Program ,
Jan. 1, 2002 .
International Organization for Standardization ( ISO ) Standards
ISO 14644 - 1: Classifi cation of air cleanliness.
ISO 14644 - 2: Specifi cations for testing and monitoring to prove continued compliance.
ISO 14644-3: Test methods.
ISO 14644 - 4: Design, construction and start - up.
ISO 14644 - 5: Operations.
ISO 14644-6: Terms and defi nitions.
ISO 14644 - 7: Separative devices (clean air hoods, gloveboxes, isolators, and
minienvironments).
ISO 14644 - 8: Classifi cation of airborne molecular contamination.
ISO 14698 - 1: Bicontamination control — General principles.
ISO 14698 - 2: Biocontamination control — Evaluation and interpretation of biocontamination
data.
ISO 14698 - 3: Biocontamination control — Methodology for measuring the effi ciency of processes
of cleaning and/or disinfection of inert surfaces bearing biocontaminated wet soiling
or biofi lms.
NORMAL DOSAGE FORMS
SECTION 4
235
4.1
SOLID DOSAGE FORMS
Barbara R. Conway
Aston University, Birmingham, United Kingdom
Contents
4.1.1 Biopharmaceutics Classifi cation System
4.1.2 Systematic Formulation Development
4.1.3 Standard and Compressed Tablets
4.1.4 Excipients in Solid Does Formulations
4.1.4.1 Diluents
4.1.4.2 Binders
4.1.4.3 Lubricants
4.1.4.4 Glidants and antiadherents
4.1.4.5 Disintegrants
4.1.4.6 Superdisintegrants
4.1.4.7 Added Functionality Excipients
4.1.4.8 Colorants
4.1.4.9 Interactions and Safety of Excipients
4.1.5 Coated Tablets
4.1.5.1 Sugar - Coated Tablets
4.1.5.2 Compression Coating and Layered Tablets
4.1.5.3 Film - Coated Tablets
4.1.5.4 Tablet Wrapping or Enrobing
4.1.6 Hard and soft gelatin capsules
4.1.6.1 Hard - Shell Gelatin Capsules
4.1.6.2 Manufacture of Hard Gelatin Shells
4.1.6.3 Hard Gelatin Capsule Filling
4.1.6.4 Soft Gelatin Capsules
4.1.6.5 Manufacture of Soft Gelatin Capsules
4.1.6.6 Dissolution Testing of Capsules
4.1.7 Effervescent Tablets
4.1.7.1 Manufacture of Effervescent Tablets
4.1.8 Lozenges
4.1.8.1 Chewable Lozenges
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
236 SOLID DOSAGE FORMS
4.1.9 Chewable Tablets
4.1.9.1 Testing of Chewable Tablets
4.1.10 Chewing Gums
4.1.10.1 Composition of Chewing Gum
4.1.10.2 Manufacture of Chewing Gum
4.1.10.3 Drug Release from Chewing Gums
4.1.10.4 Applications for Chewing Gums
4.1.11 Orally Disintegrating Tablets
4.1.11.1 Dissolution Testing of ODTs
4.1.12 Solid Dosage Forms for Nonoral Routes
References
Drug substances are most frequently administered as solid dosage formulations,
mainly by the oral route. The drug substance ’ s physicochemical characteristics, as
well the excipients added to the formulations, all contribute to ensuring the desired
therapeutic activity. Tablets and capsules are the most frequently used solid dosage
forms, have been in existence since the nineteenth century, and are unit dosage
forms, comprising a mixture of ingredients presented in a single rigid entity, usually
containing an accurate dose of a drug. There are other types of solid dosage forms
designed to fulfi ll specifi c delivery requirements, but they are generally intended for
oral administration and for systemic delivery. The major solid oral dosage form is
the tablet, and these can range from relatively simple, single, immediate - release
dosage forms to complex modifi ed - release systems. Tablets offer advantages for
both patients and manufacturers (Table 1 ). Most tablets are intended to be swallowed
whole and to rapidly disintegrate and release drug in the gastrointestinal
tract. Tablets are classifi ed by their route of administration or their function, form,
or manufacturing process. For example, some tablets are designed to be placed in
the oral cavity and to dissolve there or to be chewed before swallowing, and
there are many kinds of formulation designed for sustained or controlled release
(Table 2 ).
Solid dose formulations, including tablets, must have the desired release properties
coupled with manufacturability and aesthetics and must involve rational formulation
design. The dose of the drug and its solubility are important considerations
TABLE 1 Advantages of Tablets as a Dosage Form
Easy to handle
Variety of manufacturing methods
Can be mass produced at low cost
Consistent quality and dosing precision
Can be self - administered
Enhanced mechanical, chemical, and microbiological stability compared to liquid dosage
forms
Tamperproof
Lend themselves to adaptation for other profi les, e.g., coating for sustained release
in the design of the formulation as are the type of dosage form and its method of
preparation.
4.1.1 BIOPHARMACEUTICS CLASSIFICATION SYSTEM
Dissolution of the drug must occur before or on reaching the absorption site before
absorption can occur, and generally water - soluble drugs do not exhibit formulation
diffi culties. For poorly water - soluble drugs, the absorption rate may be dictated by
the dissolution rate, and, if dissolution is slow, bioavailability may be compromised.
The solubility of a drug should, therefore, be considered along with its dose when
designing formulations, and unsuitable biopharmaceutical properties is the major
reason for the failure of new drugs.
In 1995, the Biopharmaceutics Classifi cation System (BCS) was devised to classify
drugs based on their aqueous solubility and intestinal permeability [1] . According
to the BCS, drug substances are classifi ed as follows [2] :
Class I: high permeability, high solubility
Class II: high permeability, low solubility
Class III: low permeability, high solubility
Class IV: low permeability, low solubility
TABLE 2 Types of Solid Dosage Form
Formulation type Description
Immediate - release tablet/capsule Intended to release the drug immediately after
administration
Delayed - release tablet/capsule Drug is not released until a physical event has
occurred, e.g., change in pH
Sustained - release tablet/capsule Drug is released slowly over extended time
Soluble tablets Tablet is dissolved in water prior to administration
Dispersible tablet Tablet is added to water to form a suspension prior to
administration
Effervescent tablet Tablet is added to water, releasing carbon dioxide to
form a effervescent solution
Chewable tablet Tablet is chewed and swallowed
Chewable gum Formulation is chewed and removed from the mouth
after a directed time
Buccal and sublingual tablets Tablet is placed in the oral cavity for local or systemic
action
Orally disintegrating tablet Tablet dissolves or disintegrates in the mouth without
the need for water
Lozenge Slowly dissolving tablet designed to be sucked
Pastille Tablet comprising gelatin and glycerine designed to
dissolve slowly in the mouth
Hard gelatin capsule Two - piece capsule shell that can be fi lled with powder,
granulate, semisolid or liquid
Soft gelatin capsule (softgel) One - piece capsule containing a liquid or semisolid fi ll
BIOPHARMACEUTICS CLASSIFICATION SYSTEM 237
238 SOLID DOSAGE FORMS
A dose solubility volume can be defi ned for all drugs (i.e., the volume required
to dissolve the dose). A drug substance is considered highly soluble when the highest
dose strength is soluble in . 250 mL water over a pH range of 1 – 7.5. A drug substance
is considered highly permeable when the extent of absorption in humans is determined
to be . 90% of an administered dose, based on mass balance or in comparison
to an intravenous reference dose. A drug product is considered to be rapidly dissolving
when . 85% of the labeled amount of drug substance dissolves within 30 min
using U.S. Pharmacopeia (USP) apparatus I or II in a volume of . 900 mL buffer
solutions.
It was recognized that dissolution rate has a negligible impact on bioavailability
of highly soluble and highly permeable (BCS class I) drugs when dissolution of their
formulation is suffi ciently rapid. As a result, various regulatory agencies including
the U.S. Food and Drug Administration (FDA) now allow bioequivalence of formulations
of BCS class I drugs to be demonstrated by in vitro dissolution (often
called a biowaiver) [3] . Therefore, one of the goals of the BCS is to recommend a
class of immediate - release (IR) solid oral dosage forms for which bioequivalence
may be assessed based on in vitro dissolution tests.
4.1.2 SYSTEMATIC FORMULATION DEVELOPMENT
Systematic development approaches are needed to gather a full and detailed understanding
of marketable formulations in order to satisfy the requirements of regulatory
bodies and to provide a research database. Effi cient experimental design using
in - house or commercial software packages can ensure quality while avoiding expensive
mistakes and lost time. Information from various categories such as the properties
of the drug substance and excipients, interactions between materials, unit
operations, and equipment are required [4] . Design of experiments (DOE) and
statistical analysis have been applied widely to formulation development. Using
DOE facilitates evaluation of all formulation factors in a systematic and timely
manner to optimize the formulation and manufacturing process. Abbreviated excipient
evaluation techniques such as Plackett – Burman design can be applied to minimize
the number of experiments and identify critical components or processes.
Optimization processes can then be applied. When the formulation and manufacturing
processes of a pharmaceutical product are optimized by a systematic approach,
the scale - up and processes validation can be very effi cient because of the robustness
of the formulation and manufacturing process.
Innovations in statistical tools such as multivariate analysis, artifi cial intelligence,
and response surface methodology have enabled rational development of formulations,
and such methods allow formulators to identify critical variables without
having to test each combination.
4.1.3 STANDARD AND COMPRESSED TABLETS
The simplest tablet formulations are uncoated products that are made by direct
compression or compression following wet or dry granulation. They are a versatile
drug delivery system and can be intended for local action in the gastrointestinal
(GI) tract or for systemic effects. General design criteria for tablets are accuracy
and uniformity of drug content, stability of the drug candidate and the formulation,
optimal dissolution and availability for absorption (whether immediate or extended
release), and patient acceptability in terms of organoleptic properties and appearance.
Flocculant, low - density drugs can be diffi cult to compress and formulate into
tablets. This is a particular issue with drugs of low potency. Also some poorly water -
soluble, poorly permeable drugs or highly metabolized drugs cannot be given this
way. Additionally, local irritant effects can be harmful to the mucosa of the GI
tract.
Tablets are a popular dosage form due to their simplicity and economy of manufacture,
relative stability, and convenience in packaging, shipping, and storage. For
the patient, uniformity of dose, blandness of taste, and ease of administration ensure
their popularity. Thus, the purpose of the formulation and the identifi cation of suitable
excipients are of primary importance in the development of a successful formulation.
A well - designed formulation should contain, within limits, the stated
quantity of active ingredient, and it should be capable of releasing that amount of
drug at the intended rate and site. Tablets need to be strong enough to withstand
the rigors of manufacture, transport, and handling, and they need to be of acceptable
size, taste, and appearance. A typical manufacturing process for a tablet product
includes weighing, milling, granulation and drying, blending and lubrication, compression,
and coating. Each processing step involves several process parameters. For
a given formulation, all processing steps should be thoroughly evaluated so that a
robust manufacturing process can be defi ned, and DOE can be applied effectively
to optimize this process.
Direct compression is a simple process being more economical and less stressful
to ingredients in terms of heat and moisture, However, there are limitations governed
by the physical properties of the ingredients, and raw materials must be carefully
controlled. It is diffi cult to form directly compressed tablets containing
high - dose and poorly compactible drugs. Granulation can be employed to improve
the compaction characteristics of the powder. Granulation can also improve fl ow
properties and reduce the tendency for segregation of the mix due to a more even
particle size and bulk density. Granules can be produced by either wet or dry
methods based on the stability of the drug and excipients.
Although the basic mechanical process of producing tablets by compression has
not changed, there has been much work on improving tableting technology [5] .
Understanding of the physical and mechanical properties of powders and the compaction
process has improved and will continue to improve product design while
increases in the speed and uniformity of action of tableting presses improve the
process.
4.1.4 EXCIPIENTS IN SOLID DOSE FORMULATIONS
In addition to the active ingredients, solid oral dosage forms will also contain a range
of substances called excipients. The role of excipients is essential in ensuring that
the manufacturing process is successful and that the quality of the resultant formulation
can be guaranteed. The appropriate selection of excipients and their relative
concentrations in the formulation is critical in development of a successful product.
EXCIPIENTS IN SOLID DOSE FORMULATIONS 239
240 SOLID DOSAGE FORMS
Although they are often categorized as inert, preformulation studies can determine
the infl uence of excipients on stability, bioavailability, and processability. Excipients
are categorized into groups according to their main function, although some may
be multifunctional, and examples of common excipients used in the manufacture of
tablets and capsule are detailed in Table 3 .
4.1.4.1 Diluents
An inert substance is frequently added to increase the bulk of a tablet for processing
and handling. The lower weight limit for formulation of a tablet is usually 50 mg.
Ideally, diluents should be chemically inert, nonhygroscopic, and hydrophilic. Having
an acceptable taste is important for oral formulations, and cost is always a signifi cant
factor in excipient selection.
Lactose is a common diluent in both tablets and capsules, and it fulfi ls most of
these criteria but is unsuitable for those who are lactose intolerant. Various lactose
grades are commercially available which have different physical properties such as
particle size distribution and fl ow characteristics. This permits the selection of the
most suitable material for a particular application. Usually, fi ne grades of lactose
are used for preparation of tablets by wet granulation or when milling during processing
is carried out, since the fi ne size permits better mixing with other formulation
ingredients and facilitates more effective action of the binder [6] .
Diluents for direct compression formulations are often subject to prior processing
to improve fl owability and compression, for example, amorphous lactose, but
this can contribute to reduced stability especially under high - humidity conditions
when reversion to the crystalline form is more likely [6] .
Microcrystalline cellulose (Avicel) is purifi ed partially depolymerized cellulose,
prepared by treating . - cellulose with mineral acids. In addition to being used as a
fi ller, it is also used as dry binder and disintegrant in tablet formulations. Depending
on the preparation conditions, it can be produced with a variety of technical speci-
fi cations depending on particle size and crystallinity. It is often used as an excipient
in direct compression formulations but can also be incorporated as a diluent for
tablets prepared by wet granulation, as a fi ller for capsules and for the production
of spheres.
TABLE 3 Excipients Used in Solid Dose Formulations
Excipient Category Examples
Fillers/diluents Lactose, sucrose, glucose, microcrystalline cellulose
Binders Polyvinyl pyrrolidone, starch, gelatin, cellulose derivatives
Lubricants Magnesium stearate, stearic acid, polyethylene glycol, sodium
chloride
Glidants Fine silica, talc, magnesium stearate
Antiadherents Talc, cornstarch, sodium dodecylsulfate
Disintegrants and
superdisintegrants
Starch, sodium starch glycollate, cross - linked polyvinyl pyrrolidone
Colorants Iron oxide, natural pigments
Flavor modifi ers Mannitol, aspartame
Diluents, although commonly presumed inert, do have the ability to infl uence the
stability or bioavailability of the dosage form. For example, dibasic calcium phosphate
(both anhydrous and dihydrate forms) is the most common inorganic salt
used as a fi ller – binder for direct compression. It is particularly useful in vitamin
products as a source of both calcium and phosphorous. Milled material is typically
used in wet - granulated or roller - compacted formulations. The coarse - grade material
is typically used in direct compression formulations. It is insoluble in water, but its
surface is alkaline and it is therefore incompatible with drugs sensitive to alkaline
pH. Additionally, it may interfere with the absorption of tetracyclines [7] .
4.1.4.2 Binders
Binders (or adhesives) are added to formulations to promote cohesiveness within
powders, thereby ensuring that the tablet remains intact after compression as well
as improving the fl ow by forming granules. A binder should impart adequate cohesion
without retarding disintegration or dissolution. Binders can be added either as
a solution or as a dry powder. Binders added as dry powders are mixed with other
powders prior to agglomeration, dissolving in water or solvent added during granulation,
or added prior to compaction. Solution binders can be sprayed, poured, or
mixed with the powder blend for agglomeration and are generally more effective,
but further dry binder can be added prior to tableting. Starch, gelatin, and sugars
are used along with gums, such as acacia and sodium alginate, and are used at concentrations
between 2 and 10% w/w. Celluloses and polyvinyl pyrrolidone (PVP)
are also utilized, often as dry binders.
4.1.4.3 Lubricants
Lubricants can reduce friction between the tablet and the die wall during compression
and ejection by interposing an intermediate fi lm of low shear strength at the
interface between the tablet and the die wall. The best lubricants are those with low
shear strength but strong cohesive tendencies perpendicular to the line of shear [8] .
The hydrophobic stearic acid and stearic acid salts, primarily magnesium stearate,
are the most widely used and are included at concentrations less than 1% w/w in
order to minimize any deleterious effects on disintegration or dissolution. They
should be added after the disintegrant to avoid coating it and preferably at the fi nal
stage prior to compression to ensure mixing time is kept to a minimum. Hydrophilic
lubricants such as polyethylene glycols (PEGs) and lauryl sulfates can be used to
redress the issues with dissolution but may not be as effi cient as their hydrophobic
counterparts.
4.1.4.4 Glidants and Antiadherents
Like lubricants, glidants are fi ne powders and may be required for tablet compression
at high production speeds to improve the fl ow properties of the material
into the die or during initial compression stages. They are added in the dry state
immediately prior to compression and, by virtue of their low adhesive potential,
reduce the friction between particles. Colloidal silica is popular, as are starches
and talc.
EXCIPIENTS IN SOLID DOSE FORMULATIONS 241
242 SOLID DOSAGE FORMS
Antiadherents can also be added to a formulation that is especially prone to
sticking to the die surface (or picking). Water - insoluble lubricants such as magnesium
stearate can be used as antiadherents, as can talc and starch.
4.1.4.5 Disintegrants
Disintegrants are added to a formulation to overcome the cohesive strength imparted
during compression, thus facilitating break up of the formulation in the body and
increasing the surface area for dissolution. They can be either intragranular, extragranular,
or both, and there is still a lack of understanding concerning their precise
mechanism of action. On contact, disintegrants can draw water into the tablet, swelling
and forcing the tablet apart. Starch, a traditional and still widely used disintegrant,
will swell when wet, although it has been reported that its disintegrant action
could be due to capillary action [6] . Levels can be increased beyond the normal 5%
w/w to 15 – 20% w/w if a rapid disintegration is required. Surfactants can also act as
disintegrants promoting wetting of the formulation, and sodium lauryl sulfate can
be combined with starch to increase effectiveness.
Tablet disruption following production of carbon dioxide is another mechanism
used to enhance disintegration. This uses a mixture of sodium bicarbonate and a
weak acid such as citric acid or tartaric acid and is exploited for effervescent
formulations.
4.1.4.6 Superdisintegrants
Compared to the more traditional starch, newer disintegrants are effective at much
lower levels and comprise three groups: modifi ed starches, modifi ed cellulose, and
cross - linked povidone. Their likely mechanism of action is a combination of proposed
theories including water wicking, swelling, deformation recovery, repulsion,
and heat of wetting [9] . Superdisintegrants are so called because of the relatively
low levels required (2 – 4% w/w). Sodium starch glycollate (Primojel, Explotab) is
made by cross - linking potato starch and can swell up to 12 - fold in less than 30 s.
Crospovidone is completely insoluble in water, although it rapidly disperses and
swells in water, but does not gel even after prolonged exposure. It rapidly exhibits
high capillary activity and pronounced hydration capacity with little tendency to
form gels and has a greater surface area – volume ratio compared to other disintegrants.
Micronized versions are available to improve uniformity of mix. Croscarmellose
sodium, a cross - linked polymer of carboxymethyl cellulose sodium is also
insoluble in water, although it rapidly swells to 4 – 8 times its original volume on
contact with water [6] .
4.1.4.7 Added Functionality Excipients
Adverse physiochemical and mechanical properties of new chemical entities prove
challenging for formulation development. There is an increasing demand for faster
and more effi cient production processes. Also, biotechnological developments and
various emerging protein - based therapies are broadening the defi nition for excipient
products. Although the description of excipients from inactive ingredients is shifting
toward functionally active materials and will continue to grow in this area, the intro
duction of improved versions of long - existing excipients is probably the more successful
development. New single - component and coprocessed products have been
introduced, for example, fi ller – binders. In addition, there have been advances in the
understanding of how such substances act and hence how they can be optimally
designed. Excipients for use in direct compression product forms or physically or
chemically modifi ed excipients used in relatively new drug delivery systems, such as
patches or inhalation systems, are examples of these developments.
4.1.4.8 Colorants
Colorants are frequently used in uncoated tablets, coated tablets, and hard and soft
gelatin capsules. They can mask color changes in the formulation and are used to
provide uniqueness and identity to a commercial product. Concerns over the safety
of coloring agents in formulations generally arise from adverse effects in food substances.
Colorants are therefore subject to regulations not associated with other
pharmaceutical excipients. Legislation specifi es which colorants may be used in
medicinal products and also provides for purity specifi cations. The number of permitted
colors has decreased in recent years, and a list of approved colorants allowed
by regulatory bodies can vary from country to country.
Colorants can be divided into water - soluble dyes and water - insoluble pigments.
Some of the insoluble colors or pigments can also provide opacity to tablet coatings
or gelatin shells, which can promote stability of light - sensitive active materials. Pigments
such as the iron oxides, titanium dioxide, and some of the aluminum lakes
are especially useful for this purpose.
Water - soluble dyes are usually incorporated within the granulation process to
ensure even distribution throughout the formulation, but there can be an uneven
distribution due to migration of the dye during drying processes. Therefore, water -
soluble dyes can also be adsorbed into a carrier such as starch or lactose and dry
blended prior to the fi nal mix. Water - insoluble pigments are more popular in direct
compression and are dry blended with the other ingredients.
Lakes are largely water - insoluble forms of common synthetic water - soluble dyes
and are prepared by adsorbing the sodium or potassium salt of a dye onto a very
fi ne substrate of hydrated alumina, followed by treatment with a further soluble
aluminum salt. The lake is then purifi ed and dried. Lakes are frequently used in
coloring tablet coatings since they are more stable and have greater opacity than a
water - soluble dye [6] .
4.1.4.9 Interactions and Safety of Excipients
Because there is such a wide selection available, rational choice of the necessary
excipients and their concentration is required. Consideration must also be given to
cost, reliability, availability, and international acceptability. Although generally considered
inert, formulation incompatibility of excipients is also necessary. Lactose,
for example, can react with primary and secondary amines via its aldehyde group
by Maillaird condensation reaction [6] , and calcium carbonate is incompatible with
acids due to acid – base chemical reaction and with tetracyclines due to complexation.
Additionally, excipients can contribute to the instability of the active substance
through moisture distribution.
EXCIPIENTS IN SOLID DOSE FORMULATIONS 243
244 SOLID DOSAGE FORMS
Despite the importance of drug – excipient compatibility testing, there is no generally
accepted method available for this purpose. After identifi cation of any major
known incompatibilities, a compatibility screen needs to be proposed. Issues such
as sample preparation, storage conditions, and methods of analysis should be
addressed and factorial design applied to reduce the number if tests required.
Drug – excipient compatibility studies can be performed with minimal amounts of
materials. Usually, small amounts of each material are weighed into a glass vial, in
a ratio representative of the expected ratio in the formulation. The vials can be
sealed as is or with additional water, either in an air environment or oxygen - free
(nitrogen head space) environment, and stored in the presence or absence of ambient
light, at various temperatures. Factorial or partial factorial design experiments can
be set up to determine important binary and multiple component interaction factors.
This information helps determine which excipients should be avoided and whether
oxidation or light instability in the formulation is a consideration. Controls consisting
of the active pharmaceutical ingredient (API) alone in the various conditions
also should be run to determine whether the API is susceptible alone or must have
the mediating excipient or water additives for instability.
4.1.5 COATED TABLETS
Tablets are often coated to protect the drug from the external environment, to mask
bitter tastes, add mechanical strength, or to enhance ease of swallowing. A coating
can also be used for aesthetic or commercial purposes, improving product appearance
and identity.
4.1.5.1 Sugar - Coated Tablets
Sugar coating can be benefi cial in masking taste, odors, and colors. It is useful in
protecting against oxidation, and sugar coating was once very common due to its
aesthetic results and cheapness of materials. Use has declined in recent years due
to the complexity of the process and skills required, but advances in technology
have led to a resurgence in popularity. Typical excipients used are sucrose (although
this can be substituted with low - calorie alternatives), fi llers, fl avors, fi lm formers,
colorants, and surfactants. It is usually carried out in tumbling coating pans and
comprises several stages.
The fi rst sealing stage uses shellac or cellulose acetate phthalate, for example, to
prevent moisture from reaching the tablet core. This has to be kept to a minimum
to prevent impairment of drug release. The subcoating is an adhesive coat of gum
(such as acacia or gelatin) and sucrose used to round off the edges, and the tablets
can be dusted with substances such as kaolin or calcium carbonate to harden the
coating. A smoothing coat is built up in layers using 70% v/v sucrose syrup and often
opacifi ers such as titanium dioxide, and the tablets are dried between each application.
A colorant is added to the fi nal few layers and followed with a fi nal polishing
step which can make further embossing diffi cult. The coating is relatively brittle,
prone to chipping or cracking, and there is a substantial increase in weight, up to
50%, and size of the product.
HARD AND SOFT GELATIN CAPSULES 245
4.1.5.2 Compression Coating and Layered Tablets
A coating can be applied by compression using specially designed tablet presses.
The same process can be used to produce layered tablets which can comprise two
or even three layers if complete separation of the ingredients is required. This
process is used when physical separation of ingredients is desired due to incompatibility
or to produce a repeat - action product. The formulation can also be designed
to provide an immediate and a slow - release component. Release rates can be
controlled by modifi cation of the geometry, the composition of the core, and the
inclusion of a membrane layer.
The technique involves using a preliminary compression step to produce a relatively
soft tablet core which is then placed in a large die containing coating material.
Further coating material is added and the content compressed. A similar light compression
is used for the production of layers and a fi nal main compression step used
to bind the layers together.
4.1.5.3 Film - Coated Tablets
Film coating, although most often applied to tablets, can also be used to coat other
formulations including capsules. Film coating imparts the same general characteristics
as sugar coating but weight gain is signifi cantly less (typically up to 5%), it is
easier to automate, and it has capacity to include organic solvents if required. The
main methods involved are modifi ed conventional coating pans, side - vented pans,
and fl uid - bed coating. Celluloses are often used as fi lm - forming polymers, as detailed
in Table 4 , and usually require addition of a compatible plasticizer as glass transition
temperatures are higher than the temperatures used in the process. Polyethylene
glycol, propylene glycol, and glycerol are commonly used, and colorants and opaci-
fi ers can also be added to the coating solution. Specialist coatings such as Opadry
fx and Opaglos 2 can be used to give a high gloss fi nish to improve brand identity
and consumer recognition.
4.1.5.4 Tablet Wrapping or Enrobing 1
Recent innovations in tablet coating include the use of gelatin and non - animal -
derived coatings for tablets that require formulation of a pre - formed fi lm that is
then used to encapsulate the product (e.g., Banner ’ s Sofl et Gelcaps or Bioprogress ’
Nrobe technology). The coated formulations are tamper evident and can be designed
with different colors for branding purposes. They are reported to be preferred by
patients due to their ease of swallowing and superior taste - and odor - masking properties.
An alternative is the Press - fi t Geltabs system, which uses a high - gloss gelatin
capsule shell to encapsulate a denser caplet formulation.
4.1.6 HARD AND SOFT GELATIN CAPSULES
Capsules are solid oral dosage forms in which the drug is enclosed within a hard or
soft shell. The shell is normally made from gelatin and results in a simple, easy - to -
swallow formulation with no requirement for a further coating step. They can be
1 See http://www.banpharm.com/technologiesSofl etGelcap.cfm and http://www.fmcmagenta.com/NRobe/
tabid/145/Default.aspx .
246 SOLID DOSAGE FORMS
TABLE 4 Polymers Commonly Used in Film Coating of Tablets
Polymer Soluble in Description
Methylcellulose (MC) Cold water, GI fl uids,
and organic solvents
Low - viscosity grades best for aqueous
fi lms
Ethylcellulose (EC) Organic solvents and
GI fl uids (insoluble
in water)
Used in combination with water -
soluble agents for immediate
release
Hydroxyethyl cellulose
(HEC)
Water and GI fl uids Similar to MC with clear solutions
Hydroxypropyl
cellulose (HPC)
Cold water, GI fl uids,
and polar solvents
Results in a tacky coat and used in
combination to promote adhesion
Hydroxypropylmethyl
cellulose (HPMC)
Cold water, GI fl uids,
and alcohols
Excellent fi lm former, low - viscosity
grades best
Sodium carboxymethyl
cellulose
Water and polar
solvents
Cannot be used if presence of
moisture is a problem
Methylhydroxyethyl
cellulose (MHEC)
Water and GI fl uids Similar to HPMC but less soluble in
organic solvents
Povidone (PVP) Water, GI fl uids,
alcohol, and
isoproplyl alcohol
(IPA)
Can lead to tackiness during drying,
often brittle and hygroscopic
PEGs Water, GI fl uids, some
organic solvents
High molecular weights best for fi lm
forming and low molecular weights
used as plasticizer; can be waxy
Enteric coatings such as
poly(methacrylates)
or cellulose acetate
phthalate
Soluble at elevated
pHs
Used for delayed - release formulations
Source : Adapted from refs. 5 and 10 .
either hard or soft depending on the nature of the capsule shell, with soft capsules
possessing a fl exible, plasticized gelatin fi lm. Hard gelatin capsules are usually rigid
two - piece capsules that are manufactured in one procedure and packed in another
totally separate operation, whereas the formulation of soft gelatin capsules is more
complex but all steps are integrated.
There is a growing interest in using non - animal - derived products for formulation
of the capsule shells to address cultural, religious, and dietary requirements. HPMC
(e.g., V - caps, Quali - VC, Vegicaps) and pullulan shells (NPCaps) and starch are
alternatives.
4.1.6.1 Hard - Shell Gelatin Capsules
Although the challenges of powder blending, homogeneity, and lubcricity exist for
capsules as for tablets, they are generally perceived to be a more fl exible formulation
as there is no requirement for the powders to form a robust compact. This
means that they may also be more suitable for delivery of granular and beadlike
formulations, fragile formulations that could be crushed by the normal compaction
step. They are commonly employed in clinical trials due to the relative ease of blinding
and are useful for taste masking.
HARD AND SOFT GELATIN CAPSULES 247
Capsules are usually more expensive dosage forms than an equivalent tablet
formulation due to the increased cost of the shells and the slower production rates.
Even with modern fi lling equipment, the fi lling speeds of capsule machines are much
slower than tablet presses. However, increased costs can be offset by avoiding a
granulation step. Capsules, although smoother and easy to swallow, also tend to be
larger than corresponding tablet formulations, potentially leading to retention in
the esophagus. Humidity needs to be considered during manufacture and storage,
with moisture leading to stickiness and desiccation causing brittleness. Cross - linking
of gelatin in the formulation can also lead to dissolution and bioavailability
concerns.
Capsule excipients are similar to those required for formulation of tablets and
include diluents, binders, disintegrants, surfactants, glidants, lubricants, and dyes or
colorants. The development of a capsule formulation follows the same principles as
tablet development, and consideration should be given to the same BCS issues. The
powder for encapsulation can comprise simple blends of excipients or granules
prepared by dry granulation or wet granulation. There is a reduced requirement for
compressibility, and often the fl ow properties are not as critical as in an equivalent
tablet formulation. The degree of compressibility required is the major difference,
and capsules can therefore be employed when the active ingredient does not possess
suitable compression characteristics.
4.1.6.2 Manufacture of Hard Gelatin Shells
Gelatin is a generic term for a mixture of purifi ed protein fractions obtained either
by partial acid hydrolysis (type A gelatin) or by partial alkaline hydrolysis (type B
gelatin) of animal collagen. Type A normally originates from porcine skin while B
is usually derived from animal bones, and they have different isoelectric points
(7.0 – 9.0 and 4.8 – 5.0, respectively) [6] . The protein fractions consist almost entirely
of amino acids joined together by amide linkages to form linear polymers, varying
in molecular weight from 15,000 to 250,000. Gelatin can comprise a mixture of both
types in order to optimize desired characteristics, with bone gelatin imparting fi rmness
while porcine skin gelatin provides plasticity. Gelatin Bloom strength is measured
in a Bloom gelometer, which determines the weight in grams required to
depress a standard plunger in a 6.67% w/w gel under standard conditions. Bloom
strength and viscosity are the major properties of interest for formulation of capsules,
and Bloom strength of 215 – 280 is used in capsule manufacture.
Gelatin is commonly used in foods and has global regulatory acceptability, is a
good fi lm former, is water soluble, and generally dissolves rapidly within the body
without imparting any lag effect on dissolution. Gelatin capsules are strong and
robust enough to withstand the mechanical stresses involved in the automated fi lling
and packaging procedures.
In addition to gelatin, the shells may contain colorants, opacifi ers, and preservatives
(often parabens esters). There are eight standard capsule sizes, and the largest
capsule size considered suitable for oral use is size 0 (Table 5 ).
To manufacture the shells, pairs of molds, for the body and the cap, are dipped
into an aqueous gelatin solution (25 – 30% w/w), which is maintained at about 50 ° C
in a jacketed heating pan. As the pins are withdrawn, they are rotated to distribute
the gelatin evenly and blasted with cool air to set the fi lm. Drying is carried out by
248 SOLID DOSAGE FORMS
passing dry air over the shell as heating temperatures are limited due to the low
melting point of gelatin. The two parts are removed from the pins, trimmed, and
joined using a prelock mechanism. The external diameter of the body is usually
wider at the open end than the internal diameter of the cap to ensure a tight fi t.
They can be made self - locking by forming indentations or grooves on the inside of
both parts so that when they are engaged, a positive interlock is formed (e.g.,
Posilok, Conicap, Loxit).
Alternatively, they may be hermetically sealed using a band of gelatin around
the seam between the body and the cap (Qualicaps). This can be applied without
the application of heat and provide a tamper - evident seal. LEMS (liquid encapsulation
microspray sealing) used in Licaps is a more elegant seal in which sealing fl uid
(water and ethanol) is sprayed onto the joint between the cap and body of the
capsule. This lowers the melting point of gelatin in the wetted area. Gentle heat is
then applied which fuses the cap to the body of the Licaps capsule. The moisture
content of manufactured shells is 15 – 18% w/w and levels below 13% will result in
problems with the capsule fi lling machinery. Therefore, capsules are stored and fi lled
in areas where relative humidity is controlled to between 30 and 50%.
4.1.6.3 Hard - Gelatin Capsule Filling
The fi lling material must be compatible with the gelatin shell and, therefore, deliquescent
or hygroscopic materials cannot be used. Conversely, due the moisture
content in the capsule shells, they cannot be used for moisture - sensitive drugs. All
ingredients need to be free of even trace amounts of formaldehyde to minimize
cross - linking of gelatin.
Powders and granules are the most common fi lling materials for hard - shell gelatin
capsules, although pellets, tablets, pastes, oily liquids, and nonaqueous solutions and
suspensions have been used. Filling machines are differentiated by the way they
measure the dose of material and range in capacity from bench - top to high - output,
industrial, fully automated machines. Those that rely on the volume of the shell are
known as capsule dependent, whereas capsule - independent forms measure the
quantity to be fi lled in a separate operation. The simplest dependent method of
fi lling is leveling where powder is transferred directly from a hopper to the capsule
TABLE 5 Capsule Size and Corresponding Volume or
Weight of Fill
Size Volume (mL) Fill weight a (g)
000 1.37 1.096
00 0.95 0.760
0 0.68 0.544
1 0.50 0.400
2 0.37 0.296
3 0.30 0.240
4 0.21 0.168
5 0.13 0.104
Source : Adapted from http://capsugel.onlinemore.info/download/
BAS192 - 2002.pdf .
a Assumes a powder density of 0.8 g/cm 3 .
HARD AND SOFT GELATIN CAPSULES 249
body, aided by a revolving auger or vibration. Additional powder can be added to
fi ll the space arising, and the fi ll weight depends on the bulk density of the powder
and the degree of tamping applied.
Most automated machinery is of the independent type and compresses a controlled
amount of powder using a low compression force (typically 50 – 200 N ) to
form a plug. Most are piston - tamp fi llers and are dosator or dosing disk machines.
The powder is passed over a dosing plate containing cavities slightly smaller than
the capsule diameter, and powder that falls into the holes is tamped by a pin to form
a plug. This can be repeated until the cavity is full and the plugs (or slugs) are ejected
into the capsule shells. The minimum force required to form a plug should be used
to reduce slowing of subsequent dissolution.
In the dosator method, the plug is formed within a tube with a movable piston
that controls the dosing volume and applies the force to form the plug. The dose is
controlled by the dimensions of the dosator, the position of the dosator in the
powder bed, and the height of the powder bed. Fundamental powder properties to
ensure even fi lling are good powder fl ow, lubricity, and compressibility. The auger
or screw method, now largely surpassed, uses a revolving archimedian screw to feed
powder into the capsule shell.
A liquid fi ll can be useful when manufacturing small batches if limited quantities
of API are available. Liquid fi lls also offer improved content uniformity for potent,
low - dose compounds and can reduce dust - related problems arising with toxic compounds.
Two types of liquid can be fi lled into hard gelatin capsules: nonaqueous
solutions and suspensions or formulations that become liquid on application of heat
or shear stress. These require hoppers with heating or stirring systems. For those
formulations that are liquid at room temperature, the capsule shells need to be
sealed after fi lling to prevent leakage of the contents and sticking of the shells. It is
essential to ensure the liquid is compatible with the shell (Table 6 ).
4.1.6.4 Soft - Gelatin Capsules
Soft gelatin capsules are hermetically sealed one - piece capsules containing a liquid
or a semisolid fi ll. Like liquid - fi lled hard capsules, although the drug is presented in
a liquid formulation, it is enclosed within a solid, thus combining the attributes
TABLE 6 Liquid Excipients Compatible with Hard
Gelatin Capsules
Peanut oil Paraffi n oil
Hydrogenated peanut oil Cetyl alchohol
Castor oil Cetostearyl alcohol
Hydrogenated castor oil Stearyl alcohol
Fractionated coconut oil Stearic acid
Corn oil Beeswax
Olive oil Silica dioxide
Hydrogenated vegetable oil Polyethylene glycols
Silicone oil Macrogol glycerides
Soya oil Poloxamers
Source : Adapted from http://www.capsugel.com/products/licaps_
oil_chart.php .
250 SOLID DOSAGE FORMS
of both. Soft gelatin capsules (softgels) offer a number of advantages including
improved bioavailability, as the drug is presented in a solubilized form, and enhanced
drug stability. Consumer preference regarding ease of swallowing, convenience, and
taste can improve compliance, and they offer opportunities for product differentiation
via color, shape, and size and product line extension. The softgels can be enteric
coated for delayed release. They are popular for pharmaceuticals, cosmetics, and
nutritional products, but highly water - soluble drugs and aldehydes are not suitable
for encapsulation in softgels. Formulations are tamper evident and can be used for
highly potent or toxic drugs. However, they do require specialist manufacture and
incur high production costs.
4.1.6.5 Manufacture of Soft Gelatin Capsules
The shell is primarily composed of gelatin, plasticizer, and water (30 – 40% wet gel),
and the fi ll can be a solution or suspension, liquid, or semisolid. The size of a softgel
represents its nominal capacity in minims, for example, a 30 oval softgel can accommodate
30 minims (or 1.848 cm 3 ). Glycerol is the major plasticizer used, although
sorbitol and propylene glycol can also be used. Other excipients are dyes, pigments,
preservatives, and fl avors. Up to 5% sugar can be added to give a chewable quality.
Most softgels are manufactured by the process developed by Scherer [11] . The
glycerol – gelatin solution is heated and pumped onto two chilled drums to form two
separate ribbons (usually 0.02 – 0.04 in. thick) which form each half of the softgel.
The ribbons are lubricated and fed into the fi lling machine, forcing the gelatin to
adopt the contours of the die. The fi ll is manufactured in a separate process and
pumped in, and the softgels are sealed by the application of heat and pressure. Once
cut from the ribbon, they are tumble - dried and conditioned at 20% relative
humidity.
Fill solvents are selected based on a balance between adequate solubility of the
drug and physical stability. Water - miscible solvents such as low - molecular - weight
PEGs, polysorbates, and small amounts of propylene glycol, ethanol, and glycerin
can be used. Water - immiscible solvents include vegetable and aromatic oils, aliphatic,
aromatic, and chlorinated hydrocarbons, ethers, esters, and some alcohols.
Emulsions, liquids with extremes of pH ( < 2.5 and > 7.5), and volatile components
can cause problems with stability, and drugs that do not have adequate stability in
the solvents can be formulated as suspensions. In these instances, the particle size
needs to be carefully controlled and surfactants can be added to promote wetting.
Vegicaps soft capsules from Cardinal Health are an alternative to traditional
softgels, containing carageenan and hydroxyproyl starch. As with traditional soft
gelatin capsules, the most important packaging and storage criterion is for adequate
protection against extremes of relative humidity. The extent of protection required
also depends on the fi ll formulation and on the anticipated storage conditions.
4.1.6.6 Dissolution Testing of Capsules
In general, capsule dosage forms tend to fl oat during dissolution testing with the
paddle method. In such cases, it is recommended that a few turns of a wire helix
around the capsule be used [12] . Inclusion of enzymes in the dissolution media must
be considered on case - by - case basis. A Gelatin Capsule Working Group (including
participants from the FDA, industry, and the USP) was formed to assess the noncompliance
of certain gelatin capsule products with the required dissolution speci-
fi cations and the potential implications on bioavailability [13] . The working group
recommended the addition of a second tier to the standard USP and new drug and
abbreviated new drug applications (NDA/ANDA) dissolution tests: the incorporation
of enzyme (pepsin with simulated gastric fl uid and pancreatin with simulated
intestinal fl uid) into the dissolution medium. If the drug product fails the fi rst tier
but passes the second tier, the product ’ s performance is acceptable. The two - tier
dissolution test is appropriate for all gelatin capsule and gelatin - coated tablets and
the phenomenon may have little signifi cance in vivo.
4.1.7 EFFERVESCENT TABLETS
Effervescence is the reaction in water of acids and bases to produce carbon dioxide,
and effervescent tablets are dissolved or dispersed in water before administration.
Advantages of effervescent formulations over conventional formulations are that
the drug is usually already in solution prior to ingestion and can therefore have a
faster onset of action. Although the solution may become diluted in the GI tract,
any precipitation should be as fi ne particles that can be readily redissolved. Variability
in absorption can also be reduced. Formulations can be made more palatable
and there can be improved tolerance after ingestion. Thus, the types of drugs suited
to this formulation method are those that are diffi cult to digest or are irritant to
mucosa. Analgesics such as paracetamol and aspirin and vitamins are common
effervescent formulations. The inclusion of buffering agents can aid stability of pH -
sensitive drugs. There is also the opportunity to extend market share and to deliver
large doses of medication.
Effervescents comprise a soluble organic acid and an alkali metal carbonate salt.
Citric acid is most commonly used for its fl avor - enhancing properties. Malic acid
imparts a smoother after taste and fumaric, ascorbic, adipic, and tartaric acids are
less commonly used [14] . Sodium bicarbonate is the most common alkali, but potassium
bicarbonate can be used if sodium levels are a potential issue with the formulation.
Both sodium and potassium carbonate can also be employed. Other excipients
include water - soluble binders such as dextrose or lactose, and binder levels are kept
to a minimum to avoid retardation of disintegration. All ingredients must be anhydrous
to prevent the components within the formulation reacting with each other
during storage.
Lubricants such as magnesium stearate are not used as their aqueous insolubility
leads to cloudy solutions and extended disintegration times. Spray - dried leucine and
PEG are water - soluble alternatives [15, 16] . Both artifi cial and natural sweeteners
are used and an additional water - soluble fl avoring agent may also be required. If a
surfactant is added to enhance wetting and dissolution, the addition of an antifoaming
agent may also be considered [17] .
4.1.7.1 Manufacture of Effervescent Tablets
Essentially, effervescent formulations are produced in the same way as conventional
tablets, although due to the hygroscopicity and potential onset of the effervescence
EFFERVESCENT TABLETS 251
252 SOLID DOSAGE FORMS
reaction in the presence of water, environmental control of relative humidity and
water levels is of major importance during manufacture. A maximum of 25% relative
humidity (RH) at 25 ° C is required. Closed material - handling systems can be
used or open systems with minimum moisture content in the ventilating air.
A dry method of granulation is preferred as no liquid is involved but may not
always be possible. Wet granulation can be carried out under carefully controlled
conditions using two separate granulators for the alkaline and acid components.
Water can be added at 0.1 – 1.0% w/w, and it initiates a preeffervescent reaction. The
cycle is stopped by drying, usually by transfer into a preheated fl uidized - bed dryer.
Fluid - bed spray granulation is a process wherein granulation and drying are simultaneous
and can be useful for effervescent formulations. Water (or a binder solution) is
sprayed onto the mixture, which is suspended in a stream of hot, dry air. Organic solvents
can also be employed for granulation avoiding the need for water and are useful
for heat - labile formulations, although complex handling equipment is required.
Effervescent formulations must contain less than 0.3% w/w water and are often
quite large. Sticking due to insuffi cient lubrication can be overcome by adaptation
of punches for external lubrication or using fl at - faced punches with disks of poly
(tetrafl uoroethylene) (PTFE). Poor lubrication can also be the cause of poor fl ow
characteristics, and this can be addressed by using a constant level powder feed
system. The tablets should be stored in tightly closed containers or moisture - proof
packs. In tube arrangements, dry air is added prior to sealing and desiccants to
reduce enclosed moisture levels once the pack has been opened. Foil packaging
should be heavy gauge to minimize risk of holes, and the surrounding pocket should
be large enough to hold the tablets but minimize inclusion of air.
In - process quality control is of major importance for these formulations as are
stability testing and stress testing of packaging. Tablet disintegration and dissolution
are of prime importance, and disintegration should be carried out using representative
conditions. Hardness and friability are also important as these large tablets tend
to chip easily. Common areas for problems are that the packaging permits entry of
water, the seal is compromised or that the excipients can react with each other.
4.1.8 LOZENGES
Lozenges are tablets that dissolve or disintegrate slowly in the mouth to release
drug into the saliva. They are easy to administer to pediatric and geriatric patients
and are useful for extending drug form retention within the oral cavity. They usually
contain one or more ingredient in a sweetened fl avored base. Drug delivery can be
either for local administration in the mouth, such as anaesthetics, antiseptics, and
antimicrobials or for systemic effects if the drug is well absorbed through the buccal
lining or is swallowed. More traditional drugs used in this dosage form include
phenol, sodium phenolate, benzocaine, and cetylpyridinium chloride. Decongestants
and antitussives are in many over - the - counter (OTC) lozenge formulations, and
there are also lozenges that contain nicotine (as bitartrate salt or as nicotine polacrilex
resin), fl urbiprofen (Strefen), or mucin for treatment of dry mouth (A.S Saliva
Orthana).
Lozenges can be made by molding or by compression at high pressures, often
following wet granulation, resulting in a mechanically strong tablet that can dissolve
in the mouth. Compressed lozenges (or troches) differ from conventional tablets in
that they are nonporous and do not contain disintegrant. As the formulation is
designed to release drug slowly in the mouth, it must have a pleasant taste, smoothness,
and mouth feel. The choice of binder, fi ller, color, and fl avor is therefore most
important. The binder is particularly important in ensuring retardation of dissolution
and pleasant mouth feel. Suitable binders include gelatin, guar gum, and acacia
gum. Sugars such as sucrose, dextrose, and mannitol are preferred to lactose, and
xylitol is often included in sugar - free formulations. In order to ensure adequate
sweetness and taste masking, artifi cial sweeteners including aspartame, saccharin,
and sucralose are also included subject to regulatory guidelines.
Other variations include hard - candy - type and soft or chewable lozenges. Most
hard - candy - type lozenges contain sugar, corn syrup, acidulant, colorant, and fl avors.
They are made by heating sugars and other ingredients together and then pouring
the mixture into a mold. Corn syrup combined with sucrose and dextrose can form
an amorphous glass suitable for such formulations [18] . Colorants can be added to
enhance product appearance or to mask products of degradation. Stability and
compatibility with the drug must be established along with the other excipients.
Flavors tend to be complex entities, and stability or compatibility can pose major
formulation challenges. Acidulants such as citric and tartaric acids are often added
to enhance fl avors, thus lowering pH of the formulation as low as 2.5 – 3.0. Addition
of bases such as calcium carbonate, sodium bicarbonate, and magnesium trisilicate
is common to increase pH and enhance drug stability. For example, in vivo and in
vitro studies confi rmed that the pH of the dissolved lozenge solution was the single
most infl uential, readily adjustable formulation parameter infl uencing the activity
of cetylpyridinium chloride activity in candy - based lozenges [19] . The dosage form
needs a low moisture content (0.5 – 1.5% w/w), so water is evaporated off by boiling
the sugar mixture during the compounding process, thus limiting the process to
nonlabile drugs, and the manufacture requires specialized candy processing facilities.
Packaging also needs to protect the formulated product from moisture and
ranges from individual bunch wrapping to foil wraps.
4.1.8.1 Chewable Lozenges
Chewable lozenges are popular with the pediatric population since they are “ gummy -
type ” lozenges. Most formulations are based on a modifi ed suppository formula consisting
of glycerin, gelatin, and water. These lozenges are often highly fruit -
fl avored and may have a slightly acidic taste to cover the acrid taste associated with
glycerin. Soft lozenges typically comprise ingredients such as PEG 1000 or 1450, or a
sugar – acacia base. Silica gel can be added to prevent sedimentation, and again this
dosage form requires fl avors and sweeteners to aid compliance. Soft lozenges tend to
dissolve faster than gelatin bases and can be used if taste masking is not effective.
4.1.9 CHEWABLE TABLETS
Chewable tablets are designed to be mechanically disintegrated in the mouth.
Potential advantages of chewable tablets are mainly concerning patient convenience
and acceptance, although enhanced bioavailability is also claimed. This can be due
CHEWABLE TABLETS 253
254 SOLID DOSAGE FORMS
to a rapid onset of action as disintegrate is more rapid and complete compared to
standard formulations that must disintegrate in the GI tract. The dosage form is an
appealing alternative for pediatric and geriatric consumers. Chewable tablets also
offer convenience for consumers, avoiding the necessity of coadministration with
water, and creation of palatable formulations can increase compliance. Antacids and
pediatric vitamins are often formulated as chewable tablets, but other formulations
include antihistamines (Zyrtec), antimotility agents (Imodium Plus) and antiepileptic
agents (Epanutin Infatabs), antibiotics (Augmentin Chewable), asthma treatments
(Singulair), and analgesics (Motrin).
Constraints with these systems are that many pharmaceutical actives have an
unpleasant bitter taste that can actually reduce compliance among patients. Iron
salts, for example, can impart a rusty taste, and some antihistamines such as promethazine
HCl can have a bitter aftertaste. As such, active formulations require
very effective taste - masking strategies to provide acceptable patient tolerance and
to ensure patient adherence to their pharmaceutical regimen.
Formulation factors governing design are similar to standard formulations (e.g.,
compactability, fl ow, etc.), but disintegrants are not included. Organoleptic properties
are a major concern, especially in the design of products for children, and usage
has been limited as formulators have encountered diffi culties in achieving satisfactory
sensory characteristics.
Certain diluents are benefi cial in the formulation of chewable tablets by compression
such as mannitol, lactose, sucrose, and sorbitol. They can aid disintegration upon
chewing and can help with acceptable taste and mouth feel. Mannitol, for example,
can impart a cooling or soothing sensation. Specialist excipients with improved
sensory components such as mouth feel and lack of grittiness have been developed
for formulation of chewable tablets. For example, Avicel CE - 15 [a combination of
microcrystalline cellulose (MCC) and guar gum] can reduce grittiness, leading to a
creamier mouth feel and improved overall compatibility.
Citric acid, grape, raspberry, lemon, and cherry fl avors are often used in chewable
tablets and lozenges (Table 7 ). Flavoring agents are commonly volatile oils, and they
can be dissolved in alcohol and then sprayed onto another excipient or granules.
They are usually added immediately prior to compression to avoid loss due to their
volatile nature. Dry fl avors have advantages in terms of stability and ease of handling
and are formed by emulsifi cation of the fl avor into an aqueous solution of a
carrier followed by drying, encapsulating the fl avor within the carrier. This is useful
if the agent is prone to oxidation. Common carrier substances are acacia gum, starch,
and maltodextrin. Sweeteners such as aspartame can also be added. Low - calorie
and non - sugar - based excipients may present a marketing advantage.
Issues of taste masking for chewable formulations may be addressed by coating
in wet granulation. The granulating/coating agent should form a fl exible rather than
TABLE 7 Flavor Groups for Taste Types
Sweet Vanilla, grape, maple, honey
Sour Citrus, raspberry, anise
Salty Mixed fruit, mixed citrus, butterscotch, maple
Bitter Licorice, coffee, mint, cherry, grapefruit
Metallic Grape, lemon, lime
Source : From ref. 18 .
brittle fi lm, have no unpleasant taste of its own, not interfere with dissolution, and
be insoluble in saliva. Microencapsulation for taste masking can be achieved by
phase separation or coacervation and may also impart stability. The same taste -
masking technologies may be used to encapsulate drugs for formulation into
chewable, softchew, and fast dissolving dosage forms. Coating materials include
carboxymethylcellulose, polyvinyl alcohol (PVA), and ethylcellulose. Xylitol is the
sweetest sugar alcohol, and it has a high negative heat of solution, making it a good
candidate as an excipient for chewable tablets. There are many types of compressible
sugars today, and most of them are composed of sucrose granulated with small
amounts of modifi ed dextrins in order to make the sucrose more compressible [20] .
Modifi cations to sugar - based excipients such as spray - dried crystalline maltose and
directly compressible sucrose (95% sucrose and 5% sorbitol) to facilitate direct
compression are also aiding development in this area [21] .
4.1.9.1 Testing of Chewable Tablets
Dissolution testing for chewable tablets should be the same as that used for regular
tablets [22] . This is because patients could swallow the dosage form without adequate
chewing, in which case the drug would still need to be released to ensure
the desired pharmacological action. However, as outlined, chewable tablets will
typically have different excipients than standard formulations, including agents to
either mask or add fl avor, and may undergo a different manufacturing process.
Where applicable, test conditions would preferably be the same as used for nonchewable
tablets of the same active pharmaceutical ingredient, but because of the
nondisintegrating nature of the dosage form, it may be necessary to alter test
conditions (e.g., increase the agitation rate) and specifi cations (e.g., increase the
test duration). The reciprocating cylinder (USP apparatus 3) with the addition of
glass beads may also provide more intensive agitation for in vitro dissolution
testing of chewable tablets. As another option, mechanical breaking of chewable
tablets prior to exposing the specimen to dissolution testing could be considered.
Chewable tablets should also be evaluated for in vivo bioavailability and/or
bioequivalence.
Additional concerns in the testing of chewable tablets are organoleptic, chemical,
and physical stability. As it is a critical factor in the design of such formulations,
taste masking should be incorporated into excipient testing during preformulation
studies. Technologies like the “ electronic tongue ” can be used to match desirable
taste characteristics [23, 24] .
4.1.10 CHEWING GUMS
4.1.10.1 Composition of Chewing Gum
Medicated chewing gums are gums made with a tasteless masticatory gum base that
consists of natural or synthetic elastomers [25] . They include excipients such as
fi llers, softeners, and sweetening and fl avoring agents. Natural gum bases include
chicle and smoked natural rubber and are permitted in formulations by the FDA,
but modern gum bases are mostly synthetic in origin and approved bases include
CHEWING GUMS 255
256 SOLID DOSAGE FORMS
styrene – butadiene rubber, polyethylene, and polyvinylacetate. Gum base usually
forms about 40% of the gum, but can comprise up to 65%, and is a complex mixture,
insoluble in saliva, comprising mainly of elastomer, plasticizers, waxes, lipids, and
emulsifi ers (see Table 8 ). It will also contain an adjuvant such as talc to modify the
texture of the gum and low quantities of additional excipients including colorants
and antioxidants such as butylated hydroxyanisole. Elastomers control the gummy
texture while the plasticizers and texture agents regulate the cohesiveness of the
product. The lipid and waxes melt in the mouth to provide a cooling, lubricating
feeling while the juicy feel of the gum texture is from the emulsifi ers. The choice
and formulation of gum base will affect the release of active ingredient, and the
texture, stability, and method of manufacture of the product.
The remaining ingredients in the chewing gum itself include drug, sweeteners,
softeners, and fl avoring and coloring agents. A typical chewing gum formulation is
shown in Table 9 . The sugar is for sweetening the product while the corn syrup keeps
the gum fresh and fl exible. Softeners or fi llers are included to help blend the ingredients
and retain moisture. Sugar - free gum has sorbitol, mannitol, aspartame, or
saccharin instead of sugar. Optimized chewing gum formulations will require tailoring
for each individual product. For example, nicotine - containing gums are formulated
with the nicotine within an ion exchange resin and pH - modifying carbonates
and/or bicarbonates to increase the percentage of the drug in its free base form
in saliva.
TABLE 8 Typical Formulation of Gum Base
Ingredient Weight (%) Example
Elastomer 10 Styrene – butadiene rubber
Plasticizer 30 Rosin esters
Texture agent/fi ller 35 Calcium carbonate
Wax 15 Paraffi n wax
Lipid 7 Soya oil
Emulsifi er 3 Lecithin
Miscellaneous 1 Colorant, antioxidant
Source : From ref. 26 .
TABLE 9 Example Chewing Gum Formulations
Ingredient (%) Sugar Gum Sugar - Free Gum
Gum base 19.4 25.0
Corn syrup 19.8 —
Sorbitol, 70% — 15.0
Sugar 59.7 —
Glycerin 0.5 6.5
Sorbitol — 52.3
Flavor 0.6 1.2
Source : From ref. 26 .
4.1.10.2 Manufacture of Chewing Gum
The majority of chewing gum delivery systems today are manufactured using conventional
gum processes. The gum base is softened or melted and placed in a kettle
mixer where sweeteners, syrups, active ingredients, and other excipients are added
at a defi ned time. The gum is then sent to a series of rollers that form it into a thin,
wide ribbon. During this process, a light coating of an antisticking agent can be
added (e.g., magnesium stearate, calcium carbonate, or fi nely powdered sugar or
sugar substitute). Finally, the gum is cut to the desired size and cooled at a carefully
controlled temperature and humidity.
As the heating process involved in conventional methods may limit the applicability
of the process for formulation of thermally labile drugs, directly compressible,
free - fl owing powdered gums such as Pharmagum (SPI Pharma) and MedGumBase
(Gumbase Co) have been proposed to simplify the process. These formulations
can be compacted into a gum tablet using a conventional tablet press and have
the potential to simplify the manufacture, facilitating inclusion of a wider range
of drugs.
4.1.10.3 Drug Release from Chewing Gums
Until recently, the release of substances from chewing gums during mastication was
studied using a panel of tasters and chew - out studies. During the mastication process,
the medication contained within the gum product should be released into the saliva
and is either absorbed through the buccal mucosa or swallowed and absorbed via
the GI tract. The need for, and value of, in vitro drug release testing is well established
for a range of dosage forms, however, standard dissolution apparatus is not
suitable for monitoring release of drug from chewing gums as mastication is essential
in order to provide a renewable surface for drug release after chew action. A
number of devices to mimic the chewing action have been reported [26 – 28] . In 2000,
the European Pharmacopoeia produced a monograph describing a suitable apparatus
for studying the in vitro release of drug substances from chewing gums [25] .
The chewing machine consists of a temperature - controlled chewing chamber in
which the gum piece is chewed by two electronically controlled horizontal pistons
driven by compressed air. The two pistons transmit twisting and pressing forces to
the gum while a third vertical piston operates alternately to the two horizontal
pistons to ensure that the gum stays in the right place (see Figure 1 ). The temperature
of the chamber can be maintained at 37 ° C ± 0.5 ° C and the chew rate varied.
Other adjustable settings include the volume of the medium, distance between the
jaws and the twisting movement. The European Pharmacopoeia recommends using
20 mL of unspecifi ed buffer in a chewing chamber of 40 mL and a chew rate of 60
strokes per minute. This apparatus has been used to study release of nicotine from
commercial gums and directly compressible gums [26] .
Factors affecting the release of medicament from chewing gum can be divided
into three groups: the physicochemical properties of the drug, the gum properties,
and chew - related factors, including rate and frequency. Drugs can be incorporated
into gums as solids or liquids. For most pharmaceuticals, aqueous solubility of the
drug will be a major factor affecting the release rate. In order for drugs to be
CHEWING GUMS 257
258 SOLID DOSAGE FORMS
released, the gum would need to become hydrated; the drugs can then dissolve and
diffuse through the gum base under the action of chewing.
For treatment of local conditions, a release period less than 1 h may be desirable,
but a faster release may be required if a rapid onset of action is required for a systemically
absorbed formulation. There are a number of strategies that can be undertaken
in order to achieve the desired release rate. Decreasing the amount of the
gum base will enhance the release of lipophilic drugs and addition of excipients
designed to promote release can also be considered. Release can be sustained using,
for example, ion exchange resins as described for nicotine - containing gums. Changes
in gum texture as a consequence of changes in excipient levels provide a further
challenge to controlling the release of drugs. A quantitative measure of gum texture
during the process is possible using texture analysis techniques [26] .
4.1.10.4 Applications for Chewing Gums
The promotion of sugar - free gums to counteract dental caries by stimulation of
saliva secretion has led to a more widespread use and acceptance of gums. Medicated
gums for delivery of dental products to the oral cavity are marketed in a
number of countries, for example, fl uoride - containing gums as an alternative to
mouthwashes and tablets or chlorhexidine gum for treatment of gingivitis. The
potential use of medicated chewing gums in the treatment of oral infections has also
been reported. Gums have been prepared containing antifungal agents such as
nystatin [29] and miconazole [30] or antibiotics, such as penicillin and metronidazole
for the treatment of oral gingivitis [31] .
Chewing gum is also useful as a delivery system for agents intended for systemic
delivery. Drug that is released from the gum within the oral cavity can act locally,
be absorbed via the buccal mucosa, or swallowed with the saliva. The buccal mucosa
is well vascularized, and if a drug is absorbed by this route, then fi rst - pass metabolism
could be avoided. Associated increases in bioavailability can permit the use of
lower dosages. Like orally disintegrating tablets, chewing gum is a convenient dosage
form; it can be administered without water and to those who have diffi culty swallowing.
Although medicated gums are generally intended to be chewed for 10 –
30 min and can therefore be designed for sustained release, a fast onset of action
can result either from buccal absorption or as a consequence of the active being
FIGURE 1 Schematic of chewing chamber of in vitro chewing apparatus [26] .
Chewing pistons
Base of chewing chamber
Piston
Chewing chamber
already dissolved in the saliva prior to swallowing. Guidance can be given regarding
chewing conditions (e.g., time, frequency), but factors such as the force of chewing
and salivary fl ow will impact on drug release and the fraction of drug absorbed via
the oral mucosa. Released drug can be swallowed with the saliva, therefore leading
to multiple absorption sites, which can result in variable pharmacokinetics.
Along with nicotine replacement patches, nicotine chewing gum for smoking
cessation therapy has met with major sales success. The principal active ingredient
of currently marketed nicotine chewing gums is nicotine polacrilex USP. The nicotine
is loaded at approximately 18% w/w on an ion exchange resin (Amberlite
IRP64). Recent product variations have been launched with improved fl avors such
as mint and fruit, rather than the original peppery fl avoring, designed to reduce the
unpleasant taste and burning sensation arising from nicotine itself and fl avored
coated gums that are sweeter and easier to chew.
Other applications for chewing gum formulations include delivery of antacids
such as calcium carbonate, antiemetics for travel sickness, and vitamins and minerals.
However, the potential for a buccal delivery, a fast onset of action, and the opportunity
for product line extension makes it an attractive alternative delivery form for
other applications.
4.1.11 ORALLY DISINTEGRATING TABLETS
The demand for fast - dissolving/disintegrating tablets or fast - melting tablets that can
dissolve or disintegrate in the mouth has been growing particularly for those with
diffi culty swallowing tablets such as the elderly and children. They are referred to
using a range of terminologies: fast dissolving, orodispersible, and fast melting and
the FDA has adopted the term orally disintegrating tablets (ODTs). Patients with
persistent nausea or those who have little or no access to water could also benefi t
from ODTs. Other advantages include product differentiation and market expansion,
and applications exist in the veterinary market for oral administration to
animals.
Orally disintegrating tablets disintegrate and/or dissolve rapidly in the saliva
without the need for water, within seconds to minutes. Some tablets are designed
to dissolve rapidly in saliva, within a few seconds, and are true fast - dissolving tablets.
Others contain agents to enhance the rate of tablet disintegration in the oral cavity
and are more appropriately termed fast - disintegrating tablets, as they may take up
to a minute to completely disintegrate. Increased bioavailability using such formulations
is sometimes possible if there is suffi cient absorption via the oral cavity prior
to swallowing [32] . However, if the amount of swallowed drug varies, there is the
potential for inconsistent bioavailability. Patented orally disintegrating tablet technologies
include OraSolv, DuraSolv, Zydis, FlashTab, WOWTAB, and others. They
are generally prepared using freeze drying, compaction, or molding. Examples of
marketed products, excipients, and technologies used are given in Table 10 .
Platform technologies based on freeze drying include Zydis (Cardinal Health)
and Quicksolv (Janssen Pharmaceutica). Zydis was the fi rst ODT to be successfully
launched, and it is ideal for poorly soluble drugs. It can incorporate doses up to
400 mg, but high loadings can extend disintegration time. The porous matrix consists
of a network of water - soluble carriers and active ingredient. The maximum dose for
ORALLY DISINTEGRATING TABLETS 259
260 SOLID DOSAGE FORMS
TABLE 10 Examples of Marketed ODT Products and Technologies
Name (Company) Examples Ingredients a Technology
Zydis
(Cardinal Health)
Claritin
Reditab
Micronized loratadine (10 mg) , citric
acid, gelatin, mannitol, mint fl avor
Freeze
drying
Zydis
(Cardinal Health)
Zofran
ODT
Ondansetron (4 or 8 mg) , aspartame,
gelatin, mannitol, methylparaben
sodium, propylparaben sodium,
strawberry fl avor
Freeze
drying
Zydis
(Cardinal Health)
Zyprexa
Zydis
Olanzapine (5, 10, 15, or 20 mg) , gelatin,
mannitol, aspartame, methylparaben
sodium, propylparaben sodium
Freeze
drying
Oralsolv
(CIMA Labs Inc.)
Remeron
Soltab
Mirtazepine (15, 30, or 45 mg) ,
aspartame, citric acid, crospovidone,
hydroxypropyl methylcellulose,
magnesium stearate, mannitol,
microcrystalline cellulose,
polymethacrylate, povidone, sodium
bicarbonate, starch, sucrose, orange
fl avor
Compression
Durasolv
(CIMA Labs Inc.)
Zomig ZMT Zolmitriptan (2.5 mg) , mannitol,
microcrystalline cellulose,
crospovidone, aspartame, sodium
bicarbonate, citric acid, anhydrous,
colloidal silicon dioxide, magnesium
stearate, orange fl avor
Compression
WOWTAB
(Yamanouchi
Pharma
Technologies,
Inc.)
Benadryl
Allergy &
Sinus
Fastmelt
Diphenhydramine citrate (19 mg),
pseudoephedrine HCl (30 mg),
aspartame, citric acid, D & C red no. 7
calcium lake, ethylcellulose, fl avor,
lactitol, magnesium stearate,
mannitol, and stearic acid
Compression
molded
tablet
Flashtab
(Prographarm/
Ethypharm)
Excedrin
Quicktabs
Acetaminophen (500 mg), caffeine
(65 mg) , aminoalkyl methacrylate
copolymers, citric acid, colloidal
silicon dioxide, crospovidone,
distilled acetylated monoglycerides,
ethylcellulose, fl avors, magnesium
stearate, mannitol, methacrylester
copolymer, polyvinyl acetate,
povidone, propylene glycol, propyl
gallate, silica gel, sodium lauryl
sulfate, sucralose, talc
Compression
a Active ingredients appear in italics.
water - soluble drugs is 60 mg, and particle sizes of drug and excipients should be
below 50 . m.
Excipients used in the formulation usually include a mixture of a water - soluble
polymer and a crystalline sugar. Mannitol and natural polysaccharides such as gelatin
and alginates are used. Microencapsulation and complexation with ion exchange
resins can be combined with additional fl avors and sweeteners for taste masking of
bitter drugs. The fairly complex nature of manufacture and scale - up contributes to
a relatively high manufacturing cost. Manufacture comprises three stages. The production
sequence begins with the bulk preparation of an aqueous drug solution or
suspension and subsequent precise dosing into preformed blisters. It is the blister
that actually forms the tablet shape and is, therefore, an integral component of the
total product package. The second phase of manufacturing entails passing the fi lled
blisters through a cryogenic freezing process to control the ultimate size of the ice
crystals. This aids in ensuring porosity and the product is freeze dried. The fi nal phase
of production involves sealing the open blisters via a heat - seal process to ensure
stability and protect the fragile tablet during removal by the patient.
The manufacture of Flashdose (Fuisz Technologies/Biovail) is patented as
Shearform process and utilizes a unique spinning mechanism to produce a fl osslike
or shear - form crystalline structure, much like cotton candy. The matrix comprises
saccharides or polysaccharides which are subjected to simultaneous melting and
centrifugal force and then partially recrystallized [33] . High temperatures are
involved so the technology is only suitable for thermostable agents. Drug can then
be incorporated, either as coated or uncoated microspheres, into the sugar and the
formulation is compressed into a tablet. Manufacture of the microspheres is patented
as Ceform and will help with taste masking. The fi nal product has a very high
surface area for dissolution and it disperses and dissolves quickly once placed onto
the tongue. Like freeze - drying processes, the manufacture is expensive and resultant
formulations are friable and moisture sensitive, therefore requiring specialized
packaging.
Most commercial ODTs have been developed using mannitol as the bulk excipient
of choice because of its extremely low hygroscopicity, excellent compatibility,
good compressibility, better sweetness, and relatively slower dissolution kinetics.
Although lactose also has a relatively low aqueous solubility compared with other
excipients that have acceptable palatabilities, the dispersibility of a bulk excipient
is more important than its aqueous solubility for a successful ODT formulation.
Many of the initially marketed ODTs were prepared by the wet granulation of
mannitol followed by direct compression. However, added functionality mannitols
are now available to simplify the process of ODT manufacturing by direct
compression.
Direct compression is, as for normal tablets, the most straightforward process for
manufacturing ODTs. Conventional equipment can be used and high doses can be
incorporated. The excipients play a major role in the successful formulation and
superdisintegrants, hydrophilic polymers, and effervescent compounds are included.
Patented technologies include Orasolv and Durasolv (Cima Labs) and Ziplets
(Eurand). The OraSolv technology is best described as a fast disintegrating, slightly
effervescing tablet; the tablet matrix dissolves in less than one minute, leaving
coated drug powder. Both the coating and the effervescence contribute to taste
masking in OraSolv. The tablet is prepared by direct compression but at a low pressure,
yielding a weaker and more brittle tablet in comparison with conventional
tablets. For that reason, Cima developed a special handling and packaging system
for OraSolv called Packsolv. Acidic compounds such as citric or fumaric acid are
included in the formulation together with a carbonate or bicarbonate. An advantage
that goes along with the low degree of compaction of OraSolv is that the particle
coating used for taste masking is not compromised by fracture during processing.
DuraSolv is Cima ’ s second - generation fast - dissolving/disintegrating tablet formulation
and is also produced using direct compression but using higher compaction
ORALLY DISINTEGRATING TABLETS 261
262 SOLID DOSAGE FORMS
pressures during tableting, resulting in a stronger product. It is thus produced in a
faster and more cost - effective manner and may not require specialized packaging.
Large amounts of fi nely milled conventional fi llers are used (mannitol, lactose)
while the effervescing agents are reduced. It is best suited to potent drugs, requiring
only low doses, and the taste - masking coating can be disturbed following
compaction. DuraSolv is currently available in two products: NuLev and Zomig
ZMT.
Compression following wet or dry granulation is also employed in the manufacture
of ODTs. Patented formulations include WOWTAB and Flashtab. WOWTAB
relies on a combination of low moldable sahharides (mannitol, glucose, sucrose)
with a highly moldable saccharide (malitol, sorbitol, maltose) using conventional
granulation and tableting techniques to form a tablet of suitable mechanical properties
with desired disintegration. It is manufactured by compression of molded granules,
can accommodate a high level of drug loading (up to 50% in some cases), and
can be packed using conventional methodology.
Flashtab (Ethypharm) is the technology behind Exedrin QuickTabs and uses
swellable agents and disintegrants along with sugars and polyalcohols to achieve a
fast dispersible formulation. The manufacture involves either wet or dry granulation
of the excipients, blending with the active followed by direct compression.
4.1.11.1 Dissolution Testing of ODT s
Taste masking (drug coating) is very often an essential feature of ODTs and thus
can also be the rate - determining mechanism for dissolution/release. If taste masking
is not an issue, then the development of dissolution methods is comparable to the
approach taken for conventional tablets and pharmacopeial conditions should be
used [34] . Due to the nature of the product, the dissolution of orally disintegrating
tablets is very fast when using USP monograph conditions, and slower paddle speeds
can be used to obtain a profi le. Other media such as 0.1 N HCl can also be used.
USP 2 paddle apparatus is the most suitable and common choice for orally disintegrating
tablets, with a paddle speed of 50 rpm commonly used [34] . Faster agitation
rates may be necessary in the case of sample mounding. The method can be applied
to the ODTs (fi nished product) as well as to the bulk intermediate (in the case of
coated drug powder/granulate). A potential diffi culty for in vitro dissolution testing
may arise from fl oating particles [35] . Similarly, diffi culties can arise using USP I
due to trapping of disintegrated fragments.
A single - point specifi cation is considered appropriate for ODTs with fast dissolution
properties. For ODTs that dissolve very quickly, a disintegration test may be
used in lieu of a dissolution test if it is shown to be a good discriminating method.
If taste masking (using a polymer coating) is a key aspect of the dosage form, a
multipoint profi le in a neutral pH medium with early points of analysis (e.g., . 5 min)
may be recommended [34] .
4.1.12 SOLID DOSAGE FORMS FOR NONORAL ROUTES
Although the majority of tablets and capsules are intended for oral delivery, there
are a number of other delivery routes suitable for drug delivery by these formula
tions. Some buccal formulations have been discussed above, and tablets can also be
administered via the rectal and vaginal routes for local and systemic treatment.
Many types of product have been designed for vaginal administration with
creams, gels, and pessaries being most popular, although powders and tablets have
also been used. Despite the effectiveness of systemic vaginal absorption, the majority
of products administered by this route are for the treatment of localized infections,
especially Candida albicans , (e.g., Canestan vaginal tablets). Estradiol tablets
(Vagifem) were also designed for delivery via vaginal route to address patient preference
issues with vaginal creams. The formulations are administered with an applicator
and are designed to dissolve or erode slowly in the vaginal secretions [36] .
Bioadhesion as a means of retaining the formulation at the site of delivery is widely
accepted to retain formulations in the buccal cavity [37] and has also been reported
for the vaginal route [38] . An increased residence time may improve drug absorption
by these routes.
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264 SOLID DOSAGE FORMS
12. The United States Pharmacopeia , 26th revision, United States Pharmacopeial Convention,
Rockville, MD, 2003 .
13. Gelatin Capsule Working Group, Collaborative development of two - tier dissolution
testing for gelatin capsules and gelatin - coated tablets using enzyme - containing media,
Pharmacop. Forum , 24(5), Sept./Oct. 1998 .
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accessed Aug. 6, 2004 .
15. Rotthauser , B. , Kraus , G. , and Schmidt , P. C. ( 1998 ), Optimization of an effervescent tablet
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18. Mendes , R. W. , and Bhargava , H. ( 2002 ), Lozenges , in Swarbrick J. , and Boylan , J. V. ,
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taste - masking in a lyophilized orally disintegrating tablet formulation , Pharm. Technol.
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REFERENCES 265
267
4.2
SEMISOLID DOSAGES: OINTMENTS,
CREAMS, AND GELS
Ravichandran Mahalingam , Xiaoling Li , and Bhaskara R. Jasti
University of the Pacifi c, Stockton, California
Contents
4.2.1 Introduction
4.2.2 Ointments and Creams
4.2.2.1 Defi nition
4.2.2.2 Bases
4.2.2.3 Preparation and Packaging
4.2.2.4 Evaluation
4.2.2.5 Typical Pharmacopeial/Commercial Examples
4.2.3 Gels
4.2.3.1 Defi nition
4.2.3.2 Characteristics
4.2.3.3 Classifi cation
4.2.3.4 Stimuli - Responsive Hydrogels
4.2.3.5 Gelling Agents
4.2.3.6 Preparation and Packaging
4.2.3.7 Evaluation
4.2.3.8 Typical Pharmacopeial and Commercial Examples
4.2.4 Regulatory Requirements for Semisolids
References
4.2.1 INTRODUCTION
Semisolid dosage forms are traditionally used for treating topical ailments. The vast
majority of them are meant for skin applications. They are also used for treating
ophthalmic, nasal, buccal, rectal, and vaginal ailments. Various categories of drugs
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
268 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
such as antibacterials, antifungals, antivirals, antipruritics, local anesthetics, anti -
infl ammatories, analgesics, keratolytics, astringents, and mydriatic agents are incorporated
into these products. Drugs incorporated into semisolids either show their
activity on the surface layers of tissues or penetrate into internal layers to reach the
site of action. For example, an antiseptic ointment should be able to penetrate the
skin layers and reach the deep - seated infections in order to prevent the growth of
microbes and heal the wound.
Systemic entry of drugs from these products is limited due to various physicochemical
properties of dosage forms and biological factors. The barrier nature of
most surface biological layers such as skin, cornea and conjunctiva of the eye, and
mucosa of nose, mouth, rectum, and vagina greatly limits their entry into the systemic
circulation. Systemic delivery of drugs from topical dosages is however feasible by
suitable formulation modifi cations. Semisolid dosage forms are also used in nontherapeutic
conditions for providing protective and lubricating functions. They
protect the skin against external environments such as air, moisture, and sun rays and
hence their components do not necessarily penetrate the skin layers. Cold creams
and vanishing creams are classic examples of such semisolid preparations.
The formulation, evaluation, and regulatory feature of the three most commonly
used semisolid dosage forms, ointments, creams, and gels, are described in this
chapter.
4.2.2 OINTMENTS AND CREAMS
4.2.2.1 Defi nition
Ointments are semisolid preparations intended for topical application. They are
used to provide protective and emollient effects on the skin or carry medicaments
for treating certain topical ailments. They are also used to deliver drugs into eye,
nose, vagina, and rectum. Ointments intended for ophthalmic purposes are required
to be sterile. When applied to the eyes, they reside in the conjunctival sac for prolonged
periods compared to solutions and suspensions and improve the fraction of
drug absorbed across ocular tissues. Ophthalmic ointments are preferred for nighttime
applications as they spread over the entire corneal and conjunctival surface
and cause blurred vision.
Creams are basically ointments which are made less greasy by incorporation of
water. Presence of water in creams makes them act as emulsions and therefore are
sometimes referred as semisolid emulsions. Hydrophilic creams contain large
amounts of water in their external phase (e.g., vanishing cream) and hydrophobic
creams contain water in the internal phase (e.g., cold cream). An emulsifying agent
is used to disperse the aqueous phase in the oily phase or vice versa. As with ointments,
creams are formulated to provide protective, emollient actions or deliver
drugs to surface or interior layers of skin, rectum, and vagina. Creams are softer
than ointments and are preferred because of their easy removal from containers
and good spreadability over the absorption site.
4.2.2.2 Bases
Bases are classifi ed based on their composition and physical characteristics. The U.S.
Pharmacopeia (USP) classifi es ointment bases as hydrocarbon bases (oleaginous
OINTMENTS AND CREAMS 269
bases), absorption bases, water - removable bases, and water - soluble bases (water -
miscible bases) [1] .
Hydrocarbon bases are made of oleaginous materials. They provide emollient
and protective properties and remain in the skin for prolonged periods. It is diffi cult
to incorporate aqueous phases into hydrocarbon bases. However, powders can be
incorporated into these bases with the aid of liquid petrolatum. Removal of hydrocarbon
bases from the skin is diffi cult due to their oily nature. Petrolatum USP, white
petrolatum USP, yellow ointment USP, and white ointment USP are examples of
hydrocarbon bases.
Absorption bases contain small amounts of water. They provide relatively less
emollient properties than hydrocarbon bases. Similar to hydrocarbon bases, absorption
bases are also diffi cult to remove from the skin due to their hydrophobic nature.
Hydrophilic petrolatum USP and lanolin USP are examples of absorption bases.
Water - removable bases are basically oil - in - water emulsions. Unlike hydrocarbon
and absorption bases, a large proportion of aqueous phase can be incorporated into
water - removable bases with the aid of suitable emulsifying agents. It is easy to
remove these bases from the skin due to their hydrophilic nature. Hydrophilic ointment
USP is an example of a water - removable ointment base.
Water - soluble bases do not contain any oily or oleaginous phase. Solids can be
easily incorporated into these bases. They may be completely removed from the
skin due to their water solubility. Polyethylene glycol (PEG) ointment National
Formulary (NF) is an example of a water - soluble base.
Selection of an appropriate base for an ointment or cream formulation depends
on the type of activity desired (e.g., topical or percutaneous absorption), compatibility
with other components, physicochemical and microbial stability of the product,
ease of manufacture, pourability and spreadability of the formulation, duration of
contact, chances of hypersensitivity reactions, and ease of washing from the site of
application. In addition, bases that are used in ophthalmic preparations should be
nonirritating and should soften at body temperatures. White petrolatum and liquid
petrolatum are generally used in ophthalmic preparations. Table 1 summarizes
TABLE 1 Some Compendial Bases Used in Ointments and Creams
Name Synonyms
Offi cial
Compendia Specifi cations
Carnauba wax Caranda wax,
Brazil wax
BP, JP,
PhEur,
USPNF
Melting range 80 – 88 ° C a ; iodine value
5 – 14b ; acid value 2 – 7; saponifi cation
value 78 – 95; total ash . 0.25%
Cetyl alcohol Cetanol, Avol,
Lipocol C
BP, JP,
PhEur,
USPNF
Melting range 47 – 53 ° C b ; residue on
ignition . 0.05% b ; iodine value . 5.0;
acid value . 2.0; saponifi cation value
. 2.0 a
Cetyl ester wax Crodamol SS,
Ritachol SS,
Starfol wax
USPNF Melting range 43 – 47 ° C; acid value
. 5.0; saponifi cation value 109 – 120;
iodine value . 1.0
Emulsifying
wax
Collone HV,
Crodex A,
Lipowax PA
BP Saponifi cation value . 2.0; iodine value
. 3.0 c
270 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
Name Synonyms
Offi cial
Compendia Specifi cations
Hydrous lanolin Hydrous wool
fat, Lipolan
BP, JP,
PhEur
Melting range 38 – 44 ° C; acid value
. 0.8; saponifi cation value 67 – 79;
nonvolatile matter 72.5 – 77.5%;
iodine value 18 – 36b
Lanolin Wool fat,
purifi ed wool
fat, Corona
BP, JP,
PhEur,
USPNF
Melting range 38 – 44 ° C; loss on drying
. 0.25%; residue on ignition . 0.1%;
iodine value 18 – 36; acid value . 1.0 b
Lanolin
alcohols
Argowax,
Ritawax, wool
wax alcohol
BP, PhEur,
USPNF
Melting range . 56 ° C; loss on drying
. 0.50%; residue on ignition . 0.15%;
acid value . 2.0; saponifi cation value
. 12
Microcrystalline
wax
Petroleum
ceresin
USPNF Melting range 54 – 102 ° C; residue on
ignition . 0.10%
Paraffi n Paraffi n wax,
hard wax,
hard paraffi n
BP, JP,
PhEur,
USPNF
Melting range 47 – 65 ° C
Petrolatum Yellow soft
paraffi n,
yellow
petroleum
jelly
BP, JP,
PhEur,
USPNF
Melting range 38 – 60 ° C; residue on
ignition . 0.1%
Poloxamer Polyethylene –
propylene
glycol, Lutrol,
Pluronic
BP, PhEur,
USPNF
Melting point . 50 ° C
Polyethylene
glycol (PEG)
Macrogol,
Carbowax,
PEG, Lutrol
BP, JP,
PhEur,
USPNF
Melting range of PEG 1000, 37 – 40 ° C;
melting range of PEG 8000, 60 –
63 ° C; residue on ignition . 0.1%
Stearic acid Emersol,
Hystrene
BP, JP,
PhEur,
USPNF
Melting range . 54 ° C; iodine value . 4.0
Stearyl alcohol Lipocol S,
Cachalot, Rita
SA
BP, JP,
PhEur,
USPNF
Melting range 55 – 60 ° C; residue on
ignition 0.05% b ; iodine value . 2.0;
acid value . 2.0; saponifi cation value
. 2.0 a
White wax Bleached wax BP, JP,
PhEur,
USPNF
Melting range 62 – 65 ° C; acid value 17 –
24; saponifi cation value 87 – 104a
Yellow wax Refi ned wax BP, JP,
PhEur,
USPNF
Acid value 17 – 22 a ; saponifi cation value
87 – 102a
Note : BP, British Pharmacopoeia; JP, Japanese Pharmacopoeia; PhEur, European Pharmacopoeia;
USPNF, U.S. Pharmacopeia/National Formulary. All are USPNF specifi cations, except as indicated
below.
a European Pharmacopoeia.
b Japanese Pharmacopoeia.
c British Pharmacopoeia.
TABLE 1 Continued
OINTMENTS AND CREAMS 271
compendial status, synonym, and specifi cations of some of the bases used in ointments
and creams.
The following sections describe the source, physicochemical properties, formulation
considerations, stability, incompatibility, storage, and hypersensitivity reactions
of some of these bases.
Lanolin Lanolin is a refi ned, decolorized, and deodorized material obtained from
sheep wool. It is available as a pale yellow, waxy material with a characteristic odor.
It is extensively used in the preparation of hydrophobic ointments and water - in - oil
creams. As lanolin is prone to oxidation, antioxidants such as butylated hydroxytoluene
are generally included. Although lanolin is insoluble in water, it is miscible
with water up to 1 : 2 ratio. This property favors in preparing physically stable creams.
Addition of soft paraffi n or vegetable oil improves the emollient property of lanolin
preparations. Exposure of lanolin to higher temperature usually leads to discoloration
and rancidlike odor, and hence prolonged heating is avoided during the
preparation and preservation of lanolin - containing preparations. Gamma sterilization
or fi ltration sterilization is usually employed for sterilizing ophthalmic ointments
containing lanolin. Lanolin and some of its derivatives are reported to cause
hypersensitivity reactions and therefore are avoided in patients with known hypersensitivity.
One of the reasons for hypersensitivity reactions is free fatty alcohols.
Modifi ed lanolins containing reduced levels of free fatty alcohols are commercially
available [2, 3] .
Hydrous Lanolin Incorporation of about 25 – 30% of water into lanolin gives
hydrous lanolin. Gradual addition of water into molten lanolin with constant stirring
helps in water incorporation. It is available as a pale yellow, oily material with a
characteristic odor. The water uptake capacity of hydrous lanolin is higher than
lanolin, and it is used for preparing topical hydrophobic ointments or water - in - oil
creams with larger aqueous phase. Exposure of these preparations to higher temperatures
results in separations of oily and aqueous layers. Addition of antioxidants
and preservation in well - fi lled, airtight, light - resistant containers in a cool and dry
place improve the stability of lanolin products. Well - preserved preparations can be
stored up to two years. Hydrous lanolin that contains free fatty alcohols is avoided
in hypersensitive patients [2, 3] .
Lanolin Alcohols Lanolin alcohol is prepared from lanolin by the saponifi cation
process and is used as a hydrophobic vehicle in pharmaceutical ointments and
creams. It is composed of steroidal and triterpene alcohols and is available as a
brittle solid material pale yellow in color with a faint characteristic odor. The brittle
powder becomes plastic under warm conditions. It is practically insoluble in water
and soluble in boiling ethanol. Lanolin alcohol possesses emollient properties, which
makes it suitable for preparing dry - skin ointments, eye ointments, and water - in - oil
creams. Creams containing lanolin alcohols do not show surface darkening and
do not produce objectionable odor compared to lanolin - containing preparations.
Inclusion of about 0.1% antioxidant, however, minimizes the oxidation on storage.
Preparations containing lanolin alcohols can be stored up to two years if preserved
in well - fi lled, well - closed, light - resistant containers in a cool and dry place. As with
272 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
other lanolin bases, hypersensitivity reactions may occur in some individuals while
using preparations containing lanolin alcohols [2, 3] .
Petrolatum Petrolatum is also known as yellow soft paraffi n. It is an inert material
obtained from petroleum, which contains branched and unbranched hydrocarbons.
It is available as soft oily material and appears pale yellow to yellow in color. Various
grades of petrolatum are commercially available with varying physical properties.
All these grades are generally insoluble in water and possess emollient properties.
Concentrations up to 30% are used in creams. Petrolatum shows phase transitions
on heating to about 35 ° C. As it possesses a higher coeffi cient of thermal expansion,
prolonged heating is avoided during processing. The presence of minor impurities
can oxidize petrolatum and discolor the product. Antioxidants are therefore added
to prevent such physical changes in preparations during storage. Butylated hydroxyanisole,
butylated hydroxytoluene, or . - tocopherol is generally incorporated as an
antioxidant in petrolatum products. In addition, use of well - closed, airtight, light -
resistant containers and storage in a cool and dry place improve stability of preparations.
Minor quantities of polycyclic aromatic hydrocarbon impurities in petrolatum
sometimes cause hypersensitivity reactions. Substituting yellow soft paraffi n with
white soft paraffi n reduces such reactions [4] .
Petrolatum and Lanolin Alcohols Various quantities of lanolin alcohols are mixed
with petrolatum to form these mixtures. Wool ointment British Pharmacopoeia (BP)
2001 contains 6% lanolin alcohols and 10% petrolatum. These proportions can be
varied to alter physical properties such as consistency and melting range. They are
available as soft solids pale ivory in color and possess a characteristic odor. These
mixtures are insoluble in water, and concentrations ranging 5 – 50% are used for
preparing hydrophobic ointments. They are also used for preparing water - in - oil
emollient creams. Preparations containing petrolatum and lanolin alcohols need to
be preserved in airtight, well - closed, light - resistant containers in a cool and dry place
to avoid oxidation of impurities and discoloration. Antioxidants improve the stability
of these products. Although these mixtures are safe for topical applications,
hypersensitivity reactions may occur in some individuals due to the presence of
lanolin alcohol [5] .
Paraffi n Paraffi n is obtained by distillation of crude petroleum followed by puri-
fi cation processes. The purifi ed fraction contains saturated hydrocarbons. Paraffi n is
available as a white color solid and does not possess any specifi c odor or taste. Different
purity grades are available. Use of highly purifi ed grades can avoid batch - to -
batch variations in formulations, especially the hardness, melting behavior, and
malleability. Paraffi n is insoluble in water and is generally used to prepare hydrophobic
topical ointments and water - in - oil creams. Repeated heating and congealing
are avoided during formulation as they change the physical properties of paraffi n.
These preparations need to be preserved in well - closed container at room temperature.
Synthetic paraffi ns, which melt between 96 and 105 ° C, are sometimes used to
increase the melting point and stiffness of formulations [6] .
Polyethylene Glycol Also known as macrogol, PEG is synthesized by condensation
of ethylene oxide and water under suitable reaction conditions. Based on the
OINTMENTS AND CREAMS 273
number of oxyethylene groups present, their molecular weights vary from few hundreds
to several thousands. Usually the number that follows PEG represents their
average molecular weight. They are available as liquids or solids based on molecular
weight. PEGs 600 or less are liquids, whereas PEGs above 1000 are solids. PEG
liquids are usually clear or pale yellow in color. Their viscosity increases with
increase in molecular weight. Solid PEGs are usually white in color and available
as pastes, waxy fl akes, or free - fl owing solids based on their molecular weight. Table
2 shows the physicochemical properties of some PEGs.
PEGs are hydrophilic materials and are extensively used in the preparation of
hydrophilic ointments and creams. They are nonirritants and are easily washed
from skin surfaces. Products with varying consistency are prepared by mixing different
grades of PEGs. Excessive heating is avoided while melting PEGs. This will
prevent oxidation and discoloration of products. In addition, use of purifi ed grades
that are free from peroxide impurities, inclusion of suitable antioxidants, and
heating under nitrogen atmosphere can minimize the oxidation. PEGs are prone
to etherifi cation or esterfi cation reactions due to the presence of two terminal
hydroxyl groups. They are incompatible with some antibiotics, antimicrobial preservatives,
iron, tannic acid, and salicylic acid and also interact with plastic containers
made of polyvinyl chloride and polyethylene. PEG - containing products are
usually packed in aluminum, glass, or stainless steel containers to avoid such interactions.
Although low - molecular - weight PEGs are hygroscopic, they do not promote
microbial growth. PEG - containing products are generally stored in well - closed
containers in a cool, dry place. These products can cause stinging sensation on
mucus and some hypersensitivity reactions, especially when applied onto open
wounds [7, 8] .
Stearic Acid Stearic acid is obtained by hydrolysis of fat or hydrogenation of
vegetable oils. Compendial stearic acid contains a mixture of stearic acid and palmitic
acids. It is available as powder or crystalline solid which is white to yellowish
white in color and possesses a characteristic odor. Although stearic acid is insoluble
TABLE 2 Properties of Different Grades of PEG
Property
By Grade
200 400 600 1000 2000 3000 4000 8000
Physical
state
Liquid Liquid Liquid Solid Solid Solid Solid Solid
Average
molecular
weight
190 – 210 380 – 420 570 – 613 950 – 1050 1800 – 2200 2700 – 3300 3000 – 4800 7000 – 9000
Melting
( ° C)
— — — 37 – 40 45 – 50 48 – 54 50 – 58 60 – 63
Density
(g/cm3 )
1.11 – 1.14 1.11 – 1.14 1.11 – 1.14 1.15 – 1.21 1.15 – 1.21 1.15 – 1.21 1.15 – 1.21 1.15 – 1.21
Kinematic
viscositya
(cS)
3.9 – 4.8 6.8 – 8.0 9.9 – 11.3 16.0 – 19.0 38 – 49 67 – 93 110 – 158 470 – 900
a At 98.9 ° C.
274 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
in water, partially neutralized grades form a cream base when combined with about
10 times its weight of aqueous solvents. The appearance and consistency of these
grades are based on the proportion of alkali or triethanolamine used for neutralization.
Concentrations up to 20% are used for formulating creams and ointments.
Different grades of stearic acids are commercially available with varying stearic acid
content, melting temperature, and other physical properties. A suitable antioxidant
is included in formulations containing stearic acid. As stearic acid interacts with
metals, it is avoided in preparations which contain salts, especially divalent metals
such as calcium and zinc. It also reacts with metal hydroxides and some drugs. Compatibility
evaluation between stearic acid and other formulation components is
therefore essential when formulating newer products with stearic acid [9] .
Carnauba Wax Carnauba wax contains a mixture of esters of acids and hydroxyacids
isolated from Brazilian carnauba palm. It also contains various resins, hydrocarbons,
acids, polyhydric alcohols, and water. It is available as lumps, powder, or
fl akes which are brown to pale yellow in color and possesses a characteristic odor.
Carnauba wax is practically insoluble in water and melts at 80 – 88 ° C. Being a hard
material, it improves the stiffness of topical preparations [6] .
Cetyl Alcohol Cetyl alcohol is obtained by hydrogenolysis or esterfi cation of fatty
acids and contains not less than 90% cetyl alcohol along with other aliphatic alcohols.
It is available as fl akes or granules white in color and possesses a characteristic
odor. Different grades are commercially available with varying proportions of cetyl
alcohol, stearyl alcohol, and related alcohols. Although insoluble in water, cetyl
alcohol has good water - absorptive and emulsifying properties. This property makes
it suitable for preparing emollient ointments and creams. Its viscosity - enhancing
properties reduce coalescence of dispersed phase and improves the physical stability
of creams. Concentrations ranging from 2 to 10% are used in topical preparations
to impart emollient, emulsifying, water - absorptive, and stiffening properties. Mixtures
of petrolatum and cetyl alcohol are sometimes used for preparing creams. Such
mixtures minimize the quantity of additional emulsifying agents in preparations.
Although cetyl alcohol forms stable preparations, it is incompatible with strong
oxidizing materials and some drugs. Compatibility studies are therefore conducted
when including cetyl alcohol into formulations. Highly purifi ed grades are free from
hypersensitivity reactions [3, 10] .
Emulsifying Wax Emulsifying wax, also known as anionic emulsifying wax, is a
mixture of cetostearyl alcohol, sodium lauryl sulfate, and purifi ed water. Emulsifying
wax BP contains about 90% cetostearyl alcohol, 10% sodium lauryl sulfate, and 4%
purifi ed water. Emulsifying wax USP contains nonionic surfactants. It is available
as fl akes or solids which are white to pale yellow in color and possesses a characteristic
odor. Although emulsifying wax is insoluble in water, its emulsifying properties
help in preparing hydrophilic oil - in - water emulsions. Ointment bases are
prepared by mixing up to 50% emulsifying wax with liquid or soft paraffi ns. At
concentrations up to 10%, it forms creams. Although emulsifying wax is compatible
with many acids and alkalis, it is incompatible with many cationic materials and
polyvalent metal salts. Stainless steel vessels are preferred for mixing operations.
OINTMENTS AND CREAMS 275
Preparations containing emulsifying wax are preserved in well - closed container in
a cool, dry place [11] .
Cetyl Esters Wax Cetyl esters wax is obtained by esterifi cation of some fatty alcohols
and fatty acids. It is available as crystalline fl akes which are white to off - white
in color and possesses a characteristic aromatic odor. It is insoluble in water and
has emollient and stiffening properties. About 10% of cetyl ester wax is used for
preparing hydrophobic creams and about 20% is used for preparing topical ointments.
Various grades of cetyl esters wax are available commercially and vary in
their fatty alcohol and fatty acids content and melting range. As this wax is incompatible
with strong acids and bases, it should be avoided in certain formulations.
Cetyl ester wax – containing products are stored in well - closed containers in a cool,
dry place [6] .
Hydrogenated Castor Oil It is used as stiffening agent in hydrophobic ointments
and creams due to its higher melting point. Hydrogenated castor oil contains triglyceride
of hydroxystearic acid and is available as white color fl akes or powder. It
is insoluble in water and melts at 85 – 88 ° C. Different grades with varying compositions
and physical properties are commercially available. Products can be prepared
at higher temperatures, as hydrogenated castor oil is stable up to 150 ° C. It is compatible
with other waxes obtained from vegetable and animal sources. Preparations
containing hydrogenated castor oil need to be preserved in well - closed containers
in a cool and dry place [12] .
Microcrystalline Wax Microcrystalline wax is obtained from petroleum by solvent
fractionation and dewaxing procedures. It contains many straight - chain and
branched - chain alkanes, with carbon chain lengths ranging from 41 to 57. It is available
as fi ne fl akes or crystals which are white or yellow in color. Microcrystalline
wax is insoluble in water and possesses a wide melting range (54 – 102 ° C). High -
melting and stiffening properties of microcrystalline wax make it suitable for preparing
ointments and cream with higher consistency. Acids, alkalis, oxygen, and light
do not affect its stability [6] .
Stearyl Alcohol Reduction of ethyl stearate in the presence of lithium aluminum
hydride yields stearyl alcohol, which contains not less than 90% of 1 - octadecanol.
It is available as fl akes or granules which are white in color and possesses a characteristic
odor. It is insoluble in water and melts at 55 – 60 ° C. Stearyl alcohol has stiffening,
viscosity - enhancing, and emollient properties and hence is used in the
preparation of hydrophobic ointments and creams. Its weak emulsifying properties
help in improving the water - holding capacity of ointments. Hypersensitivity reactions
are sometimes observed due to the presence of some minor impurities. Stearyl
alcohol preparations are compatible with acids and alkalis and are preserved in
well - closed containers in a cool and dry place [6] .
White Wax White wax is a bleached form of yellow wax which is usually obtained
from the honeycomb of bees and hence is known as bleached wax or white bees wax .
It contains about 70% esters of straight - chain monohydric alcohols, 15% free acids,
276 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
12% carbohydrates, and 1% free wax alcohols and stearic esters of fatty acids. It is
available as granules or sheets which are white in color and possesses a characteristic
odor. White wax is insoluble in water and melts between 61 and 65 ° C. It has stiffening
and viscosity - enhancing properties and therefore is used in hydrophobic ointments
and oil - in - water creams. Although it is thermally stable, heating to above
150 ° C results in reduction of its acid value. White wax is incompatible with oxidizing
agents. The presence of small quantities of impurities results in hypersensitivity
reactions in rare occasions. Preparations are stored in well - closed, light - resistant
containers in a cool, dry place [13] .
Yellow Wax Yellow wax, also known as yellow beeswax, is obtained from honey
combs. It contains about 70% esters of straight - chain monohydric alcohols, 15%
free acids, 12% carbohydrates, and 1% free wax alcohols and stearic esters of fatty
acids. It is available as noncrystalline pieces which are yellow in color and possesses
a characteristic odor. It is practically insoluble in water and melts at 61 – 65 ° C. It is
used in the preparation of hydrophobic ointments and water - in - oil creams because
of its viscosity - enhancing properties. Concentrations up to 20% are used for producing
ointments and creams. It is incompatible with oxidizing agents. Esterifi cation
occurs while heating to 150 ° C and hence should be avoided during preparation.
Hypersensitivity reactions sometimes occur on topical application of yellow wax –
containing ointments and creams due to the presence of some minor impurities.
These products are preserved in well - closed, light - resistant containers [13] .
Combinations of bases are sometimes used to acquire better stability. Gelling
agents such as carbomers and PEG are also included in some ointment and cream
preparations. Table 3 shows examples of cream bases used in some commercial
cream preparations.
4.2.2.3 Preparation and Packaging
In addition to the base and drug, ointments and creams may also contain other
components such as stabilizers, preservatives, and levigating agents. Usually levigation
and fusion methods are employed for incorporating these components into the
base. Levigation involves simple mixing of base and other components over an ointment
slab using a stainless steel ointment spatula. A fusion process is employed only
when the components are stable at fusion temperatures. Ointments and creams
containing white wax, yellow wax, paraffi n, stearyl alcohol, and high - molecular -
weight PEGs are generally prepared by the fusion process. Selection of levigation
or the fusion method depends on the type base, the quantity of other components,
and their solubility and stability characteristics.
Oleaginous ointments are prepared by both levigation and fusion processes.
Small quantities of powders are incorporated into hydrocarbon bases with the aid
of a levigating agent such as liquid petrolatum, which helps in wetting of powders.
The powder component is mixed with the levigating agent by trituration and is then
incorporated into the base by spatulation. All solid components are milled to fi ner
size and screened before incorporating into the base to avoid gritty sensation of the
fi nal product. Roller mills are used for producing large quantities of ointments in
pharmaceutical industries. Uniform mixing can be obtained by the geometric dilution
procedure, which usually involves stepwise dilution of solids into the ointment
OINTMENTS AND CREAMS 277
TABLE 3 Cream Bases Present in Some Commercial Creams
Commercial Name Drug Cream Base (s) Used
Dritho - Calp,
Psoriatec
Anthralin, 0.5%, 1.0% White petrolatum, cetostearyl alcohol
Temovate E Clobetasol propionate,
0.05%
Propylene glycol, glyceryl monostearate,
cetostearyl alcohol, glyceryl stearate,
PEG 100 stearate, white wax
Eurax Crotamiton, 10% Petrolatum, propylene glycol, cetyl
alcohol, carbomer - 934
Topicort Desoximetasone,
0.25%
White petrolatum USP, isopropyl
myristate NF, lanolin alcohols NF,
mineral oil USP, cetostearyl alcohol NF
Apexicon, Maxifl or,
Psorcon
Difl orasone diacetate,
0.05%
Hydrophilic vanishing cream base of
propylene glycol, stearyl alcohol, cetyl
alcohol
Lidex Cream, Vanos Fluocinonide, 0.05%,
0.10%
Polyethylene glycol 8000, propylene
glycol, stearyl alcohol
Carac Fluorouracil, 0.5%,
1.0%, 5.0%
Carbomer - 940, PEG 400, propylene
glycol, stearic acid
Halog Halcinonide, 0.1% Polyethylene and mineral oil gel base
with PEG 400, PEG 6000, PEG 300,
PEG 1450
Cortaid, Anusol-Hc,
Proctosol HC
Hydrocortisone, 2.5%
water washable
Petrolatum, stearyl alcohol, propylene
glycol, carbomer - 934
Monistat - Derm Miconazole nitrate,
2%
Water - miscible base consisting of pegoxol
7 stearate, peglicol 5 oleate, mineral oil,
butylated hydroxyanisole
base. The fusion method is followed when the drugs and other solids are soluble in
the ointment bases. The base is liquefi ed, and the soluble components are dissolved
in the molten base. The mixture is then allowed to congeal by cooling. Fusion is
performed using steam - jacketed vessels or a porcelain dish. The congealed mixture
is then spatulated or triturated to obtain a smooth texture. Care is taken to avoid
thermal degradation of the base or other components during the fusion process.
Absorption - type ointments and creams are prepared by incorporating large
quantities of water into hydrocarbon bases with the aid of a hydrophobic emulsifying
agent. Water - insoluble drugs are added by mechanical addition or fusion methods.
As with oleaginous ointments, levigating agents are also included to improve wetting
of solids. Water - soluble or water - miscible agents such as alcohol, glycerin, or propylene
glycol are used if the drug needs to be incorporated into the internal aqueous
phase. If the drug needs to be incorporated into the external oily phase, mineral oils
are used as the levigating agent. Incorporation of water - soluble components is
achieved by slowly adding the aqueous drug solution to the hydrophobic base using
pill tile and spatula. If the proportion of aqueous phase is larger, inclusion of additional
quantities of emulsifi er and application of heat may be needed to achieve
uniform dispersion. Care must be taken to avoid excessive heating as it can result
in evaporation aqueous phase and precipitation of water - soluble components and
formation of stiff and waxy product.
278 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
Water - removable ointments and creams are basically hydrophilic - type emulsions.
They are prepared by fusion followed by mechanical addition approach. Hydrocarbon
components are melted together and added to the aqueous phase that
contains water - soluble components with constant stirring until the mixture congeals.
A hydrophilic emulsifying agent is included in the aqueous phase in order to obtain
stable oil - in - water dispersion. Sodium lauryl sulfate is used in the preparation of
hydrophilic ointment USP.
Water - soluble ointments and creams do not contain any oily phase. Both water -
soluble and water - insoluble components are incorporated into water - soluble bases
by both levigation and fusion methods. If the drug and other components are water
soluble, they are dissolved in a small quantity of water and incorporated into the
base by simple mixing over an ointment slab. If the components are insoluble in
water, aqueous levigating agents such as glycerin, propylene glycol, or a liquid PEG
are used. The hydrophobic components are mixed with the levigating agent and then
incorporated into the base. Heat aids incorporation of a large quantity of hydrophobic
components.
A wide range of machines are available for the large - scale production of ointments
and creams. Each of these machines is designed to perform certain unit
operations, such as milling, separation, mixing, emulsifi cation, and deaeration.
Milling is performed to reduce the size of actives and other additives. Various fl uid
energy mills, impact mills, cutter mills, compression mills, screening mills, and tumbling
mills are used for this purpose. Alpine, Bepex, Fluid Air, and Sturtevant are
some of the manufacturers of these mills. Separators are employed for separating
materials of different size, shape, and densities. Either centrifugal separators or
vibratory shakers are used for separation. Mixing of the actives and other formulation
components with the ointment or cream base is performed using various types
of low - shear mixers, high - shear mixers, roller mills, and static mixers. Mixers with
heating provisions are also used to aid in the melting of bases and mixing of components.
Chemineer, Fryma, Gate, IKA, Koruma (Romaco), Moorhouse - Cowles,
Ross, and Stokes Merrill are some of the manufacturers of semisolids mixers.
Creams are produced with the help of low - shear and high - shear emulsifi ers.
These emulsifi ers are used to disperse the hydrophilic components in the hydrophobic
dispersion phase (e.g., water - in - oil creams) or oleaginous materials in aqueous
dispersion medium (oil - in - water creams). Bematek, Fryma, Koruma (Romaco),
Lightnin, Moorhouse, and Ross supply various types of emulsifi ers. Entrapment of
air into the fi nal product due to mixing processes is a common issue in the large -
scale manufacturing of semisolid dosage forms. Various offl ine and in - line deaeration
procedures are adopted to minimize this issue. Effective deaeration is generally
achieved by using vacuum vessel deaerators. Some of the recent large - scale machines
are designed to perform heating, high - shear mixing, scrapping, and deaeration processes
in a single vessel. Figure 1 shows the design feature of a semisolid production
machine manufactured by Ross.
Various low - and high - shear shifters are used to transfer materials from the production
vessel to the packaging machines. In the packaging area, various types of
holders (e.g., pneumatic, gravity, and auger holders), fi llers (e.g., piston, peristaltic
pump, gear pump, orifi ce, and auger fi llers), and sealers (e.g., heat, torque, microwave,
indication, and mechanical crimping sealers) are used to complete the unit
OINTMENTS AND CREAMS 279
operations. These equipments are supplied by various manufacturers, namely Bosch,
Bonafacci, Erweka, Fryma - Maschinenbau, IWKA, Kalish, and Norden.
Sterility of ointments, especially those intended for ophthalmic use, is achieved
by aseptic handling and processing. Improper processing, handling, packing, or use
of ophthalmic ointments lead to microbial contaminations and eventually result in
ocular infections. In general, the empty containers are separately sterilized and fi lled
under aseptic condition. Final product sterilization by moist heat sterilization or
gaseous sterilization is ineffective because of product viscosity. Dry - heat sterilization
is associated with stability issues. Strict aseptic procedures are therefore practiced
when processing ophthalmic preparations. Antimicrobial preservatives such as
benzalkonium chloride, phenyl mercuric acetate, chlorobutanol, or a combination
of methyl paraben and propyl paraben are included in ophthalmic ointments to
retain microbial stability.
Packaging An ideal container should protect the product from the external atmosphere
such as heat, humidity, and particulates, be nonreactive with the product
components, and be easy to use, light in weight, and economic [14] . As tubes made
of aluminum and plastic meet most of these qualities, they are extensively used for
packaging semisolids. Aluminum tubes with special internal epoxy coatings are
commercially available for improving the compatibility and stability of products.
Various modifi ed plastic materials are used for making ointment tubes. Tubes made
FIGURE 1 Semisolid production machine with heat jacketed vessel, high - shear mixer,
scrapper, vacuum attachments, and control station. (Courtesy of Ross, Inc.)
280 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
of low - density polyethylene (LDPE) are generally soft and fl exible and offer good
moisture protection. Tubes made of high - density polyethylene (HDPE) are relatively
harder but offer high moisture protection. Polypropylene containers offer
high heat resistance. Plastic containers made of polyethylene terephthalate (PET)
are transparent and provide superior chemical compatibility. Ointments meant for
ophthalmic, nasal, rectal, and vaginal applications are supplied with special application
tips for the ease of product administration.
A recent method known as blow fi ll sealing (BFS) performs fabrication of
container, fi lling of product, and sealing operations in a single stage and hence is
gaining greater attention. The products can be sterile fi lled, which makes BFS a
cost - effective alternative for aseptic fi lling. All plastic materials are suitable for
BFS processing. In most cases, monolayered LDPE materials are used for making
small - size containers. If the product is not compatible with the LDPE or sensitive
to oxygen, barrier layers are added to the container wall by coextrusion methods.
As the container is formed inside the BFS machine, upstream handling problems
are avoided. The BFS machine can hand the container off to any secondary packaging
operation that needs to be performed. Typically a secondary overwrap is added
to the containers prior to cartooning. An additional advantage of BFS containers is
the integrated design of the applicator into the product container. Figure 2 shows
some of the custom - designed BFS containers for topical products.
4.2.2.4 Evaluation
Ointments and creams are evaluated for various pharmacopeial and nonpharmacopeial
tests to ascertain their physicochemical, microbial, in vitro, and in vivo
characteristics. These tests help in retaining their quality and minimizing the batch -
to - batch variations. The USP recommends storage and labeling, microbial screening,
minimum fi ll, and assays for most ointments and creams. Tables 4 and 5 summarize
the compendial requirements for some pharmacopeial ointments and creams.
FIGURE 2 Custom - designed LDPE containers made by BFS process for packaging topical
products. (Courtesy of Rommelag USA, Inc.)
OINTMENTS AND CREAMS 281
TABLE 4 USP Specifi cations for Some Offi cial Ointments
Drug Quality Control Tests Packaging and Storage Requirements
Acyclovir Staphylococcus aureus,
Pseudomonas aeruginosa ,
minimum fi ll, limit of
guanine, and assay
Tight containers; store between 15 and
25 ° C in a dry place
Alclometasone
dipropionate
S. aureus, P. aeruginosa ,
minimum fi ll, and assay
Collapsible tubes or tight containers,
store at controlled room temperature
Amphotericin B Minimum fi ll, water, and
assay
Collapsible tubes or other well - closed
containers
Anthralin Assay Tight containers; in a cool place; protect
from light
Bacitracin Minimum fi ll, water, and
assay
Well - closed containers containing not
more than 60 g; controlled temperature
Benzocaine S. aureus, P. aeruginosa ,
minimum fi ll, and assay
Tight containers; protect from light; avoid
prolonged exposure to temperatures
exceeding 30 ° C
Betamethasone
valerate
S. aureus, P. aeruginosa ,
minimum fi ll, and assay
Collapsible tubes or tight containers;
avoid exposure to excessive heat.
Clioquinol Assay Collapsible tubes or tight, light - resistant
containers
Clobetasol
propionate
S. aureus, P. aeruginosa,
Escherichia coli,
Salmonella species, total
aerobic microbial count,
minimum fi ll, and assay
Collapsible tubes or in tight containers;
store at controlled room temperature;
do not refrigerate
Erythromycin Minimum fi ll, water, and
assay
Collapsible tubes or in tight containers at
controlled room temperature
Fluocinolone
acetonide
S. aureus, P. aeruginosa , and
assay
Tight containers
Gentamycin
sulfate
Minimum fi ll, water, and
assay
Collapsible tubes or in tight containers;
avoid exposure to excessive heat
Hydrocortisone
valerate
S. aureus, P. aeruginosa ,
total microbial count,
minimum fi ll, and assay
Tight container; store at room
temperature
Ichthammol Assay Collapsible tubes or in tight containers;
avoid prolonged exposure to
temperatures exceeding 30 ° C
Lidocaine S. aureus, P. aeruginosa ,
minimum fi ll, and assay
Tight containers
Mometasone
furoate
S. aureus, P. aeruginosa, E.
coli, Salmonella species,
minimum fi ll, and assay
Well - closed containers
Nitrofurazone Completeness of solution
and assay
Tight, light - resistant containers; avoid
exposure to direct sunlight, strong
fl uorescent lighting, and excessive heat
Nitroglycerine Minimum fi ll, homogeneity,
and assay
Tight containers
Nystatin Minimum fi ll, water, and
assay
Well - closed containers at controlled
room temperature
Tetracycline
hydrochloride
Minimum fi ll, water, and
assay
Well - closed containers at controlled
room temperature
Zinc oxide Minimum fi ll, calcium,
magnesium, other foreign
substances, and assay
Tight containers; avoid prolonged
exposure to temperatures exceeding
30 ° C
282 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
TABLE 5 USP Specifi cations for Some Offi cial Creams
Cream Quality Control Tests Packaging and Storage Requirements
Alclometasone
dipropionate
Microbial limits, minimum
fi ll, and assay
Collapsible tubes or tight containers; store
at controlled room temperature
Amphotericin B Minimum fi ll and assay Collapsible tubes or other well - closed
containers
Benzocaine Microbial limits, minimum
fi ll, and assay
Tight containers, protected from light,
and avoid prolonged exposure to
temperatures exceeding 30 ° C
Betamethasone
dipropionate
Minimum fi ll and assay Collapsible tubes or tight containers; store
at 25 ° C; excursions permitted between
15 and 30; protect from freezing
Ciclopirox
olamine
Minimum fi ll, pH, content
of benzyl alcohol, and
assay
Collapsible tubes at controlled room
temperature
Clobetasol
propionate
Microbial limits, minimum
fi ll, pH, and assay
Collapsible tubes or tight containers; store
at controlled room temperature; do not
refrigerate
Clotrimazole Assay Collapsible tubes or tight containers at a
temperature between 2 and 30 ° C
Desoximetasone Minimum fi ll, pH, and
assay
Collapsible tubes at controlled room
temperature
Dibucaine Microbial limits, minimum
fi ll, and assay
Collapsible tubes or in tight, light - resistant
containers
Dienestrol Minimum fi ll and assay Collapsible tubes or in tight containers
Difl orasone
diacetate
Microbial limits, minimum
fi ll, and assay
Collapsible tubes, preferably at controlled
room temperature
Fluocinolone
acetonide
Microbial limits, minimum
fi ll, and assay
Collapsible tubes or in tight containers
Fluorouracil Microbial limits, minimum
fi ll, and assay
Tight containers and stored at controlled
room temperature
Gentamycin
sulfate
Minimum fi ll and assay Collapsible tubes or in other tight
containers; avoid exposure to excessive
heat
Hydrocortisone
butyrate
Microbial limits, minimum
fi ll, pH, and assay
Well - closed containers
Hydroquinone Minimum fi ll and assay Well - closed, light - resistant containers
Lindane pH and assay Tight containers
Meclocycline
sulfosalicylate
Minimum fi ll and assay Tight containers, protected from light
Miconazole
nitrate
Minimum fi ll and assay Collapsible tubes or tight containers; store
at controlled room temperature
Monobenzone Assay Tight containers; avoid exposure to
temperatures higher than 30 ° C
Nystatin Minimum fi ll and assay Collapsible tubes or in other tight
containers; avoid exposure to excessive
heat
Prednisolone Minimum fi ll and assay Collapsible tubes or in tight containers
Tetracaine
hydrochloride
Microbial limits, minimum
fi ll, pH between 3.2 and
3.8, and assay
Collapsible, lined metal tubes
Triamcinolone
acetonide
Microbial limits, minimum
fi ll, and assay
Tight containers
OINTMENTS AND CREAMS 283
Packaging and Storage The USP recommends packaging and storage requirements
for each offi cial ointment and cream. Generally collapsible tubes, tight containers,
or other well - closed containers are recommended for packing. They are
stored in either a cool place or at controlled room temperatures. In some cases,
special storage conditions are recommended: for example, protect from light, avoid
exposure to excessive heat, avoid exposure to direct sunlight, avoid strong fl uorescent
lighting, do not refrigerate, and avoid prolonged exposure to temperatures
exceeding 30 ° C.
Minimum Fill This test is performed to compare the weight or volume of product
fi lled into each container with their labeled weight or volume. It helps in assessing
the content uniformity of product. A minimum - fi ll test is applied only to those
containers that contain not more than 150 g or mL of preparation. It is performed
in two steps. Initially, labels from the product containers are removed. After washing
and drying the surface, their weights are recorded ( W1 ). In the second step, the entire
product from each container is removed. After cleaning and drying, the weight of
empty containers is recorded ( W2 ). The difference between total weight ( W1 ) and
empty - container weight ( W2 ) gives the weight of product. The USP recommends
that the average net content of 10 containers should not be less than the labeled
amount. If the product weight is less than 60 g or mL, the net content of any single
container should not be less than 90% of the labeled amount. If the product weight
is between 60 and 150 g or mL, the net content of any single container should not
be less than 95% of the labeled amount. If these limits are not met, the test is
repeated with an additional 20 containers. All semisolid topical preparations should
meet these specifi cations [15] .
Water Content The presence of minor quantities of water may alter the microbial,
physical, and chemical stability of ointments and creams. Titrimetric methods
(method I) are usually performed for determining the water content in these preparations.
These methods are based on the quantitative reaction between water and
anhydrous solution of sulfur and iodine in the presence of a buffer that can react
with hydrogen ions. Special titration setups and reagents (Karl Fischer, KF) are used
in these determinations. In the direct method (method Ia), about 35 mL of methanol
is titrated with suffi cient quantity of KF reagent to the electrometric or visual endpoint
(color change from canary yellow to amber). This blank titration helps to
consume any moisture that may be present in the reaction medium. A known quantity
of test material (ointment or cream) is added to the reaction medium, mixed,
and again titrated with KF reagent to the reaction endpoint. The water content is
determined by considering the volume of KF reagent consumed and its water
equivalence factor. In the residual titration method (method Ib), a known excess
quantity of KF reagent is added to the titration vessel, which is then back titrated
with standardized water to the electrometric or visual endpoint. In the coulometric
titration method (method Ic), the sample is dissolved in anhydrous methanol and
injected into the reaction vessel that contains the anolyte, and the coulometric reaction
is performed until the reaction endpoint. In some cases, methanol is replaced
with other solvents. The maximum allowable limit of water in ointment preparations
varies between 0.5 and 1.0%. The limit of water in bacitracin, chlortetracycline
hydrochloride, and nystatin ointments is not more than 0.5%, whereas amphotericin
284 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
B, erythromycin, gentamycin sulfate, neomycin sulfate, and tetracycline hydrochloride
ointments may contain up to 1% moisture [15] .
Metal Particles This test is required only for ophthalmic ointments. The presence
of metal particles will irritate the corneal or conjunctival surfaces of the eye. It is
performed using 10 ointment tubes. The content from each tube is completely
removed onto a clean 60 - mm - diameter petridish which possesses a fl at bottom. The
lid is closed and the product is heated at 85 ° C for 2 h. Once the product is melted
and distributed uniformly, it is cooled to room temperature. The lid is removed after
solidifi cation. The bottom surface is then viewed through an optical microscope at
30. magnifi cation. The viewing surface is illuminated using an external light source
positioned at 45 ° on the top. The entire bottom surface of the ointment is examined,
and the number of particles 50 . m or above are counted using a calibrated eyepiece
micrometer. The USP recommends that the number of such particles in 10 tubes
should not exceed 50, with not more than 8 particles in any individual tube. If these
limits are not met, the test is repeated with an additional 20 tubes. In this case, the
total number of particles in 30 tubes should not exceed 150, and not more than 3
tubes are allowed to contain more than 8 particles [15] .
Leakage Test This test is mandatory for ophthalmic ointments, which evaluates
the intactness of the ointment tube and its seal. Ten sealed containers are selected,
and their exterior surfaces are cleaned. They are horizontally placed over absorbent
blotting paper and maintained at 60 ± 3 ° C for 8 h. The test passes if leakage is not
observed from any tube. If leakage is observed, the test is repeated with an additional
20 tubes. The test passes if not more than 1 tube shows leakage out of 30
tubes [15] .
Sterility Tests Ophthalmic semisolids should be free from anaerobic and aerobic
bacteria and fungi. Sterility tests are therefore performed by the membrane fi ltration
technique or direct - inoculation techniques. In the membrane fi ltration method,
a solution of test product (1%) is prepared in isopropyl myristate and allowed to
penetrate through cellulose nitrate fi lter with pore size less than 0.45 . m. If necessary,
gradual suction or pressure is applied to aid fi ltration. The membrane is then
washed three times with 100 - mL quantities of sterile diluting and rinsing fl uid and
transferred aseptically into fl uid thioglycolate (FTG) and soybean – casein digest
(SBCD) medium. The membrane is fi nally incubated for 14 days. Growth on FTG
medium indicates the presence of anaerobic and aerobic bacteria, and SBCD
medium indicates fungi and aerobic bacteria. Absence of any growth in both these
media establishes the sterility of the product. In the direct - inoculation technique, 1
part of the product is diluted with 10 parts of sterile diluting and rinsing fl uid with
the help of an emulsifying agent and incubated in FTG and SBCD media for 14
days. In both techniques, the number of test articles is based on the batch size of
the product. If the batch size is less than 200 the containers, either 5% of the containers
or 2 containers (whichever is greater) are used. If the batch size is more than
200, 10 containers are used for sterility testing [15] .
Microbial Screening Semisolid preparations are required to be free from any
microbial contamination. Hence, most of the topical ointments are screened for the
OINTMENTS AND CREAMS 285
presence of Staphylococcus aureus and Pseudomonas aeruginosa . In some cases,
screening for Escherichia coli, Salmonella species, and total aerobic microbial counts
is recommended by the USP. For instance, clobetasol propionate ointment USP and
mometasone furoate ointment USP are screened for all these organisms. In addition,
preparations meant for rectal, vaginal, and urethral applications are tested for yeasts
and molds [15] .
Test for S. aureus and P. aeruginosa The test sample is mixed with about 100 mL
of fl uid soybean – casein digest (FSBCD) medium and incubated. If microbial growth
is observed, it is inoculated in agar medium by the streaking technique. Vogel –
Johnson agar (VJA) medium is used for S. aureus screening, and cetrimide agar
(CA) medium is used for screening P. aeruginosa . The petridishes are then closed,
inverted, and incubated under appropriate conditions. The appearance of black
colonies surrounded by a yellow zone over VJA medium and greenish colonies in
CA medium indicates the presence of S. aureus and P. aeruginosa , respectively.
Various other agar media are also available for screening these organisms. A coagulase
test is then performed for confi rming the presence of S. aureus and oxidase and
pigment tests for confi rming P. aeruginosa .
Test for Salmonella Species and E. coli The test sample is mixed with about 100 mL
of fl uid lactose (FL) medium and incubated. If microbial growth is observed, the
contents are mixed and 1 mL is transferred to vessels containing 10 mL of fl uid
selinite cystine (FSC) medium and fl uid tetrathionate (FT) medium and incubated
for 12 – 24 h under appropriate conditions. To identify the presence of Salmonella ,
samples from the above two media are streaked over brilliant green agar (BGA)
medium, xylose lysine desoxycholate agar (XLDA) medium, and bismuth sulfi te
agar (BSA) medium and incubated. The appearance of small, transparent or pink -
to - white opaque colonies over BGA medium, red colonies with or without black
centers over XLDA medium, and black or green colonies over BSA medium indicates
the presence of Salmonella . It is further confi rmed in triple sugar iron agar
medium. The presence of E. coli is screened by streaking the samples from FL
medium over MacConkey agar medium. The appearance of brick red colonies indicates
the presence of E. coli . It is further confi rmed using Levine eosin methylene
blue agar medium.
total aerobic microbial counts The plate method or multiple - tube method is
performed to estimate the total count. About 10 g or 10 mL of the test sample is
dissolved or suspended in suffi cient volume of phosphate buffer (pH 7.2), fl uid
soybean casein digest (FSBCD) medium, or fl uid casein digest – soy lecithin –
polysorbate 20 medium to make the fi nal volume 100 mL. In the plate method, about
1 mL of this diluted sample is mixed with molten soybean – casein digest agar
(SBCDA) medium and solidifi ed at room temperature. The plates are inverted and
incubated for two to three days. The number of colonies that are on the surface of
nutrient media are counted. The multiple tube method is performed using sterile
fl uid SBCD medium. The number of colonies formed should not exceed the limits
specifi ed in an individual monograph. For example, clobetasol propionate ointment
USP and hydrocortisone valerate ointment USP contains less than 100 colony -
forming units (CFU) per gram of sample.
286 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
Test for Yeasts and Molds The plate method is used for testing molds and yeast in
semisolids. The procedure is similar to that of the total count test. Instead of SBCDA
medium, Sabouraud dextrose agar (SDA) medium or potato dextrose agar (PDA)
medium is used. Samples are incubated for fi ve to seven days at 20 – 25 ° C to identify
the presence of yeasts and molds.
Assay The quantity of drug present in a unit weight or volume of ointment or
cream is determined by various methods. Spectrophotometric, titrimetric, chromatographic,
and in some cases microbial assays are performed. Selection of a particular
method is based on the nature of drug, its concentration in the product, interference
between the drug and other formulation components, and offi cial requirements.
Although spectrophotometric methods are accurate and easy to perform, the complexity
of ointment matrix sometimes reduced the specifi city of analysis compare
to liquid chromatographic methods. The USP prescribes high - performance liquid
chromatographic (HPLC) assays for many offi cial ointments due to its specifi city,
accuracy, and precision. For example, amcinonide, anthralin, betamethasone dipropionate,
clobetasol propionate, dibucaine, nitroglycerine, hydrocortisone, and triamcinolone
acetonide are assayed by HPLC methods. These methods involve extraction
of drug from the formulation matrix using suitable solvents followed by chromatographic
separation using suitable reversed - phase columns followed by ultraviolet
(UV) detection. Clioquinol preparation is assayed by gas chromatography. The USP
also recommends potentiometric titrations (benzocaine, lidocaine, and ichthammol)
and complexometric titrations (zinc oxide) for some semisolid preparations.
Microbial assays are recommended for certain preparations containing antibiotics
such as amphotericin B, bacitracin, chlortetracycline hydrochloride, gentamycin
sulfate, neomycin sulfate, and nystatin. These tests evaluate the potency of an antibiotic
by means of its inhibitory effects on specifi c microorganism. Two types of
microbial assays are performed to determine the antibiotic potency. They are known
as cylindrical plate or plate assays and turbidimetric or tube assays. The plate
method measures the extent of growth inhibition of a particular microorganism in
solidifi ed agar medium in the presence of the test antibiotic (commonly known as
zone of inhibition ). The tube method measures the turbidity of a liquid medium that
contains a particular organism in the presence and absence of the test antibiotic.
These methods involve extracting drug from the formulation matrix, diluting the
drug to a known concentration, and measuring the zone of inhibition or turbidity.
In Vitro Drug Release Studies These studies are conducted to ascertain release
of drug from the formulation matrix. Open - chamber diffusion cells such as Franz
cells are used for performing in vitro studies. These cells consist of a donor side and
a receiver side separated by a synthetic membrane such as cellulose acetate/nitrate
mixed ester, polysulfone, or polytetrafl uoroethylene. The membranes are usually
pretreated with the receiver fl uid to avoid any lag phase in drug release. The receiver
side is fi lled with a known volume of release medium and is heated to 32 ± 0.5 ° C
by circulating warm water through an outer jacket. Aqueous buffers are used for
water - soluble drugs. Phosphate buffer solution of pH 5.4 is considered most appropriate
for dermatological products as it mimics the pH of skin. Hydroalcoholic or
other suitable medium may also be used for sparingly water soluble drugs. A known
quantity of the test product is applied uniformly over the membrane on the donor
OINTMENTS AND CREAMS 287
side and samples are withdrawn from the receiver side at different time intervals.
After each sampling, an equal volume of fresh medium is replaced to the receiver
side. The sampling time points are different for different formulations; however, at
least fi ve samples are withdrawn during the study period for determining the release
rate. A typical sample time sequence for a 6 - h study is 0.5, 1.0, 2.0, 4.0, and 6.0 h.
The receiver samples are analyzed by a suitable analytical method to quantify the
amount of drug released from the formulation at different time intervals. The slope
of the straight line which is obtained from a plot of cumulative amount drug release
across 1 - cm 2 membrane versus the square root of time represents the release rate.
Experiments are conducted in hexaplicate to obtain statistically signifi cant results
[16] .
In Vivo Bioequivalence Studies In vivo studies are conducted to establish the
biological availability or activity of the drug from a topically applied semisolid formulation.
Dermatopharmacokinetic studies, pharmacodynamic studies, or comparative
clinical trials are generally conducted to assess the bioequivalence of topical
products [16, 17] .
Dermatopharmacokinetic (DPK) studies are applicable for topical semisolid
products that contain antifungals, antivirals, corticosteroids, and antibiotics and
vaginally applied products. They are not applicable for ophthalmic, otic, and other
products that damage stratum corneum. DPK studies involve measurement of drug
concentrations in stratum corneum, drug uptake, apparent steady state, and elimination
after application of the test product onto skin. These studies are conducted in
healthy human subjects adopting crossover design. The test and the reference products
are applied onto eight to nine sites in the forearm. The surface area of each
site is based on the strength of drug, extent of drug diffusion, exposure time, and
sensitivity of the analytical technique. The application site is washed and allowed to
normalize for at least 2 h prior to drug application. A known amount of product is
applied onto these selected sites. At appropriate time intervals, the excess of drug
from each area is removed using cotton swabs or soft tissue papers. Care is taken
to avoid stratum corneum damage during sample collection. Stripping of stratum
corneum is performed using adhesive tape - strips (e.g., D - Squame, Transpore). In the
elimination phase, the excess drug is removed at the steady - state time point, and
the stratum corneum is harvested at succeeding times over 24 h. The drug content
from strips from each time point are extracted using suitable solvents and quantifi ed
by a validated analytical method. A stratum corneum drug levels – time curve is
developed, and pharmacokinetic parameters such as maximum concentration at
steady state ( Cmax - ss ), time to reach Cmax - ss ( Tmax - ss ), and areas under the curve for the
test and standard (AUC test and AUC reference ) are computed. DPK studies are performed
in either one or two occasions. If performed in one occasion, both arms of
a single subject are used to compare the test and reference products. If performed
in two occasions, a wash - out period of at least 28 days is allowed to rejuvenate the
harvested stratum corneum.
Pharmacodynamic (PD) studies are also performed to estimate the bioavailability
and bioequivalence of drugs from topically applied semisolids. In this case,
known therapeutic responses from drug products such as skin blanching due to
vasoconstrictor effects caused by corticosteroids and transepidermal water loss
caused by retinoids are measured and compared between the test and reference
288 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
products. Comparative clinical studies are rarely conducted due to the diffi culties
involved in performing the study, variability in study results, and their poor
sensitivity.
4.2.2.5 Typical Pharmacopeial/Commercial Examples
The vast majority of topical ointments and creams are meant for dermatological
applications. They are used to treat various skin conditions such as eczema, dermatitis,
allergies, infl ammatory and pruritic manifestations, minor skin wounds, pain,
insect bites, psoriasis, herpes and other infections of the skin (e.g., impetigo), acne,
and precancerous and cancerous skin growths. Similarly, ophthalmic conditions such
as infections, infl ammation, allergy, and dry - eye symptoms are treated with semisolid
preparations. Products are also available for certain eye examinations. Vaginal preparations
are available for treating genital herpes, yeast infections, and vaginosis
caused by bacteria and to reduce menopausal symptoms (e.g., vaginal dryness), and
rectal preparations are available for treating minor pain, itching, swelling, and discomfort
caused by hemorrhoids and other problems of the anal area. Tables 6 and
7 show some of the commercially available compendial ointment and cream preparations
used for treating various topical ailments.
4.2.3 GELS
4.2.3.1 Defi nition
Gels are semisolid preparations that contain small inorganic particles or large
organic molecules interpenetrated by a liquid. Gels made of inorganic materials are
usually two - phase systems where small discrete particles are dispersed throughout
the dispersion medium. When the particle size of the dispersed phase is larger, they
are referred to as magmas. Gels made of organic molecules are single - phase systems,
where no apparent physical boundary is seen between the dispersed phase and the
dispersion medium. In most cases, the dispersion medium is aqueous. Hydroalcoholic
or oleaginous dispersion media are also used in some cases. Unlike dispersed
systems like suspensions and emulsions, movement of the dispersed phase is
restricted in gels because of the solvated organic macromolecules or interconnecting
three - dimensional networks of particles.
Gels are attractive delivery systems as they are simple to manufacture and
suitable for administering drugs through skin, oral, buccal, ophthalmic, nasal,
otic, and vaginal routes. They also provide intimate contact between the drug and
the site of action or absorption. With the advancement in polymer science, gel - based
systems that respond to specifi c biological or external stimuli like pH, temperature,
ionic strength, enzymes, antigens, light, magnetic fi eld, ultrasound, and electric
current are being designed and evaluated as smart delivery systems for various
applications.
4.2.3.2 Characteristics
Gels may appear transparent or turbid based on the type of gelling agent used. They
exhibit different physical properties, namely, imbibition, swelling, syneresis, and
TABLE 6 Examples of Compendial/Commercial Ointments
Drug a Category Indication
Commercial
Names
Strength(s)
Available
(%)
Acyclovir Antiviral Genital herpes,
herpes infections
of the skin, and
oral herpes
Zovirax 5
Atropine
sulfate (oph)
Mydriatic Relax muscles in the
eye, treat
infl ammation of
certain parts of
the eye (uveal
tract), and used
for certain eye
exams
Atropisol,
Isopto
Atropine
0.5, 1
Bacitracin First - aid antibiotic Treat or prevent skin
infections
Baciguent
Oint,
Bacitracin
Top
500 units/g
Bacitracin
(oph)
Antibiotic Treat or prevent eye
infections
Ak - Tracin,
Bacticin
500 units/g
Benzocaine Antipruritic and
local anesthetic
Itching, minor skin
wound pain, and
insect bites
Americaine 20
Ciprofl oxacin
(oph)
Antibiotic Eye infections Ciloxan 0.3
Clobetasol
propionate
Anti - infl ammatory
agent
Relieve
infl ammatory
and pruritic
manifestations of
corticosteroid -
responsive
dermatoses
Temovate
Ointment
0.05
Erythromycin Antibiotic Treatment of acne Akne - Mycin 2.0
Erythromycin
(oph)
Antibiotic Infections of eye or
ear
Erythromycin
Ophthalmic
0.5
Gentamicin
sulfate (oph)
Antibacterial Infections of eye or
ear
Gentamicin
Sulfate
0.3
Hydrocortisone Anti - infl ammatory
agent
Minor pain, itching,
swelling, and
discomfort caused
by hemorrhoids
and other
problems of
anal area
Cortaid,
Anusol - HC,
Proctosol
HC
2.5
Mupirocin Antibiotic Treat certain skin
infections (e.g.,
impetigo)
Bactroban 2.0
Sodium
chloride
(oph)
Miscellaneous Treat fl uid
accumulation in
cornea of eye
causing swelling
Muro - 128,
Sochlor
2.0
a oph: ophthalmic ointment.
GELS 289
290 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
TABLE 7 Examples of Compendial/Commercial Creams
Drug a Category Indication
Commercial
Products
Strength(s)
Available
(%)
Alclometasone
dipropionate
Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash
Aclovate 0.05
Amcinonide Anti -
infl ammatory
Lymphomas of the skin,
atopic dermatitis,
contact dermatitis,
and skin rash
Cyclocort 0.1
Amphotericin B Antifungal
antibiotic
Treat skin infection due
to a Candida yeast
and diaper rash
Fungizone 3.0
Anthralin Keratolytic Long - term psoriasis Dritho - Calp,
Psoriatec
0.5, 1.0
Betamethasone
dipropionate
Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash
Diprolene
AF
0.05
Butoconazole
nitrate (vag)
Antifungal Vaginal yeast infections Gynazole - 1 2.0
Clindamycin
phosphate
(vag)
Antibiotic Vaginosis caused by
bacteria
Clindesse 2.0
Clobetasol
propionate
Anti -
infl ammatory
Infl ammatory
and pruritic
manifestations of
corticosteroid -
responsive
dermatoses
Temovate E
Cream
0.05
Clotrimazole Antifungal Ringworm of groin
area, athlete ’ s foot,
ringworm of the body,
fungal infection of
the skin with yellow
patches, skin infection
due to a candida
yeast, and diaper rash
Lotrimin 1.0
Crotamiton Scabicidal and
antipruritic
Scabies infection and
itching
Eurax 10.0
Desoximetasone Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash
Topicort 0.25
Dienestrol Estrogen Reduce menopause
symptoms such as
vaginal dryness
Ortho -
Dienestrol
0.01
Difl orasone
diacetate
Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash
Apexicon,
Maxifl or
0.05
Fluocinonide Anti -
infl ammatory
and
antipruritic
Psoriasis, eczema,
dermatitis, allergies,
and rash
Lidex, Vanos 0.05, 0.10
Fluorouracil Anticancer Precancerous and
cancerous skin
growths
Fluoroplex,
Carac,
Efudex
0.5, 1.0, 5.0
thixotropy. Imbibition refers to the uptake of water or other liquids by gels without
any considerable increase in its volume. Swelling refers to the increase in the volume
of gel by uptake of water or other liquids. This property of most gels is infl uenced
by temperature, pH, presence of electrolytes, and other formulation ingredients.
Syneresis refers to the contraction or shrinkage of gels as a result of squeezing out
of dispersion medium from the gel matrix. It is due to the excessive stretching of
macromolecules and expansion of elastic forces during swelling. At equilibrium, the
system still maintains its physical stability because the osmotic forces of swelling
balance the expanded elastic forces of macromolecules. On cooling, the osmotic
pressure of the system decreases and therefore the expanded elastic forces return
to normal. This results in shrinkage of the stretched molecules and squeezing of
dispersion medium from the gel matrix. Addition of osmotic agents such as sucrose,
glucose, and other electrolytes helps in retaining higher osmotic pressure even at
lower temperatures and avoids syneresis of gels. Thixotropy refers to the non -
Newtonian fl ow nature of gels, which is characterized by a reversible gel - to - sol
formation with no change in volume or temperature [18] .
4.2.3.3 Classifi cation
Gels are classifi ed as hydrogels and organogels based on the physical state of the
gelling agent in the dispersion. Hydrogels are prepared with water - soluble materials
or water - dispersible colloids. Organogels are prepared using water - insoluble oleaginous
materials.
Hydrogels Natural and synthetic gums such as tragacanth, sodium alginate, and
pectin, inorganic materials such as alumina, bentonite, silica, and veegum, and
organic materials such as cellulose polymers form hydrogels in water. They may
either be dispersed as fi ne colloidal particles in aqueous phase or completely dissolve
in water to gain gel structure. Gums and inorganic gelling agents form gel
structure due to their viscosity - increasing nature. Organic gelling agents which are
generally high - molecular - weight cellulose polymer derivatives produce gel structure
Drug a Category Indication
Commercial
Products
Strength(s)
Available
(%)
Halcinonide Anti -
infl ammatory
and
antipruritic
Eczema, dermatitis,
allergies, and rash
Halog 0.1
Mometasone
furoate
Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash
Elocon 0.1
Naftifi ne
hydrochloride
Antifungal Jock itch, athlete ’ s feet,
or ringworm
Naftin 1
Nystatin Antifungal Fungal skin infections Mycostatin 100,000
units/g
a vag, vaginal cream.
TABLE 7 Continued
GELS 291
292 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
because of their swelling and chain entanglement properties. The swollen molecular
chains are held together by secondary valence forces, which help in retaining their
gel structure. The physical strength of the gel structure is based on the quantity of
gelling agent, nature and molecular weight of gelling agent, product pH, and gelling
temperature. Generally high - molecular - weight polymers at higher concentrations
produce thick gels. The gel - forming temperature ( gel point ) varies with different
polymers. Generally natural gums form gel at lower temperatures. Gelatin, a natural
protein polymer, forms gel at about 30 ° C. If the temperature is increased, gel consistency
is not obtained even at higher concentrations of gelatin. On the other hand,
polymers such as methylcellulose gain gel structure only when the temperature is
above 50 ° C due to its decreased solubility and precipitation. Knowledge of the gel
point for each gelling agent is therefore essential for preparing physically stable
hydrogels.
Organogels Organogels are also known as oleaginous gels. They are prepared
using water - insoluble lipids such as glycerol esters of fatty acids, which swell in water
and form different types of lyotropic liquid crystals. Widely used glycerol esters of
fatty acids include glycerol monooleate, glycerol monopalmito stearete, and glycerol
monolinoleate. They generally exist as waxes at room temperature and form cubic
liquid crystals in water and increase the viscosity of dispersion. Waxes such as carnauba
wax, esparto wax, wool wax, and spermaceti are used in cosmetic organogel
preparations. A large quantity of water is entrapped between the three - dimensional
lipid bilayers. The equilibrium water content in organogels is about 35%. The structural
properties of the lipid, quantity of water in the system, solubility of drug
incorporated, and external temperature infl uence the nature of the liquid crystalline
phase. The bipolar nature of organogels allows incorporation of both hydrophilic
and lipophilic drugs. Release rates can be controlled by altering the hydrophilic and
hydrophobic components. Biodegradability of these waxes by the lipase enzyme in
the body makes organogels suitable for parenteral administration.
The water present in the gel framework can be completely removed with some
gelling agents. Gelatin sheets, acacia tears, and tragacanth ribbons are generally
prepared by removal of water from their respective gel matrix. These dehydrated
gel frameworks are called as xerogels.
4.2.3.4 Stimuli - Responsive Hydrogels
The three - dimensional networks of hydrophilic polymers absorb a large quantity of
water and form soft structures which resemble biological tissues. Swelling properties
of these hydrogels can be altered by various physicochemical parameters. Physical
factors such as temperature, pH, and ionic strength of the swelling medium
and chemical factors such as the structure of polymer (linear/branched) and chemical
modifi cations (cross - linking) can be altered to tailor their swelling rate. This
feature makes them very attractive for drug delivery and biomedical applications
[19 – 23] .
pH-Responsive Hydrogels Some polymers show pH - dependent swelling and
gelling characteristics in aqueous media. A polymer that exhibits such phase transition
properties is very useful from the point of drug delivery. Methacrylic acids
(e.g., carbomers) that contain many carboxylic acid groups exist as solution at lower
pH conditions. When the pH is increased, they undergo a sol - to - gel transition. This
is because of the increase in the degree of ionization of acidic carboxylic groups at
higher pH conditions, which in turn results in electrostatic repulsions between
chains and, increased hydrophilicity and swelling. Conversely, polymers that contain
amine - pendant groups swell at lower pH environment due to ionization and repulsion
between polymer chains. The ionic strength of surrounding fl uids signifi cantly
infl uences the equilibrium swelling of these pH - responsive polymers. Higher ionic
strength favors gel – counter ionic interactions and reduces the osmotic swelling
forces.
Thermoresponsive Hydrogels A dispersion which exists as solution at room temperature
and transforms into gel on instillation into a body cavity can improve the
administration mode and help in modulating the drug release. Many polymers with
thermoresponsive gelling properties are currently being synthesized and evaluated.
A triblock copolymer that consists of polyethylene glycol – polylactic acid, glycolic
acid – polyethylene glycerol (PEG – PLGA – PEG) is solution at room temperature
and gels at body temperature. Poloxamers, which are made of triblock poly(ethylene
oxide) – poly(propylene oxide) – poly(ethylene oxide), exhibit gelatin properties at
body temperatures. Similarly, xyloglucan and xanthan gum aqueous dispersions are
solutions at room temperature and become gel at body temperature. These are
considered convenient alternatives for rectal suppository formulations which usually
cause mucosal irritations due to their physical state. The physicochemical properties
of these chemically modifi ed thermoresponsive hydrogels are altered by changing
the ratio of hydrophilic and hydrophobic segments, block length, and polydispersity.
ReGel by MacroMed contains a triblock copolymer PLGA – PEG – PLGA, undergoes
sol - to - gel transition on intratumoral injection, and releases paclitaxel for six
weeks.
Ionic-Responsive Hydrogels Administration of sodium alginate aqueous drops
into the eye results in alginate gelation due to its interaction with calcium ions in
the tear fl uid. Alginate with high guluronic acid and deacetylated gellan gum
(Gelrite) show sol - to - gel conversions in the eye due to their interaction with cations
in the tear fl uid. Timolol maleate sterile ophthalmic gel - forming solution (Timoptic -
XE) that contains Gelrite gellan gum is commercially available.
4.2.3.5 Gelling Agents
A large number of gelling agents are commercially available for the preparation of
pharmaceutical gels. In general, these materials are high - molecular - weight compounds
obtained from either natural sources or synthetic pathways. They are water
dispersible, possess swelling properties, and improve the viscosity of dispersions. An
ideal gelling agent should not interact with other formulation components and
should be free from microbial attack. Changes in the temperature and pH during
preparation and preservation should not alter its rheological properties. In addition,
it should be economic, readily available, form colorless gels, provide cooling sensation
on the site of application, and possess a pleasant odor. Based on these factors,
gelling agents are selected for different formulations. Table 8 summarizes the
GELS 293
294 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
TABLE 8 Some Compendial Gelling Agents Used in Gels
Name
Molecular
Weight
Gelling
Strength
(%) Synonyms
Offi cial
Compendia
Bentonite 359.16 10 – 20 Magnabrite,
mineral soap,
Polargel, Veegum
HS
BP, JP, PhEur,
USPNF
Carbomer 7 . 10 5 – 4 . 10 9 0.5 – 2.0 Acritamer,
Carbopol,
polyacrylic acid
BP, PhEur,
USPNF
Carboxymethyl
cellulose sodium
90,000–700,000 3.0–6.0 Akucell, Aquasorb,
Sodium CMC,
Tylose CB
BP, JP, PhEur,
USPNF
Carrageenan . 1,000,000 0.3 – 2.0 Gelcarin, Genu,
Hygum Marine
colloids
USPNF
Colloidal silicon
dioxide
60.08 2.0 – 10.0 Aerosil, colloidal
silica, fumed
silica
BP, PhEur,
USPNF
Gelatin 15,000 – 25,0000 10.0 – 20.0 Cryogel, Solugel BP, JP, PhEur,
USPNF
Glyceryl behenate 1059.8 1.0 – 15.0 Docosanoic acid,
glycerol behenate
BP, PhEur,
USPNF
Guar gum . 220,000 1.0 – 5.0 Galactosal, Guar
fl our, Jaguar gum,
Meyproguar
BP, PhEur,
USPNF
Hydroxypropyl
cellulose
50,000–1,250,000 2.0 – 5.0 Hyprolose, klucel,
Methocel
BP, JP, PhEur,
USPNF
Hydroxypropylmethyl
cellulose
10,000–1,500,000 1.0 – 10.0 Hypromellose BP, JP, PhEur,
USPNF
Magnesium aluminum
silicate
— 5.0 – 15.0 Veegum,
aluminosilicic
acid, Carrisorb,
Magnabite
BP, PhEur,
USPNF
Methylcellulose 10,000 – 220,000 1.0 – 5.0 Benecel, Methocel,
Metolose
BP, JP, PhEur,
USPNF
Poloxamer 2090 – 17,400 15.0 – 20.0 Lutrol, Monolan,
Pluronic,
Supronic
BP, phEur,
USPNF
Polyvinyl alcohol . 20,000 – 200,000 2.5 – 10.0 Airvol, Elvanol,
PVA, vinyl
alcohol
USP
Povidone 2500–3,000,000 2.0 – 20.0 Kollidon, Plasdone,
Polyvidone, PVP
BP, JP, PhEur,
USPNF
Sodium alginate 20,000 – 240,000 10.0 – 20.0 Algin, alginic acid,
sodium salt,
Protanal
BP, PhEur,
USPNF
Tragacanth 840,000 1.0 – 8.0 Gum Benjamin,
Gum dragon,
Trag, Tragant
BP, JP, PhEur,
USPNF
Note : BP, British Pharmacopoeia; JP, Japanese Pharmacopoeia; PhEur, European Pharmacopoeia; USPNF, U.S.
Pharmacopeia/National Formulary
molecular weight, gelling strength, synonyms, and compendial status of some of
these agents.
The following sections briefl y describe the source, physicochemical properties,
formulation, and preservation of some pharmacopeial gelling agents.
Alginic Acid Alginic acid is tasteless and odorless and occurs as a yellowish white
fi brous powder. The main source for this naturally occurring hydrophilic colloidal
polysaccharide is different species of brown sea weed, known as Phaeophyceae. It
consists of a mixture of d - mannuronic acid and l - glucuronic acids. It is used in gels
due to its thickening and swelling properties. Alginic acid is insoluble in water;
however, it absorbs 200 – 300 times its own weight of water and swells. The viscosity
of alginic acid gels can be altered by changing the molecular weight and concentration.
Addition of calcium salts increases the viscosity of alginic acid gels. Its viscosity
decreases at higher temperature. Depolymerization due to microbial attack also
results in viscosity reduction. Inclusion of an antimicrobial preservative avoids
depolymerization and viscosity reduction during storage [6] .
Bentonite Bentonite is a naturally occurring colloidal hydrated aluminum silicate
and contains traces of calcium, magnesium, and iron. It is odorless, available as fi ne
crystalline powder, and is cream to grayish in color. The particles are negatively
charged. Its high water uptake and swelling and thickening properties make it suitable
for preparing gels. It swells to about 12 - fold when it comes in contact with
water. The viscosity of bentonite dispersion increases with increase in concentration.
The gel - forming properties increase with addition of alkaline materials such as
magnesium oxide and decrease with addition of alcohol or electrolytes. Use of hot
water and stirring improve wetting and dispersion of bentonite particles in the
preparation of the gel. Mixing with magnesium oxide or zinc oxide prior to addition
helps in dispersion of bentonite in water. Prior trituration of bentonite with glycerin
also helps in easy dispersibility in water. These dispersions are generally left for
about 24 h to complete the swelling process. At lower concentration (10%) bentonite
suspension exhibits the properties of shear thinning systems and at high concentrations
(about 50 – 60%) it forms gel with fi nite yield strength [24] .
Carbomer Carbomers are one of the widely used gelling agents in topical preparations
due to their extensive swelling properties. They are obtained by cross -
linking acrylic acid with allyl sucrose or allyl pentaerythritol. Various grades with
varying degree of cross - linking and molecular weight are commercially available.
Carbomers are generally available as hygroscopic powders, are white in color, and
possess a characteristic odor. Presence of about 60% carboxylic acid in its composition
makes them acidic. Carbomer 934P, 971P, 974P, and so on, are used for preparing
clear gels. Aqueous dispersions of carbomers are usually low viscous, and on
neutralization they form high - viscous gels. Basic materials such as sodium hydroxide,
potassium hydroxide, sodium bicarbonate, and borax are being used for neutralizing
carbomer dispersions. About 0.4 g of sodium hydroxide is used to neutralize
1 g of carbomer dispersion. The viscosity of gels depends on the molecular weight
of carbomer and its degree of cross - linking. Inclusion of antioxidants, protection
from light, and preservation at room temperature help in retaining their viscosity
for prolonged periods. Microbial stability of carbopol gels can be improved by
GELS 295
296 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
adding antimicrobial preservatives. These gels are prone to discoloration in the
presence of large amounts of electrolytes, strong acids, and cationic polymers. Glass,
plastic, and resin - lined containers which possess good corrosion - resistant properties
are used for packing carbomer gels [6] .
Carboxymethylcellulose Calcium (Calcium CMC) A calcium salt of polycarboxymethyl
ether of cellulose, calcium CMC is obtained by carboxymethylation of
cellulose and conversion into calcium salt. Different molecular grades are prepared
by changing the degree of carboxymethylation. It is available as a fi ne powder, white
to yellowish white in color, and hygroscopic in nature. Calcium CMC has swelling
and viscosity - enhancing properties in water. It can swell twice its volume in water
[25] .
Carboxymethylcellulose Sodium (Sodium CMC) A sodium salt of polycarboxymethyl
ether of cellulose, sodium CMC is obtained by treating alkaline cellulose
with sodium monochloroacetate. It is available as white - colored granular powder.
Various viscosity grades of sodium CMC commercially available basically differ in
their degree of substitution. The degree of substitution represents the average
number of hydroxyl groups that are substituted per anhydroglucose unit. It is readily
dispersible in water and forms clear gels. The aqueous solubility of CMC sodium is
governed by the degree of substitution. Higher concentrations generally yield
thicker gels. Although the viscosity of gels is stable over a wide range of pH (4 – 10),
a fall in pH below 2 or a rise to above 10 results in physical instability and viscosity
reduction. Higher viscosity is obtained at neutral pH conditions. Exposure of gels
to higher temperature also results in viscosity reduction. Preservation at optimum
temperature and inclusion of an antimicrobial preservative improve the physical
and microbial stability of CMC sodium gels [25] .
Carrageenan Extraction of some red seaweed belonging to the Rhodophyceae
class with water or aqueous alkali yields carrageenan. It is a hydrocolloid and mainly
contains sodium, potassium, calcium, magnesium, and ammonium sulfate esters of
galactose and copolymers of 3, 6 - anhydrogalactose. They differ in their ester sulfate
and anhydrogalactose content. It is available as a coarse to fi ne powder which is
yellow to brown in color. It is odorless and tasteless. Carrageenan is soluble in hot
water and forms gels at 0.3 – 2.0%. . - Carrageenan and . - carrageenan show good
gelling properties [26] .
Colloidal Silicon Dioxide Colloidal silicon dioxide is a fumed silica obtained by
vapor hydrolysis of chlorosilanes. It is available as nongritty amorphous powder
which is bluish white in color. It is tasteless and odorless and possesses low tapped
density. Although insoluble in water, it readily forms a colloidal dispersion due to
its fi ne particle size, higher surface area, and water - adsorbing properties. The bulk
density and particle size of colloidal silicon dioxide can be altered by changing the
method of manufacture. Transparent gels can be formed by mixing with other materials
that possess similar refractive index. Under acidic and neutral pH conditions,
it shows viscosity - increasing properties. This property is lost at higher pH conditions
because of its dissolution. Viscosity of gels is not generally affected by temperature
[27] .
Ethylcellulose Ethylcellulose is a synthetic polymer made of . - anhydroglucose
units connected by acetyl linkages. It is obtained by ethylating alkaline cellulose
solution with chloroethane. Ethylcellulose is available as a free - fl owing powder
which is tasteless and white in color. Although it is insoluble in water, it is incorporated
into topical preparations due to its viscosity - enhancing properties. Ethanol or
a mixture of ethanol and toluene (2 : 8) is used as a solvent. A decrease in the ratio
of alcohol increases the viscosity. The viscosity of the dispersion is increased by
increasing the concentration of ethylcellulose or by using a high - molecular - weight
material. As ethylcellulose is prone to photo - oxidation at higher temperature, and
gels are prepared and preserved at room temperature and dispensed in airtight
containers [28] .
Gelatin Gelatin is a protein obtained by acid or alkali hydrolysis of animal tissues
that contain large amounts of collagen. Based on the method of manufacture, it is
named type A or type B gelatin. Type A is obtained by partial acid hydrolysis and
type B is obtained by partial alkaline hydrolysis. They differ in their pH, density,
and isoelectric point. Gelatin is available as yellow - colored powder or granules. It
swells in water and improves the viscosity of dispersions. Different molecular weights
and particle size grades are commercially available. Gels can be prepared by dissolving
gelatin in hot water and cooling to 35 ° C. Temperature greatly infl uences the
viscosity and stability of gelatin dispersions. It transforms to a gel at temperatures
above 40 ° C and undergoes depolymerization above 50 ° C. The viscosity of gelatin
gel is also affected by microbes [29] .
Guar Gum Guar gum is a high - molecular - weight polysaccharide obtained from
the endosperms of guar plant. It mainly contains d - galactan and d - mannan. It is
available as powder which is odorless and white to yellowish white in color. It readily
disperses in water and forms viscous gels. The viscosity of gel is infl uenced by the
particle size of material, pH of the dispersion, rate of agitation, swelling time, and
temperature. Viscosity reduces on long - time heating. Maximum viscosity can be
achieved within 2 – 4 h. Gels are stable at pH between 7 and 9 and show liquifi cation
below pH 7. Addition of antimicrobial preservatives improves the microbial stability
of guar gum gels. Rheological properties of these gels can be modifi ed by incorporating
other plant hydrocolloids such as tragacanth and xanthan gum [30] .
Hydroxyethyl Cellulose ( HEC) HEC is a partially substituted poly(hydroxyethyl)
ether of cellulose. It is obtained by treating alkali cellulose with ethylene oxide. HEC
is available as a powder and appears light tan to white in color. Different viscosity
grades of HEC are commercially available which differ in their molecular weights.
Clear gels are prepared by dissolving HEC in hot or cold water. Dispersions can be
prepared quickly by altering the stirring rate of dispersion, temperature, and pH.
Slow stirring at room temperature during the initial stages favors wetting. Increasing
the temperature at this stage increases the rate of dispersion. Although HEC dispersions
are stable over a wide pH range, maintaining basic pH improves the dispersion.
The preservation temperature, formulation pH, and microbial attack infl uence the
rheological properties of HEC dispersions. Viscosity reduces at higher temperature,
but reverts to the original value on returning to room temperature. Lower
and higher pH of the vehicle usually results in hydrolysis or oxidation of HEC,
GELS 297
298 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
respectively. Some of the enzymes secreted by microbes decrease the viscosity of
HEC dispersions. The presence of higher levels of electrolytes may also destabilize
HEC dispersions. Inclusion of a suitable antimicrobial preservative is essential to
retain the viscosity of HEC gels [31] .
Hydroxyethylmethyl Cellulose ( HEMC) HEMC is a partially o - methylated and
o - (2 - hydroxyethylated) cellulose. It is available as powder or granules which are
white, grayish white, or yellowish white in color. Various viscosity grades of HEMC
are commercially available, and form viscous colloidal dispersions or gels in cold
water which has a pH in the range of 5.5 – 8 [6] .
Hydroxypropyl Cellulose ( HPC) HPC is a partially substituted poly(hydroxypropyl)
ether of cellulose. It is obtained by treating alkali cellulose with propylene oxide at
higher temperatures. It is available as tasteless and odorless powder which is yellowish
or white in color. Different viscosity grades are commercially available.
Gradual addition of HPC powder into vigorously stirred water yields clear viscous
dispersions or gels below 38 ° C. Increase in temperature destabilizes the dispersion
and leads to precipitation. The viscosity of dispersions can be increased by increasing
the concentration of HPC or by using high - molecular - weight grades. Inclusion
of a cosolvent such as dichloromethane or methane produces viscous dispersion or
gels with modifi ed texture. The viscosity of HPC dispersions can be increased by
mixing with an anionic polymer. High concentrations of electrolytes destabilize
HPC dispersions. HPC dispersions are neutral in pH (6 – 8). They undergo acid
hydrolysis at lower pH and oxidation at higher pH. Both processes decrease the
dispersion viscosity. In addition, certain enzymes produced by microbes degrade
HPC and reduce its viscosity. Addition of an antimicrobial preservative is therefore
recommended for HPC gels. Preservation of these gels from light can further
improve its physical stability [25] .
Hydroxypropylmethyl Cellulose ( HPMC) HPMC is a partly o - methylated and
o - (2 - hydroxypropylated) cellulose obtained by treating alkali cellulose with chloromethane
and propylene oxide. It is available as odorless and tasteless granular or
fi brous powder which is creamy white or white in color. HPMC is soluble in cold
water. Aqueous dispersions are prepared by dispersing material in about 25% hot
water (80 ° C) under vigorous stirring. On complete hydration of HPMC, a suffi cient
quantity of cold water is added and mixed. The gel point of HPMC dispersions varies
from 50 to 90 ° C. Gels are stable over a wide pH range (3 – 11). The viscosity HPMC
dispersions depends on the concentration of material used, its molecular weight,
vehicle composition, presence of preservatives, and so on. Viscous gels can be prepared
using high concentrations of high - molecular - weight grades. Inclusion of
organic solvents such as ethanol or dichloromethane improves the viscosity. Addition
of an antimicrobial preservative (e.g., benzalkonium chloride) minimizes microbial
spoilage of HPMC gels [25] .
Glyceryl Behenate Glyceryl behenate is a mixture of glycerides of fatty acids
which is obtained by esterifi cation of glycerin with behenic acid. It may also contain
arachidic acid, stearic acid, erucic acid, lignoceric acid, and palmitic acid. It is available
as a waxy mass or powder, possesses a faint odor, and is white in color. It is
practically insoluble in water and soluble in dichloromethane and chloroform. It is
used as a viscosity - increasing agent in silicon gels [6] .
Glyceryl Monooleate ( GMO) GMO is a mixture of glycerides of fatty acids
obtained by esterifi cation of glycerol with oleic acid. It may also contain linoleic
acid, palmitic acid, stearic acid, linolenic acid, arachidic acid, and eicosenoic acid. It
is available as a partially solidifi ed or oily liquid. GMO is insoluble in water. Self -
emulsifying grades that contain an anionic surfactant disperse easily and swell in
water. The nonemulsifying grades are used as emollients in topical preparations and
self - emulsifying grades are used as emulsifi ers in aqueous emulsions [6] .
Magnesium Aluminum Silicate ( MAS) MAS is also known as veegum. It is a
polymeric complex of magnesium, aluminum, silicon, oxygen, and water and is
obtained from silicate ores. Based on the ratio of aluminum and magnesium and
viscosity, it is classifi ed as types IA, IB, IC, and IIA. It is available as fi ne powder
that is odorless, tasteless, and off - white to creamy white in color. Although MAS is
insoluble in water, it swells to a large extent and produces viscous colloidal dispersions.
Use of higher concentration, addition of electrolytes, and heating of dispersion
usually improve the viscosity [32] .
Methylcellulose ( MC) MC is a long - chain cellulose polymer with methoxyl substitutions
at positions 2, 3, and 6 of the anhydroglucose ring. It is synthesized by
methylating alkali cellulose with methyl chloride. The degree of substitution of
methoxy groups infl uences the molecular weight, viscosity, and solubility characteristics
of MC. It is available as powder or granules and is odorless, tasteless, and white
to yellowish white in color. MC is insoluble in hot water but slowly swells and forms
viscous colloidal dispersions in cold water. Gels can be prepared by initially mixing
the methylcellulose with half the volume of hot water ( . 70 ° C) followed by addition
of the remaining volume of cold water. Viscosity of these dispersions can be increased
by using high - concentration or high - molecular - weight grades of methylcellulose.
Higher processing or preservation temperatures reduce the viscosity of formulations,
which regain their original state on cooling to room temperature. MC aqueous
dispersions show pH values of 5 – 8. Reduction in pH to less than 3 leads to acid -
catalyzed hydrolysis of glucose – glucose linkages and results in low viscosity. Antimicrobial
preservatives are generally included to enhance the microbial stability of
dispersions. Salting out is observed when high concentrations of electrolytes are
added to methylcellulose dispersions. The viscosity of methylcellulose dispersions is
also infl uenced by the presence of formulation excipients and drugs [25] .
Poloxamer Poloxamers are copolymers of ethylene oxide and propylene oxide.
Different molecular weight grades that are different in physical form, solubility, and
melting point are available. Poloxamer 124 is a colorless liquid, whereas poloxamers
188, 237, 338, and 407 are solids at room temperature. All poloxamer grades are
freely soluble in water and form gels at higher concentrations. The pH of aqueous
liquids ranges between 5 and 7.5 [33] .
Polyethylene Oxide Polyethylene oxide is a nonionic homopolymer of ethylene
oxide synthesized by polymerization of ethylene oxide. It is available as a free -
GELS 299
300 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
fl owing powder white to off - white in color with a slight ammonia odor. Various
molecular weight grades of polyethylene oxide are commercially available. They
swell in water and form viscous dispersions or gels based on the concentration and
grade used. Inclusion of alcohol improves the rheological stability of polyethylene
oxide dispersions [6] .
Polyvinyl Alcohol ( PVA ) PVA is a synthetic polymer prepared by hydrolysis of
polyvinyl acetate. It is available as a granular powder which is odorless and white
in color. Mixing with water at room temperature, heating for about 5 min at 90 ° C,
followed by cooling with constant mixing yield aqueous dispersions or gels. Higher
viscosities can be obtained by using high - viscosity grades. Addition of borax improves
the gelling properties of PVA, whereas inorganic salts destabilize these dispersions.
The pH of PVA dispersions ranges between 5 and 8. Physical and chemical decompositions
occur at lower and higher pH conditions. Incorporation of an antimicrobial
preservative and storage at room temperature improve its stability [6] .
Povidone Povidone is a synthetic polymer consisting of 1 - vinyl - 2 - pyrrolidinone
units. It is available as a fi ne powder and appears white to creamy - white in color.
Various molecular weight grades of povidone are available which differ in their
degree of polymerization. Povidone is soluble in water and forms viscous solutions
and gels based on the concentration and viscosity grade used. Decomposition occurs
when dispersions are heated to about 150 ° C. The pH of aqueous dispersions ranges
from 3 to 7. The microbial stability of povidone aqueous dispersions can be increased
by adding preservatives [6] .
Propylene Carbonate ( PC) PC is prepared by reacting propylene chlorohydrin
with sodium bicarbonate. It is available as a clear liquid with a faint odor. Mixtures
of PC and propylene glycol are good solvents for corticosteroids in topical preparations.
It is incompatible with strong acids, bases, and amines. The pH of 10% aqueous
dispersion is 6.0 – 7.5 [34] .
Propylene Glycol Alginate ( PGA) PGA is a propylene glycol ester of alginic acid
obtained by treating alginic acid with propylene oxide. It is available as granular or
fi brous powder which is odorless, tasteless, and white to yellowish - white in color.
PGA is soluble in water and forms viscous colloidal dispersions. The viscosity of
these dispersions is based on the concentration of PGA, temperature, and pH. Its
aqueous solubility decreases at higher temperatures. The aqueous dispersions are
acidic in nature and more stable at pH 3 – 6. Higher pH leads to saponifi cation. As
these dispersions are prone to microbial spoilage, antimicrobial preservatives are
generally included [6] .
Sodium Alginate Sodium alginate is obtained by extraction of alginic acid from
brown seaweed followed by neutralization with sodium bicarbonate. Alginic acid is
composed of d - mannuronic acid and l - guluronic acid. It is available as a powder
which is tasteless, odorless, and white to yellowish - brown in color. Sodium alginate
forms viscous gels in water. Dispersing agents such as glycerol, propylene glycol,
sucrose, and alcohol are added to improve dispersion. The presence of low concentration
of electrolytes improves the viscosity, whereas at high concentrations salting
out takes place. The viscosity of gel is based on the concentration of sodium alginate,
temperature, pH, and other additives. Various viscosity grades of sodium alginate
are commercially available. Aqueous dispersions are stable at pH 4 – 10. Precipitation
or decrease in viscosity is observed when the pH is below or above these values.
Autoclaving or heating above 70 ° C results in depolymerization and decrease in
viscosity. Inclusion of preservative is essential to maintain the microbial stability of
sodium alginate topical gels [35] .
Tragacanth Tragacanth is a polysaccharide polymer obtained from some Astragalus
species. It is composed of two polysaccharides: bassorin (water insoluble) and
tragacanthin (water soluble). It is available as odorless powder white to yellowish
in color and possesses mucilaginous taste. Tragacanth swells about 10 times its
weight in water and forms viscous solutions or gels. Tragacanth is usually added with
vigorous stirring to avoid lump formation. Wetting agents such as glycerin, propylene
glycol, and 95% ethanol are used in initial stages to improve wetting and dispersion
of tragacanth in water. The viscosity of tragacanth dispersions is infl uenced by
the processing temperature and formulation pH. High temperature usually increases
the viscosity of gels. Tragacanth dispersions show higher viscosity at pH 8 and starts
decreasing at higher pH. These gels usually contain preservatives such as benzoic
acid or a combination of methyl and propyl parabens for effective preservation from
microbial attack. The viscosity of tragacanth dispersions reduces in the presence of
strong mineral and organic acids and sodium chloride [6] .
4.2.3.6 Preparation and Packaging
Gels are relatively easier to prepare compare to ointments and creams. In addition
to the gelling agent, medicated gels contain drug, antimicrobial preservatives, stabilizers,
dispersing agents, and permeation enhancers. Some of the factors discussed
below are essential to obtain a uniform gel preparation.
Order of Mixing The order of mixing of these ingredients with the gelling agent
is based on their infl uence on the gelling process. If they are likely to infl uence the
rate and extent of swelling of the gelling agent, they are mixed after the formation
of gel. In the absence of such interference, the drug and other additives are mixed
prior to the swelling process. In this case, the effects of mixing temperature, swelling
duration, and other processing conditions on the physicochemical stability of the
drug and additives are also considered. Ideally the drug and other additives are
dissolved in the swelling solvent, and the swelling agent is added to this solution
and allowed to swell.
Gelling Medium Purifi ed water is the most widely used dispersion medium in the
preparation of gels. Under certain circumstances, gels may also contain cosolvents
or dispersing agents. A mixture of ethanol and toluene improves the dispersion of
ethylcellulose, dichloromethane and methanol increase the viscosity of hydroxypropyl
cellulose dispersions, alcohol improves the rheological stability of polyethylene
oxide gels, and inclusion of glycerin, propylene glycol, sucrose, and alcohol improves
the dispersion of sodium alginate dispersions. Borax is included in polyvinyl alcohol
gels and magnesium oxide, zinc oxide, and glycerin are included in bentonite gel as
GELS 301
302 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
dispersing agents. Care should be taken to avoid the evaporation or degradation of
these cosolvents and dispersing agents during the preparation of gels.
Processing Conditions and Duration of Swelling The processing temperature, pH
of the dispersion, and duration of swelling are critical parameters in the preparation
of gels. These conditions vary with each gelling agent. For instance, hot water is
preferred for gelatin and polyvinyl alcohol, and cold water is preferred for methylcellulose
dispersions. Carbomers, guar gum, hydroxypropyl cellulose, poloxamer,
and tragacanth form gels at weakly acidic or near - neutral pH conditions (pH 5 – 8).
Gelling agents such as carboxymethyl cellulose sodium, hydroxypropylmethyl cellulose,
and sodium alginate form gels over a wide pH range (4 – 10). Hydroxyethyl
cellulose forms gel at alkaline pH condition. A swelling duration of about 24 – 48 h
generally helps in obtaining homogeneous gels. Natural gums need about 24 h and
cellulose polymers require about 48 h for complete hydration.
Removal of Entrapped Air Entrapment of air bubbles in the gel matrix is a
common issue, especially when the swelling process involves a mixing procedure or
the drug and other additives are added after the swelling process. Positioning the
propeller at the bottom of the mixing container minimizes this issue to a larger
extent. Further removal of air bubbles can be achieved by long - term standing, low -
temperature storage, sonication, or inclusion of silicon antifoaming agents. In large -
scale production, vacuum vessel deaerators are used to remove the entrapped air.
Packaging Being viscous and non - Newtonian systems, gels need high attention
during packing into containers. Usually they are packed into squeeze tubes or jars
made of plastic materials. Aluminum containers are also used when the product pH
is slightly acidic. Pump dispensers and prefi lled syringes are sometimes used for
packing gels. As most of the gels contain an aqueous phase, preservation in airtight
containers helps in protecting them from microbial attack. Usually they are preserved
at room temperature and protected from direct sunlight and moisture.
In large - scale production, different mills, separators, mixers, deaerators, shifters,
and packaging machines are used. Most of this equipment is similar to those discussed
under ointments and creams. Figure 3 shows a “ one - bowl ” vacuum processing
machine manufactured by FrymaKoruma - Rheinfelden (Romaco) for the
preparation gels. Batch sizes ranging from 15 to 160 L are processed using this
machine. It uses an extremely effi cient high - shear rotor/stator system for homogenizing
and a counterrotating mixing system for macromixing. The raw materials are
drawn into the multichamber system of the homogenizer by vacuum and then mixed
and pumped into the homogenizing zone. The product which enters the vessel is
mixed, sheared, and recirculated. All the entrapped air is removed during the recirculation.
The machine also has an insulated jacket for controlling the processing
temperature.
4.2.3.7 Evaluation
Various pharmacopeial and nonpharmacopeial tests are carried out to evaluate the
physicochemical, microbial, in vitro, and in vivo characteristics of gels. These tests
are meant for assessing the quality of gel formulations and minimizing the batch -
to - batch variations. Some of the tests recommended by the USP for gels include
minimum fi ll, pH, viscosity, microbial screening, and assay. In some cases sterility
and alcohol content are also specifi ed. The USP also recommends storage for each
compendial gel formulation. Table 9 shows the quality control tests and storage
requirements that are specifi ed for some pharmacopeial gels. The procedures for
minimum fi ll, microbial screening, sterility, assay, in vitro drug release, and in vivo
bioequivalence are similar to those of ointments and creams. The procedures for
additional tests such as homogeneity, surface morphology, pH, alcohol content,
rheological properties, bioadhesion, stability, and ex vivo penetration are described
below.
Homogeneity and Surface Morphology The homogeneity of gel formulations is
usually assessed by visual inspection and the surface morphology by using scanning
electron microscopy. Generally, the swollen gel is allowed to freeze in liquid nitrogen
and then lyophilized by freeze drying. It is assumed that the morphologies of
the swollen samples are retained during this process. The lyophilized hydrogel is
gold sputter coated and viewed under an electron microscope.
pH Many gelling agents show pH - dependent gelling behavior. They show highest
viscosity at their gel point. Determination of pH is therefore important to maintain
consistent quality. As conventional pH measurements are diffi cult and often give
erratic results, special pH electrodes are used for viscous gels. Flat - surface digital
FIGURE 3 Vacuum processing machine used for preparation of gels. (Courtesy of
FrymaKoruma - Rheinfelden, Switzerland.)
GELS 303
304 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
TABLE 9 USP Specifi cations for Some Offi cial Gels
Drug Quality Control Tests Packaging and Storage Requirements
Aminobenzoic
acid
Minimum fi ll, pH (4.0 – 6.0),
alcohol content, and assay
Tight, light - resistant containers
Benzocaine Microbial limits, minimum fi ll,
and assay
Well - closed containers
Benzoyl peroxide pH (2.8 – 6.6), and assay Tight containers
Betamethasone
benzoate
Microbial limits, minimum fi ll,
and assay
Tight containers; store at 25 ° C,
excursions permitted between 15
and 30 ° C; protect from freezing
Clindamycin
phosphate
Minimum fi ll, pH (4.5 and
6.5), and assay
Tight containers
Desoximetasone Minimum fi ll, alcohol content,
and assay
Collapsible tubes at controlled room
temperature
Dexamethasone Minimum fi ll and assay Collapsible tubes; keep tightly closed;
avoid exposure to temperatures
exceeding 30 ° C
Dyclonine
hydrochloride
pH (2.0 and 4.0), and assay Collapsible, opaque plastic tubes or in
tight, light - resistant glass containers
Erythromycin Minimum fi ll and assay Tight containers
Fluocinonide Minimum fi ll and assay Collapsible tubes or tight containers
Hydrocortisone Minimum fi ll and assay Tight containers
Lidocaine
hydrochloride
Sterility, minimum fi ll, pH
(7.0 – 7.4), and assay
Tight containers
Metronidazole Minimum fi ll, pH (4.0 and
6.5), and assay
Laminated collapsible tubes at
controlled room temperature
Naftifi ne
hydrochloride
Microbial limits, minimum fi ll,
pH (5.5 – 7.5), content of
alcohol, and assay
Tight containers
Salicylic acid Alcohol content and assay Collapsible tubes or tight containers,
preferably at controlled room
temperature
Sodium sulfi de pH (11.5 – 13.5) and assay Tight containers at controlled room
temperature or in a cool place
Stannous fl uoride Viscosity, pH (2.8 – 4.0),
stannous ion content, total
tin content, and assay
Well - closed containers
Tolnaftate Minimum fi ll and assay Tight containers
pH electrodes from Crison, combination electrodes that contain a built - in temperature
probe, a bridge electrolyte chamber and movable sleeve junction from
Mettler, and combination pH puncture electrodes with spear - shaped tip from
Mettler are some commercially available pH measurement systems for semisolid
formulations.
Alcohol Content Alcohol levels in some gel preparations are determined by gas
chromatographic (GC) methods. Desoxymetasone gel USP and naftifi ne hydrochloride
gel USP contain 18 – 24% and 40 – 45% (w/w) of ethyl alcohol, respectively. In a
desoxymetasone gel, the sample is dissolved in methanol and injected into a gas
chramatograph for quantitative analysis. Isopropyl alcohol is used as an internal
standard. In naftifi ne hydrochloride gel, n - propyl alcohol is used as an internal
standard and water is used as the diluting solvent [15] .
Rheological Studies Viscosity measurement is often the quickest, most accurate,
reliable method to charactreize gels. It gives an idea about the ease with which gels
can be processed, handled, or used. Some of the commonly used tests for characterizing
rheology of gels are yield stress, critical strain, and creep. Yield stress refers to
the stress that must be exceeded to induce fl ow. This helps in characterizing the fl ow
nature of non - Newtonian systems. Critical strain or gel strength refers to the minimum
energy needed to disrupt the gel structure. When samples are subjected to increasing
stress, viscosity is maintained as long as the gel structure is maintained. When the
gel ’ s intermolecular forces are overcome by the oscillation stress, the sample breaks
down and the viscosity falls. The higher the critical strain, the better the physical
integrity of gel systems. Creep or recovery helps in assessing the strength of bonds
in a gel structure. This is assessed by determining relaxation times, zero - shear viscosity,
and viscoelastic properties.
Based on the nature of the test material, different techniques are employed to
measure the rheological parametrs of gels. Very sophisticated automatic equipment
is commercially available for measurements. Cup - and - bob viscometers and cone -
and - plate viscometers are widely used for viscous liquids and gels. They measure
the frictional force that is created when gels start fl owing. These viscometers are
usually calibrated with certifi ed viscosity standards before each measurement.
General - purpose silicone fl uids which are less sensitive to temperature change are
used as standards. The viscosity of gels is affected by the experimental temperature
and shear rate and the gels exhibit liquid - or solidlike properties. Hence the viscosity
of these non - Newtonian systems are recorded at several shear rates under controlled
temperatures. The USP specifi es the operating conditions for each gel formulation.
Commercially available viscometers include Brookfi eld rotational
viscometers, Haake rheometers, Schott viscoeasy rotational viscometers, Malvern
viscometers, and Ferranti - Shirley cone - and - plate viscometers.
Bioadhesion This test is performed to assess the force of adhesion of a gel with
biological membranes. The bioadhesive property is preferred for ophthalmic, nasal
buccal, and gastroretentive gel formulations. Drugs applied as solutions, viscous
solutions, and suspensions drain out from these biological locations within a short
time and only a limited fraction of drug elicits the pharmacological activity. Products
with higher bioadhesion thus help in increasing the contact time between drugs and
absorbing surface and improve their availability. The bioadhesive properties of
gels are measured using various custom - designed equipment. All the equipment,
however, measures the force required to detach the gel from a biological surface
under controlled experimental conditions (e.g., temperature, wetting level, contact
time, contact force, surface area of tissue). A typical bioadhesion measurement
system consists of a moving platform and a static platform. A tissue from a particular
biological region is fi xed onto these platforms and a known quantity of the test
product is uniformly applied to the tissue surface of the lower static platform. The
upper moving platform is allowed to contact with the product surface with a known
contact force. After allowing for a short contact time, the moving platform is
GELS 305
306 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
separated from the product with a constant rate. The force required to detach the
mucosal surface from the product is recorded. The analog signals generated by
precision load cells are then converted to digital signals through data acquisition
systems and processed using specifi c software programs.
Stability Studies Being dispersed systems containing water in their matrix, gels
are prone to physical, chemical, and microbial stability issues. Syneresis is a commonly
observed physical stability problem with gels. It involves squeezing out dispersion
medium due to elastic contraction of polymeric gelling agents. This results
in shrinkage of gels. Syneresis can be determined by heating the gels to a higher
temperature followed by rapid cooling using an ice water bath at room temperature.
The sample is preserved at 4 ° C for about a week, and water loss from the gel matrix
is measured. Water loss is measured by weighing the mass of the gel matrix after
centrifugation. Absence of syneresis indicates higher physical stability of gels. The
chemical stability of drugs in the gel matrix is determined using stability - indicating
analytical methods. Studies are conducted at accelerated temperature, moisture, and
light conditions to determine the possible degradation of drug in the gel.
Ex Vivo Penetration Ex vivo studies are carried out to examine the permeation
of drug from gels through the skin or any other biological membrane. As with in
vitro release studies, ex vivo penetration is conducted using vertical diffusion cells
or modifi ed cells with fl ow - through design. In this case, the receiver side is fi lled
with phosphate buffer solution of pH 7.4 to simulate the biological pH of human
blood. Skin samples from different animal sources such as rats, rabbits, pigs, and
human cadavers are used for screening dermatological products. The stratum
corneum layer of the skin is separated from the dermis before mounting onto the
diffusion cells. The epidermis is separated by immersing the skin sample in normal
saline or purifi ed water which is maintained at 60 ° C for 2 min followed by immersion
into cold water for 30 s. Careful peeling helps in the separation of the epidermis
layer from the dermis. This layer is mounted between the donor and receiver sides
and studies are conducted after application of test gel over the surface of the stratum
corneum in the donor side. Samples are withdrawn at different time intervals and
analyzed for drug permeation by suitable analytical techniques.
4.2.3.8 Typical Pharmacopeial and Commercial Examples
Gels are becoming popular dosage forms for delivering various categories of drugs
for treating dermatological, oral, ophthalmic, vaginal, and other conditions. Many
dermatological gels are used for treating mild to moderate acne, eczema, dermatitis,
allergies, rash, and psoriasis and for removal of common warts. Oral gels are available
for relieving painful mouth sores, treating tooth decay, preventing tooth plaque,
and relieving infl ammation of the gums, and vaginal gels are available for treating
certain type of vaginal infections (e.g., bacterial vaginosis). Some special types of
gels are available for preventing or controlling pain during certain medical procedures,
numbing and treating urinary tract infl ammation (urethritis), and numbing
mucous membranes. Table 10 shows some of the commercially available compendial
gels.
TABLE 10 Examples of Compendial/Commercial Gels
Drug a Category Indication
Commercial
Products
Strength
(%)
Benzocaine Local
anesthetic
In mouth to relieve pain
or irritation caused by
many conditions
Oratect Gel,
Num Zit
Gel
7.5, 10
Benzoyl
peroxide
Keratolytic Mild to moderate acne Persa - Gel, 5
Benzagel
10
5.0, 10
Betamethasone Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash
Diprolene 0.05
Clindamycin
phosphate
(vag)
Antibiotic Certain types of vaginal
infection (e.g., bacterial
vaginosis)
Cleocin T 1.0
Desoximetasone Anti -
infl ammatory
Eczema, dermatitis,
allergies, and rash.
Topicort 0.05
Dyclonine
hydrochloride
Antipruritic
and local
anesthetic
Relieve painful mouth
sores
Dyclone 0.5, 1.0
Erythromycin Antibiotic Acne and skin infection
due to bacteria
Erygel 2.0
Fluocinonide Anti -
infl ammatory
Psoriasis, eczema,
dermatitis, allergies,
and rash
Lidex 0.05
Lidocaine
hydrochloride
Local
anesthetic
Prevent and control pain
during certain medical
procedures, numb and
treat urinary tract
infl ammation
(urethritis), and numb
mucous membranes
Xylocaine,
Anestacon
2.0
Metronidazole
(vag)
Antifungal Certain types of bacterial
infections in the vagina
Metrogel 0.75, 1.0
Naftifi ne
hydrochloride
Antifungal Fungal infections of skin
such as jock itch,
athlete ’ s feet, or
ringworm
Naftin 1.0
Salicylic acid Keratolytic Removal of common
warts
Sal - Plant
Gel
17.0
Stannous
fl uoride
Fluoride Treat tooth decay, prevent
tooth plaque and
infl ammation of gums
Flo - Gel,
Gel - Kam
0.4
Tolnaftate Antifungal Skin infections such as
athlete ’ s foot, jock itch,
and ringworm
Tolnaftate 1.0
a vag: vaginal gel.
GELS 307
308 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
TABLE 11 Description on SUPAC Guidelines for Nonsterile Semisolid Dosage Forms
Type of Change Level Description
Change in
components
and
composition
1 Partial deletion or deletion of color, fragrance, or fl avor; up to
5% excipient change in approved amount; change in supplier
for structure forming or technical - grade excipient
2 Excipient changes from 5 to 10%; change in supplier for
structure forming excipient, which is not covered under level
1; change in technical grade of structure forming excipient;
change in particle size of drug if drug is in suspension
3 Qualitative and quantitative changes in excipient not covered
under levels 1 and 2; any change in crystallinity of drug if
drug is in suspension
4.2.4 REGULATORY REQUIREMENTS FOR SEMISOLIDS
Regulatory agencies such as the Center for Drug Evaluation and Research (CDER)
have at the Food and Drug Administration (FDA) have set forth guidelines for
various pharmaceutical activities to ensure the identity, strength, quality, safety, and
effi cacy of semisolid drug products. A manufacturer of semisolid formulations needs
to fulfi ll these requirements at the time of fi ling for investigational new drug (IND),
abbreviated new drug application (ANDA), or abbreviated antibiotic drug application
(AADA). Standard chemistry, manufacturing, and control (CMC) tests are
necessitated for all dermatological drug products. Additional information on polymorphic
form, particle size distribution, and other characteristics is needed for
submitting an NDA. When an ANDA for a semisolid product is fi led, the manufacturer
should meet the standards of compendial requirements if available and match
the important in vitro and in vivo characteristics of the reference listed drug (RLD).
If such information is not available, appropriate in vitro release methods are submitted
to ensure batch - to - batch consistency. Even at later stages, if changes are
made for an approved semisolid product with respect to its components, composition,
equipment, process, batch size, and manufacturing site, the formulator should
submit necessary details to the regulatory agency.
A typical guideline that defi nes the types and levels of scale - up and postapproval
changes (SUPAC) is outlined in Table 11 . Based on the type and level of change,
the manufacturer needs to submit application and compendial product release
requirements, executed batch records, accelerated and long - term stability data, identifi
cation and assay for new preservative, preservative effectiveness test at lowest
specifi ed level, validation methods to support absence of interference of preservative
with other tests, in vitro release test, and in vivo bioequivalence data to the
FDA. When changes are made with respect to the quality and quantity of excipients
or crystallinity of drug, especially if the drug is in suspension, in vivo bioequivalence
studies are recommended. As routine pharmacokinetic studies do not produce
measurable quantities of drug in blood, plasma, urine, and other extracutaneous
biological fl uids, dermatopharmacokinetic (DPK) studies and pharmacodynamic or
comparative clinical trials are recommended to establish bioequivalence of topical
products. Table 12 shows specifi c requirements for various SUPAC levels. If bioavailability
or bioequivalence data of a highest strength product are already available,
TABLE 12 SUPAC Requirements for Nonsterile Semisolid Dosage Forms
Parameter
Change
Level
Requirements a
A B C D E F G H I J K
Change in
components
and
composition
1 • •
2 • • • •
3 • • • • •
Change in
preservative
components
and
composition
1 • •
2 • •
3 • • • • •
Change in
manufacturing
equipment
1 • •
2 • • • •
Change in
manufacturing
process
1 •
2 • • • •
Change in batch
size
1 • • •
2 • • •
REGULATORY REQUIREMENTS FOR SEMISOLIDS 309
Type of Change Level Description
Change in
preservative
components
and
composition
1 Less than 10% quantitative change in preservative
2 10 – 20% quantitative change in preservative
3 Deletion or more than 20% quantitative change in
preservative; inclusion of a different preservative
Change in
manufacturing
equipment
1 Change to automated or mechanical equipment for transfer of
ingredients; use of alternative equipment of same design and
operating principles
2 Use of alternative equipment of different design and operating
principles; change in type of mixing equipment
Change in
manufacturing
process
1 Changes in process within approved application ranges;
addition of formulation additives
2 Changes in process outside approved application ranges;
process of combining phases
Change in batch
size
1 Batch size changes upto 10 times of pivotal clinical trial or
biobatch
2 Batch size changes above 10 times of pivotal clinical trial or
biobatch
Change in
manufacturing
site
1 Changes within existing facility
2 Changes within same campus or facilities in adjacent city
blocks
3 Change to different campus; change to a contract manufacturer
TABLE 11 Continued
310 SEMISOLID DOSAGES: OINTMENTS, CREAMS, AND GELS
in vitro release data are used to evaluate the in vivo bioequivalence of a lower
strength product [16] .
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Parameter
Change
Level
Requirements a
A B C D E F G H I J K
Change in
manufacturing
site
1 •
2 • • • •
3 • • • • •
a Only those highlighted with black circles:
A: Application/compendial product release requirement.
B: Executed batch records.
C: long - term stability data for fi rst production batch.
D: 3 - month accelerated stability data for 1 batch and long - term data for fi rst production batch.
E: 3 - month accelerated stability data for 1 batch and long - term data for fi rst 3 production batches if signifi
cant information is available or 3 - month accelerated stability data for 3 batches and long - term data
for fi rst 3 production batches if signifi cant information is not available.
F: 3 - month accelerated stability data for 1 batch and long - term data for fi rst production batch if signifi -
cant information is available or 3 - month accelerated stability data for 3 batches and long - term data for
fi rst 3 production batches if signifi cant information is not available.
G: In vitro release test.
H: In vivo bioequivalence.
I: Preservative effectiveness test at lowest specifi ed preservative level.
J: Identifi cation and assay for new preservative; validation methods to support absence of interference
with other tests.
K: Location of new site.
TABLE 12 Continued
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9. Mores , L. R. ( 1980 ), Application of stearates in cosmetic creams and lotions , Cosmet.
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10. Mapstone , G. E. ( 1974 ), Crystalization of cetyl alcohol from cosmetic emulsions , Cosmet.
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32 – 36 .
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30. Goldstein , A. M. , Alter , E. N. , and Seaman , J. K. ( 1973 ), Guar gum , in Whistler , R. L. , Ed.,
Industrial Gums , 2nd ed. , Academic , New York , pp. 303 – 321 .
31. Gauger , L. J. ( 1984 ), Hydroxyethylcellulose gel as a dinaprostone vehicle , Am. J. Hosp.
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33. Cabana , A. , Ait - Kadi , A. , and Juhasz , J. ( 1997 ), Study of the gelation process of polyethylene
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313
4.3
LIQUID DOSAGE FORMS
Maria V. Rubio - Bonilla 1 , Roberto Londono 1 , and Arcesio Rubio 2
1 Washington State University, Pullman, Washington
2 Caracas, Venezuela
Contents
4.3.1 Introduction
4.3.2 Generalities
4.3.2.1 Dosage Form
4.3.2.2 Liquid Dosage Form
4.3.2.3 Dispersed Systems
4.3.2.4 Solutions
4.3.2.5 Manufacturing of Nonparenteral Liquid Dosage Forms
4.3.2.6 Optimizing Drug Development Strategies
4.3.2.7 Unit Operation or Batch
4.3.2.8 Batch Management
4.3.2.9 Steps of Liquids Manufacturing Process
4.3.2.10 Protocols
4.3.3 Approaches
4.3.4 Critical Aspects of Liquids Manufacturing Process
4.3.4.1 Physical Plant
4.3.4.2 Equipment
4.3.4.3 Particle Size of Raw Materials
4.3.4.4 Compounding: Effects of Heat and Process Time
4.3.4.5 Uniformity of Oral Suspensions
4.3.4.6 Uniformity of Emulsions
4.3.4.7 Microbiological Quality
4.3.4.8 Filling and Packing
4.3.4.9 Stability
4.3.4.10 Process Validation
4.3.5 Liquid Dosage Forms
References
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
314 LIQUID DOSAGE FORMS
4.3.1 INTRODUCTION
Liquid dosage forms are designed to provide the maximum therapeutic response in
a target population with diffi culty swallowing tablets and capsules and/or to produce
rapid therapeutic effects. The major ingredient in most liquid dosage forms is water.
While it is the safest and most palatable solvent option, water quality is signifi cant
for the stability of pharmaceutical dosage forms. Furthermore, the Food and
Drug Administration (FDA) “ Guide to Inspections of Dosage Form Drug Manufacturer
’ s — CGMPRs ” considers microbial contamination due to inappropriate
design and control of purifi ed water systems as the most common problem of liquid
dosage forms. Solutions and dispersions studied in this chapter are chemically,
microbiologically, and/or physically unstable systems that require a high level of
organizational management of manufacturing processes in order to maintain a state
of apparent stability, at least until the expiration date [1] .
The pharmaceutical industry manufactures dosage forms in large - scale formulations.
The decision to scale up is based on the economics of the production process
related to costs of materials, personnel, equipment, and control [2] . To reduce costs
of wastes and to obtain high - quality and effi cacious drug products, the strategic plan
to be applied during the process has to be developed carefully. In fact, the variables
that affect product quality are identifi ed and understood in the process instead of
tested into the fi nal product [3] .
Commercial liquid dosage forms reach large - scale production after being preformulated
at the laboratory level followed by formulation at the small scale and then
at the pilot plant scale. Due to the complexity of the manufacturing process, scale - up
from pilot to commercial production is not a simple extrapolation. The approaches
to the four levels of production are different. Most of the formulation ingredients
are analyzed, studied, and selected at the laboratory scale. While small - scale production
is more focused on the liquid preparation procedure with higher amounts of
ingredients, the main issues at the pilot plant scale are the design of infrastructure
and reduction of costs. Commercial production introduces problems that are not a
major issue on a small scale, for instance, materials handling and storage, pulverizing,
mixing, dissipation of the generated heat during production, time control, personnel
administration, and bottle - fi lling capabilities. Furthermore, purifi ed water is essential
for the manufacturing of these products as well as on - site packing capabilities
[2] .
The organization and advance of the pharmaceutical industry should be focused
on three main points of project development based on quality by design (QbD):
product objective (design of experiment, DoE), production resources (process analytical
technology, PAT), and product acceptability (quality system) [3] . Manufacturers
of liquid dosage forms must ensure safety, effi cacy, stability, elegance, and
acceptability of the fi nal drug product while achieving development and clinical
milestones [1] . Achieving the desirable clinical attributes of the product effi cacy
means ensuring potency stability by confi rming the functionability of the manufacturing
process and quality system. The stability and safety of the product are
goals to be reached through chemical and microbiological stability by establishing
and updating manufacturing and quality control protocols of product development.
Time control and aesthetic considerations refl ect the physical stability through
product elegance and acceptability. Flavoring, sweetening, coloring, and texturing
are both challenges and opportunities. They are challenges because “ no single
correct method exists to solve signifi cant problems of elegance ” ; they are opportunities
because “ they enable a pharmacist to prepare a product more easily
accepted by the patient ” [4] . Although the most important characteristic of a
dosage form is effi cacy, there are other characteristics that remain important subjects
for the manufacturing of liquid dosage forms such as safety as well as chemical,
physical, and microbiological stabilities. From a pharmaceutical point of view,
stability problems are the main causes of safety complaints. Despite its signifi cance,
some companies decide to outsource stability services [5] . To solve or minimize
stability problems in drug products, it is necessary to analyze and enhance the
development of critical control points in each operation of the full manufacturing
process as well as expected variances and tolerance limits.
Except some aqueous acids, water in aqueous solutions is an excellent media for
microbiological growth, such as molds, yeast, and bacteria. Typical microorganisms
affecting drug microbiological stability are Pseudomonas, Escherichia coli Salmonella
, and Staphylococcus [1] . Defi cient methods or an insuffi cient preservative
system may be the principal causes of microbiological contamination in the pharmaceutical
industry of liquid manufacturing [6] .
Chemical instability reactions appear with or without microbiological contribution
through reactions such as hydrolysis, oxidation, isomerization, and epimerization.
Interactions between ingredients and ingredients with container closure
materials are established as the principal causes of these reactions [1] , for instance,
the hydrolysis of cefotaxime sodium, the oxidation of vitamin C, the isomerization
of epinephrine, and the epimerization of tetracycline [7] .
In most cases, physical instabilities are consequences of previous chemical instabilities.
Physical instabilities can arise principally from changes in uniformity of
suspensions or emulsions, diffi culties related to dissolution of ingredients, and
volume changes [6] . For instance, some cases where physical stability has been
affected are cloudiness, fl occulence, fi lm formation, separation of phases, precipitation,
crystal formation, droplets of fog forming on the inside of container, and swelling
of the container [8] .
Although commercial oral solution and emulsion dosage forms rarely present
bioequivalence issues, some bioequivalence problems have been reported for oral
suspensions such as phenytoin [9] . The possibility of microbiological contamination
and physicochemical instabilities during the manufacturing process also needs
to be carefully considered. To approach the stability problems of liquid dosage
forms, in this chapter, the main critical aspects during the manufacturing process
are based on FDA inspection. From physical plant systems to batching management
and packing, the potential sources of microbiological, chemical, and physical instabilities
will be analyzed using defi nitions, case - by - case explanations, and practical
examples.
Final product stability, which determines the therapeutic activity and uniformity
among other characteristics of the fi nal product, refl ects the dynamic of the production
process. Conceptualization of stability issues is important to determine the
changes to enhance the design space as well as protocols of manufacturing and
quality control [3] . An information technology (IT) solution like Enterprice Resource
Planning (ERP) may support the pharmaceutical industry ’ s current challenges of
organization [10] .
INTRODUCTION 315
316 LIQUID DOSAGE FORMS
4.3.2 GENERALITIES
4.3.2.1 Dosage Form
According to the FDA: “ A dosage form is the physical form in which a drug is produced
and dispensed. In determining dosage form, FDA examines such factors as
(1) Elegance: physical appearance of the drug product, (2) Stability: physical form
of the drug product prior to dispensing to the patient, (3) Acceptability: the way the
product is administered, (4) Effi cacy: frequency of dosing, and (5) Safety: how pharmacists
and other health professionals might recognize and handle the product ”
[11] . The term dosage form is different from “ dose, ” which is defi ned as a specifi c
amount of a therapeutic agent that can be taken at one time or at intervals.
4.3.2.2 Liquid Dosage Form
The physical form of a drug product that is pourable displays Newtonian or pseudoplastic
fl ow behavior and conforms to its container at room temperature. In contrast,
a semisolid is not pourable and does not fl ow at low shear stress or conform
to its container at room temperature [12] . According to its physical characteristics,
liquid dosage forms may be dispersed systems or solutions.
4.3.2.3 Dispersed Systems
Dispersed systems are dosage forms composed of two or more phases, where one
phase is distributed in another [2] . If a dispersed system is formed by liquid phases,
then it is known as an “ emulsion. ” In contrast, the dispersed system is named a
“ suspension ” when the liquid dosage form is accomplished by the distribution of a
solid phase suspended in a liquid matrix. The solid phase of a suspension is usually
the drug substance, which is insoluble or very poorly soluble in the matrix [12] .
4.3.2.4 Solutions
A solution refers two or more substances mixed homogeneously [2] . Although solubility
refers to the concentration of a solute in a saturated solution at a specifi c
temperature, in pharmacy, solution liquid dosage forms are unsatured to avoid
crystallization of the drug by seeding of particles or changes of pH or temperature
[13] . The precipitation of drug crystals is one of the most important physical instabilities
of solutions that may affect its performance [14] . Water is the most used
solvent in solutions manufacturing; however, there are also some commercial nonaqueous
solutions in the pharmaceutical market [1] .
4.3.2.5 Manufacturing of Nonparenteral Liquid Dosage Forms
The manufacturing of liquid dosage forms with market - oriented planning includes
the following stages with respect to special good manufacturing practice (GMP)
requirements: planning of material requirements, liquid preparation, fi lling and
packing, sales of drug products, vendor handling, and customer service [15] . From
the viewpoint of product stability, each stage of the process includes critical batches
that are more decisive than others. Also, each decisive batch contains one or several
unit operations that are more critical than others. The FDA inspection focuses on
those critical unit operations to ensure the safety and stability of the liquid dosage
forms [6] .
4.3.2.6 Optimizing Drug Development Strategies
According to Sokoll [16] : The phases of drug development include discovery, preclinical
development, clinical development, fi ling for licensure, approval/licensure and post -
approval. Discovery typically includes basic research, drug identifi cation and early -
stage process and analytical method development. . . . Emerging companies that review
their pipeline objectively and strike a balance between properly resourcing and developing
their lead candidates in the clinic while nurturing their next generation of drug
candidates will have the best chance for success and sustainability.
4.3.2.7 Unit Operation or Batch
A “ batch ” job or operation is defi ned as a unit of work. Raw materials, semifi nished
drug products (bulk), and fi nished drug products are handled in batches. Each different
type of material used during the process, such as product packing, should be
managed by batches. This applies also to process aids and operation facilities [15] .
4.3.2.8 Batch Management
The batch management of production simplifi es the process and makes it easier to
control the status of transformation between raw and fi nal products [2] . Some of
the data used to follow the material performance around and out of the product
manufacturing process are batch - where - used - list, initial status, batch determinations,
master data, and expiration date check [15] .
The functionality of the overall process to manufacture liquid dosage forms
depends on the successful linkage of one unit operation to another. To use mathematical
formulations to scale up the manufacturing process, it is necessary to divide
the process into stages, batches, and unit operations. Each single unit operation is
scalable, but the composite manufacturing process is not. Production problems
result from attempts to follow a process scale - up instead of a unit operation scale -
up. By using mathematical formulations, it is possible to understand the level of
similarity between two scale sizes. In addition, nonlinear similarities between two
scale sizes might require the use of conversion factors to achieve an extrapolation
point for the scale [2] .
4.3.2.9 Steps of Liquids Manufacturing Process
Establishing short - term goals makes it easier to measure effi ciency as well as evaluate
the diffi culties [2] . Based on these concepts, the problems of manufacturing
liquid dosage forms can be approached as problems in one or more batches of the
following process steps [6, 15] :
Planning of Material Requirements Research and development of protocols and
selection of materials; acquisition and analysis of raw materials; physical plant
GENERALITIES 317
318 LIQUID DOSAGE FORMS
design, building, and installation; equipment selection and acquisition; personnel
selection and initial training; and monitoring information system.
Liquid Preparation Research and development of protocols concerning liquid
compounding; scale - up of the bulk product compounding; physical plant control
and maintenance; equipment maintenance and renovation; continuous
training of personnel and personnel compensation plan; and supervision of
system reports.
Filling and Packing Research and development of protocols concerning fi lling
and packing; scale - up of the fi nished drug product fi lling and packing; physical
plant control and maintenance; equipment maintenance and renovation; continuous
training of personnel and personnel compensation plan; and supervision
of system reports.
Sales of Drug Products Research and development of protocols concerning
product storage; distribution process; continuous training of personnel and
personnel compensation plan; and supervision of system reports.
Vendor Handling Research and development protocols concerning precautions
to maintain product stability; control of vendor stock; and sales system
reports.
Customer Service Research and development of protocols concerning home
storage and handling to maintain product stability; relations with health insurance
companies and health care professionals; educational materials for patient
counseling; and customer service system reports.
4.3.2.10 Protocols
Protocols are patterns developed by repeating procedures and fi xing the identifi ed
problems each time that the procedure is followed. Therefore, protocols are dynamic
entities that originally can be developed at a laboratory level but must be adjusted
in every new step of the scal - up process. When the manufacturing process moves
up in scale, the number of people affected by the protocol increases geometrically.
Initially, the information can be obtained from library references, personal tests,
interpersonal training, and previous laboratory protocols. However, when the production
is scaled up, the information required to fi ne tune the process comes from
monitoring the process itself [2] .
4.3.3 APPROACHES
Quality by Design is a systemic approach that applies the scientifi c method to the
process. QbD theory contains components of management, statistics, psychology,
and sociology. The FDA ’ s new century has identifi ed the QbD approach as its “ key
component ” based on process quality control before industry end results [3, 17] .
The cooperation between industry members and regulators is increased when the
industry explains clearly what it is doing and the agency can understand the formulation
and production process. In these cases, regulatory relief appears when industry
explores its issues and receives active guidance and programs from the FDA.
The agency takes the role of facilitator, or even partner of the industry, in order to
improve the strength of the process and formulation [3, 17] .
To apply QbD as a systemic approach, the company starts by understanding,
step by step, the space design, the design of the dosage form, the manufacturing
process, and the critical process parameters to be controlled in order to reach the
new building block which is the expectation of variances within those critical
process parameters that can be accepted. This approach allows the establishment
of priorities and fl exible boundaries in the process [3] . Infl exible specifi cations
allow uncontrolled small variances that can follow the butterfl y effect of the theory
of chaos by producing unpredictable large variations in the long - term behavior of
the product shelf - life [18, 19] . In contrast, fl exibility, with knowledge of potential
variances, reduces changes in the approved spaces and manufacturing protocols
[3, 17] .
According to the FDA [6] , critical parameters during the manufacturing process
of nonparenteral liquid dosage forms may appear in the design of physical plant
systems, equipment, protocols of usage and maintenance, raw materials, compounding,
microbiological quality control, uniformity of suspensions and emulsions, and
fi lling and packing [6] .
Process isolation and installation of an appropriate air fi ltration system in the
physical plant may reduce product exposition to chemical and microbiological
contaminations. In addition, the use of a suitable dust removal system as well as
a heating, ventilation, and air conditioning system (HVACS) may help to repress
product chemical instabilities [6] .
The equipment of sanitary design, including transfer lines, as well as appropriate
cleaning and sanitization protocols may reduce chemical and microbiological contaminations
in the fi nal product. Chemical instabilities may be reduced by weighting
the right amount of liquids instead of using a volumetric measurement, avoiding the
common use of connections between processes, and using appropriate batching
equipment [6] .
Particle sizes of raw materials are critical to control dissolution in solutions as
well as uniformity in suspensions and emulsions. Temperature control during compounding
is important since heat helps to support mixing and/or fi lling operations,
but, in contrast, high - energy mixers may produce adverse levels of heat that affect
product stability. Too much heat may cause chemical and physical instabilities such
as change of particle size or crystallization of drugs in suspensions, dissolution and
potency loss of drugs in suspensions, oxidation of components, and activation of
microbiological growth after degradation of compounds as well as precipitation of
dissolved compounds in solution [20] . In addition, uniformity of suspensions depends
on viscosity and segregation factors while solubility, particle size, and crystalline
form determine uniformity of emulsions. Application of pharmaceutical GMP for
product processes and storage assures microbiological quality. A defi cient deionizer
water - monitoring program and product preservative system facilitate microbial contamination.
Filling uniformity is indispensable for potency uniformity of unit - dose
products and depends on the mixing operation. Calibration of provided measuring
devices and the use of clean containers will allow administering the right amount
of the expected components in the liquid dosage form [6] .
Principal product specifi cations are microbial limits and testing methods, particle
size, viscosity, pH, and dissolution of components. Process validation requires control
of critical parameters observed during compounding and scale - up. Product stability
examination is based on chemical degradation of the active components and interac-
APPROACHES 319
320 LIQUID DOSAGE FORMS
tions with closure systems, physical consequences of moisture loss, and microbial
contamination control [6] .
4.3.4 CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS
4.3.4.1 Physical Plant
Heating, Ventilation, and Air Conditioning System The manufacturer has to
warrant adequate heating, ventilation, and air conditioning in places where labile
drugs are processed [6] .
The effect of long processing times at suboptimal temperatures should be considered
at the production scale in terms of the consequences on the physical or chemical
stability of individual ingredients and product. A pilot plant or production scale
differs from laboratory scale in that their volume - to - surface - area ratio is relatively
large. Thus, for prolonged suboptimal temperatures, jacketed vessels or immersion
heaters or cooling units with rapid circulation times are absolutely necessary [2] .
For heat - labile drugs, uncontrolled temperature increments can activate auto -
oxidation chains when the drug product ingredients react with oxygen and generate
free radicals but without drastic external interference. Vitamins, essential oils, and
almost all fats and oils can be oxidized. A good example of a heat - labile drug solution
is clindamycin, which has to be stored at room temperature and away from
excess heat and moisture [19] . Auto - oxidation chains are fi nished when free radicals
react with each other or with antioxidant molecules (quenching). The tocopherols,
some esters of gallic acid, as well as BHA and BHT (butylated hydroxyanisole and
butylated hydroxytoluene) are common antioxidants used in the pharmaceutical
industry [1] .
Isolation of Processes To minimize cross - contamination and microbiological contamination,
the manufacturer may develop special procedures for the isolation of
processes. The level of facilities isolation depends on the types of products to be
manufactured. For instance, steroids and sulfas require more isolation than over -
the - counter (OTC) oral products [6] . To minimize exposure of personnel to drug
aerosols and loss of product, a sealed pressure vessel must be used to compound
aerosol suspensions and emulsions [21] . An example of cross - contamination with
steroids was the controversial case of a topical drug manufactured for the treatment
of skin diseases. High - performance liquid chromatography/ultraviolet and mass
spectrometry (HPLC/UV, HPLC/MS) techniques were used by the FDA for the
detection of clobetasol propionate, a class 1 superpotent steroid, as an undeclared
steroid in zinc pyrithione formulations. The product was forbidden and a warning
was widely published [22] .
Dust Removal System The effi ciency of the dust removal system depends on the
amount and characteristics of dust generated during the addition of drug substance
and powdered excipients to manufacturing vessels [6] . Pharmaceutical industries
usually generate some type of dust or fume during processing. Important factors for
selecting dust collectors are maintenance, surrogate test, economics, and containment.
In addition, reentrainment of the fi ne particles, vertical or horizontal position,
effi ciency, pressure resistant, service life time, as well as sealing capacity to work
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 321
through the bag are signifi cant factors concerning fi lter selection of dust removal
systems. Some examples of dust collection applications in the manufacture of liquid
dosage forms are handling and pulverization of raw materials, spray dryers, and
general room ventilation [23] .
Air Filtration System The effi ciency of the air fi ltration system has to be demonstrated
by surface or air - sampling data where the air is recirculated [6] . To monitor
the levels of contamination in the air, there are commercial automatic samplers for
microbiological contamination or gas presence. Air trace environmental samplers
for pharmaceutical industries are based on the slit - to - agar impaction technique for
the presence of viable microorganisms. Automatic samplers for compressed gas
analyze the presence of a specifi ed gas in 1 m 3 by absorbing air at a fi xed fl ow rate
for a sampling period of 1 h or a different adjusted time. These solutions to the
sampling needs of the pharmaceutical industry are robust, require low maintenance,
and are easy to use. This allows for validation of sampling data at the moment of
application fi lling to support the process control. Sampling time and selection of
microbiological growth media or analysis technique are important components to
consider when developing a sampling plan [24] .
4.3.4.2 Equipment
Sanitary Design Pumps, valves, fl owmeters, and other equipment should be easily
sanitized. Some examples of identifi ed sources of contamination are ball valves,
packing in pumps, and pockets in fl owmeters [6] .
The sanitary design and performance of equipment make it accessible for inspection,
cleaning, and maintenance. It has to be cleanable at a microbiological level and
its performance during normal operations should contribute to sanitary conditions.
The materials used in the design have to assure hygienic compatibility with other
equipment, the product, the environment, other systems such as electrical, hydraulics,
steam, air, and water, as well as the method and products used for cleaning and
sanitation. The equipment should be self - draining to assure product or liquid collection.
Small niches, for example, pits, cracks, corrosion, recesses, open seams, gaps,
lap seams, protruding ledges, inside threads, bolt rivets, and dead ends, as well as
inaccessible cavities of equipment such as entrap and curlers must be eliminated
whenever possible; otherwise they have to be permanently sealed. Enclosures, for
example, push buttons, valve handles, switches, and touch screens, should be prepared
for a hygienic design of maintenance. Standards have been developed by the
American Meat Institute [25] .
Standard Operating Procedures for Cleaning Production Equipments Current
GMPs are defi ned as the basic principles, procedures, and resources required to guarantee
an environment appropriate for manufacturing products of adequate quality
[26] . To minimize cross - contamination and microbiological contamination, it is GMP
for a manufacturer to create and pursue written standard operating procedures
(SOPs) to clean and sanitize production equipment in a way that avoids contamination
of in - progress and upcoming batches. When the drug is known as a potent generator
of allergic reactions, such as steroids, antibiotics, or sulfas, cross - contamination
becomes an issue of safety [20] . In addition, validation and data analysis procedures,
322 LIQUID DOSAGE FORMS
FIGURE 1 Mixing and fi lling lines for pharmaceutical dosage forms. Positive indoor pressure
of 5 psi over outdoor pressure assures constant airfl ow from inside to outside in order
to reduce entrance of contaminating agents.
UV Rays
Collector Tank
Continuous or Batch
GENERAL DIAGRAM “A”
INDUSTRIAL MANUFACTURING PLANT
FOR PHARMACEUTICAL LIQUID DOSAGE FORMS Decontamination
Camera
Restricted access area
Pressure = Atmospheric pressure + 5 PSIG
Bottling
Equipment
Packing
Filters
Compressor
Homogenizator
Main
Pump
Filters
Mixer
Dosing
Pumps
Primary Components Tanks
Restriction
Gate
Compressor
Purified
Water
Air
recycle
including drawings of the manufacturing and fi lling lines [6] , are especially important
for clean - in - place (CIP) systems, as indicated in Figures 1 and 2 .
Many companies have problems with standardizing operating procedures for
cleaning steps and materials used [6] . Appropriate SOPs are necessary to determine
the scope of the problem in investigations about possible cross - contaminations or
mix - ups. The best approach to validate a SOP is to test it, use it as a training tool,
and observe the results obtained by different persons. This includes the worst - case
situation in order to enhance the step - by - step writing methodology as well as standardizing
the materials used. A typical SOP contains a header to present the SOP
title, date of issue, date of last review, total number of pages, responsible person,
and approval signature. Typically, a SOP includes position of responsible person,
SOP purpose and scope, defi nitions, equipment and materials, safety concerns, step -
by - step procedure, explanation of critical steps, tables to keep data, copies of forms
to fi ll, and references [26] . The forms to keep the records must show the date, time,
product, and lot number of each batch processed. However, the most important
points of the SOP are equipment identity, cleaning method(s) with documentation
of critical cleaning steps, materials approved for cleaning that have to be easily
removable, names and position of persons responsible for cleaning and inspection,
inspection methods, and maintenance and cleaning history of the equipment [20] .
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 323
Cleaning and Sanitizing Transfer Lines Pipes should be hard, easily cleaned, and
sanitized. To avoid moisture collection and microbiological contamination, hoses
should be stored in a way that allows them to drain rather than be looped. For
example, transfer lines are an important source of contamination when fl exible
hoses are handled by operators, lying on the fl oor, and after they are placed in
transfer or batching tanks [6] .
Heat is considered one of the most effi cient physical treatments for sanitizing
pharmaceutical equipment and could be used for sanitizing hoses that have already
been cleaned. The recirculation of hot water at a temperature of 95 o C for at least
100 min allows bacteria elimination [14] .
Due to the important amount of insoluble residues left on piping and transfer
lines after emulsion manufacturing, such as topical creams and ointments, equipment
cleaning becomes diffi cult to address. To avoid cross - contamination, some
manufacturers have decided to dedicate lines and hoses to specifi c products.
FIGURE 2 Mixing and fi lling lines for pharmaceutical dosage forms. Using this hydropneumatic
system, instead of the mechanical system in Figure 1 , the liquid moves by the
pressure generated in a compressed air tank.
Hydropneumatic
vessel
UV Rays
Restricted access area
Pressure = Atmospheric pressure + 5 PSIG
Compressor
Collector Tank
Continuous or Batch
GENERAL DIAGRAM “B”
INDUSTRIAL MANUFACTURING PLANT
FOR PHARMACEUTICAL LIQUID DOSAGE FORMS
OF LOW VISCOSITY
Restriction
Gate
Air
Recycle
Pressure
pump
Decontamination
Camera
Bottling
Equipment
Homogenizator Dosing
Pumps
Primary Components Tanks Purified
Water
Homogenizator
Mixing
Control
Booster
Pump
Mixer
Packing
Filters
Compressor
Restriction
Gate
Filters
324 LIQUID DOSAGE FORMS
However, these decisions have to appear in the written production protocols and
SOPs [20] .
Sampling Cleaned Surfaces for Presence of Residues The cleaning method is
validated by sampling the cleaned surfaces of the equipment for the existence of
residues. The equipment characteristics and residue solubility are factors to support
the selection of the sampling method to be used [6] . There are two acceptable
general types of sampling methods: direct surface sampling by swabbing of surfaces
and rinse sampling with a routine production in - process control. Although surface
residues will not be identical on each part of the surface, statistically the most
advantageous is direct surface sampling because it allows evaluation of the hardest
areas to clean as well as insoluble or “ dried - out ” residues by physical removal. The
type of sampling material and solvent used for extraction from the sampling material
should be validated in order to determine their impact on the test data. The
second method, rinse sampling, is used for larger surfaces or inaccessible systems.
Contaminants that are physically occluded and insoluble residues are disadvantages
of the rinse sampling method. To validate this cleaning process, direct measurement
of the contaminant in the rinse water has to be tested instead of a simple
test for water quality. Routine production in - process control is used as indirect
testing for large equipment that has to be cleaned by the rinse sampling method.
The uncleaned equipment has to give an unacceptable result for the indirect
test [27] .
Establishing Appropriate Limits on Levels of Postequipment Cleaning
Residues Very low levels of residue are possible to be determined since technological
advances offer more sensitive analytical methods. The manufacturer should
know the toxicological information of the materials used and potential amounts of
residues after exposure to the equipment surface. Accordingly, the manufacturer
has to establish proper limits of residues after equipment cleaning and scientifi cally
justify these limits. The established limits must be clinically and pharmaceutically
safe, realistic, viable, and verifi able [20] . The sensitivity of the analytical method will
determine the logic of the established limits since absence of residues could indicate
a low sensitivity of the analytical method or a poor sampling procedure. Sometimes
thin - layer chromatography (TLC) screening must be used in addition to chemical
analyses. Some practical levels established by manufacturers include 10 ppm of
chemicals, 1/1000 of the biological activity levels met on a normal therapeutic dose,
and no visible residues of particles determined organoleptically [27] .
Connections Connectors and manifolds should not be for common use. For
example, sharing connectors in a water supply, premix, or raw material supply tanks
may be a source of cross - contamination [6] .
Time between Completion of Manufacturing and Initiation of Cleaning The time
that may elapse from completion of a manufacturing operation to initiation of
equipment cleaning should also be stated where excessive delay may affect the
adequacy of the established cleaning procedure. For example, residual product may
dry and become more diffi cult to clean [20] .
SOPs are an example of defi ciency in many manufacturers regarding time limitations
between batch cleaning and sanitization [6] . Lack of communication between
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 325
departments responsible for the production at different levels is the main cause of
time control problems. Typically each department, from human resources to fi nances,
manufacturing, and warehouse, has its own computer system optimized for the
particular ways that the department does its work. Therefore, time control becomes
a primordial issue when labile materials are transferred from one department to
another [28] .
To facilitate communication between different departments, some useful softwares
have been developed. For example, ERP is an integrated approach which may
have positive payback if the manufacturer installs it correctly. An ERP is a type
of software that can improve communication between planning and resources. The
software attempts to integrate all departments and functions in a company onto a
single computer system that can serve each particular need, such as fi nance, human
resources, manufacturing management, process manufacturing management, inventory
management, purchasing management, quality management, and sales management.
Each department has its own software, except now the software is linked
together, so that, for example, someone in manufacturing can look into the maintenance
software to see if specifi c batch cleaning and sanitization have been scheduled
or realized and someone in fi nance can review the warehouse software to see if a
specifi c order has been shipped. The information is online and not in someone ’ s
heads or on papers that can be misplaced. People in different departments may see
the same information, update it if they are allowed to do, and make right decisions
faster. However, the software is less important than the changes companies make
in the ways they work. Reorganization and training are the keys of ERP ’ s success
to fi x integration problems. There are three different ways to install an ERP: big
bang, franchising strategy, and slam dunk. Big bang is the most ambitious way
whereby companies install a single ERP across the entire company. By the franchising
strategy, departments do not share many common processes across, whereas
slam dunk is focused on just a few key processes [28] .
Weight in Formulations Flow properties of liquids rarely vary due to their constant
density at a constant temperature. Oral solutions and suspensions are formulated
on a weight basis (gravimetry) in order to be able to measure the fi nal volume
by weight before fi lling and packing. Volumetric measurements of liquid amounts
to be used for manufacturing liquid dosage forms have shown greater variability
than weighted liquids. For instance, the inaccurate measurement of the fi nal volume
by using dip sticks or a line on a tank may cause further analytical errors and
potency changes [6] .
The importance of selecting gravimetry instead of volumetry to measure liquid
amounts in the pharmaceutical industry of liquid dosage forms is well illustrated by
the volume contraction of water – ethanol and volume expansion of ethyl acetate –
carbon disulfi de liquid mixtures as well as a CS2 – ethyl acetate system. The National
Formulary (NF) diluted alcohol is a typical example of the volume nonadditivity of
liquid mixtures [29] . This solution is prepared by mixing equal volumes of alcohol
[U.S. Pharmacopeia (USP)] USP and purifi ed water (USP). The fi nal volume of this
solution is about 3% less than the sum of the individual volumes because of the
contraction due to the mixing phenomenon [1] . In addition, molecular interactions
of surfactants in mixed monolayers at the air – aqueous solution interface and in
mixed micelles in aqueous media also cause some contraction of volume upon
mixing [30] .
326 LIQUID DOSAGE FORMS
Location of Bottom Discharge Valve in Batching Tank The bottom discharge
valve should be located exactly at the bottom of the tank. In some cases valves have
been found to be several inches to a foot above the bottom of the tank [6] .
For a tank suspected of having substantial deposits at the bottom, a fi ber - optic
camera can be inserted in the tank to provide a view and positive confi rmation of
the tank bottom condition. These camera and light vision systems are sanitary equipment
able to provide a computational real - time visual inspection of the inside tank
under process conditions or pressure vessel. In addition, they are used to control
several parameters during the manufacturing process, such as product level and
thickness, solids level, uniformity of suspensions, foam, and interface and/or cake
detection [31] .
Batching Equipment to Mix Solution Ingredients of solutions have to be completely
dissolved. For instance, it has been observed that some low - solubility drugs
or preservatives can be kept in the “ dead leg ” below the tank, and the initial samples
have reduced potency [6] . When there is inadequate solubility of the drug in the
chosen vehicle, the dose is unable to contain the correct amount of drug in a manageable
size unit, that is, one teaspoonful or one tablespoonful. Thus, ingredients
as well as handling and storage conditions should be chosen to manage the problem
[14] .
In solutions, the most important physical factors that infl uence the solubility of
ingredients are type of fl uid, mixing equipment, and mixing operations. Generalized
Newtonian fl uids are ideal fl uids for which the ratio of the shear rate to the shear
stress is constant at a particular time. Unfortunately, in practice, usually liquid
dosage forms and their ingredients are non - Newtonian fl uids in which the ratio of
the shear rate to the shear stress varies. As a result, non - Newtonian fl uids may not
have a well - defi ned viscosity [32] .
When all the ingredients are miscible liquids, the combination and distribution
of these components to obtain a homogeneous mixture are called blending. Whenever
possible, ingredients should be added together and the impeller mixer often is
located near the bottom of the vessel [21] . Mixing of high - viscosity materials requires
higher velocity gradients in the mixing zone than regular blending operations. In
fact, the fundamental laws of physics regarding the performance of Newtonian fl uids
in the production process may be studied using computational tools. For example,
VisiMix is a software that is routinely used to calculate shear rates [2] .
Finally, if it is determined that there is a bigger problem of insolubility
coming from the formulation, then addition of cosolvents, surfactants, as well as
the preparation of the ionized form of an acid or base, drug derivatization, and
solid - state manipulation are approaches to manipulating the solubility of the drug
[14] .
Batching Equipment to Mix Suspension In the case of suspensions, the fl ow necessary
to overcome settling in a satisfactory suspension depends on the mixing
equipment and is predicted by Stokes ’ s law. Thus, to use the Stokes ’ s law, suspensions
are considered as Newtonian fl uids if the percentage of solids is below 50%.
Mixing equipment uses a mechanical device that moves through the liquid at a given
velocity. Dispersing and emulsifying equipment is categorized as “ high - shear ” mixing
equipment. The maximum shear rate with such equipment occurs very close to the
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 327
mixing impeller. Therefore, the diameter of the impeller and the impeller speed
directly infl uence the power applied by the mixer to the liquid [21] .
Batching Equipment to Mix Emulsion The most common problems of mixing
emulsions are removing “ dead spots ” of the mixture and scrapping internal walls of
the mixer. Dead spots are quantities of ingredients that are not mixed and become
immobile. Where dead spots are present, that quantity of the formula has to be
recirculated or removed and not used. If the inside walls of the mixer keep residual
material, operators should use hard spatulas to scrape the walls; otherwise the
residual material will become part of the next batch. In both cases, the result may
be nonuniformity. Stainless steel mixers have to include blades made of hard plastic,
such as Tefl on, to facilitate the scrapping of the mixer walls without damaging the
mixer. Scrapper blades should be fl exible enough to remove internal material but
not too rigid to avoid damaging the mixer [20] . The mixing will be successful if the
macroscale mixing offers suffi cient fl ow of components in all areas in the mixing
tank and the microscopic examination shows a correct particle size distribution
[33] .
4.3.4.3 Particle Size of Raw Materials
Raw materials in Solution The types of raw materials used to be part of solutions
are presented in Table 1 . They have different purposes and can be cosolvents, electrolytes,
buffers, antioxidants, preservatives, coloring, fl avoring and sweetener agents,
among others.
Particle Size of Raw Materials in Solution Particle size is affected by the breaking
process of the particle, crystal form, and/or salt form of the drug. The particle
size can affect the rate of dissolution of raw materials in the manufacturing process.
Raw materials of a fi ner particle size may dissolve faster because they have a larger
surface area in contact with the solvent than those of a larger particle size when the
product is compounded [6] . Mixing faster causes the particle to break down and
dissolve more quickly. In addition, hydrated particles are less soluble than their
anhydrous partners [37] .
TABLE 1 Solutions: pharmaceutical excipients
Purpose Agent
Protecting the active product
ingredients
- Buffers
- Antioxidants
- Preservatives
Maintaining the appearance - Colorings
- Stabilizers
- Cosolvents
- Antimicrobial preservatives
- Electrolytes
Taste/Small Masking - Sweeteners
- Flavorings
Source : From ref. 4, 34, 35, 36
328 LIQUID DOSAGE FORMS
Solid drugs may occur as pure crystalline substances of defi nite identifi able shape
or as amorphous particles without defi nite structure. In addition, when a drug particle
is broken up, the total surface area is increased as well as its rate of dissolution.
The amorphous form of a chemical is usually more soluble than the crystalline form
while the crystalline form usually is more stable than the amorphous form [37] .
Processing conditions used for providers to obtain raw materials can dramatically
impact their quality and stability; for instance, the presence of different polymorphs
may depend on the thermal history of freezing, concentration of solvents, and drying
conditions [38] . The polymorphism of a crystalline form is the capacity of a chemical
to form different types of crystals, depending on the conditions of temperature,
solvents, and time followed for its crystallization. Among different polymorphs, only
one crystalline form is stable at a given temperature and pressure. Over time, the
other crystalline forms, called metastable forms, will be transformed into stable
forms. Transformations longer than the shelf - life of metastable forms into stable
forms of a drug are very common in fi nal products and compromise its stability and
effi cacy to different extents depending on quality control [37] .
While the metastable forms offer higher dissolution rates, many manufacturers
use a particular amorphous, crystalline, salt, or ester form of a drug with the solubility
needed to be dissolved in the established conditions, for instance, to prepare a
chloramphenicol ophthalmic solution [39] . Thus, the selection of amorphous or
crystalline form of a drug may be of considerable importance to facilitate the formulation,
handling, and stability [37] .
However, the dissolution rate of an equal sample of a slowly soluble raw material
usually will increase with increasing temperature or rate of agitation as well as with
reduce viscosity, changes of pH or nature of the solvent. In addition, other alternative
mechanisms to enhance the solubility of insoluble drugs are: 1) hydrophilization:
the reduction in contact angle or angle between the liquid and solid surface
[40] , which can be accessed by intensive mixing of the hydrophobic drug with a small
amount of methylcellulose solution [41] ; 2) the formation of microemulsions: by
covering small particles with surfactants to obtain micromicelles that are visible only
in the form of an opalescence; and, 3) the formation of complexing compounds: by
adding a soluble substance to form soluble reversible complexes. However, the last
method is used with some restrictions [42] .
Raw Materials in Suspension The types of raw materials used to be part of suspensions
are presented in Table 2 . They have different purposes and can be wetting
agents, salt formation ingredients, buffers, polymers, suspending agents, fl occulating
agents, electrolytes, antioxidants, poorly soluble Active Product Ingredients, preservatives,
coloring, fl avoring and sweetener agents, among others.
Particle Size of Drug in Suspension The physical stability of a suspension can be
enhanced by controlling the particle size distribution [43] . Uncontrolled changes of
drug particle size in a suspension affect the dissolution and absorption of the drug
in the patient. Drug substances of fi ner particle size may be absorbed faster and
bigger particles may not be absorbed. Aggregation or crystal growth is evaluated
by particle size measurements using microscopy and a Coulter counter [21] or preferably
techniques that allow samples to be investigated in the natural state. Allen
[44] offers an academic and industrial discussion about particle characterization.
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 329
Powder properties and behavior, sampling, numerous potential particle size measuring
devices, available equipment as well as surface and pore size are his principal
themes.
Particles are usually very fi ne (1 – 50 . m). For instance, topical suspensions use
less than 25 . m particle size [6] . The particle size of the drug is the most important
consideration in the formulation of a suspension, since the sedimentation rate of
disperse systems is affected by changes in particle size. Finer particles become interconnected
and produce particle aggregation followed by the formation of nonresuspendable
sediment, known as caking of the product. The two main causes of
aggregation and caking are energetic bonding and bonding through shared material.
A statistical wide distribution of particle sizes gives more compact packing and
energetic bonding than narrower distributions. It has been observed that heat treatments
can cause agglomeration of particles, not only due to energetic bonding but
also by formation of crystal bridges. Also, when the application of shear forces to
mix and homogenize the suspension uses too high energy inputs, then the probability
for aggregation increases [43] .
Examples of oral suspensions in which a specifi c and well - defi ned particle size
specifi cation for the drug substance is important are phenytoin suspension, carbamazepine
suspension, trimethoprim and sulfamethoxazole suspension, and hydrocortisone
suspension [6] .
There are some useful methods to improve the physical stability of a suspension,
such as decreasing the salt concentration, addition of additives to regulate the
osmolarity, as well as changes in excipient concentrations, unit operations in the
process, origin and synthesis of the drug substance, polymorphic behavior of
the drug substance crystals, and other particle characteristics. However, methods
based on changes of the particle properties and the surfactants used are the most
successful [43] .
TABLE 2 Suspensions: pharmaceutical excipients
Purpose Agent
Facilitating the connection between Active
Product Ingredient and vehicle
- Wetting agents particle size ( > 0.1 . m)
- Salt formation ingredients
- Sugars
Protecting the Active Product Ingredients - Buffering – systems
- Polymers
- Antioxidants
- Poorly soluble drugs
Maintaining the suspension appearance - Colorings
- Suspending agent
- Flocculating agent
- Antimicrobial preservatives
- Electrolytes
Masking the unpleasant taste/smell - Sweeteners
- Flavorings
- Poorly soluble Active Product Ingredient
Source : From ref. 4, 34, 35, 36
330 LIQUID DOSAGE FORMS
To approach physical stability problems of suspensions, effectiveness and stability
of surfactants as well as salt concentrations must be checked with accelerated aging.
In addition, unit operations affecting particle size distribution, surface area, and
surfactant effectiveness should be approached, taking into account that different
types of distributions, for instance, volume or number weighted, give a different
average diameter for an equal sample [43] .
Raw Materials in Emulsions The types of raw materials used to be part of emulsions
are presented in table 3 . They have different purposes and can be buffers,
polymers, emulsifying agents, penetration enhancers, gelling agents, stabilizers, antioxidants,
preservatives, coloring, fl avoring and sweetener agents, among others.
Particle Size in Emulsions When a solid drug is suspended in an emulsion, the
liquid dosage form is known as a coarse dispersion. In addition, a colloidal dispersion
has solid particles as small as 10 nm – 5 . m and is considered a liquid between
a true solution and a coarse dispersion [44] .
4.3.4.4 Compounding: Effects of Heat and Process Time
Oxygen Oxygen removal for processing materials that require oxygen to degrade
is possible by methods such as nitrogen purging, storage in sealed tanks, as well
as special instructions for manufacturing operations [6] . For instance, sealing glass
ampules containing a liquid dosage form with heat under an inert atmosphere is a
packing mechanism used to prevent oxidation. Some aspects of oxygen sensitiveness
that should be taken into account are the necessity of water and headspace deoxygenation
in ampules before sealing, the avoidance of multidose vials that facilitate
oxygen contact with the product after opened, and rubber stoppers for vial sealing
that are permeable to oxygen as well as release additives to catalyze oxidative reac-
TABLE 3 Emulsions: pharmaceutical excipients
Purpose Agent
Particle Size - Solid particles (10 nanometers to 5 micrometers size)
- Droplet particles (0.1 – 1.0 micrometers size)
Protecting the Active Product
Ingredients
- Buffering - Systems
- Polymers
- Antioxidants
- Distribution pattern (O/W, W/O)
Maintaining the appearance - Colorings
- Emulsifying agents
- Penetration enhancers
- Gelling agents
- Stabilizers
- Antimicrobial preservatives
Taste/smell Masking - Sweeteners
- Flavorings
- Relation oil vs. water
Source : From ref. 4, 34, 35, 36
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 331
tions. Rubber stoppers soften and get sticky over time because all rubber products
degrade as sulfur bonds induced during vulcanization revert. Connors et al. [45]
present the oxygen content of water at different temperatures and an interesting
discussion of calculations for the case of captopril as an oxygen - sensitive drug.
Dissolution of Drugs in Solutions Although some compounds, such as poloxamers,
decrease their aqueous solubility with an increase in temperature [46] , usually,
drugs dissolve more quickly when the temperature increases because particle vibration
is augmented and the molecules move apart to form a liquid. Chemical instabilities
by oxidation due to high temperature or prolonged periods of heat exposure
can occur when trying to increase the dissolution of poorly soluble raw materials.
To control such instabilities, charts of time and amount of temperature treatments
to dissolve materials as well as tests of dissolution are required [6] . In addition,
precipitations and other reactions may occur between salts in solution and can be
anticipated by using heat - of - mixing data and activation energy calculations for
decomposition reactions. Connors et al. [45] provide examples of calculations about
effects of temperature on chemical stability of pharmaceuticals in solution. Regarding
the instability of the product, the reasons to limit temperature amounts can go
from controlling fi nal concentration changes to controlling burn - on/fouling when
too - high temperatures are applied [45] . Usually salts are more soluble in water and
alcohol than weak acids or bases. The reason salts are not always the best choice to
increase the solubility of a drug is its permeability. Oral drug absorption depends
not only on solubility and dissolution but also on permeability through the cellular
membrane. Drugs have to be able to dissolve not only in the aqueous fl uids of the
body before reaching the intestinal wall but also in the lipophilic environment of
the cellular membrane in order to reach the internal part of the cell and interfere
with its functionability. Therefore, the cosolvent approach is essential if the drug
presents problems in dissolving in the media. The dielectric constant of a solvent is
a relative measure of its polarity. Comparing the hydroxyl – carbon ratio of the
solvent molecule allows establishing the relative polarity of the cosolvent as determined
by its dielectric constant [47] . Remington describes the formulations of some
solutions, such as the ferrous sulfate syrup, amantadine hydrochloride syrup, phenobarbital
elixir, and theophylline elixir [1] .
Potency of Drugs in Suspension To avoid degradation of the suspended drug substance
by high temperature or prolonged periods of heat exposure, it is necessary
to record the time and amount of temperature treatments on charts [6] . The rate
of dissolution of a suspended drug increases with the increase in temperature. The
potency stability of a suspended drug depends on the concentration of the dissolved
drug since drug decomposition occurs only in solution [48] . The goal is to avoid
the dissolution of suspensions. Changing the pH of the vehicle or replacing the drug
with a less soluble molecule may result in enhanced potency stability of the suspended
drug [48] .
For instance, when the chemical stability of a suspension of ibuprofen powder
and other ibuprofen – wax microspheres was studied with a modifi ed HPLC procedure
for three months, the amount of drug released from the microspheres was
affected by the medium pH, type of suspending agent, and storage temperature
without observing chemical degradation of the drug [49] .
332 LIQUID DOSAGE FORMS
Temperature Uniformity in Emulsions During the preparation of emulsions, heat
may be increased as part of the manufacturing protocol or mixing operation system.
Temperature measurements should be monitored and documented continuously
using a recording thermometer if the temperature control is critical or using a hand -
held thermometer if it is not a critical factor. Temperature may be critical in the
manufacturing process depending on the thermosensitivity of the drug product and
excipients as well as the type of mixer used. To guarantee the temperature uniformity
during the mixing operation, manufacturers may consider the relation between
the container size, mixer speed, blade design, viscosity of the contents, and rate of
heat transfer [20] .
Fong - Spaven and Hollenbeck [50] studied the apparent viscosity as a function
of the temperature from 25 to 75 ° C of an oil – water emulsion stabilized with 5%
triethanolamine stearate (TEAS) using a Brookfi eld digital viscometer. They
observed that the viscosity decreased when the temperature reached about 48 ° C,
but surprisingly viscosity increased to a small peak at 54 ° C and then continued
decreasing after that peak. The viscosity peak was attributed to a transitional gellike
arrangement molecular structure of TEAS that is destroyed as soon as the temperature
continues increasing, the TEAS crystalline form reappears, and viscosity again
decreases [36] .
Microbiological Control To avoid chemical instabilities that yield microbiological
and physical instabilities, as a result of high temperature or prolonged periods of
exposure, it is necessary to record the time and amount of temperature treatments
on charts [6] .
Product Uniformity Charts of storage and transfer operation times for the bulk
product are required to control the risk of segregation. Transfers to the fi lling line
and during the fi lling operation are the most critical moments to keep the suspension
uniformity [6] . The implementation of an ERP for time scheduling is the best
solution for time control and organization of resources. However, it could be diffi -
cult due to the reluctance of people to change [10] . The constant fl ow of the liquid
through the piping, the constant mixing of the bulk product in the tank, as well as
the transfer of small amounts near the end of the fi lling process to a smaller tank
during the fi lling process may minimize segregation risks [6] .
Final Volume Excess heating produces variations of the fi nal volume over time
[6] . Although increasing solute concentration can elevate the boiling point and
reduce evaporation of water, changes in drug concentration are undesirable because
they yield different fi nal products. Regarding the instability of the product, the
reasons to limit temperature amounts can go from controlling fi nal concentration
changes to controlling burn - on/fouling when too - high temperatures are applied
[36] .
A solution is a liquid at room temperature that passes into the gaseous state when
heated at very high temperature, forming a vapor with determined vapor pressure,
through a process called vaporization. The kinetic energy is not evenly distributed
between the molecules of the liquid. When the liquid is in a closed container at a
constant temperature, the molecules with the highest kinetic energy leave the surface
of the liquid and become gas molecules. Some of the gas molecules remain as gas
and others condense and return to the liquid. When, at a determined temperature,
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 333
the rate of condensation equals the rate of vaporization, the equilibrium vapor pressure
is reached. However, vapor pressure increases with increases in liquid temperature,
resulting in more molecules leaving the liquid surface and becoming gas
molecules [51] .
Storage Charts of time and temperature of storage are important to control the
increased levels of degradedness [6] . Shelf life is defi ned as the amount of time in
storage that a product can maintain quality and is equivalent to the time taken to
reach 90% of the composition claim or have 10% degradation. The availability of
an expiration date is assumed under specifi ed conditions of temperature. Based on
zero - and fi rst - order reaction calculations, Connors et al. [45] show the estimation
methods to determine the shelf life of a drug product at temperatures different from
the one specifi ed under standard conditions.
4.3.4.5 Uniformity of Oral Suspensions
Keeping the particles uniformly distributed throughout the dispersion is an important
aspect of physical stability in suspensions. Based on Stokes ’ s law for dilute
suspensions where the particles do not interfere with one another, there are different
factors that control the velocity of particle sedimentation in a suspension, for
instance, particle diameter, densities of the dispersed phase and the dispersion
medium, as well as viscosity of the dispersion medium [36] . Remington describes
the formulation of trisulfapyrimidines oral suspension [1] . In addition, Lieberman
et al. [42, 48] are also good sources of typical formulations for suspensions.
Viscosity Depending upon the viscosity, many suspensions require continuous or
periodic agitation during the fi lling process [6] .
Segregation in Transfer Lines When the stored bulk of a nonviscous product is
transferred to fi lling equipment through delivery lines, some level of segregation is
expected. The manufacturer has to write the procedures and diagrams for line setup
prior to fi lling the product [6] . Delivery lines of suspensions increase the tendency
of particles of the same size to assemble together. However, slightly increasing the
global mixing in the lines can easily reverse the segregation without enhancing the
global mixing [52] . Shear stress versus rate of shear can be plotted to determine
the fl ow pattern of a specifi c suspension as pseudoplastic, Newtonian, or dilatant.
The type of fl ow is determined by the slope of the plot. While shaking increases the
yield stress and causes particles fl ow, the cessation of shear and rest rebuilds the
order of the system. A good - quality suspension is known as a thixotrophic system
and is obtained when the particles at rest avoid or show reduced sedimentation. The
rheogram of a thixotrope system presents a typical hysteresis or curve representing
different shear stresses over time [33] .
Quality Control The GMPs for suspensions include testing samples at different
checkpoints in the procedure, at the beginning, middle, and end, as well as samples
from the bulk tank. The uniformity will be successful only if, on microscopic analysis,
the components are dispersed to the expected particle size distribution established
by product development. Visual and microscopic examinations should consist of
looking for verifi cation of foam formation, segregation, and settling, although testing
334 LIQUID DOSAGE FORMS
for viscosity is important to determine agitation during the fi lling process. Samples
used for tests should not be combined again with the lot [6, 33] .
4.3.4.6 Uniformity of Emulsions
Remington describes the following three typical formulas of emulsions: type A
gelatin, mineral oil emulsion (USP), and oral emulsion (O/W) containing an insoluble
drug [1] . In addition, Lieberman et al. [42, 50] are also good sources of typical
formulations for emulsions. The components of the emulsion system may present
physical and chemical instabilities refl ected on the distribution of an active ingredient,
component migration from one phase to another, polymorphic changes in
components, and chemical degradation of components [33] .
Solubility The soluble active ingredient should be added to the liquid phase that
will be its carrier vehicle. Data of solubility have to be determined as part of the
process validation. Potency uniformity has to be tested by demonstrating satisfactory
distribution in the emulsifi ed mix [20] .
Particle Size Regarding globule diameter in emulsions, the size – frequency distribution
of particles in an emulsion over time may be the only method for determining
stability [36] . Drug activity and potency uniformity of insoluble active ingredients
depend upon control of particle size and distribution in the mix [6] . In addition,
aggregation of the internal phase droplets, formation of larger droplets, and phase
separation are categorized as emulsion system instabilities that are refl ected in the
particle size distribution of the emulsion. The measurement of particle size distribution
over time allows the characterization of the emulsion stability and determines
the rheological behavior of the emulsion. Well - accepted approaches to determine
particle size distribution include microscopy, sedimentation, chromatography, and
spectroscopy. However, these analyses are problematic in a multiphase emulsion
[33] .
Crystalline Form Uncontrolled temperature or shear can induce changes in component
crystallinity or solubility. For this reason, analytes originally present in each
phase of the product should be counted as well possible interactions with the container
or closure and the processing equipment analyzed. Some techniques used to
obtain information about the emulsion system and its components are microscopic
examination, macro - and microlaser Raman, and rheological studies [33] . The FDA
guidance offers the following example: “ in one instance, residual water remaining
in the manufacturing vessel, used to produce an ophthalmic ointment, resulted in
partial solubilization and subsequent recrystallization of the drug substance; the
substance recrystallized in a larger particle size than expected and thereby raised
questions about the product effi cacy ” [20] .
4.3.4.7 Microbiological Quality
Microbial Specifi cations These specifi cations are determined by the manufacturer.
The USP Chapters 61, 62, and 1111 present the microbial limits to assess the
signifi cance of microbial contamination in a dosage form [53] . However, the USP
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 335
does not determine specifi c methods for water - insoluble topical products. The
microbial specifi cations are presented as a manufacturer ’ s document that details the
methods to isolate and identify the organisms as well as the number of organisms
permitted and action levels to be taken when limits are exceed and the potential
causes are investigated [6] . The Pharmaceutical Microbiology Newsletter (PMF)
presents several articles to discuss topics such as microbial identifi cation, methods,
data analysis, and preservation as well as topics related to USP and FDA regulations
[54] . To minimize the differences about microbial limits and test methods, the USP
is trying to harmonize the standards with the European Pharmacopoeia (EP) [55] .
Microbial Test Methods The selected microbial test methods determine specifi c
sampling and analytical procedures. When the product has a potential antimicrobial
effect and/or preservative, the spread technique on microbial test plates must be
validated. In addition, the personnel performing the analytical techniques have to
be qualifi ed and adequately trained for this purpose [6] .
Usually, total aerobic bacteria, molds, and yeasts are counted by using a standard
plate count in order to test the microbial limits. The microbial limit test may be
customized by performing a screening for the occurrence of Staphylococcus aureus,
Pseudomonas aeruginosa, Pseudomonas cepacia, Escherichia coli, and Salmonella
sp. [56] .
Investigation of Exceeded Microbiological Limits A high number of organisms
may indicate defi ciencies in the manufacturing process, such as excessive high
temperature, component quality, inadequate preservative system, and/or container
integrity. Information about the health hazards of all organisms isolated from the
product has different meanings depending on the type of dosage form and group
of patients to be treated. For instance, in oral liquids, pseudomonads are usually a
high - risk contamination. Examples presented by the FDA are Nystatin antifungal
suspension, used as prophylaxis in AIDS patients [57] ; antacids, with which P. aeruginosa
contamination can promote gastric ulceration [58] ; and the presence of
Pseudomona putida , which could indicate the presence of other signifi cant contaminants
such as P. aeruginosa [6] .
Deionizer Water - Monitoring Program Deionizing systems must be controlled in
order to produce purifi ed water, required for liquid dosage forms and USP tests and
assays [1] . The monitoring program has to include the manufacturer ’ s documentation
about time between recharging and sanitizing, microbial quality and chlorine
levels of feed water, establishment of water microbial quality specifi cations, conductivity
monitoring intervals, methods of microbial testing, action levels when microbial
limits are exceeded, description of sanitization and sterilization procedures for
deionizer parts, and processing conditions such as temperature, fl ow rates, use
and sanitization frequency, and regenerant chemicals for ion exchange resin beds
[6, 59] .
Effectiveness of Preservative Manufacturing controls and shelf life must ensure
that the specifi ed preservative level is present and effective as part of the stability
program [6] . Depending on the type of product, the selection of the preservative
system is based on different considerations, such as site of use, interactions,
336 LIQUID DOSAGE FORMS
spectrum, stability, toxicity, cost, taste, odor, solubility, pH, and comfort. The
USP and other organizations describe methods to validate the preservative
system used in the dosage form. Compounds used as preservatives are alcohols,
acids, esters, and quaternary ammonium compounds, among others. For instance, to
preserve ophthalmic liquid dosage forms, these products are autoclaved or fi ltrated
and require an antimicrobial preservative to resist contamination throughout
their shelf life, such as chlorobutanol, benzalkonium chloride, or phenylmercuric
nitrate [1] .
4.3.4.8 Filling and Packing
Constant Mixing during Filling Process Due to the tendency of suspensions to
segregate during transport through transfer lines, special attention is required on
suspension uniformity during the fi lling process. Appropriate constant mixing of the
bulk to keep homogeneity during the fi lling process and sampling of fi nished products
and other critical points are indispensable conditions to assure an acceptable
quality level during the fi lling and packing process [20] .
Mixing Low Levels of Bulk Near End of Filling Process Constant mixing during
the fi lling process includes mixing low levels of bulk near the end of the fi lling
process. Large - size batches of bulk suspension require the transfer of the residual
material to a smaller tank in order to assure appropriate mixing of components
before fi lling and packing the containers [20] .
Potency Uniformity of Unit - Dose Products Products manufactured have to be of
quality at least as good as the established acceptable quality level (AQL). The quality
level should be based on the limits specifi ed by the USP. However, when the bulk
product is not properly mixed during fi lling and packing processes, liquid dosage
forms, and specially suspensions, are not homogeneous and unit - dose products
contain very different amounts of the active component and potency. For these
reasons, fi nished products have to be tested to assure that the fi nal volume and/or
weight as well as the amount of active ingredient are within the specifi ed limits [6] .
Calibration of Provided Measuring Devices Measuring devices consist of droppers,
spoons for liquid dosage forms, and cups labeled with both tsp and mL. Measuring
devices have to be properly calibrated in order to assure the right amount
of ingredients per volume to be administered [6] .
Container Cleanliness of Marketing Product The previous cleanliness of containers
fi lled with the product will depend on their transportation exposure, composition,
and storage conditions. Glass containers usually carry at least mold spores of
different microorganisms, especially if they are transported in cardboard boxes.
Other containers and closures made with aluminum, Tefl on, metal, or plastic usually
have smooth surfaces and are free from microbial contamination but may contain
fi bers or insects [45] . Some manufacturers receive containers individually wrapped
to reduce contamination risks and others use compressed air to clean them. However,
the cleanliness of wrapped containers will depend on the provider ’ s guarantee of
the manufacturing process and compressed - air equipment may release vapors or
oils that have to be tested and validated [6] .
CRITICAL ASPECTS OF LIQUIDS MANUFACTURING PROCESS 337
4.3.4.9 Stability
The typical stability problems are color change, loss of active component, and clarity
changes for solutions; inability to resuspend the particles and loss of signifi cant
amounts of the active component for suspensions; and creaming and breaking
(or coalescence) for emulsions [1] . These instabilities are usually related to the
following:
Active and Primary Degradant. A liquid dosage form is stable while it remains
within its product specifi cations. When chemical degradation products are
known, for stability study and expiration dating, the regulatory requirements
for the primary degradant of a active component are chemical structure, biological
effect and signifi cance at the concentrations to be determined, mechanism
of formation and order of reaction, physical and chemical properties,
limits and methods for quantitating the active component and its degradant
molecule at the levels expected to be present, and pharmacological action or
inaction [45] . Examples of drugs in liquid dosage forms that are easily degraded
are vitamins and phenothiazines [6] .
Interactions with Closure Systems. Elastomeric and plastic container and closure
systems release leachable compounds into the liquid dosage form, such as
nitrosamines, monomers, plasticizers, accelerators, antioxidants, and vulcanizing
agents [44] . Each type of container and closure with different composition
and/or design proposed for marketing the drug or physician ’ s samples has to
be tested and stability data should be developed. Containers should be stored
upright, on their side, and inverted in order to determine if container – closure
interactions affect product stability [6, 45] .
Moisture Loss. When the containers are inappropriately closed, part of the
vaporized solvent is released and the concentration and potency of the active
component may be increased [6] .
Microbiological Contamination. Inappropriate closure systems also increase the
possibilities of microbial contamination when opening and closing containers
[6] .
4.3.4.10 Process Validation
Objective Process validation has the objectives of identifying and controlling
critical points that may vary product specifi cations through the manufacturing
process [6] .
Amount of Data To validate the manufacturing process, the manufacturer has to
design and specify in the protocol the use of data sheets to keep information about
the control of product specifi cations from each batch in - process as well as fi nished -
product tests. Some formats are common to different products, though each type of
product has some specifi c information to be kept on special sheets. Thus, the amount
of data varies from one type of product to another [6] .
Scale - Up Process Data obtained using special batches for the validation of the
scale - up process are compared with data from full - scale batches and batches used
for clinical essays [6] .
338 LIQUID DOSAGE FORMS
Product Specifi cations The most important specifi cations or established limits for
liquid dosage forms are microbial limits and test methods, medium pH, dissolution
of components, viscosity, as well as particle size uniformity of suspended components
and emulsifi ed droplets. Effectiveness of the preservative system depends on
the dissolution of preservative components and may be affected by the medium pH
and viscosity. In addition, dissolved oxygen levels are important for components
sensitive to oxygen and/or light [6] .
Bioequivalence or Clinical Study In the patient, the general or systemic circulation
is responsible for carrying molecules to different tissues of the body. To assure
the expected bioactivity of a product, the amount of drug that reaches the systemic
circulation per unit of time is analyzed and is known as bioavailability. Bioequivalence
is the comparison of the bioavailability of a product with a reference product.
While oral solutions may not always need bioequivalence studies because they are
considered self - evidente, suspensions usually require bioequivalence or clinical
studies in order to demonstrate effectiveness. However, OTC suspension products
such as antacids are exempt from these studies [6] .
Control of Changes to Approved Protocol The manufacturing process of a specifi c
product is validated and approved internally by the quality control unit and externally
by the FDA. Any change in the approved protocol has to be documented to
explain the purpose and demonstrate that the change will not unfavorably affect
product safety and effi cacy. Factors include potency and/or bioactivity as well as
product specifi cations. However, the therapeutic activity and uniformity of the
product are the main concerns after formulation and process changes [20] .
4.3.5 LIQUID DOSAGE FORMS *
Douche A liquid preparation, intended for the irrigative cleansing of the vagina,
that is prepared from powders, liquid solutions, or liquid concentrates and
contains one or more chemical substances dissolved in a suitable solvent or
mutually miscible solvents.
Elixir A clear, pleasantly fl avored, sweetened hydroalcoholic liquid containing
dissolved medicinal agents; it is intended for oral use.
Emulsion A dosage form consisting of a two - phase system comprised of at least
two immiscible liquids, one of which is dispersed as droplets (internal or dispersed
phase) within the other liquid (external or continuous phase), generally
stabilized with one or more emulsifying agents. (Note: Emulsion is used as a
dosage form term unless a more specifi c term is applicable, e.g. cream, lotion,
ointment.).
Enema A rectal preparation for therapeutic, diagnostic, or nutritive purposes.
Extract A concentrated preparation of vegetable or animal drugs obtained by
removal of the active constituents of the respective drugs with a suitable menstrua,
evaporation of all or nearly all of the solvent, and adjustment of the
residual masses or powders to the prescribed standards.
* The defi nitions in this section are from ref. 11 .
For Solution A product, usually a solid, intended for solution prior to
administration.
For Suspension A product, usually a solid, intended for suspension prior to
administration.
For Suspension, Extended Release A product, usually a solid, intended for suspension
prior to administration; once the suspension is administered, the drug
will be released at a constant rate over a specifi ed period.
Granule, Effervescent A small particle or grain containing a medicinal agent in
a dry mixture usually composed of sodium bicarbonate, citric acid, and tartaric
acid which, when in contact with water, has the capability to release gas, resulting
in effervescence.
Inhalant A special class of inhalations consisting of a drug or combination of
drugs, that by virtue of their high vapor pressure can be carried by an air
current into the nasal passage where they exert their effect; the container from
which the inhalant generally is administered is known as an inhaler.
Injection A sterile preparation intended for parenteral use; fi ve distinct classes
of injections exist as defi ned by the USP.
Injection, Emulsion An emulsion consisting of a sterile, pyrogen - free preparation
intended to be administered parenterally.
Injection, Solution A liquid preparation containing one or more drug substances
dissolved in a suitable solvent or mixture of mutually miscible solvents that is
suitable for injection.
Injection, Solution, Concentrate A sterile preparation for parenteral use which,
upon the addition of suitable solvents, yields a solution conforming in all
respects to the requirements for injections.
Injection, Suspension A liquid preparation, suitable for injection, which consists
of solid particles dispersed throughout a liquid phase in which the particles
are not soluble. It can also consist of an oil phase dispersed throughout an
aqueous phase, or vice - versa.
Injection, Suspension, Liposomal A liquid preparation, suitable for injection,
which consists of an oil phase dispersed throughout an aqueous phase in such
a manner that liposomes (a lipid bilayer vesicle usually composed of phospholipids
which is used to encapsulate an active drug substance, either within a
lipid bilayer or in an aqueous space) are formed.
Injection, Suspension, Sonicated A liquid preparation, suitable for injection,
which consists of solid particles dispersed throughout a liquid phase in which
the particles are not soluble. In addition, the product is sonicated while a gas
is bubbled through the suspension and these result in the formation of microspheres
by the solid particles.
Irrigant A sterile solution intended to bathe or fl ush open wounds or body
cavities; they ’ re used topically, never parenterally.
Linament A solution or mixture of various substances in oil, alcoholic solutions
of soap, or emulsions intended for external application.
Liquid A dosage form consisting of a pure chemical in its liquid state. This
dosage form term should not be applied to solutions.
LIQUID DOSAGE FORMS 339
340 LIQUID DOSAGE FORMS
Liquid, Extended Release A liquid that delivers a drug in such a manner to allow
a reduction in dosing frequency as compared to that drug (or drugs) presented
as a conventional dosage form.
Lotion An emulsion, liquid dosage form. This dosage form is generally for
external application to the skin.
Lotion/Shampoo A lotion dosage form which has a soap or detergent that is
usually used to clean the hair and scalp; it is often used as a vehicle for dermatologic
agents.
Mouthwash An aqueous solution which is most often used for its deodorant,
refreshing, or antiseptic effect.
Oil An unctuous, combustible substance which is liquid, or easily liquefi able, on
warming, and is soluble in ether but insoluble in water. Such substances,
depending on their origin, are classifi ed as animal, mineral, or vegetable oils.
Rinse A liquid used to cleanse by fl ushing.
Soap Any compound of one or more fatty acids, or their equivalents, with an
alkali; soap is detergent and is much employed in liniments, enemas, and in
making pills. It is also a mild aperient, antacid and antiseptic.
Solution A clear, homogeneous liquid dosage form that contains one or more
chemical substances dissolved in a solvent or mixture of mutually miscible
solvents.
Solution, Concentrate A liquid preparation (i.e., a substance that fl ows readily
in its natural state) that contains a drug dissolved in a suitable solvent or
mixture of mutually miscible solvents; the drug has been strengthened by the
evaporation of its nonactive parts.
Solution, for Slush A solution for the preparation of an iced saline slush, which
is administered by irrigation and used to induce regional hypothermia (in
conditions such as certain open heart and kidney surgical procedures) by its
direct application.
Solution, Gel Forming/Drops A solution, which after usually being administered
in a drop - wise fashion, forms a gel.
Solution, Gel Forming, Extended Release A solution that forms a gel when it
comes in contact with ocular fl uid, and which allows at least a reduction in
dosing frequency.
Solution/Drops A solution which is usually administered in a drop - wise
fashion.
Spray A liquid minutely divided as by a jet of air or steam.
Spray, Metered A non - pressurized dosage form consisting of valves which allow
the dispensing of a specifi ed quantity of spray upon each activation.
Spray, Suspension A liquid preparation containing solid particles dispersed in
a liquid vehicle and in the form of coarse droplets or as fi nely divided solids
to be applied locally, most usually to the nasal - pharyngeal tract, or topically
to the skin.
Suspension A liquid dosage form that contains solid particles dispersed in a
liquid vehicle.
Suspension, Extended Release A liquid preparation consisting of solid particles
dispersed throughout a liquid phase in which the particles are not soluble; the
suspension has been formulated in a manner to allow at least a reduction in
dosing frequency as compared to that drug presented as a conventional dosage
form (e.g., as a solution or a prompt drug - releasing, conventional solid dosage
form).
Suspension/Drops A suspension which is usually administered in a dropwise
fashion.
Syrup An oral solution containing high concentrations of sucrose or other
sugars; the term has also been used to include any other liquid dosage form
prepared in a sweet and viscid vehicle, including oral suspensions.
Tincture An alcoholic or hydroalcoholic solution prepared from vegetable
materials or from chemical substances.
Notes :
1. A liquid is pourable; it fl ows and conforms to its container at room temperature.
It displays Newtonian or pseudoplastic fl ow behavior.
2. Previously the defi nition of a lotion was “ The term lotion has been used to
categorize many topical suspensions, solutions, and emulsions intended for
application to the skin. ” The current defi nition of a lotion is restricted to an
emulsion.
3. A semisolid is not pourable; it does not fl ow or conform to its container at
room temperature. It does not fl ow at low shear stress and generally exhibits
plastic fl ow behavior.
4. A colloidal dispersion is a system in which particles of colloidal dimension
(i.e., typically between 1 nm and 1 . m) are distributed uniformly throughout a
liquid.
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342 LIQUID DOSAGE FORMS
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SPECIAL/NEW DOSAGE FORMS
SECTION 5
347
5.1
CONTROLLED - RELEASE
DOSAGE FORMS
Anil Kumar Anal
Living Cell Technologies (Global) Limited, Auckland, New Zealand
Contents
5.1.1 Introduction
5.1.2 Rationale
5.1.3 General Design Principle for Controlled - Release Drug Delivery Systems
5.1.4 Physicochemical and Biological Factors Infl uencing Design and Performance of
Controlled - Release Formulations
5.1.4.1 Physicochemical Factors
5.1.4.2 Biological Factors
5.1.5 Controlled - Release Oral Dosage Forms
5.1.5.1 Anatomical and Physiological Considerations
5.1.5.2 Fundamentals of Controlled - Release Oral Dosage Forms
5.1.5.3 Factors Infl uencing Oral Controlled - Release Dosage Forms
5.1.6 Design and Fabrication of Controlled - Release Dosage Forms
5.1.6.1 Microencapsulation
5.1.6.2 Nanostructure - Mediated Controlled - Release Dosage Forms
5.1.6.3 Liposomes
5.1.6.4 Niosomes
5.1.7 Technologies for Developing Transdermal Dosage Forms
5.1.8 Ocular Controlled - Release Dosage Forms
5.1.9 Vaginal and Uterine Controlled - Release Dosage Forms
5.1.10 Release of Drugs from Controlled - Release Dosage Forms
5.1.10.1 Time - Controlled - Release Dosage Forms
5.1.10.2 Stimuli - Induced Controlled - Release Systems
5.1.11 Summary
References
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
348 CONTROLLED-RELEASE DOSAGE FORMS
5.1.1 INTRODUCTION
Therapeutic value and pharmaeconomic value have in recent years become major
issues in defi ning health care priorities under the pressure of cost containment [1] .
The improvement in drug therapy is a consequence of not only the development of
new chemical entities but also the combination of active substances and a suitable
delivery system. The treatment of an acute disease or chronic illness is mostly
accomplished by delivery of one or more drugs to the patient using various pharmaceutical
dosage forms. Tablets, pills, capsules, suppositories, creams, ointments,
liquids, aerosols, and injections are in use as drug carriers for many decades. These
conventional types of drug delivery systems are known to provide a prompt release
of the drug. Therefore, to achieve as well as to maintain the drug concentration
within the therapeutically effective range needed for treatment, it is often necessary
to take this type of drug several times a day, resulting in the signifi cant fl uctuation
in drug levels [2] . For all categories of treatment, a major challenge is to defi ne the
optimal dose, time, rate, and site of delivery. Recent developments in drug delivery
techniques make it possible to control the rate of drug delivery to sustain the duration
of therapeutic activity and/or target the delivery of drug to a specifi c organ or
tissue. Many investigations are still going on to apply the concepts of controlled
delivery for a wide variety of drugs [3] .
5.1.2 RATIONALE
The basic rationale for controlled drug delivery is to alter the pharmacokinetics and
pharmacodynamics of pharmacologically active moieties by using novel drug delivery
systems or by modifying the molecular structure and/or physiological parameters
inherent in a selected route of administration. It is desirable that the duration
of drug action become more a design property of a rate - controlled dosage form and
less, or not at all, a property of the drug molecules ’ inherent kinetic properties.
The rationale for development and use of controlled dosage forms may include
one or more of the following arguments [4] :
• Decrease the toxicity and occurrence of adverse drug reactions by controlling
the level of drug and/or metabolites in the blood at the target sites.
• Improve drug utilization by applying a smaller drug dose in a controlled - release
form to produce the same clinical effect as a larger dose in a conventional
dosage form.
• Control the rate and site of release of a drug that acts locally so that the drug
is released where the activity is needed rather than at other sites where it may
cause adverse reactions.
• Provide a uniform blood concentration and/or provide a more predictable drug
delivery.
• Provide greater patient convenience and better patient compliance by signifi -
cantly prolonging the interval between administrations.
However, there are also disadvantages attached to the use of controlled - release
dosage forms. These include higher cost of manufacturing, unpredictability, poor in
vitro/in vivo correlation, reduced potential, and poor systemic availability in general
and the effective release period is infl uenced and limited by the gastrointestinal
(GI) residence time [5] . The transit time of a dosage form through the GI tract is
dependent on the physical characteristics of the formulation as well as on physiological
factors such as stomach emptying time and effect of food on the absorption
process.
Only drugs with certain properties are suitable for controlled - release dosing.
Characteristics that may make a drug unsuitable for controlled - release dosing
include a long or short elimination half - life, a narrow therapeutic index, a large dose,
low/slow solubility, extensive fi rst - pass clearance, and time course of circulating drug
levels different from that of the pharmacological effect. The ideal drug delivery
should be inert, biocompatible, mechanically strong, comfortable for the patient,
capable of achieving high drug loading, simple to administer, and easy to fabricate
and sterilize [6] . A range of materials have been employed to control the release of
drugs and other active substances. Controlled - release dosage forms have been
developed for over four decades. One of the fi rst practically used controlled - release
oral dosage forms was the Spansule capsule, which was introduced in the 1950s.
Spansule capsules were manufactured by coating a drug onto nonpareil particles
and further coating with glyceryl stearate and wax. Subsequently, ion exchange
resins were proposed for application as sustained - release delivery systems of
ac cessible drug. Since then numerous products have been introduced and
commercialized.
5.1.3 GENERAL DESIGN PRINCIPLE FOR CONTROLLED - RELEASE
DRUG DELIVERY SYSTEMS
In the drug delivery system, the pharmacodynamics of active molecules becomes
more a function of design and less one of inherent kinetic properties. Therefore, a
deep understanding of the design of controlled - release systems of the pharmacokinetics
and pharmacodynamics of the drug is required [7] . The conventional tablet
or capsule provides only a single and transient burst of drug. A modifi cation introduced
to the molecular structure of the drug (often used to decrease the elimination
rate) or a system for modifi ed release rate is the common approaches used to
increase the interval between two doses. The objective of both these approaches is
to decrease the fl uctuations in plasma levels during multiple dosing. This allows the
dosing interval to increase without compromising the required dosage levels. If the
half - life of a drug is less than 6 h or the passage time in the smaller intestinal track
is decreased, there might not be enough time to allow proper absorption, thus
making frequent dosing compulsory. For other routes, where the residence time is
not a constraint, dosing intervals can be as long as months or even years.
A controlled - release drug delivery system serves primarily two functions [8] .
First, it involves the transport of the drug to a particular part of the body. This may
be accomplished in two ways, parenterally and nonparenterally. Second, the release
of active ingredients occurs in a controlled manner, depending on the preparation
of dosage forms. This determines the rate at which a drug is made available to the
body once it has been delivered. Controlled drug delivery occurs when a biomaterial,
either natural or synthetic, is judiciously combined with a drug or other active
CONTROLLED-RELEASE DRUG DELIVERY SYSTEMS 349
350 CONTROLLED-RELEASE DOSAGE FORMS
agent in such a way that the active agent is released from the material in a predesigned
manner. To be successfully used in controlled drug delivery formulations, a
material must be chemically inert and free of leachable impurities. It must also have
an appropriate physical structure, with minimal undesired aging, and be readily
processable.
Controlled - release systems provide numerous benefi ts over conventional dosage
forms. Conventional dosage forms are not able to control either the rate of drug
delivery or the target area of administration and provide an immediate or rapid
drug release. This necessitates frequent administration in order to maintain a therapeutic
level. As a result, as shown in Figure 1 , drug concentrations in the blood
fl uctuate widely. The concentrations of drug remain at a maximum value, which may
represent a toxic level, or a level at which undersized side effects might occur, and
a minimum value, below which the drug is no longer effective. The duration of
therapeutic effi cacy is dependent upon the frequency of administration, the half - life
of the drug, and the release rate of dosage forms. In contrast, controlled - release
dosage forms not only are able to maintain therapeutic levels of drug with narrow
fl uctuations but also make it possible to reduce the frequency of drug administration.
The drug concentrations, as shown in Figure 1 , released from controlled - release
dosage forms fl uctuate within the therapeutic range over a longer period of time.
The plasma concentration profi le depends on the preparation technology, which
may generate different release kinetics, resulting in different pharmacological and
pharmacokinetic responses in the blood or tissues.
The primary objectives of controlled drug delivery are to ensure safety and to
improve effi cacy of drugs as well as patient compliance. This is achieved by better
control of plasma drug levels and less frequent dosing. For conventional dosage
forms, only the dose ( D ) and dosing interval ( . ) can vary above which undesirable
or side effects are elicited. As an index of this window, the therapeutic index (TI)
can be used. This is often defi ned as the ratio of lethal dose (LD 50 ) to median effective
dose (ED 50 ). Alternatively, it can be defi ned as the ratio of maximum drug
concentration ( C max ) in blood that can be tolerated to the minimum concentration
( C min ) needed to produce an acceptable therapeutic response.
FIGURE 1 Theoretical plasma concentration after administration of various dosage forms:
( a ) standard oral dose; ( b ) oral overdose; ( c ) IV injection; ( d ) controlled - release system.
Toxic level
Minimum
effective level
c
a
b
d
Drug concentration in blood
Time
Different types of modifi ed release systems can be defi ned [4, 8] :
• Sustained release (extended release) that permits a reduction in dosing frequency
as compared to the situation in which the drug is presented as a conventional
form
• Delayed release when the release of the active ingredient comes sometimes
other than promptly after administration
• Pulsatile release when the device actively controls the dosage released following
predefi ned parameters
In general, the sustained - release dosage form is designed to maintain therapeutic
blood or tissue levels of the drug for an extended period of time. This is accomplished
by attempting to obtain zero - order release from the designed dosage form.
Zero - order release constitutes drug release from the dosage form that is independent
of the amount of drug in the delivery system at a constant release rate. Systems
that are designed for prolonged release can also be attributed as achieving
sustained - release delivery systems. Repeat - action tablets are an alternative method
of sustained release in which multiple doses of drugs are contained within a dosage
form and each dose is released at a periodic interval, while delayed - release systems
may not be sustaining, since often the function of these dosage forms is to maintain
the drug within the dosage form.
5.1.4 PHYSICOCHEMICAL AND BIOLOGICAL FACTORS
INFLUENCING DESIGN AND PERFORMANCE OF
CONTROLLED - RELEASE FORMULATIONS
A number of variables, such as drug properties including stability, solubility, partitioning
characteristics. charge and protein binding behavior, routes of drug delivery,
target sites, acute or chronic therapy, the disease, and the patient, must be considered
to establish the criteria for designing controlled - release products [9] . The performance
of a drug in its release pattern from the dosage form as well as in the body
proper is a function of its properties. These properties can at times prohibit placement
if the drug is in a controlled - release form, restrict the route of drug administration,
and signifi cantly modify performance for one reason or another. There is
no clear distinction between physicochemical and biological factors since the biological
properties of a drug are a function of its physicochemical properties while
biological properties result from typical pharmacokinetic studies on the absorption,
distribution, metabolism, and excretion (ADME) characteristics of a drug as well
as those resulting from pharmacological studies.
5.1.4.1 Physicochemical Factors
Physicochemcial properties, such as aqueous solubility, partition coeffi cient and
molecular size, drug stability, and protein binding, are those that can be determined
from in vitro experiments.
CONTROLLED-RELEASE FORMULATIONS 351
352 CONTROLLED-RELEASE DOSAGE FORMS
Ionization, p K a
, and Aqueous Solubility Most drugs are weak acids or bases. It
is important to note the relationship between the p K a of the compound and the
absorptive environment. Delivery systems that are dependent on diffusion or dissolution
will likewise be dependent on the solubility of drug in the aqueous media.
Since drugs must be in solution before they can be absorbed, compounds with very
low aqueous solubility usually have the oral bioavailability problems because of
limited GI transit time of the undissolved drug particles and they are limited at the
absorption site. Unfortunately, for many of the drugs and bioactive compounds, the
site of maximum absorption occurs at the site where solubility of these compounds
is least.
The drug (e.g., tetracycline) for which the maximum solubility is in the stomach
but high absorption takes place in the intestinal region may be poor candidates for
controlled - release systems, unless the system is capable of retaining the drug in the
stomach and gradually releasing it to the small intestine or unless the solubility is
made higher and independent of the external environment by encapsulating those
compounds in a membrane system. Other compounds, such as digoxin [10] , with
very low solubility, are inherently sustained, since their release over the time course
of a dosage form in the gastrointestinal tract is limited by dissolution of the drug.
Although the action of a drug can be prolonged by making it less soluble, this may
occur at the expense of consistent and incomplete bioavailability.
The choice of mechanism for oral sustained/controlled - release systems is limited
by the aqueous solubility of the drug. Thus, diffusional systems are poor choices for
low aqueous - soluble drugs since the driving force for diffusion, the concentration
in aqueous solution, will be low. The lower limit for the solubility of a drug to be
formulated in a controlled - release system has been reported to be 0.1 mg/mL [11] .
Partition Coeffi cient and Molecular Size Following administration, drugs and
other bioactive compounds must traverse a variety of membranes to gain access to
the target area. The partition coeffi cient and molecular size infl uence not only the
permeation of drug across biological membranes but also diffusion across or through
a rate - controlling membrane or matrix. The partition coeffi cient is generally defi ned
as the ratio of the fraction of drug in an oil phase to that of an adjacent aqueous
phase. Drugs with extremely high partition coeffi cient (i.e., those that are highly oil
soluble) readily penetrate the membranes but are unable to proceed further, while
the excessive high aqueous - soluble compounds, having low oil/water partition coef-
fi cients, cannot penetrate the membranes. A balance in the partition coeffi cient is
needed to give an optimum fl ux for permeation through the biological and rate -
controlling membranes. The ability of drugs to diffuse through membranes, also
known as diffusivity, is related to its molecular size by the following equation:
log log log D s V k s M k V V = . + = . + M M
where D is the diffusivity, M is the molecular weight, V is the molecular volume,
and s V , s M , k V , and k M are constants in a particular medium. Generally, there is smaller
diffusivity with the denser medium.
Drug Stability The stability of drug in the environment where it is to be exposed
is an essential physicochemical factor to be considered before designing controlled
dosage forms [12] . For example, orally administered drugs are subjected to both
acid – base hydrolysis and enzymatic degradation [13] . For drugs that are unstable in
the stomach, the dosage forms can be designed in so that they can be placed in a
slowly soluble form or have their release delayed until they reach the intestine. This
type of approach can be ineffective and the drug may be unstable in the small
intestine or undergo extensive gut - wall metabolism. To obtain better bioavailability
for such types of drugs, which are unstable even in the intestine, a different route
of administration (e.g., transdermal with controlled - release dosage forms) can be a
better option [14] . A transdermal patch of nitroglycerin is a good example. The
details for transdermal dosage forms will be described later in this chapter.
5.1.4.2 Biological Factors
A drug, being a chemical/biological agent or a mixture of chemical and biological
agents, is recognized as a xenobiotic by the human body. Subsequently, the drug will
be prevented from entering the body and/or eliminated after its entry. As a result,
the defense mechanisms of the human body become barriers to the delivery of
drugs. A drug may encounter physical, physiological, enzymatic, or immunological
barriers on its way to the site of action. Hence, the design of controlled - release
product should be based on a comprehensive picture of drug disposition. This would
entail a complex examination of the ADME characteristics of the drug. The details
of these effects on various controlled - release dosage systems will be given in the
following sections of this chapter.
5.1.5 CONTROLLED - RELEASE ORAL DOSAGE FORMS
Oral drug delivery is the preferred route for drug administration because of its
convenience, economy, and high patient compliance compared with several other
routes. About 90% of the drugs are administered via the oral route [15, 16] . For the
oral controlled administration of drugs, several research and development activities
have shown encouraging signs of progress in the development of programmable
controlled - release dosage forms as well as in the search for new approaches to
overcome the potential problems associated with oral drug administration. Many
oral drugs are perceived as “ patient friendly ” for compliance, often requiring that
the medication only needs to be taken once a day. The most prescribed drugs in the
United States that use oral drug delivery technologies include Lipitor (atorvastatin
calcium), manufactured by Pfi zer, and AstraZeneca ’ s Toprol - XL (metoprolo succinate).
These potential developments and recently developed approaches are discussed
here along with an overview of GI physiology.
5.1.5.1 Anatomical and Physiological Considerations
Anatomically, the alimentary canal can be divided into a conduit region and
digestive and absorptive regions. The conduit region includes the mouth, pharynx,
esophagus, and lower rectum. The digestive and absorptive regions include the
stomach, small intestine, and all parts of the large intestine except the very distal
region.
CONTROLLED-RELEASE ORAL DOSAGE FORMS 353
354 CONTROLLED-RELEASE DOSAGE FORMS
The role of the stomach in drug and nutrition absorption is very limited, and it
acts primarily as a reception area for oral dosage forms. Nonionic, lipophilic molecules
of moderate size can be absorbed through the stomach only to a limited extent
owing to the small epithelial surface area and the short duration of contact with the
stomach epithelium in comparison with the intestine [17] . The transit time in the GI
tract varies from one person to another and also depends upon the physical properties
of the object ingested and the physiological conditions of the alimentary canal
(Table 1 ). After passing through the stomach, the next organ that a drug or bioactive
compound encounters is the small intestine. The intestinal epithelium is composed
of absorptive cells (enterocytes) interspersed with goblet cells (specialized for mucus
secretion) and a few enteroendocrine cells (that release hormones). The enterocytes
of intestinal epithelium are the most important cells in view of the absorption of
drugs and nutrients [18] . Histologically, colonic mucosa resembles the small intestinal
mucosa, the absence of villi being the major difference [19] . The microvilli of
the large intestine enterocytes are less organized than those of the small intestine.
The resulting decrease in the surface area of the colon leads to a low absorption
potential in comparison with the small intestine. However, the colonic residence
time is longer than that for the small intestine, providing extended periods of time
for the slow absorption of drugs and nutrients [20] . Figure 2 shows the various
physiological processes encountered by an orally administered drug during the
course of GI transit.
5.1.5.2 Fundamentals of Controlled - Release Oral Dosage Forms
Oral controlled drug delivery is a system that provides the continuous delivery of
drugs at predictable and reproducible kinetics for a predetermined period throughout
the course of GI transit [21] . Also included are systems that target the delivery
of a drug to a specifi c region within the gastrointestinal tract (GIT) for either local
or a systemic action. All the oral controlled drug delivery systems have limited utilization
in the GI controlled administration of drugs if the systems cannot remain
in the vicinity of the absorption site for the lifetime of the drug delivery. In the
exploration of oral controlled - release dosage forms, one encounters three areas of
potential challenges [22] :
1. Drug Delivery System To develop a viable oral controlled - release drug delivery
system capable of delivering a drug at a therapeutically effective rate to a
desirable site for the duration required for optimal effi cacy.
2. Modulation of GI Transit Time To modulate the GI transit time so that the
drug delivery system developed can be transported to a target site or to the
TABLE 1 Gastrointestinal Tract: Physical Dimensions and
Dynamics
Region Surface area (m 2 ) pH
Transit Time
Fluid Solid
Stomach 0.1 – 0.2 1.2 50 min 8 h
Small intestine 100 6.8 2 – 6 h 4 – 9 h
Large intestine 0.5 – 1 7.5 2 – 6 h 3 – 72 h
vicinity of an absorption site and reside there for a prolonged period of time
to maximize the delivery of a drug dose.
3. Minimization of Hepatic First - Pass Elimination If the drug to be delivered
is subjected to extensive hepatic fi rst - pass elimination, preventive measures
should be devised to either bypass or minimize the extent of hepatic metabolic
effect.
With most orally administered drugs, targeting is not a primary concern, and it
is usually intended for drugs to permeate to the general circulation and perfuse to
other body tissues, except it is medicated intentionally for localized effects in the
GIT. There is a general assumption that increasing concentration at the absorption
site will increase the rate of absorption and, therefore, increase circulating blood
levels, which in turn promotes greater concentrations of drug at the site of action.
If toxicity is not an issue, therapeutic levels can thus be extended as shown in Figure
3 . In essence, “ drug delivery ” by these systems usually depends on release from
specifi c types of dosage forms and permeation through an epithelial membrane to
the blood. Still, the biological and physicochemical factors that come across play
important roles in the design of such systems. The physicochemical properties have
been described earlier in this chapter while biological factors involved with oral
dosage forms will be described below.
FIGURE 2 Physical model illustrating various physiological processes during gastrointestinal
transit.
Stomach
Liver
Jejunum
Pportal vein
Ileum
Colon
Rectum
Complexation
Absorption
S
y
s
t
e
m
i
c
c
i
r
c
u
l
a
t
i
o
n
Adsorption
Hydrolytic
Enzymatic
Degradation within
wall
Hydrolytic
Enzymatic
Microbial
Enterohepatic
recycling
Absorption
Absorption
Absorption
Absorption
Degradation
CONTROLLED-RELEASE ORAL DOSAGE FORMS 355
356 CONTROLLED-RELEASE DOSAGE FORMS
The degree to which a delivery system can achieve standard release profi les for
a variety of chemically and physically diverse, pharmaceutically active molecules is
a measure of a delivery system ’ s effi cacy and fl exibility (Figure 3 ). Among the most
challenging profi les, linear, zero - order release of highly soluble actives over a 12 –
24 - h period could be considered a reasonable performance standard against which
delivery systems may be judged.
5.1.5.3 Factors Infl uencing Oral Controlled - Release Dosage Forms
Biological Half - Life The usual goal of an oral controlled - release dosage form is
to maintain therapeutic blood levels, as shown in Figure 3 , over an extended period
of time. A drug must be absorbed and enter the circulation at approximately the
same rate at which it is eliminated. The elimination rate is quantitatively described
by the half - life ( t 1/2 ). Each drug has its own characteristic elimination rate, which is
the sum of all elimination process, including metabolism, urinary excretion, and all
other processes that permanently remove drug from the bloodstream.
Therapeutic compounds with short half - lives are excellent candidates for controlled/
sustained - release preparations, since this can reduce dosing frequency [23] .
In general, drugs with half - lives shorter than 2 h, such as furosemide or levodopa,
are poor candidates for controlled - release preparations. Compounds with longer
half - lives, such more than 8 h, also do not need to be in the form of controlled
release, since their effect is already sustained. Digoxin, warfarin and phenytoin are
some examples [24 – 26] . However, drugs having even longer half - lives can be used
in other forms of modifi ed release, such as pulsatile release.
Gastrointestinal Tract and Absorption The design of a controlled - release dosage
form should be based on a comprehensive picture of drug disposition. Both the
pharmacokinetic property and biological response parameter have a useful range
for the design of sustained - and controlled - release products. The potential problems
inherent in oral controlled - release oral dosage forms generally relate to (i) interac-
FIGURE 3 Profi le of drug level in blood: ( a ) traditional dosing of tablets; ( b ) controlled
drug delivery dose.
(a)
Maximum desired level
Minimum effective level
Dose Dose Dose Dose
Time
(b)
Maximum desired level
Minimum effective level
Time
Drug level
Drug level
tions between the rate, extent, and location that the dosage form releases the drug
and (ii) the regional differences in GI physiology [27] .
Total GI transit time in the normal population varies from 5 to 36 h, with an
average total transit time of approximately 24 h [28] . There is still much more to
explore about the physiological processes involved and factors that infl uence gastric
emptying, intestinal transit, and colon residence. One of the major factors is food
administration, which delays the “ housekeeper wave, ” causing delay in gastric emptying.
A high - fat meal may delay gastric emptying from 3 to 5 h, and the total GI
transit delay is largely a function of delay in gastric emptying in this case. If we
presume that the transit time of most drugs and devices in the absorptive areas of
the GI tract is about 8 – 12 h, the maximum half - life for absorption should be approximately
3 – 4 h; otherwise, the device will pass out of the potential absorptive regions
before drug release is complete. That many controlled - release products are having
somewhat lesser bioavailability than their conventional dosage forms may be due
to incomplete release of the dosage form or release at such a slow rate that the drug
has passed the actual site of absorption. Compounds that demonstrate a true lower
absorption rate constant will probably be poor candidates for controlled - release
dosage forms [28] .
An understanding of the behavior of dosage forms in the stomach has been
gained largely from scintographic studies in which phases of a meal and formulations
are labeled with different nucleotides, particularly technetium - 99 and indium -
111 [29] . Such studies have demonstrated that retention times of formulations in
the stomach are dependent on the size of formulations and whether or not the formulation
is taken with a meal. Enteric - coated or enteric matrix tablets may be
retained for a considerable time if dosed with heavy meals or breakfast. Multiparticulate
dosage forms will empty more slowly in the presence of food, and because
the dosage forms mix evenly with the food, the entry into the small intestine will
be strongly infl uenced by the caloric density and bulk of the ingested meal. The
rate of gastric emptying, therefore, predicts the absorption behavior [29, 30] . However,
the absorption of drugs from small, soft gelatin capsules is sometimes less
predictable [31] .
In the small intestine, contact time with the absorptive epithelium is limited, and
the small intestinel transit time is 3.5 – 4.5 h [32] . The scintographic data show that
many prolonged release products, particularly those intended for twice - or once -
daily administration, actually release some of their drug contents in the colon where
it may be absorbed into the systemic circulation for higher bioavailability. It is
anticipated that conditions of dissolution, absorption, and metabolism in the distal
portions of the intestine are different than in the proximal regions, due to differences
in pH, lumen fl uid, mucosal morphology, and motility.
For most formulations, colonic absorption represents the only real opportunity to
increase the interval between doses. Transit through the lower part of the gut is
quoted at about 24 h, but in reality only the ascending colonic environment has suffi -
cient fl uid to facilitate dissolution. In the cecum, the fermentation of soluble fi ber
produces fatty acids and gas [33] . The gas rises into the transverse colon and can form
temporary pockets, restricting access of water to the formulation. Consequently,
distal release of drug is associated with poor spreading, reduced surface area, and
restricted absorption. In the colon, water availability is also low past the hepatic
fl exure, as the ascending colon is extremely effi cient at water absorption [34] .
CONTROLLED-RELEASE ORAL DOSAGE FORMS 357
358 CONTROLLED-RELEASE DOSAGE FORMS
5.1.6 DESIGN AND FABRICATION OF CONTROLLED - RELEASE
DOSAGE FORMS
5.1.6.1 Microencapsulation
Microencapsulation has been the subject of massive research efforts since its inception
around 1950. Today, it is the mechanism utilized by approximately 65% of all
sustained - release systems [35] . The technique ’ s popularity can be attributed mainly
to its wide variety of applications. Hundreds of drugs have been microencapsulated
and used as controlled - release systems. Some examples are Arthritis Bayer,
Dexatrim Capsules, and Dimetapp Elixir.
Microencapsulation provides more effi cient drug delivery because it increases
the ability of the drug to interact with the body. The active ingredient of a drug is
encapsulated into a particle that may be as small as 1 . m. The greatest feature of
microencapsulation is the control provided by the choice of coating [36 – 38] . This
control allows microencapsulation to be a controlled - release device. Microcapsules
can be engineered to gradually release drugs to the body. To achieve this type of
delivery, equilibrium is established which will monitor the liberation of medicine
from those microcapsules. A microcapsule may be opened by many different
means. Release mechanisms include fracture by heat, solvation, diffusion, and pressure
[39] . A coating may also be designed to open specifi c areas of the body. A
microcapsule containing drugs that will be consumed by GI fl uids must not be fractured
until after it passes through the stomach [36 – 39] . A coating can therefore be
used that is able to withstand stomach acids and allow the drug to pass through the
stomach.
Methods of Microencapsulation
1. Air Suspension This method, known as the Wurster process or fl uidized - bed
coating, involves dispersing solid particulate core materials in a supporting air
stream and the spray coating of the suspended materials [40] . The design of the
chamber and its operating parameters effect a recirculating fl ow of the particles
through the coating zone of the chamber, where a coating material, usually a polymer
solution, is sprayed onto the fl uidized particles. The cyclic process is repeated until
the desired coat thickness is obtained.
2. Pan Coating This process has been around for many decades and is commonly
associated with sugar coating. It is essential that the particles be greater than
600 . m for effective coating. The process has been extensively utilized for the preparation
of controlled - release beads.
The coating by this method is applied as a solution or as an atomized spray to the
desired solid core material in the coating pan [41] . Warm air is passed over the coated
materials as the coatings are applied in the coating pans to remove the coating
solvent. Sometimes the fi nal solvent removal is carried out in a drying oven.
3. Multiorifi ce Centrifugal Process This is a mechanical process involving the
use of centrifugal forces to hurl a material particle through an enveloping microencapsulation
membrane to effect mechanical encapsulation [42] . The microcapsules
are then hardened and congealed. This method is capable of microencapsulating
liquids and solids dispersed in a liquid.
DESIGN AND FABRICATION OF CONTROLLED-RELEASE DOSAGE FORMS 359
4. Coacervation Phase Encapsulation by coacervation is the one of the more
popular methods commonly studied. The process consists of three steps carried out
under continuous agitation [43] :
Step 1 The core material is dispersed in a solution of coating polymer, the solvent
for the polymer being the liquid manufacturing vehicle phase.
Step 2 Deposition of the coating, accomplished by controlled, physical mixing of
the coating material and the core material in the manufacturing vehicle.
Step 3 Rigidization of the coating by thermal, cross - linking, or desolvation techniques
to form self - sustaining microcapsules.
Since the core materials are microencapsulated while being dispersed in
some liquid manufacturing vehicle, subsequent drying operations are usually
required.
5. Solvent Evaporation Technique [39, 44, 45] The process is carried out in a
liquid manufacturing vehicle. The core material to be encapsulated is dissolved
or dispersed in the coating polymer solution. With agitation, the core coating
material mixture is dispersed in the liquid manufacturing vehicle phase to obtain
the appropriate microcapsule size. The microcapsules can be used in suspension
form, coated onto substrates or isolated as powders. A schematic of the emulsifi cation/
solvent evaporation technique to prepare drug - loaded microparticles is shown
in Figure 4 .
6. Spray Drying Spray drying, by defi nition, is the transformation of feed from
a fl uid state to a dried particulate form by spraying the feed into a hot drying
medium [44, 46] . The feed can be a solution, suspension, or paste. A schematic is
shown in Figure 5 .
Spray drying consists of four process stages:
(i) Atomization of feed into spray
(ii) Spray – air contact (mixing and fl ow)
(iii) Drying of spray (moisture evaporation)
(iv) Separation of dried product from the air
FIGURE 4 Schematic of microspheres prepared by emulsifi cation/solvent evaporation
method.
Polymer
(e.g., chitosan)
solution + drug
14 h
37°C
1000
rpm
Cross linking
agent, e.g,
tripolyphosphate
Filter wash
n-Hexane
Microsphere
Span 85
Cottonseed oil
360 CONTROLLED-RELEASE DOSAGE FORMS
7. Spray Congealing [47] Spray congealing is similar to spray drying in that it
involves dispersing the core material in a liquefi ed coating substance. Coat solidifi cation
is accomplished by congealing the molten coating material or by solidifying a
dissolved coating material by introducing the coat – core material mixture into a
nonsolvent. Removal of the nonsolvent is then achieved by sorption, extraction, or
evaporation techniques. Waxes, fatty acids and alcohol, and polymers and sugars,
which are solids at room temperature but melt at high temperatures, are applicable
to spray congealing methods.
5.1.6.2 Nanostructure - Mediated Controlled - Release Dosage Forms
The effi ciency of drug delivery to various parts of the body is directly affected by
particle size. Nanostructure - mediated drug delivery, a key technology for the realization
of nanomedicine, has the potential to enhance drug bioavailability, improve
the timed release of drug molecules, and enable precision drug targeting [48] .
Nanoscale drug delivery systems can be implemented within pulmonary therapies,
as gene delivery vectors, and in stabilization of drug molecules that would otherwise
degrade too rapidly. Additional benefi ts of using targeted nanoscale drug carriers
are reduced drug toxicity and effi cient drug distribution.
Anatomic features such as the blood – brain barrier, the branching pathways of
the pulmonary system, and the tight epithelial junctions of the skin make it diffi cult
for drugs to reach many desired physiological targets. Nanostructured drug carriers
will help to penetrate or overcome these barriers to drug delivery [49] . Greater
uptake effi ciency has also been shown for GI absorption and transcutaneous per-
FIGURE 5 Schematic of spray drying method.
Exhaust tube
Smell
clamp
Cyclone
Chamber head
To compressor (atomizer)
Sample tube
To compressor (de-blocker)
Main chamber
Collection tube
Jet nozzle
Pump
Sample
Collection bottle
DESIGN AND FABRICATION OF CONTROLLED-RELEASE DOSAGE FORMS 361
meation, with particles around 100 and 50 nm in size, respectively. However, such
small particles traveling in the pulmonary tract may help with delivery to the pulmonary
extremities. For instance, the outer layers of the carrier architecture may
be formulated to biodegradable as the carrier travels through the pulmonary tract.
As the drug carrier penetrates further into the lung, additional shedding will allow
the encapsulated drug to be released. Biodegradable nanoparticles of gelatin and
human serum albumin (HSA) show promise as drug carriers.
Advantages of nanostructure - mediated drug delivery include the ability to deliver
drug molecules directly into cells and the capacity to target tumors within healthy
tissue [50] . The mechanisms of cellular uptake of external particulates include
calthrin - and caveoli - mediated endocytosis, pinocytosis, and phagocytosis. However,
phagocytosis may not play a role in the uptake of nanoscale particles because of
the small size of such particles.
Nanoscale drug delivery architectures are able to penetrate tumors due to the
discontinuous, or “ leaky, ” nature of the tumor microvasculature, which typically
contains pores ranging from 100 to 1000 nm in diameter. The microvasculature of
healthy tissue varies by tissue type, but in most tissues, including the heart, brain,
and lung, there are tight intercellular junctions less than 10 nm. Therefore, tumors
within these tissue types can be selectively targeted by creating drug delivery nanostructures
greater than the intercellular gap of the healthy tissue but smaller than
the pores found within the tumor vasculature.
Various nanoscale architecture can be designed, including solid spheres, hollow
spheres, tubes, porous particles, solid particles, and branched structures (Table 2 ). To
achieve such nanostructures, different fabrication methods are used depending on
the types of material. The methods used for nanoscale assembly include molecular
self - assembly, bioaggregation, nanomanipulation, photochemical patterning, molecular
imprinting, layer - by - layer electrsostatic deposition, and vapor deposition.
TABLE 2 Nanoscale Drug Delivery Technologies
Drug
Delivery
Technology Materials Nanostructure Forms
Biologicals Lipids, peptides, nucleic acids,
polysaccharides, viruses
Vesicles, nanotubes, rings,
nanoparticles, nanocapsules,
nanospheres
Polymeric poly(lactic acid), poly(glycolic acid),
poly(alkylcyanoacrylate),
poly(3 - hydroxybutanoic acid),
Poly(organophosphazene),
poly(ethylene glycol),
poly(caprolactone), poly(ethylene
oxide), poly(amidoamine),
poly(l - glutamic acid),
poly(propylene imine)
Vesicles, nanospheres,
nanoparticles, micelles,
dendrimers
Silicon based Silicon, silicon dioxide Porous nanoparticles, nanoneedles
Carbon based Carbon Nanotubes
Metallic Gold, silver, palladium, platinum Nanoparticles, nanoshells
362 CONTROLLED-RELEASE DOSAGE FORMS
Manufacturing and Characterization of Nanoparticles/Nanocapsules/
Nanospheres Production of nanoparticles of soft materials is much more diffi cult
and challenging than that of hard materials because of the high stickiness of the
former. The bulk pharmaceuticals are available in solids of large sizes, which often
can be easily solubilized in solvent to obtain particular sizes. Hence, there are two
extremes of sizes: molecular size (each particle containing one molecule) and larger
sizes (e.g., each particle containing on the order of 10 18 molecules). To obtain
nanoparticles in the range of 50 – 300 nm of drug delivery, one requires on the order
of 10 4 – 10 8 molecules in each particle. This size has to be achieved from either solution
- phase (single - molecule) or millimeter - size particles (10 18 molecules).
Pearl/Ball Milling Technology for Production of Drug Nanocrystals There are two
different drug nanocrystal products, prepared by using pearl/ball milling technology.
The Rapamune coated tablet is the more convenient formulation, introduced by
Wyeth Pharmaceuticals in 2002. The Emend, introduced in 2003 by MSD, Sharp and
Dohme Gmbh, is a capsule composed of sucrose, microcrystalline cellulose, hyperlose,
and sodium dodecylsulfate [51] .
Traditional equipment used for micronization of drug powders such as rotor -
stator colloid mills or jet mills are of limited use for the production of nanocrystals.
For example, jet milling leads to a drug powder with a size range of roughly 0.1 –
20 . m, containing only a very small fraction of about 10% in the nanometer range.
However, it has been shown when running a pearl mill over a suffi ciently long
milling time that drug nanosuspensions can be obtained [52 – 54] . These mills consist
of a milling container fi lled with fi ne milling pearls or larger sized balls. The container
can be static and the milling material is moved by a stirrer; alternatively, the
complete container is moved in a complex movement leading consequently to
movement of the milling pearls.
The different milling materials available include traditional steel, glass, and zircon
dioxide as well as new special polymers such as hard polystyrene. A general problem
associated with this technology is the erosion from the milling materials during
manufacturing [55] . Surfactants and stabilizers have to be added for the physical
stability of the produced nanosuspensions. In the production process, the coarse
drug powder is dispersed by high - speed stirring or homogenization in a surfactant/
stabilizer solution to yield macro - and nanosuspensions. The choice of surfactant or
stabilizers depends not only on the properties of the particles to be suspended
(e.g., affi nity of surfactant/stabilizer to the crystal surface) but also on the physical
principles (e.g., electrostatic or steric stabilization) and the route of administration.
In general, steric stabilization is recommended as it is less susceptible to electrolytes
in the gut and blood.
There are number of pearl mills available on the market, ranging from
laboratory - scale to industrial - scale volumes. The ability of large - scale production is
an essential prerequisite for the introduction of a product to the market. One advantage
of pearl mills, apart from their low cost, is their ability in scaling up.
Nanoparticles/Nanoemulsions/Nanospheres Prepared by High - pressure Homogenization
High - pressure homogenization is a technology that has been applied for
many years in various areas of the production of emulsions and suspensions. A distinct
advantage of this technology is its ease in scaling up, even to very large volumes.
DESIGN AND FABRICATION OF CONTROLLED-RELEASE DOSAGE FORMS 363
In the pharmaceutical industry, parenteral emulsions such as Intralipid and Lipofundin
(mean droplet diameter 200 – 400 nm) are generally produced by this technology
[56] . Typical pressures for the production of drug nanosuspensions are 1000 – 1500
bars (corresponding to 100 – 150 MPa); the number of required homogenization
cycles varies from 10 to 20 depending on the properties of the drug. Most of the
homogenizers used are based on the piston gap principle; an alternative can be jet
stream technology [57] .
In the piston gap homogenizer, the liquid is forced through a tiny homogenization
gap, typically in the range of 5 – 20 . m (depending on the pressure applied and the
viscosity of the dispersion medium). Using a Micron Lab 40, the suspension is supplied
from a metal cylinder by a piston, and the cylinder diameter is approximately
3 cm. The suspension is moved by the piston having an applied pressure between
100 and 1500 bars. The piston gap homogenizer corresponds to a tube system in
which the tube diameter narrows from 3 to 20 . m. The Microfl uidizer (Microfl uidics
Inc.) is based on the jet stream principle [58] . Two streams of liquid collide, diminution
of droplets or crystals is achieved mainly by particle collision, but occurrence
of cavitation is achieved mainly by particle collision.
Nanoparticles/Nanocapsules Obtained by Interfacial Polymerization Nanoparticles/
nanocapsules can be obtained by fast polymerization of a monomer at the interface
between the organic and the aqueous phase of an emulsion. Alkylcyanoacrylates
have been proposed for the preparation of both oil - and water - containing nanocapsules
[59] . These monomers polymerize within a few seconds, initiated by hydroxyl
ions from equilibrium dissociation of water or by nucleophilic groups of any compound
of the polymerization medium.
1. Formation of Nanocapsules/Nanospheres Containing Oil Core This type of
nanocapsule is preferred for the encapsulation of lipophilic and oil - soluble compounds.
The general procedure for the preparation of oil - containing nanocapsules
by interfacial polymerization of alkylcyanoacrylates consists of preparing a very fi ne
oil - in - water (O/W) emulsion with an additional water - miscible organic solvent such
as ethanol or acetone [60, 61] . These solvents are used to disperse the oil as very
small droplets in the aqueous phase, which contains a hydrophilic surfactant. The
solvent also serves as a vehicle for the monomer. Gallardo et al. [62] proposed a
mechanism to explain nanocapsule formation. An important factor is that the
organic solvent must be completely water miscible so that the formation of
small enough oil droplets occurs spontaneously while the solvent is diffusing toward
the aqueous phase and the water is diffusing toward the organic phase. The polymerization
of monomer is also induced by contact with hydroxyl ions from the water
phase, which should be very fast to allow effi cient formation of a thin layer of coating
around the oil droplet and thus achieve effective encapsulation of drugs.
The organic phase containing the oil, the monomer, and the bioactive compounds,
dissolved in the water - miscible organic solvent, is injected into the aqueous phase
containing water and a hydrophilic surfactant under strong magnetic stirring. The
nanocapsules/nanospheres are formed immediately to give a milky suspension. The
organic phase is then removed under reduced pressure using a rotary evaporator.
The most commonly used materials for the preparation of oil - loaded nanocapsules
are given in Table 3 .
364 CONTROLLED-RELEASE DOSAGE FORMS
Nanocapsule/nanosphere size ranges between 200 and 350 nm were observed to
be affected by both the oil – ethanol ratio and the oil – monomer ratio [63, 64] . It is
also infl uenced by the particular oil, water - miscible organic solvent, and nonionic
surfactant in the aqueous phase. The pH of the aqueous phase and the temperature
also affect the size distribution.
2. Nanocapsules/Nanospheres Containing Aqueous Core Obtained by Interfacial
Polymerization Nanocapsules/nanospheres with an aqueous core are a recent
technology developed for the effi cient encapsulation of water - soluble compounds,
which are generally very diffi cult to include within carrier systems. This type of
nanocapsule/nanosphere may also be obtained by interfacial polymerization, but in
this case monomers are added to a water - in - oil (W/O) emulsion. Anionic polymerization
of the cyanoacrylates in the water phase is initiated at the interface by
nucleophiles such as hydroxyl ions in the aqueous phase, leading to the formation
of nanocapsules/nanospheres with an aqueous core. A typical procedure as described
by Lambert et al. [65] consists of preparing an aqueous phase composed of ethanol
(20% v/v) in water (pH 7.4) which is emulsifi ed in an organic phase containing
Miglyol oil and Montane 80. Slow addition of cyanoacrylic monomer under mechanical
stirring (about 4 h) allows the polymerization to occur. Thus the water droplets
are surrounded by the polymer - forming nanocapsules with an aqueous core dispersed
in an oily phase.
Polymeric Nanocapsules/Nanospheres/Nanoparticles
1. Manufacturing by Interfacial Nanodeposition/Nanoprecipitation The nanoprecipitation
procedure generally consists of a water - miscible organic phase such as
an alcohol or a ketone containing oil (with or without lipophilic surfactant) with an
aqueous phase containing a hydrophilic surfactant. The polymer, which may be
preformed synthetic, semisynthetic, or natural, is solubilized in the organic phase
(or in a phase in which the polymer is soluble). After addition of the organic phase
to the aqueous phase, the polymer diffuses with the organic solvent and is stranded
at the interface between oil and water. The driving force for the formation of
nanocapsules/nanospheres is the rapid diffusion of the organic solvent in the
aqueous phase inducing interfacial nanoprecipitation of the polymer around droplets
of the oily phase. The polymers which may be used to manufacture nanoparticles
by this method include natural polymers such as gum arabic, chitosan, alginate,
gelatin, ethylcellulose, hydroxypropyl methylcellulose (HPMC), and hydroxypropyl
methylcellulose phthalate (HPMCP); semisynthetic polymers such as diacyl . -
cyclodextrin; and synthetic polymers such as poly( d , l - lactide), poly( . - caprolactone),
TABLE 3 Main Components Used to Prepare Oil - Containing Nanocapsules/
Nanospheres by Interfacial Polymerization
Components Examples
Oil Miglyol, Lipiodol, benzylbenzoate
Monomers Ethylcyanoacrylate, isobutylcyanoacrylate, n - butylcyanoacrylate,
isohexylcyanoacrylate
Organic solvents Ethanol, acetone, acetonitrile, n - butanol, isopropanol
Surfactants Poloxamer 188, poloxamer 238, poloxamer 407, Triton X100, Tween 80
DESIGN AND FABRICATION OF CONTROLLED-RELEASE DOSAGE FORMS 365
and poly(alkylcyanoacrylate), which are most commonly employed [65] . Similarly,
a broad range of oils is suitable for the preparation of nanocapsules/nanospheres,
including vegetable or mineral oils and pure compounds such as ethyl oleate and
benzyl benzoate. The criteria for the selection of these compounds are the nontoxicity
and the low solubility of the oil in the polymers and vice versa.
Both hydrophilic and lipophilic surfactants can be used to stabilize the polymeric
nanoparticles. Generally the lipophilic surfactant is a natural lecithin of relatively
low phosphotidylcholine content, whereas the hydrophilic one is synthetic: anionic
(lauryl sulfate), cationic (quaternary ammonium), or more commonly nonionic
[poly(oxyethylene) - poly(propylene)glycol]. Nanoparticles can be prepared in the
absence of surfactants, but there are lots more chances to get aggregated during
storage.
2. Nanoparticles Obtained by Multiple Emulsion/Solvent Evaporation Method
The multiple emulsion/solvent evaporation method was initially developed for the
preparation of microcapsules. This method consists in fi rst dissolving the drug in an
aqueous solution with or without a surfactant and the polymer in a volatile organic
solvent that is not miscible to water. Polymers used for the formation of such types
of particles have been mainly poly(lactide - co - glycolide) and poly( . - caprolactone)
[66, 67] . The inner water phase is then poured into the organic phase. This mixture
is generally emulsifi ed forming the fi rst inner emulsion or the primary W/O emulsion,
which is then mixed vigorously into an aqueous phase (outer water phase) that
contains an emulsifi er forming the water - in - oil - in - water (W/O/W) multiple emulsion.
The resulting multiple nanoemulsion is continuously stirred and the solvent is
allowed to evaporate, inducing precipitation of polymer and, thereby, the formation
of solid drug loaded nanoparticles.
5.1.6.3 Liposomes
Liposomes were discovered in the mid - 1960s [68] and were originally studied as cell
membrane models. They have since gained recognition in the fi eld of drug delivery.
Liposomes are formed by the self - assembly of phospholipid molecules in an aqueous
environment. The amphophilic phospholipid molecules form a closed bilayer sphere
in an attempt to shield their hydrophobic groups from the aqueous environment
while still maintaining contact with aqueous phase via the hydrophilic head group.
The resulting closed sphere may encapsulate aqueous soluble drugs within the
central aqueous compartment or lipid - soluble drugs within the bilayer membrane.
Alternatively, lipid - soluble drugs may be complexed with other polymers (e.g.,
cyclodextrin) and subsequently encapsulated within the liposome aqueous compartment.
The encapsulation within/association of drugs with liposomes alters the drug
pharmacokinetics.
Attractive Biological Properties of Liposomes [69]
• Liposomes are biocompatible.
• Liposomes can entrap hydrophilic bioactive compounds in their internal compartment
and hydrophobic into the membrane.
• Liposome - incorporated bioactives are protected from the inactivating effect of
external conditions yet do not cause undesirable side reactions.
366 CONTROLLED-RELEASE DOSAGE FORMS
• Liposomes provide a unique opportunity to deliver pharmaceuticals into cells
or even inside individual cellular compartments.
• The size, charge, and surface properties of liposomes can be easily changed by
adding new ingredients to the lipid mixture before liposome preparation and/or
by variation of preparation methods.
The clinical applications of liposomes are well known (Table 4 ). The initial
success achieved with many liposome - based drugs has fueled further clinical investigations.
One of the drawbacks of the use of liposomes is the fast elimination from
the blood and capture of liposomal preparations by the cells of the reticuloendothelial
system (RES), primarily in the liver.
There are a number of different types of liposomal vesicles [69] :
• Multilamellar Vesicles These range in size from 500 to 5000 nm and consist of
several concentric bilayers.
• Small Unilamellar Vesicles These are around 100 nm in size and are formed
by a single bilayer.
• Large Unilamellar Vesicles These range in size from 200 to 800 nm.
• Long Circulating Liposomes Different methods have been suggested to
achieve long circulation of liposomes in vivo, including coating the liposome
surface with inert, biocompatible polymers, such as polyethylene glycol (PEG),
which form a protective layer over the liposome surface and slow down its
recognition by opsonins and therefore subsequent clearance of liposomes. An
important feature of protective polymers is their fl exibility, which allows a relatively
smaller number of surface - grafted polymer molecules to create an impermeable
layer over the liposome surface. These types of modifi ed liposomes
demonstrate dose - dependent, nonsaturable , long - linear kinetics, and increased
bioavailability.
• Immunoliposomes To increase liposomal drug accumulation in the desired
tissues and organs, the use of targeted liposomes with surface - attached ligands
capable of recognizing and binding to cells of interest has been suggested [70] .
Immunoglobulins (Ig) of the IgG class and their fragments are the most widely
TABLE 4 Liposomal Drugs Approved for Clinical Application or Undergoing Clinical
Evaluation
Active Drug Product Name Applications
Daunorubicin DaunoXome Sarcoma
Doxorubicin Mycet Breast cancer
Doxil/Caelyx Sarcoma, ovarian cancer, breast cancer
Amphotericin B AmBisome Fungal infections
Cytarabine DepoCyt Lymphomatous meningitis
Vincristine Onco TCS Non - Hodgkin ’ s lymphoma
Lurtotecan NX211 Ovarian cancer
Nystatin Nyotran Topical antifungal agent
All - trans retinoic acid Altragen Leukemia, carcinomas
DNA plasmid encoding HLA - B7
and .2 - microglobulin
Allovectin - 7 Metastatic melanoma
used targeting moieties for liposomes, which can be attached, without affecting
liposomal integrity or the antibody properties, by covalent bonding to the liposome
surface or by hydrophobic insertion into the liposomal membrane after
modifi cation with hydrophobic residues.
5.1.6.4 Niosomes
The success achieved with liposomal formulations stimulated the search for other
vesicle - forming amphiphiles. Nonionic surfactants were among the fi rst alternative
materials studied and a large number of surfactants have since been found to self -
assemble into closed bilayer vesicles which may be used for drug delivery [71] .
Anticancer niosomes are expected to accumulate within tumors. The niosomal
encapsulation of methotrexate and doxorubicin increases drug delivery to the tumor
and tumoricidal activity. Unlike nonstealth liposomes, doxorubicin niosomes (size
800 nm) possessing a triglycerol or doxorubicin niosomes (size 200 nm) possessing
a muramic acid surface are not taken up signifi cantly by the liver. As such, these
triglycerol niosomes accumulate in the tumor. However, muramic acid vesicles do
accumulate in the spleen. Uptake by the liver and spleen make niosomes ideal for
targeting diseases manifesting in these organs. One such condition is leishmaniasis,
and number of studies have shown that niosomal formulations of sodium stibogluconate
improve parasite suppression in the liver, spleen, and bone marrow.
Niosomes may also be used as depot systems for short - acting peptide drugs on
intramuscular administration [72] .
Niosomal antigens are potent stimulators of the cellular and humoral immune
response. The formulation of antigens as a niosome in W/O emulsions further
increases the activity of antigens. The controlled - release property of these types
of emulsion formulations is responsible for enhancing the immunological
responses.
5.1.7 TECHNOLOGIES FOR DEVELOPING TRANSDERMAL
DOSAGE FORMS
Continuous intravenous infusion at a programmed rate has been recognized as a
superior model of drug delivery not only to bypass the hepatic fi rst - pass elimination
but also to maintain a constant, prolonged, and therapeutically effective drug level
in the body. A closely monitored intravenous infusion can provide both the advantages
of direct entry of drugs into the systemic circulation and control of circulating
drug levels. However, such a mode of drug delivery entails certain risks and therefore
necessitates hospitalization of patients and close medical supervision of the
medication. Recently there has been an increasing awareness that the benefi ts of
intravenous drug infusion can be easily duplicated, without its potential hazards, by
continuos transdermal drug administration through intact skin [73] .
Advances in transdermal delivery systems (TDSs) and the technology involved
have been rapid because of the sophistication of polymer science, which now allows
incorporation of polymeric additives in TDSs in adequate quantity. Drugs with
which transdermal therapy was pioneered include scopolamine, nitroglycerine, isosorbide
dinitrite, clonidine, estradiol, nicotine, and testosterone [74] .
TECHNOLOGIES FOR DEVELOPING TRANSDERMAL DOSAGE FORMS 367
368 CONTROLLED-RELEASE DOSAGE FORMS
Advantages of Transdermal Drug Delivery System [75]
• Avoids GI absorption (pH effects, enzymatic activity, drug interactions)
• Substitute for oral route
• Avoids fi rst - pass effect (drug deactivation by digestive and liver enzymes)
• Multiday therapy with a single application
• Extends the activity of the drugs with short half - lives
• Provides capacity to terminate drug effects rapidly
• Rapid identifi cation of medication in emergency
Limitations of Transdermal Drug Delivery [75]
• Not for all drugs
• Limited time that the patch can remain affi xed
• Variable intra - and interindividual percutaneous effi ciency absorption
effi ciency
• Variable adhesion to different skin types
• Skin rashes and sensitization
• Bacterial and enzymatic drug metabolism under the patch
• Complex technology/high cost
Skin Site for Transdermal Drug Administration The skin is one of the most
extensive and readily accessible organs of the human body. The skin of an average
adult body covers a surface area of approximately 2 m 2 and receives about one - third
of the blood circulating through the body. It is elastic, rugged, and, under normal
physiological conditions, self - regenerating. It serves as a barrier against physical and
chemical attacks and shields the body from invasion by microorganisms.
Microscopically the skin is a multilayered organ composed of, anatomically, many
histological layers, but it is generally described in terms of three tissue layers: the
epidermis, the dermis, and the subcutaneous fat tissue.
Microscopic sections of the epidermis show two main parts: the stratum corneum
and the stratum germinativum. The stratum corneum forms the outermost layer of
the epidermis and consists of many layers of compacted, fl attened, dehydrated,
keratinized cells in stratifi ed layers. In normal stratum corneum, the cells have a
water content of only approximately 20% compared to the normal physiological
level of 70% in the physiologically active 10% (w/w) to maintain fl exibility and
softness. It becomes rough and brittle, resulting in so - called dry skin, when its moisture
content decreases at a rate faster than can be resupplied from the underlying
tissues. The stratum corneum is responsible for the barrier function of the skin. It
also behaves as the primary barrier to percutaneous absorption. The thickness of
this layer is mainly determined by the extent of stimulation of the skin surface by
abrasion and weight bearing; hence thick palms and soles develop.
Several technologies have been successfully developed to provide rate control
over the release and skin permeation of drugs. These technologies can be classifi ed
into four basic approaches which are described below [73, 75] .
Polymer Matrix Diffusion - Controlled Transdermal Drug Delivery ( TDD )
System In this approach, the drug reservoir is formed by homogeneously dispersing
the drug solids in a hydrophilic or lipophilic polymer matrix, and the medicated
polymer formed is then molded into medicated disks with a defi ned surface area
and controlled thickness. This drug - reservoir - containing polymer disk is then
mounted onto an occlusive baseplate in a compartment fabricated from a drug -
impermeable plastic baking (Figure 6 a ).
FIGURE 6 Cross - sectional view of several TDSs: ( a ) poly(sebacic anhydride) (PSA) matrix
device; ( b ) membrane - moderated TDS; ( c ) adhesive - controlled TDS; ( d ) microreservoir - type
TDS; ( e ) matrix dispersion – type TDS.
(a)
Impermeable
backing
Adhesive matrix reservoir containing
drug Drug reservoir
Drug-impermeable metallic plastic
laminate
(b)
Rate-controlling polymeric membrane
Adhesive layer
(c)
Drugimpermeable metallic
plastic laminate
Adhesive layer
Drug reservoir layer
Rate-controlling adhesive
membrane
Metallic
laminate
Occlusive baseplate
(aluminum foil disc) Adhesive foam pad
(flexible polyurethane)
(d)
Adhesive rim
Microscopic drug reservoirs
Polymer matrix
Absorbent pad Drug-impermeable
plastic backing
(e) Occlusive baseplate
(aluminum foil disc)
Adhesive rim
r i o v r e s e r g u r D
TECHNOLOGIES FOR DEVELOPING TRANSDERMAL DOSAGE FORMS 369
370 CONTROLLED-RELEASE DOSAGE FORMS
Polymer Membrane Permeation - Controlled TDD System In this system, the drug
reservoir is sandwiched between a drug - impermeable backing laminate and a rate -
controlling polymeric membrane (Figure 6 b ). The drug molecules are permitted to
release only through the rate - controlling polymeric membrane.
Drug Reservoir Gradient - Controlled TDD System The rate of drug release from
this type of drug reservoir type is gradient controlled. In this system the thickness
of the diffusion path through which the drug molecule diffuses increases with time
(Figure 6 c ).
Microreservoir Dissolution - Controlled TDD System This type of drug delivery
system can be considered a hybrid of the reservoir and matrix dispersion - type drug
delivery systems. In this approach, the drug reservoir is formed by fi rst suspending
the drug solids in an aqueous solution of water – miscible solubilizer (e.g., PEG), and
then homogeneously dispersing the drug suspension with controlled aqueous solubility,
in a lipophilic polymer, by high - shear mechanical force, to form thousands of
unleachable microscopic drug reservoirs. This thermodynamically unstable dispersion
is quickly stabilized by immediately cross - linking the polymer chains in situ,
which produces a medicated polymer disk with a constant surface area and a fi xed
thickness. Mounting the medicated disk at the center of an adhesive pad then produces
a TDD system (Figure 6 d and e ).
5.1.8 OCULAR CONTROLLED - RELEASE DOSAGE FORMS
The eye is unique in its therapeutic challenges. The eye drop dosage form is easy to
instill but suffers from the inherent drawback that the majority of the medication
it contains is immediately diluted in the tear as soon as the eye drop solution is
instilled. Usually less than 10% of a topically applied dose is absorbed into the eye,
leaving the rest of the dose to potentially absorb into the bloodstream, resulting in
unwanted side effects [76] . The objectives of most of the controlled delivery system
are to maintain the drug in the precorneal area and allow its diffusion across the
cornea. Polymeric matrices seem to reduce the drainage signifi cantly, but other
newer methods of controlled - release dosage forms can also be used.
The sustained release of artifi cial tears has been achieved by a hydroxypropylcellulose
polymer insert [77] . However, the best known application of diffusional
therapy in the eye, Ocusert - Pilo, as shown in Figure 7 , is a relatively simple structure
with two rate - controlling membranes surrounding the drug reservoir containing
FIGURE 7 Schematic of Ocusert intraocular device for controlled release of pilocarpine.
White annular
rings
Pilocarpine reservoir
Rate-controlling
ethylene–vinyl
acetate copolymer
membranes
pilocarpine. The unit is placed in the eye and resides in the lowe cul - de - sac, just
below the cornea. Since the device itself remains in the eye, the drug is released into
the tear fi lm. The advantage of such a device is that it can control intraocular pressure
for up to a week. Controlled release is achieved with less drug and fewer side
effects, since the release of drug is zero order. However, it is diffi cult to keep it in
the eye for a longer time and can cause discomfort.
The prodrug administration is also getting attention as ocular controlled - release
dosage forms. Since the corneal surface presents an effective lipoidal barrier, especially
to hydrophilic compounds, it seems reasonable that a prodrug that is more
lipophilic than the parent drug will be more successful in penetrating this barrier.
Recently, dipivalyl epinephrine (Dividephrine), a dipivalyl ester of epinephrine, has
been formulated [78] . Epinephrine itself is poorly absorbed owing to its polar characteristics
and is highly metabolized. The prodrug form is 10 times as effective at
crossing the cornea and produces substantially higher aqueous humor levels.
New sustained technologies are also gaining much interest in ocular delivery, as
in other routes. Liposomes as drug carriers have achieved enhanced ocular delivery
of certain drugs, antibiotics, and peptides. Prolonged delivery of pilocarpine can be
achieved with a polymeric dispersion or submicrometer emulsions [79] .
5.1.9 VAGINAL AND UTERINE CONTROLLED - RELEASE
DOSAGE FORMS
Controlled - release devices for vaginal and uterine areas are most often for the
delivery of contraceptive steroid hormones. The advantages are prolonged release,
minimal side effects, and increased bioavailability. First - pass metabolism that inactivates
many steroid hormones can also be avoided.
Therapeutic levels of the medroxyprogestrone vaginal ring have been achieved
at a total dose that is one - sixth the required oral dose [80] . The sustained release of
progesterone from various polymers given vaginally has also been found useful in
cervical ripening and the induction of labor. A possible new use of the vaginal route
is for long - term delivery of antibodies. When various antibodies, including monoclonal
IgG, were administered from polymer vaginal rings in test animals, antibody
concentrations remained high over a month in vaginal secretions and detectable in
blood serum [81] .
The hormone - releasing devices in uterus have a closer resemblance to controlled
release because they involve the release of a steroid compound by diffusion [82, 83] .
Progesterone, the active ingredient, is dispersed in the inner reservoir, surrounded
by ethlene/vinyl acetate copolymer membrane. The release of progesterone from
this system is maintained almost constant for about a year [84 – 86] .
5.1.10 RELEASE OF DRUGS FROM CONTROLLED - RELEASE
DOSAGE FORMS
There are three primary mechanisms by which active agents can be released
from a delivery system: diffusion, degradation, and swelling followed by diffusion.
Any or all of these mechanisms may occur in a given release system. Probable
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 371
372 CONTROLLED-RELEASE DOSAGE FORMS
mechanisms of drug release from controlled - release dosage forms are briefl y
described in Table 5 . Diffusion occurs when a drug or other active agent passes
through the polymer that forms the controlled - release device. The diffusion can
occur on a macroscopic scale — as through pores in the polymer matrix — or on a
molecular level — by passing between polymer chains [4, 87] .
Table 5 describes the probable mechanisms of drug delivery from controlled -
release dosage forms under contain environmental conditions. A polymer and active
agent have generally been mixed to form a homogeneous system, also referred to
as a matrix system. Diffusion occurs when the drug passes from the polymer matrix
into the external environment. As the release continues, its rate normally decrease
with this type of system, since the active agent has a progressively longer distance
to travel and therefore requires a longer diffusion time to release. The drug release
is accomplished only when the polymer swells. Because many of the potentially most
useful pH - sensitive polymers swell at high pH values and collapse at low pH values,
the triggered drug delivery occurs upon an increase in the pH of the environment.
Such materials are ideal for systems such as oral delivery, in which the drug is not
released at low pH values in the stomach but rather at high pH in the upper small
intestine.
In reservoir systems, a reservoir — whether solid drug, dilute solution, or highly
concentrated drug solution with in polymer matrix — is surrouned by a fi lm or membrane
of a rate - controlling material [5] . The only structure effectively limiting the
TABLE 5 Probable Mechanism of Drug Delivery from Hydrogels with Certain
Environmental Conditions
Stimulus Hydrogel Mechanism
pH Acidic or basic
hydrogel
Change in pH, swelling – diffusion, erosion or
burst release of drug
Ionic strength Ionic hydrogel Change in ionic strength, change in
concentration of ions inside gel, change in
swelling, release of drug
Chemical species Hydrogel containing
electron - accepting
groups
Electron - donating compounds, formation of
charge transfer complex, change in swelling,
release of drug
Enzyme – substrate Hydrogel containing
immobilized
enzymes
Substrate present, enzymatic conversion,
product changes, swelling of gel, release of
drug
Magnetic Magnetic particles
dispersed in
alginate
microspheres
Applied magnetic fi eld, change in pores in gel,
change in swelling, release of drug
Thermal Thermoresponsive
hydrogel
Change in temperature, change in polymer –
polymer and water – polymer interactions,
change in swelling, release of drug
Electrical Polyelectrolyte
hydrogel
Apply electric fi eld, membrane charging,
electrophoresis of charged drug, change in
swelling, release of drug
Ultrasound
irradiation
Ethylene – vinyl
alcohol hydrogel
Ultrasound irradiation, temperature increase,
release of drug
release of the drug is the polymer layer surrounding the reservoir. Since this polymer
coating is essentially uniform and of a nonchanging thickness, the diffusion rate of
the active agent can be kept fairly stable throughout the lifetime of the delivery
system.
For the diffusion - controlled systems ddescribed thus far, the drug delivery device
is fundamentally stable in the biological environment and does not change its size
through either swelling or degradation [4] . It is also possible for a drug delivery
system to be designed so that it is incapable of releasing its agent or agents until it
is placed in an appropriate biological environment. Swelling - controlled - release
systems are initially dry and, when placed in the body, will absorb water or other
body fl uids and swell. The swelling increases the aquenous solvent content with the
formulation as well as the polymer mesh size, enabling the drug to diffuse through
the swollen network into the external environment. Most of the materials used in
swelling - controlled - release systems are based on hydrogels, which are polymers that
well without dissolving when placed in water or other biological fl uids.
5.1.10.1 Time - Controlled - Release Dosage Forms
To achieve a drug release which is independent of the environment (e.g., pH, enzymatic
activity, intestinal motility), the lag time prior to the drug release has to be
controlled primarily by the delivery system. The release mechanisms employed
include bulk erosion of polymers in which drug release by diffusion is restricted,
surface erosion of layered devices composed of alternation drug - containing and
drug - free layers, osmotically controlled rupture, and enzymatic degradation of liposomes.
The device environment may modulate the release profi le of any of these
systems and may depend on factors such as the amount of free moisture, regional
blood fl ow, and various cellular activities at the site [88, 89] .
Systmes with Eroding or Soluble Barrier Coatings These types of delivery systems
comprise reservoir devices coated with a barrier layer. the barrier dissolves or
erodes after a specifi ed lag period, after which the drug is released rapidly from the
reservoir core. In general, the lag time prior to drug release from a reservoir - type
device can be controlled by the thickness of the coating layer, for example, the
Chronotropic systems, which consists of a drug - containing core layered with hydroxy
propyl methyl cellulose (HPMC), optionally coated with an outer enteric coating.
The lag time prior to drug release is controlled by the thickness and the viscosity
grade of the HPMC layer. After erosion or dissolution of the HPMC layer, a distinct
pulse was observed. The Chronotropic system [90, 91] is an oral dosage form designed
to achieve time - controlled delivery. This system has been developed keeping in mind
the interaction between GI fl uid and coating polymer, which causes time - or site -
controlled release. The reaction causes the liberation of drugs by the mechanism of
swelling of polymer, increased permeability, and dissolution/erosion phenomena.
This system probably works better for poorly water soluble drug because highly
water soluble drugs could diffuse through the swollen HPMC layer prior to complete
erosion.
The TIME CLOCK system for the oral dosage should enable fast and complete
release of drug after a predetermined lag time [92] . A tablet was made containing
the drug molecule and bulking agents (lactose, polyvinlpyrrolidine, corn starch, and
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 373
374 CONTROLLED-RELEASE DOSAGE FORMS
magnesium stearate). This core was coated with a hydrophobic dispersion of carnauba
wax, bees ’ wax, poly(oxyethylene) sorgitan monooleate, and HPMC in water.
By altering the coating thickness, the lag time could be proportionally modulated.
In vitro results indicated that the drug core was dissolved immediately after direct
immersion in water and release was completed within 30 min, while a rapid release
was observed after a certain lag time for the TIME CLOCK system with the hydrophobic
coating. In vivo results revealed that drug disintergration was modulated by
the coating thickness of the drug core as well as the food intake before drug administration.
This approach may also be used to control the release onset time. Since
the drug core is formulated with soluble ingredients, shell dissolution/distintegration
becomes the key factor to control the lag time. Furthermore, drug release is independent
of normal physiological conditions, such as pH, digestive state, and anatomical
position at the time of release. This approach could be applicable for oral
as well as for implant systems. Figure 8 illustrates the theoretical description of drug
release from surface - eroding polymeric controlled - release dosage forms [93] .
Systems with Rupturable Coatings This class of reservoir - type pulsatile release
system is based on rupturable coatings. The drug is released from a core after rupturing
of a surrounding polymer layer, caused by a pressure buildup within the
system, as shown in Figure 9 [93] . The pressure necessary to rupture the coating can
be achieved with gas - producing effervescent excipients, inner osmotic pressure, or
swelling agents.
An effervescent mixture of citric acid and sodium bicarbonate was incorporated
in a tablet core coated with ethyl cellulose. The carbon dioxide development after
water penetration into the core resulted in a pulsatile release after rupture of the
coating, which was strongly dependent on the mechanical properties of the coating
layer: The weak and nonfl exible ethyl cellulose fi lm ruptured suffi ciently when
FIGURE 8 Theoretical controlled release from a surface - eroding polymeric system.
( Adapted from ref. 93 with permission of Elsevier Copyright 1999 .)
Polymer degradation
with MW decrease
until critical MW
High-molecular-weight drug in
bulk-eroding polymer
(limited release by diffusion)
Surface release
Porous polymer matrix
Release of remaining entrapped drug
(booster release)
Completion of polymer
degradation by fragmentation
and macrophage uptake
compared with more fl exible fi lms. The lag time before release increased with
increasing coating level and increasing hardness of the core tablet. The effectiveness
of so - called superdistintegrants, which are highly swellable agents, was demonstrated
for a capsule - based system consisting of a drug containing a core capsule,
and swelling layer, and a rupturable polymeric layer. Croscarmellose, sodium starch
glycolate, or low - substituted hydroxypropyl cellulose (L - HPC) were used as swelling
substances, which resulted in a complete fi lm rupture followed by a rapid drug
release. The lag time is controlled by the composition of the outer polymer layer:
Water - soluble polymers such as HPMC increase the permeability and therefore
reduce the lag time. The swelling energy of several excipients decreased in the following
order: croscamellose sodium > L - HPC > crospovidone > HPMC. Bothe solid
and liquid drug formulations could be delivered with this system [94 – 96] .
A novel capsule was made from ethyl cellulose for the time - controlled release
of drugs in the colon [97] . Initially the ethyl cellulose capsule was prepared using a
gelatin capsule with ethyl cellulose, followed by dissolution of the gelatin in water.
The thickness of the ethyl cellulose capsule body was varied and the effect of wall
thickness on the release of drugs in the capsules was investigated. Ethyl cellulose
capsules contained a large number of mechanically made micropores (400 . m) at
the bottom. Also located in the bottom of the capsule body was a swellable layer
consisting of L - HPC. Above the swellable layer was the drug reservoir, which contained
a mixture of the model drug, fl uorescein, and a bulking agent, such as lactose
or starch. The capsule was thus capped and sealed with a concentrated ethyl cellulose
solution. After administration of drug - containing capsule, water molecules
penetrated the capsule through the micropores in the bottom of the capsule body.
Hydration and swelling of HPC induced an increase in the internal osmotic pressure,
which resulted in the “ explosion ” of the capsule and a burstlike drug release was
observed. By altering the thickness of the capsule, the lag time of the drug release
could be altered. A similar approach for the pulsatile release of drug was reported
in which a hydrostatic pressure was generated inside the capsules.
FIGURE 9 Theoretical controlled release from an osmotically driven system. ( Adapted
from ref. 93 with permission of Elsevier Copyright 1999 .)
Surface erosion of outer
drug-containing layer
Drug release from outer layer
as surface erodes
No drug release
Drug-free layer
Surface erosion of
drug-free layer
Drug release from core
(booster release)
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 375
376 CONTROLLED-RELEASE DOSAGE FORMS
Systems with Capsular Structure Several single - unit pulsatile dosage forms with
a capsular design have been developed. Most of them consist of an insoluble capsular
body which contains the drug and a plug which is removed after a predetermined
lag time because of swelling, erosion, or dissolution.
The Pulsincap system consists of a water - insoluble capsule body fi lled with the
drug formulation. The capsule half is closed at the open end with a swellable hydrogel
plug. The dimension and the position of the plug can control the lag time prior
to the release. In order to assure a rapid release of the drug content, effervescent
agents or disintegrants can be included in the drug formulation, in particular with
water - insoluble drugs. The system is coated with an enteric layer which dissolves
upon reaching the higher pH region of the small intestine. This system comprises
insoluble capsules and plugs. The plugs consist either of swellable materials, which
are coated with insoluble but permeable polymers (e.g., polymethacrylates), or of
erodible substances, which are compressed (e.g., HPMC, polyvinyl alcohol, polyethylene
oxide) or prepared by congealing of melted polymers (saturated polyglycolated
glycerides of glyceryl monooleate). The erosion of the plug can also be
controlled enzymatically: A pectin plug can be degraded by pectinolytic enzymes
being directly incorporated into the plug [98 – 100] .
Linkwitz et al. [101] described the delivery of agents from osmotic systems based
on the technology of an expandable orifi ce. The system is in the form of a capsule
from which the drug is delivered by the capsule ’ s osmotic infusion of moisture from
the body. The delivery orifi ce opens intermittently to achieve a pulsatile delivery
effect. The orifi ce forms in the capsule wall, which is constructed of an elastic material,
preferably elastomer (e.g., styrene – butadiene copolymer), which strectches
under apressure dufferential caused by the pressure rise inside the capsule as the
osmotic infusion progessses. The orifi ce is small enough that when the elastic wall
is relaxed, the fl ow rate of drug through the orifi ce is substantially zero, but when
the elastic wall is stretched due to the pressure differential across the wall exceeding
a threshold, the orifi ce expands suffi ciently to allow the release of the drug at a
phsiologically required rate. This osmotically driven delivery device as an implant
can used in the anal – rectal passageway, in the cervical canal, as an artifi cial gland,
in the vagina, as ruminal bolus, and the like.
A core - shelled cylindrical dosage form is available comprising a hydrophobic
polycarbonate coating and a cylindrical core of alternating polyanhydride isolating
layer and drug - loaded poly[ethyl glycinate) (benzyl amino acethydroxamate) phosphazene]
(PEBP) layer for a programmable drug delivery system for single - dose
vaccine and other related applications [102] . The pulsatile release of model compounds
[fl uorescein isothiocyanate (FITC) – dextran and myoglobin] whith a certain
lag time (18 – 118 h) was achieved on the basis of the pH - sensitive degradation of
PEBP and its cooperative interaction with polyanhydrides. In another experiment,
Jiang and Zhu [103, 104] designed laminated devices comprising of polyanhydrides
as isolating layers and pH - sensitive complexes of poly(sebacic anhydride) - b -
polyethylene glycol (PSA - b - PEG) and poly(trimellitytylimdoglycine - co - sebacic
anhydride) -b - polyethylene glycol [P(TMA - gly - co - SA) - b - PEG] as protein - loaded
layers. The release of model proteins [bovine serum albumin (BSA) and myoglobin]
showed a typical pulsatile fashion. The lag time prior to the release correlated with
the hydrolytic druation of polyanhydrides, which varied from 30 to 165 h depending
on polymer type and isolating layer thickness.
5.1.10.2 Stimuli - Induced Controlled - Release Systems
Several polymeric delivery systems undergo phase transitions and demonstrate
marked swelling – deswelling change in reponse to environmental changes, including
solvent composition ionic strength, temperature, electric fi elds, and light [105] .
Responsive drug release from those systems results from the stimuli - induced
changes in the gels or micelles, which maydeswell, swell, or erode in response to
the respecive stimuli. The mechanisms of drug release include ejection of the drug
from the gel as the fl uid phase synerses out and drug diffusion along a concentration
gradient.
pH-Responsive Drug Release Dosage Forms pH - sensitive enteric coatings
have been used routinely to deliver drug to the small intestine. These polymer
coatings are insensitive to the acidic conditions of the stomach yet dissolve at
the higher pH environment of the small intestine. This pH differential has
also been attempted for colonic delivery purposes, although the polymers
used for colonic targeting tend to have a threshold pH for dissolution that is
higher than for those used in conventional enteric coating applications
[104 – 106] .
The synthesis and characterization of series of novel azo hydrogels for colon -
targeting drug delivery have been described. The colon specifi city is achieved dure
to the presence of pH - sensitive monomers and azo cross - linking agents in the
hydrogel structures. Most commonly, copolymers of methacrylic acid and methylmethacrylate
that dissolve at pH 6 (Eudragit L) and pH 7 (Eudragit S) have been
extensively investigated [106, 107] . This approach is based on the assumption that
gastrointestinal pH increases progressively from the small intestine to the colon.
The pH in the distal small intestine is usually around 7.5, while the pH in the proximal
colon is closer to 6.
To overcome the premature release of drugs, a copolymer of methacrylic acid,
methylmethacrylate, and ethyl acetate (Eudragit ES), which dissolves at a slower
rate and at a higher threshold pH (7 – 7.5), has been developed [108] . The trn A series
of in vitro dissolution studies with this polymer have highlighted clear benefi ts over
the Eudragit S polymer for colon targeting. A gamma scintigraphy study comparing
the in vivo performance of these various polymers revealed that Eudragit S (coating
over the tablets) was superior to the older polymers in terms of retarding drug
release in the small intestine, although, in some cases, the coated tablets did not
break up at all. pH - sensitive delivery systems are commercially available for mesalazine
(5 - iminosalicylic cid) (Asacol and Salofalk) and budesonide (Budenofalk and
Entocort) for the treatment of ulcerative colitis and Crohn ’ s disease, respectively
[109] .
Natural polysaccharides are being used for the development of solid dosage
forms for pH - dependent delivery and for targeting the release of drugs in colon
[110] . Various major approaches utilizing polysaccharides are fermentable coating
of the drug core, embedding of the drug in biodegradable matrix, and formation of
drug – saccharide conjugate (prodrugs). A large number of polysaccharides have
already been studied for their potential in these types of delivery systems, such as
chitosan, alginate, pectin, chondroitin sulfate, cyclodextrin, dextrans, guar gum,
inulin, amylose, and locust bean gum [111] .
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 377
378 CONTROLLED-RELEASE DOSAGE FORMS
A pectin - and - galactomannan coating was developed by Lee et al. [112] . It consists
of a conventional tablet coated with pectin and galactomannan. The coating
from aqueous solutions of pectin and galactomannan was shown to be strong, elastic,
and insoluble in gastric fl uid. Figure 10 shows the plasma concentration profi le of
nifedifi ne from pectin – galactomannan - coated tablets and associated in vivo transit
and disintegration characteristics. The mean plasma concentration of nifedifi ne was
negligible for more than 5 h postdose and then increased rapidly.
CODES Technology CODES is a unique colon - specifi c drug delivery technology
that was designed to avoid the inherent problems associated with pH - or time -
dependent systems [113, 114] . The design of CODES exploited the advantages of
certain polysaccharides that are only degraded by microorganisms available in the
colon [115] . This is coupled with a pH - sensitive polymer coating. Since the degradation
of polysaccharides occurred only in the colon, this system exhibited the
capability to achieve colon delivery consistently and reliably. As schematically presented
in Figure 11 , one typical confi guration consists of a core table coated with
three layers of polymer coatings. The fi rst coating (next to the core tablet) is an
acid - soluble polymer (e.g., Eudragit E) and an outer coating is enteric with a
HPMC barrier layer in between the oppositely charged polymers. The polysaccharides,
degradable by enteroorganisms, generate organic acid, including mannitol,
maltose, lactulose, and fructooligosaccharides. During the transit through the
GI tract, CODES remains intact in the stomach, but the enteric and barrier coatings
disolve in the intestines. In vivo performance of CODES in beagle dogs was
studied using acetaminophen as the model drug and lactulose as the matrix - forming
excipient in the core tablet. Compared with enteric - coated tablet, the onset of
acetaminophen release form CODES was delayed more than 3 h, as shown in
Figure 12 .
FIGURE 10 Nifedipine plasma concentration – time profi le from pectin – galactomannan -
coated tablets. ( Adapted from ref. 110 with permission of Elsevier Copyright 2002 .)
0
1
2
3
4
5
6
7
8
9
10
Nifedipine concentration (ng/mL)
0 3 6 9 12 15 16 24 21
Time (h)
Initial disintegration
Complete disintegration
Colon arrival time
COOES
Enteric-coated core
1400
1200
1000
800
600
400
200
0
Time (h)
APAP plasma concentration (ng/mL)
0 2 4 6 8 10 12 14 16
FIGURE 11 Conceptual design of CODES technology. ( Adapted from ref. 114 with permission
of Elsevier Copyright 2002 .)
In stomach
In small intestime
In colon
Enter coating
Acid-soluble
polymer coating
Lactulose
Microflora
Organic acid
FIGURE 12 Percentage of acetaminophen released from CODES and enteric - coated core
tablets in beagle dogs. ( Adapted from ref. 114 with permission of Elsevier Copyright 2002 .)
Thermoresponsive Drug Release Dosage Forms Temperature is the most widely
utilized triggering signal for a variety of modulated or pulsatile drug delivery systems.
The use of temperature as a signal has been justifi ed by the fact that the body temperature
often deviates from the physiological temperature (37 ° C) in the presence
of pathogens or pyrogens. This deviation sometimes can be a useful stimulus that
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 379
380 CONTROLLED-RELEASE DOSAGE FORMS
achivates the release of therapeutic agents from various temperature - responsive
drug delivery systems for diseases accompanying fever. The drug delivery systems
that are responsive to temperature utilize various polymer properties, including the
thermally reversible coil/globule transition of polymer molecules, swelling change
of networks, glass transition, and crystalline melting.
Thermoresponsive hydrogels have been investigated as possible drug delivery
carriers for stimuli - responsive drug delivery systems [116 – 118] . The common characteristics
of temperature - sensitive polymers are the presence of hydrophobic
groups, such as methyl, ethyl, and propyl groups. Of the many temperature - sensitive
polymers, poly( N - isopropylacrylamide) (PIPPAm) is probably the most extensively
used. PIPPAm cross - linked gels have shown thermoresponsive, discontinuous swelling/
deswelling phases — swelling, for example, at temperatures below 32 ° C while
shrinking above this temperature. A sudden temperature increase above the transition
temperature of these gels resulted in the formation of a dense, shrunken layer
on the gel surface, which hindered water permeation from inside the gel into the
environment. Drug release from the PIPPAm hydrogels at temperatures below 32 ° C
was governed by diffusion, while above this temperature drug release was stopped
completely, due to the “ skin layer ” formation on the gel surface (on – off drug release
regulation).
Swelling – deswelling kinetics of conventional cross - linked hydrogels are normally
the reciprocal of the square of the gel dimension. This mobility of the cross -
linked chains in the gel is affected by the surrounding chains and the
swelling – deswelling phases of the gel are governed by the collective diffusions of
the network chains. Thus, to accelerate structural changes of the gel in response
to external stimuli, several approaches have been developed which form porous
structure within the gel and decrease gel size. Kaneko et al. [119, 120] introduced
a method to accelerate gel swelling – deswelling kinetics based on the molecular
design of the gel structure by grafting the free mobile linear PIPPAm chains within
the cross - linked PIPPAm hydrogels. These novel graft - type PIPPAm gels had the
same transition temperature as conventional cross - linked PIPPAm gels and existed
in the swollen state below the transition temperature, while above this temperature,
they shrank. Adense skin layer formed on the conventional PIPPAm gels
upon temperature change above the transition temperature, which limited the
complete shrinkage of the gel. In contrast, the PIPPAm - grafted gels showed rapid
deswelling kinetics without the formation of a skin layer on the gel surface. This
is probably due to the rapid dehydration of the graft chains formed by hydrophobic
aggregation on the three - dimensional cross - linked gel chains. The low - molecular
- weight compounds released immediately from conventional PIPPAm gels after
a temperature increase, after which the release was teminated due to the formation
of a dense impermeable skin layer on the surface. In comparison, 65% of the drug
was released in one burst from free PIPPAm - grafted hydrogels with a graft molecular
weight (MW) of 9000 following the temperature increase. Graft - type gels with
a molecular weight of 4000 showed oscillating drug release profi les. The release of
high - molecular - weight compound (e.g., dextran, MW 9300) from PIPPAm graft -
type gels was shown to burst after a temperature increase of 40 ° C. The difference
in drug release profi les for two graft - type gels is probably due to the different
strengths of aggregation forces between the formed hydrophobic cores within the
graft - type gels. That is, the high - molecular - weight graft chains formed more hydro
phobic cores within the gels upon the temperature increase, which induced rapid
gel deswelling.
Temperature - sensitive hydrogels can also be placed inside a rigid capsule containing
holes or apertures. The on – off release is achieved by the reversible volume
change of temperature - sensitive hydrogels. Such a device is called a squeezing
hydrogel device because the drug release is affected by the hydrogel dimension. In
addition to temperature, hydrogels can be made to respond to other stimuli, such
as pH. In this type of system, the drug release rate was found to be proportional to
the rate of squeezing of the drug - loaded polymer.
Clinical applications of thermosensitive hydrogels based on NIPAAm and its
derivatives have limitations [121] . The monomers and cross - linkers used in the synthesis
of the hydrogels are still not known to be biocompatible and biodegradable.
The observation that acrylamide - based polymers activate platelets upon contact
with blood, together with the unclear metabolism of poly(NIPAAm), requires
extensive toxicity studies before clinical applications can merge.
Recently some studies have been conducted on anocomposite hydrogels for
photothermally modulated drug delivery. Gold nanoshells can be designed to absorb
light strongly at desired wavelengths, in particular, in the near infrared between 800
and 1200 nm, where tissue is relatively transparent [122] . When optically absorbing
gold nanoshells are embedded in a matrix material, illuminating them at their resonance
wavelength causes the nanoshells to transfer heat to their local environment.
This photothermal effect can be used to optically modulate drug release from a
nonshell polymer composite drug delivery system. To accomplish photothermally
modulated release, the matrix polymer material must be thermally responsive.
The authors observed the pulsatile release of insulin and other proteins in response
to near - infrared irradiation when gold nanoshells were embedded in NIPAAm - co -
acrylamide hydrogels [122] .
Electroresponsive Release An electric fi eld as an external stimulus has advantages,
such as the availability of equipment, which allow precise control with regard
to the magnitude of current, duration of electric pulses, interval between pulses, and
so on. Electrically responsive delivery systems are prepared from polyelectrolytes
(polymers which contain relatively high concentration of ionizable groups along the
backbone chain) and are thus pH responsive as well as electroresponsive. Under
the infl uence of an electric fi eld, electroresponsive hydrogels generally deswell or
bend, depending on the shape of the gel that lies parallel to the electrodes, whereas
deswelling occurs when the hydrogel lies perpendicular to the electrodes. Synthetic
(e.g., acrylate and methacrylae derivatives) as well as naturally occurring polymers
(including hyaluronic acid, chondroitin sulfate, agarose, carbomer, xanthan gum,
and calcium alginate separately or in combination) have been used. Complex multicomponent
gels or interpenetrating networks have been prepared in order to
enhance the gels or interpenetrating networks have been prepared in order to
enhance the gel ’ s electroresponsiveness [123] . Electrically enhanced transdermal
delivery provides the time - dependent delivery. Ionotophoresis, the electromigrational
movement of charged molecules through the skin under a low - voltage
and continuous driving force, has been reported for a number of bioactive compounds,
such as leutinizaing hormone relesing hormone (LHRH), testosterone, and
buserelin.
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 381
382 CONTROLLED-RELEASE DOSAGE FORMS
Electronic Microelectromechanical System for Controlled Release Electronic
microelectromechanical devices are manufactured using standard microfabrication
techniques that are used to create silicon chips for computers, and they often have
moving parts or components that enable some physical or analytical function to be
performed by the device. Microfabrication techniques, the same processing techniques
used to make microprocessors for computers and other microelectronic
devices, have been used increasingly to produce microscale devices whose primary
functions are mechanical, chemical and optical in nature. Such devices are commonly
referred to as microelectromechanical systems (MEMS) and are found in
ink - jet printers, automotive applications, and microtube engines in the aerospace
industry. MEMS for biological applications are classifi ed as either microfl uidic
devices or nonmicrofl uidic devices. The ultimate goal of MEMS is to develop a
microfabricated device with the ability to store and release multiple chemical substances
on demand by a mechanism devoid of moving its parts [124, 125] . A wide
variety of microreservoirs, micropumps, cantilevers, rotors, channels, valves, sensors,
and other structures have been fabricated, typically from the materials that have
been demonstrated to be biocompatible and can be sterilely fabricated and hermetically
sealed. The digital capabilities of MEMS may allow greater temporal control
over drug release compared to traditional polymer - based systems, while the batch -
processing techniques used in the microelectronics industry can lead to greater
device uniformity and reproducibility than is currently available to the pharmaceutical
industry. The use of MEMS for drug delivery necessitates the existence of drug
depot or supply within or on the device. One straightforward approach to achieve
this drug reservoir is the fabrication of silicon microparticles that contain an internal
reservoir loaded with drug. These devices could be used for oral drug delivery, with
release of the drug triggered by binding of a surface - functionalized molecule to cells
in the digestive tract.
The completely implantable minipump made by Minimed has a pulsatile, radio -
controlled injection rate through a catheter into the intraperitoneal region [126] .
One study found that patients with the implantable pump did not differ from control
subjects on any meansure of psychosocial function but that puump users monitored
their blood glucose levels more frequently and had lower average blood glucose
levels. Even though this type of device may improve patient ’ s mbility and reduce
infections by eliminating transcutaneous catheters, they may still be hampered by
their size, cost, ability to deliver only drugs in solution, and limited stability of some
drugs in solution at 37 ° C. Ikemoto and Sharpe [127] have developed a stepmotor
micropump for the injection of nanoliter volumes of d - amphetamine solution into
discrete brain regions of freely moving rats that was well tolerated. This micropump
delivered a reliable volume of 50 nL per infusion over an hour at a rate of one infusion
per minute.
Another development in MEMS technology is the microchip. The microchip
consists of an array of reservoirs that extend through an electrolyte - impermeable
substrate. The prototype microchip is made of silicon and contains a number of drug
reservoirs. Each reservoir is sealed at one end by a thin gold membrane of material
that serves as an anode in an electrochemical reaction and dissolves when an electric
potential is applied to it in an electrolyte solution. The reservoirs are fi lled with any
combination of drug or drug mixtures in any form (i.e., solid, liquid, or gel) through
the opening opposite the anode membrane by ink - jet printing or microinjection and
are then sealed with a waterproof material. A cathode is also required for the electrochemical
reaction to take place, and the cathode is usually made of the same
conductive material as the anode to simplify the fabrication procedure. The device
is submerged in an electrolyte solution containing ions and upon electric stimulation
forms a soluble complex with the anode in its ionic form. When release is desired,
an electric potential is applied between an anode membrane and a cathode, and the
gold membrane anode is dissolved within 10 – 20 s and allows the drug in the reservoir
to be released. This electric potential causes oxidation of the anode material to
form a soluble complex with the electrolytes which when dissolves allowing release
of the drug. Complex release patterns (such as simultaneous constant and pulsatile
release) can be achieved from the microchips. The microchip has the ability to
control both release time and release rate. The rate of release from a reservoir is a
function of the dissolution rate of the materials in the reservoir, the diffusion rate
of these materials out of the reservoir, or both. Therefore, the release rate from an
individual reservoir can be tailored to a particular application by proper selection
of the materials placed inside the reservoir [e.g., pure drug(s), drugs with polymers]
[124, 125] .
A microchip with insulin - fi lled reservoirs could eventually provide a better alternative
for the treatment of insulin - dependent diabetes mellitus (IDDM) [125] .
Because the microchip is capable of being programmed as well as integrated with
other electronic devices, it is supposable that the microchip could be incorporated
into a closed - loop biofeedback system. An electronic apparatus that continuously
measures the blood glucose levels could provide the stimulus to the microchip and
result in release of insulin into the bloodstream. Although such a system could still
not perfectly mimic an endogenous system of healthy person, it could practically
meet the needs of IDDM patients. Pulsatile release of synthetic gonadotropin –
releasing hormone (GnRH) can be achieved with a programmed microchip. A subcutaneous
implanted microchip containing 1000 drug reservoirs would be adequate
to administer a month ’ s worth of drug therapy. The implanted microchip would be
a convenient means to achieve the desired pharmacotherapeutic outcome of ovulation
without interfering with the patient ’ s daily activities or causing phlebitis.
While microchip drug delivery would be the most technologically advanced
delivery system, it has itself limited storage capacity for therapeutic drugs [125] .
Because most applications of this technology require implantation within bodily
tissues, the question arises, “ What would be done when the chip runs out of drug? ”
Some sort of procedure would be required to retrieve the empty chip cartridge once
it has emptied. Due to the limited quantity of drug that can be stored on one
chip, this technology is only ideal for potent drugs. If a larger dose of a medication
is required, the chip would not be adequate for dispensing larger quantities
of drug.
Magnetically Induced Release Magnetic carriers receive their magnetic response
to a magnetic fi eld from incorporated materials such as magnetite, iron, nickel, and
cobalt. For biomedical applications, magnetic carriers must be water based, biocompatible,
nontoxic, and nonimmunogenic. Earlier, Langer et al. [128] embedded magnetite
or iron beads into a drug - fi lled polymer matrix and then showed that they
could activate or increase the release of the drug from the polymer by moving a
magnet over it or by applying an oscillating magnetic fi eld. When the frequency of
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 383
384 CONTROLLED-RELEASE DOSAGE FORMS
the applied fi eld was increased from 5 to 11 Hz, the release of BSA from ethylenevinylacetate
copolymer (EVAc) matrices slowed in a linear fashion. The rate of
release could be modulated by altering the position, orientation, and magnetic
strength of the embedded materials as well as by changing the amplitude of frequency
of the magnetic fi eld. The micromovement within the polymer produced
microcracks in the matrix and thus made the infl ux of liquid, dissolution, and effl ux
of the drug. Done repeatedly, this would allow the pulsatile delivery of insulin.
Another mechanistic approach based on magnetic attraction is the slowing down
of oral drugs in the gastrointestinal system. This is possible by fi lling an additional
magnetic component into capsules or tablets. The speed of travel through the
stomach and intestines can then be slowed down at specifi c positions by an external
magnet, thus changing the timing and/or extent of drug absorption into stomach or
intestines. Slowing down the passage of magnetic liposomes with a magnet actually
increased the blood levels of drug. Babincova et al. [129] developed magnetoliposomes
for triggered release of drug. In their delivery systems, they entrapped
dextran – megnetite and model drug 6 - carboxyfl uorescein in the liposomes and used
laser to trigger the release of drug. The magnetite absorbs the laser light energy to
heat the lipid bilayer above the gel – liquid crystal - phase transsition temperature Tc ,
which is 41 ° C for dipalmitoyl - phosphatidylcholine. Liposomes made from this lipid
release their content as soon as the temperature reaches this level. They have also
suggested that the absorption of laser energy by magnetite particles provides a
means for localized heating and controlled release of liposome with a single laser
pulse. This may have potential applications for selective drug delivery, especially to
the eyes and skin. Even though the magnetic - modulated therapeutic approach
is promising, it still needs very careful attention for a number of physical and
magnetism - related properties. The magnetic force, which is defi ned by its fi eld
and fi eld gradient, needs to be large and carefully shaped to activate the delivery
system within the target area. The magnetic materials should be tissue stable
and compatible.
Chemically Induced Release
Gluose - Responsive Insulin Release Device A decrease in or the absence of insulin
secretion from pancreatic islets is the cause of diabetes mellitus. An effective glucose -
responsive insulin delivery system should be composed of a glucose - sensing component
and an insulin - releasing component. The sensing component detects a change
in the glucose level and produces a signal that affects the releasing component. The
magnitude of the signal increases with increasing glucose concentration, and so does
the rate of insulin release. Based on this principle, various polymer - based glucose -
responsive delivery systems have been designed, most of which are hydrogels that
can alter their volume and degree of hydration in response to glucose concentration.
Several systems have already been developed which are able to respond to glucose
concentration changes, such as glucose oxidase (GOD), which catalyzes glucose
oxidation [130] . Glucosylated insulin bound to concanavalin (Con) A was released
through exchange with external glucose, due to the difference in their binding constants.
This system needs direct injection of microcapsules into the peritoneal cavity
of patients, which may cause undesirable side effects arising from the immune
response to Con A if Con A was directly exposed to immune systems after breakage
of microcapsules. Obaidat and Park [131] prepared a copolymer of acrylamide and
allyl glucose. The side - chain glucose units in the copolymer were bound to Con A.
These hydrogels showed a glucose - responsive, sol – gel phase transition dependent
upon the external glucose concentration. The nonlinear dependence of this sol – gel
phase transition with regard to the glucose concentration was due not only to the
increased binding affi nity of allyl glucose to Con A compared to native glucose but
also to the cooperative interaction between glucose - containing copolymer and Con
A. Kataoka et al. [132] developed glucose and thermoresponsive hydrogels using
acrylamidophenylboronic acid and N - isopropylacrylamide (IPAAm). The obtained
gels, containing 10 mol % phenylboronic acid moieties, showed a transition temperature
of 22 ° C in the absence of glucose. Below this temperature, the gels existed in
a swollen state. The introduction of glucose to the medium altered the transition
temperature of the gels in such a way that the transition temperature increased with
increasing glucose concentration to reach 36 ° C at 5 g/L glucose concentration.
Boronic acid was in equilibrium between the undissociated and dissociated forms.
With increasing glucose concentration, the equilibrium shifted to increase the
amount of dissociated boronate groups and gels became more hydrophilic. Although
all of the glucose - sensitive insulin delivery systems are elegant and highly promising,
many improvements need to be made for them to become clinically useful. First of
all, the response of these systems upon changes in the environment occurs too
slowly. In clinical situations, these systems need to respond to ever - changing glucose
concentrations at all times, requiring hydrogels that can respond reproducibility and
with rapid - response onset times on a long - term basis. An additional constant is that
all the components used in the systems must be biocompatible.
Chemotactic Factor - Induced Controlled - Release Systems With physical or chemical
stress such as injury and broken bones, an infl ammation reaction takes place at
the injured site. At the infl ammatory sites, infl ammation-responsive phagocytic cells
such as macrophages and polymorphonuclear cells play a role in healing the injury.
During infl ammation, hydroxyl radicals (OH • ) are produced from the cells. Yui and
co - workers [133, 134] developed infl ammatory - induced hydroxyl radicals and
designed drug delivery system which responded to the hydroxyl radicals
and degraded in a limited manner. They used hyaluronic acid (HA), a linear aminopolysaccharide
composed of repeating disaccharide subunits of N - acetyl - d -
glucosamine and d - guluronic acid. In the body, HA is mainly degraded by
hyaluronidase, or hydroxyl radicals. Degradation of HA via the enzyme is very low
in a normal state of health. Degradation via hydroxyl radicals, however, is usually
dominant and rapid when HA is injected at infl ammatory sites. These authors prepared
cross - linked HA with ethyleneglycol diglydylether or polyglycerol polygluycidalether
. These HA gels degraded only when the hydroxyl radicals were generated
through the reaction between the iron (Fe 2+ ) ions and the hydrogen peroxide in
vitro. Thus, a surface erosion type of degradation was achieved. When microspheres
were incorporated in the HA hydrogels as a model drug, these microspheres were
released when hydroxyl radicals induced HA gel degradation. Furthermore,
degradation of HA in vivo tests showed that HA gels are degraded only when
infl ammation was induced by surgical incision. Control HA gels were stable over
100 days. It is possible to treat locally in infl ammatory diseases such as rheumatoid
arthritis using anti - infl ammatory drug incorporated in HA gels [135] .
RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS 385
386 CONTROLLED-RELEASE DOSAGE FORMS
5.1.11 SUMMARY
As pharmaceutical scientists have increased their knowledge of pharmacokinetics
and pharmacodynamics, it has become apparent that these factors can result in more
effi cacious drugs. The number of new drug entities appearing on the market yearly
has declined and pharmaceutical manufacturers have shown a renewed interest in
improving existing dosage forms and developing more sophisticated drug delivery
systems, including those employing the principles of controlled drug release.
Current research in this area involves numerous new and novel systems, many
of which have strong therapeutic potential. In this chapter, we have tried to emphasize
the importance of oral routes as well as others, such as ocular, transdermal,
intrauterine, and vaginal. The various microencapsulation, nanoencapsulation, and
liposome technologies and the release of drugs and bioactive compounds from such
products have been described.
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393
5.2
PROGRESS IN DESIGN OF
BIODEGRADABLE POLYMER - BASED
MICROSPHERES FOR PARENTERAL
CONTROLLED DELIVERY OF
THERAPEUTIC PEPTIDE/PROTEIN
Shunmugaperumal Tamilvanan *
University of Antwerp, Antwerp, Belgium
Contents
5.2.1 Introduction
5.2.2 Peptide/Protein - Loaded Microsphere Production Methods
5.2.2.1 Phase Separation (A Traditional Technique)
5.2.2.2 Double Emulsion (A Hydrous Technique)
5.2.2.3 Spray Drying (An Anhydrous Technique)
5.2.2.4 New Trends in Production Methods
5.2.3 Analytical Characterization of Peptide/Protein - loaded Microspheres
5.2.4 Immune System Interaction with Injectable Microspheres
5.2.5 Excipient Inclusion: Injectable Peptide/Protein - Loaded Microspheres
5.2.5.1 Solubility - and Stability - Increasing Excipients
5.2.5.2 Preservation - Imparting Excipients
5.2.6 Peptide/Protein Encapsulated into Biodegradable Microspheres: Case Study
5.2.6.1 Vaccines
5.2.6.2 Proteins
5.2.7 Conclusion
References
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
* Current address: Department of Pharmaceutics, Arulmigu Kalasalingam College of Pharmacy, Anand
Nagar, Krishnankoil, India
394 BIODEGRADABLE POLYMER-BASED MICROSPHERES
5.2.1 INTRODUCTION
At the cellular level, deoxyribonucleic and ribonucleic acids (DNA and RNA, respectively)
serve as an endogenious vehicle not only to store genetic information but
also to transfer genetic information from one generation to their offsprings of all
known living organisms. In addition, utilizing the rule of complementary base
pairing, the DNA undergoes replication and transcription processes to produce
respectively a new double - stranded DNA molecule and a complementary single -
stranded RNA molecule. Following the translation process, peptide and protein are
synthesized/constructed in ribosomal subunits through peptidic linkages between
available 20 amino acids. The peptide and protein thus constructed perform a wide
variety of functions and each cell contains several thousands of different proteins.
Peptide - and protein - mediated, important physiological and biological processes of
the human body include ligands/hormones for signaling, enzymes for biotransformation
reactions, receptors for pharmacological response elucidation, antibodies in
immune system interactions, transcription, and translation. Hence these molecules
play a vital role to ensure proper development and functioning of entire organs of
the human body.
Webster ’ s New World Dictionary defi nes a drug as “ any substances used as a medicine
or as an ingredient in a medicine. ” Indeed, peptides and recombinant proteins
are highly potent, relatively macromolecular and promising therapeutic agents that
emerged out from the signifi cant development of biotechnic and biogenetic engineering
technologies. Peptide and protein therapeutics include semisynthetic vaccines,
monoclonal antibodies, growth factors, cytokines, soluble receptors, hormones, and
enzymes. The advent of recombinant DNA technology allowed the possibility of the
commercial production of proteins for pharmaceutical applications from the early
1980s and, in fact, manufacture of therapeutic proteins represented the fi rst true
industrial application of this technology [1] . During the 1980s the term biopharmaceutical
became synonymous with therapeutic protein produced by recombinant DNA
technology (or, in the case of a small number of therapeutic monoclonal antibodies,
by hybridoma technology ). Clinical evaluation of nucleic acid – based drugs used for
the purposes of gene therapy and antisense technology commenced in the 1990s, and
today the term biopharmaceutical also encompasses such (as - yet - experimental)
drugs [2] . The fi rst such recombinant therapeutic protein (insulin) was approved for
general medical use only 24 years ago. Today there are in excess of 100 such products
approved in some world regions at least, with 88 having re ceived approval within
the European Union (EU). This represents 36% of all new drug approvals since the
introduction of the new centralized European drug approval system in 1995 [3] . Over
the coming decade, therefore, in the region of a dozen new therapeutic proteins
should, on average, gain regulatory approval each year. While EU fi gures are diffi cult
to locate, the American Association of Pharmaceutical Researchers and Manufacturers
(PhRMA) estimates that there are currently some 371 biotechnology medicines
in development [4] . Out of these 371 biotechnology medicines, as estimated by
PhRMA, more than 300 are protein based, with recombinant vaccines and monoclonal/
engineered antibodies representing the two most promising categories. Incidentally,
all 88 biopharmaceu tical products currently approved within the EU are
protein based. Of the proteins thus far approved, hormones and cytokines represent
the largest product categories (23 and 18 products, respectively). Hormones approved
include several recombinant insulins, displaying both native and modifi ed amino acid
sequences. In addition, several recombinant gonadotrophins [follicle - stimulating
hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotrophins
(hCG)] have been approved for the treatment of various forms of subfertility/infertility.
Cytokines approved include a range of recombinant hematopoietic factors,
including multiple erythropoietin - based products used for the treatment of anemia
as well as a colony - stimulating factor aimed at treating neutropenia. Additional
approved cytokines include a range of interferon - based products, mainly used to
treat cancer and various viral infections, most notably hepatitis B and C, and a
recombinant tumor necrosis factor (TNF) used as an adjuvant therapy in the treatment
of some soft tissue cancers. Blood - related approved therapeutic proteins
include a range of recombinant blood coagulation factors used to treat hemophilia,
recombinant thrombolytics, and recombinant anticoagulants. Additional product
categories include a range of subunit vaccines containing at least one recombinant
component [mainly hepatitis B surface antigens (HBsAg)] and a variety of monoclonal
antibody – based products indicated for the treatment/detection of various
cancers or the prevention of organ transplant rejection. In summary, ailments that
can be treated more effectively by this new class of therapeutic agents include cancers,
autoimmune disease, memory impairment, mental disorders, hypertension, and
certain cardiovascular and metabolic diseases [5, 6] .
Poor absorption and easy degradation by endogenous proteolytic enzymes
present in eye tissues, nasal mucosa, and gastro intestinal tract and low transdermal
bioavailabilities due to relatively large size make the peptide/protein molecules to
be administered only through parenteral routes either by multiple injections or
infusion therapy in order to achieve desired therapeutic plasma levels for prolonged
periods of time. Nevertheless, because of remarkably short half - lives within the in
vivo arena, the therapeutic usuage of most of the peptide/protein is practically possible
only through daily multiple injections under close medical supervision. Hence,
the commercial success of peptides/proteins as therapeutic agents depends mainly
on development of novel drug delivery systems which could potentially reduce
the injection frequencies and thus eliminate the accompanying serious problem of
patient compliance.
Among the several technologies that have been suggested for reducing injection
frequencies of therapeutic peptide/protein, microspheres prepared from biodegradable
polymers are widely recognized for controlled drug delivery following parenteral
administration. Polyester polymers such as poly(lactic acid) (PLA), poly(glycolic
acid) (PGA), and their copolymer poly(lactic acid - co - glycolic acid) (PLGA) are
used routinely for the preparation of injectable microspheres after taking into consideration
their well - known biocompatibility, controlled biodegradability, absorbability,
and no toxicity of degradation products [7] . Furthermore, the PLGA types
and related poly(hydroxyalkanonates) have a long history of medical and pharmaceutical
use in fi elds as diverse as sutures, bone fi xatives, artifi cial skins and cartilages,
dental materials, materials for bone generation, drug delivery, and many
others, as reviewed by Ueda and Tabata [8] . In conjunction with a long safety record
of PLGA polymers, at least 12 different peptide/protein - loaded PLGA microsphere
products are available in the market from nine different companies worldwide
for the treatment of some life - threatening diseases (Table 1 ). In recent years,
poly(. - caprolactone) (PCL) has been investigated as an alternative to PLGA to
make microspheres [9, 10] . A glimpse of ongoing research activities utilizing biodegradable
polymer - based microspheres for various peptide/protein is shown in
INTRODUCTION 395
396 BIODEGRADABLE POLYMER-BASED MICROSPHERES
Table 2 (incorporating refs. 12 – 35). However, overcoming the propensity for peptides/
proteins to undergo degradation processes during incorporation into the biodegradable
microspheres or after injection into the body awaiting release is one of
the key hurdles in bringing microencapsulated systems for these drugs to market.
This partially explains the limited and only a countable number of formulations
available on the market. Furthermore, irrespective of the various microencapsulation
techniques adopted to prepare peptide/protein - loaded microspheres, several
transfer - required processes such as fi ltration, centrifugation, and vacuum or freeze
drying are necessary to obtain a fi nal product, and these processes might be obstacles
when scaling up the manufacturing technique to produce suffi cient quantities
of sterile material for clinical trial and, ultimately, commercialization [11] .
This chapter encompasses investigations made progressively on the design of
injectable peptide/protein - loaded PLGA microspheres. It covers an update on the
state of art of the manufacturing of peptide/protein - loaded microspheres through
both conventional and newer microencapsulation techniques, different analytical
methods used for microsphere characterization, immune system interaction with
microspheres following parenteral administration, and potential application of
microspheres having therapeutic peptides/proteins. Special emphasis is given particularly
on various instability problems and investigated mechanistic ways to
obviate the possible instability problems of peptide/protein drug during microsphere
preparation as well as its release from the microspheres. It should be added
that although the chapter focuses mainly on PLGA microspheres, many of the
destabilization mechanisms and stabilization approaches described herein can be
valid to some extent for other polymeric delivery systems, too.
TABLE 1 Currently Marketed Preparations (Injectable Microspheres) Containing
Peptide/Protein Molecules
Commercial
Name API Polymer Company Indication
Lupron Depot Leuprolide PLGA or PLA TAP Prostate cancer,
endometriosis
Enantone Depot Leuprolide PLGA or PLA Takeda Prostate cancer,
endometriosis
Trenantone Leuprolide PLGA or PLA Takeda Prostate cancer,
endometriosis
Enantone Gyn Leuprolide PLGA or PLA Takeda Prostate cancer,
endometriosis
Sandostatin LAR Octreotide PLGA - glucose Novartis Acromegaly
Nutropin Somatropin PLGA Genentech Growth
defi ciencies
Trelstar Depot Triptorelin PLGA Pfi zer Prostate cancer
Decapeptyl SR Triptorelin PLGA or PLA Ipsen - Beaufour Prostate cancer
Decapeptyl Triptorelin PLGA Ferring Prostate cancer
Suprecur MP Buserelin PLGA Aventis Endometriosis
Somatuline LA Lanreotide PLGA Ipsen - Beaufour Acromegaly
Parlodel LAR Bromocriptine PLGA - Glu Novartis Parkinsonism
Abbreviations: PLA: polylactide; PLGA: poly(lactide - co - glycolide); API: active pharmaceutical ingredient;
PLGA - Glu: poly( d , l - lactide - co - glycolide - d - glucose).
TABLE 2 Injectable Peptide/Proteins/Vaccines Encapsulated in Biodegradable
Microspheres
Peptides, Protein, Vaccine Technique Polymer Reference
Vaccine
SPf 66 malaria vaccine Double emulsion PLGA 28
Multivalent vaccines of
Haemophilus infl uenzae
type b (Hib), diphtheria
toxoid (DT), tetanus
toxoid (TT), pertussis
toxin (PT)
Spray drying PLGA 29
Rotavirus Double emulsion PLG 30
Polypeptides and Proteins
Insulin Double emulsion PLA polyethylene
glycol (PEG)
12
Recombinant human
epidermal growth factor
(rhEGF)
Double emulsion PLA 13
Ribozyme Double emulsion PLA, PLGA 14
Vapreotide (somatostatin
analogue)
Spray drying PLGA 15
Insulinlike growth factor - 1
(IGF - 1)
Double emulsion PLGA - PEG 16
Ornitide acetate
leuteinizing hormone
releasing hormone
[(LHRH) antagonist]
Dispersion/solvent
extraction/evaporation
PLA, PLGA 17
Vascular endothelial
growth factor (VEGF)
Single emulsion PLGA/PEG 18
Human chorionic
gonadotropin (hCG)
Double emulsion PLA, PLGA 19
Calcitonin Double emulsion PLGA 20
FITC - bovine serum
albumin (BSA)
Double emulsion Poly( . - caprolactone) 9
Levonorgestrel and
ethinylestradiol
Double emulsion Poly( . - caprolactone) 10
Recombinant human bone
morphogenetic protein
Double emulsion PLGA 21 – 24
Transforming growth factor
beta
Double emulsion PLGA or
PLGA - PEG
25 – 27
Recombinant human
erythropoietin (rhEPO)
Modifi ed double
emulsion
LPLG - PEO - LPLG 31
Protein - C Double emulsion PLA 32
Ovalbumin Double emulsion PLGA 33
Human serum albumin Double emulsion PLA 34
Bovine serum albumin Nonaqueous oil - in - oil
(o/o) emulsion
PLG 35
Abbreviations: FITC: fl uroscein isothiocyanate; PLG: poly(lactide - co - glycolide); LPLG - PEO - LPLG:
copoly(l - lactic - co - glycolic acid - b - oxyethylene - b - l - lactic - co - glycolic adic); PEO: polyethylenenoxide.
INTRODUCTION 397
398 BIODEGRADABLE POLYMER-BASED MICROSPHERES
5.2.2 PEPTIDE/PROTEIN - LOADED MICROSPHERE
PRODUCTION METHODS
The development of delivery systems for therapeutic peptides/proteins depends on
biophysical, biochemical, and physiological characteristics of these molecules, including
molecular size, biological half - life, immunogenicity, conformational stability,
dose requirement, site and rate of administration, pharmacokinetics, and pharmacodynamics
[36] . Unlike conventional drug molecules, the unique conformational
structure of peptidic/proteinic therapeutic agents poses a great challenge right from
the beginning of the selection of suitable microencapsulation techniques to make
microspheres. Table 3 lists the considerations to be taken before choosing a particular
encapsulation technique. Apart from the traditional phase separation technique,
other techniques suitable for peptide/protein - loaded microsphere production can
be divided into two main categories: during microsphere preparation, those involved
in utilizing a hydrous environment such as emulsion - based methods and those based
on an anhydrous environment such as spray freeze drying, spray drying, freeze
drying, grinding, jet milling, liquid - phase antisolvent precipitation, and supercritical
CO2 - based methods [37 – 40] . In the following sections, the various production techniques
to make injectable peptide/protein - loaded microspheres are briefl y introduced;
however, a detailed discussion is beyond the scope of this chapter.
5.2.2.1 Phase Separation (A Traditional Technique)
Polymer phase separation or coacervation is an excellent technique for the encapsulation
of water - soluble drugs including peptide/protein into a fi nal microsphere
product [41] . The peptide/protein molecule is dispersed in solid form into solution
containing dichloromethane and PLGA. Silicone oil is added to this dispersion at
a defi ned rate, reducing the solubility of polymer in its solvent. The polymer - rich
liquid phase (coacervate) encapsulates the dispersed peptide/protein molecules and
embryonic microspheres are subjected to hardening and washing using heptane.
The process is quite sensitive to polymer properties, and residual solvent is also an
important issue. Decapeptyl [triptorelin, a luteinizing hormone releasing hormone
(LHRH) analogue] [42] and Somatuline LA (lanreotide, a somatostatin analogue)
[43] are microsphere commercial products developed by this technique (Table 1 ).
TABLE 3 Factors in Selection of Microencapsulation
Method to Prepare Peptide/Protein-Loaded Microspheres
Optimal peptide loading
High microsphere yield
Batch content uniformity
Interbatch reproducibility
Peptide stability during preparation and release
Size uniformity
Adjustable release profi le
Low burst release
Flowability of fi nal product
Residual solvent and polymer monomer control
Sterilization (both aseptic and terminal)
5.2.2.2 Double Emulsion (A Hydrous Technique)
Oil - in - water (o/w) and water - in - oil - water (w/o/w) are the two hydrous techniques
representing respectively the single - and double - emulsion formation during microsphere
preparation. However, the w/o/w technique is most commonly employed
[44] . In this process, peptides/proteins in aqueous solution are emulsifi ed with
nonmiscible organic solution of polymer to form a w/o emulsion. Dichloromethane
serves as organic solvent and the o/w primary emulsion is formed using either high -
speed homogenization or ultrasonication. This primary emulsion is then rapidly
transferred to an excess of aqueous medium containing a stabilizer, usually polyvinyl
alcohol. Again homogenization or intensive stirring is necessary to initially form a
double emulsion of w/o/w. Subsequent removal (by evaporation) of organic solvent
by heat, vacuum, or both results in phase separation of polymer and core to produce
microspheres. Instead of solvent evaporation, solvent extraction with a large quantity
of water with or without a stabilizer can also be undertaken to yield mi crospheres
containing peptide/protein. Although the w/o/w microencapsulation technique
seems to be conceptually simple to carry out, the particle formation process is quite
complicated, and a host of process parameters infl uence the properties of peptide/
protein - loaded PLGA microspheres [45] . In spite of that, different peptides and
proteins such as bovine serum albumin (BSA) or ovalbumin (OVA), insulin, recombinant
human insulinlike growth factor - 1 (rhIGF - 1), recombinant human epidermal
growth factor (rhEGF), human chorionic gonadotropin (hCG), protein C, recombinant
human bone morphogenetic protein (rhBMP), and calcitonin, along with antigens
and other therapeutically relevant proteins such as recombinant human
erythropoietin (rhEPO), have been successfully encapsulated (see Table 2 ) by the
w/o/w double - emulsion technique. Lupron Depot/Enantone Depot/Trenantone/
Enantone Gyn (all having leuprolide acetate, a LHRH analogue) are very popular
commercial microsphere products produced by this technique [46, 47] , available
both in the EU and United States, for the treatment of either prostate cancer of
man or infertility (endometriosis) of women (Table 1 ).
5.2.2.3 Spray Drying (An Anhydrous Technique)
Spray drying is a rapid, convenient technique which can be conducted under aseptic
conditions. First, a polymer — prevalently PLGA is applied — is dissolved in a volatile
organic solvent such as dichloromethane or acetone. The protein is suspended as a
solid or emulsifi ed as aqueous solution in this organic solution by homogenization.
After that, the resulting dispersion is atomized through a (heated) nozzle into a
heated airfl ow. The organic solvent evaporates, thereby forming microspheres with
dimensions of typically 1 – 100 . m. The microspheres are collected in a cyclone separator.
For the complete removal of the organic solvent, a vacuum drying or lyophilization
step can follow downstream.
The internal structure of the resulting polymeric microspheres depends on the
solubility of the peptide/protein in the polymer before being spray dried leading to
the formation of reservoir - or matrix - type products (see Figure 1 ). When the initial
dispersion is solution, the fi nal product obtained following spray drying is matrix or
monolithic type, that is, polymer particles with a dissolved or dispersed nature
of the active ingredient (defi ned as microspheres). Conversely, when the initial
PEPTIDE/PROTEIN-LOADED MICROSPHERE PRODUCTION METHODS 399
400 BIODEGRADABLE POLYMER-BASED MICROSPHERES
dispersion is in suspension, the product obtained is reservoir type, that is, a distinct
polymeric envelope/shell encirculating a liquid core of dissolved active ingredient
(defi ned as microcapsules). Recombinant human erythropoietin [48] and bromocriptine
mesylate, Parlodel Depot [49] , are examples of microspheres (matrix type)
obtained by the spray drying technique.
5.2.2.4 New Trends in Production Methods
Several issues such as reducing cost, reducing scale - up diffi culties, improving protein
stability, allowing for terminal sterilization, and eliminating the need for organic
solvents during addition of the peptide/protein motivate the development of new
methods to manufacture microspheres. Moreover conventional microencapsulation
methods involve relatively harsh conditions that are not generally tolerated by
peptide/protein molecules without stabilization. Therefore, new and improved processes
shielding the peptide/protein from deleterious conditions have been proposed
and evaluated.
Modifi ed Conventional Methods The w/o/w solvent evaporation or extraction is
probably one of the most widely used methods for peptide and protein microencapsulation
[44] , despite its many drawbacks. Improvements and alternatives have
therefore been proposed such as oil in water (o/w), * o/w (the asterisk including
cosolvent) and oil in oil (o/o) [50] .
Utilising a modifi ed w/o/w method, the rhIGF - 1 was encapsulated into PLGA
microspheres after increasing the pH of the protein solution from 4.5 to 5.5 – 6.0,
where rhIGF - 1 formed a viscous gel [51] . High entrapment effi ciency of fully bioactive
protein was achieved, and 92 – 100% of pure, monomeric, and bioactive rhIGF - 1
was released in vitro over 21 days. The lowering of the rhIGF - 1 solubility at pH
5.5 – 6.0 probably restricted its conformational fl exibility and changes upon exposure
to the polymer solvent. Without pH adjustment, approximately 10 – 32% of rhIGF - 1
was lost upon solvent exposure, due to degradation and aggregation. Elsewhere, a
w/o 1 /o 2 system was investigated for encapsulating different proteins and peptides,
with the o 1 and o 2 phases consisting of acetonitrile/dichloromethane and liquid
paraffi n/Span 80, respectively [52] . The acetonitrile mediated the partial mixing of
the w and o 1 phases and subsequent protein/peptide precipitation, which was a
FIGURE 1 Polymeric delivery systems: (a) reservoir systems (microcapsules); (b) matrix
systems (microspheres).
Drug
Drug
(b) (a)
Polymer
Polymer
prerequisite for microencapsulation. The proteins BSA, tetanus toxoid (TT), and
lysozyme did precipitate at low acetonitrile concentration, resulting in effi cient
microencapsulation (more than 90%), while a decapeptide and a linear gelatine did
not precipitate so rapidly, resulting in poor entrapment. TT and lysozyme released
during the burst phase (15%) maintained their bioactivity, although lack of further
release suggested aggregation within the microspheres.
Another approach consisted of dispersing the protein antigen in a mineral oil
before encapsulation into PLGA microspheres by a o 1 /o 2 /w method [53] . The mineral
oil (o 1 ) was intended as a barrier to protect the antigen during emulsifi cation with
the polymer solution and from exposure to moisture during release. Over 92%
of enzyme - linked imunosorbert assay (ELISA) reactive TT was released from the
reservoir - type microspheres in a pulsatile pattern, proceeding with an initial burst
and followed by a second release pulse between 14 and 35 or 35 and 63 days, depending
on the polymer type used. The latter stage of release was ascribed to TT diffusion
through the oily phase, once an appreciable loss of polymer mass had occurred. The
authors claimed the mineral oil was the key to protecting the solid antigen during
polymer erosion, where acidic degradants and moisture would otherwise have led
to antigen inactivation.
To improve solvent extraction, a novel method using a static micromixer was
presented where a w 1 /o dispersion (aqueous BSA in organic PLGA solution) is fed
into an array of microchannels and the extraction fl uid (w 2 ) into a second array of
interdigitated channels [54] . The two fl uids, transported separately through the channels,
are discharged through an outlet slit where alternating fl uid lamellae are
formed with the w 1 /o fl uid lamella disintegrating into microdroplets, which harden
quickly to form microspheres. This process offers easy scale - up, methodological
robustness, continuous production, and a simple setup, making it ideally suited
for aseptic production, a strongly needed feature for microsphere vaccine
formulations.
ProLease Technology (Cryogenic Spray Drying) A variation of the conventional
spray drying method is a cryogenic method which will described below. A novel
low - temperature spraying technique (called ProLease technology) for preparing
PLA and PLGA microspheres has been reported by Khan et al. [55] and the group
at Alkermes [56, 57] . The method relies on the use of stabilizing and release controlling
agents, low processing temperature, and nonaqueous microencapsulation. Typically,
a protein powder is micronized, possibly with a stabilizer, by spray freeze
drying and then suspended in an organic polymer solution. The suspension is atomized
into a vessel containing liquid N 2 underlaid by frozen ethanol (extraction
solvent). The atomized droplets freeze in the liquid N 2 and deposit on the surface
of the frozen ethanol. As liquid N 2 evaporates, the frozen ethanol liquefi es ( Tm
approximately . 110 ° C) so that the frozen polymeric droplets will transfer into the
ethanol where the polymer solvent is extracted, yielding solid microspheres [58, 59] .
To date, the ProLease system has been effectively applied to the encapsulation of
zinc - complexed human growth hormone in PLGA microspheres, resulting in a one -
month effect after one single injection [37, 57, 60] . As a reference, the recombinant
human growth hormone (rhGH) was unstable in contact with ethyl acetate or
dichloromethane [61] . The only protein - containing PLGA microspheres, Nutropin
Depot, is produced by this novel technique. However, this product containing rhGH
PEPTIDE/PROTEIN-LOADED MICROSPHERE PRODUCTION METHODS 401
402 BIODEGRADABLE POLYMER-BASED MICROSPHERES
marketed initially in the United States in 1999 was pulled from the market voluntarily
by the manufacturer in June 2004 because of high costs of production and
commercialization ( http://www.gene.com/gene/news/press-releases/ , accessed May
25, 2006).
ProLease technology was also used for encapsulating recombinant human vascular
endothelial growth factor (rhVEGF) and rhIGF - 1 [62, 63] . Both proteins were
stabilized in aqueous solution, prior to spray freeze drying, and encapsulated (9 –
20% w/w) into PLGA microspheres. The microspheres also contained ZnCO 3 (3 –
6% w/w) as release modifi er. The resistance of rhIGF - 1 to aggregation and oxidation,
determined from in vitro release studies, hardly changed. Protein, released in
an almost pulsatile fashion over 21 days, was composed of predominantly monomeric
rhIGF - 1 with only minor amounts ( . 6%) of degradants forming toward day 21.
Similarly, the integrity of rhVEGF dimer released over 21 days was good and its
bioactivity remained largely unaffected, regardless of the extent of aggregation and
degradation. In view of these studies, ProLease technology appears to have potential
for sustaining antigen stability and release from microspheres.
Techniques Using Supercritical Fluids Generally, the application of supercritical
(SC) fl uids for the encapsulation of peptides and proteins has been fueled by the
recognition that the established methods implicate some drawbacks. The application
of supercritical fl uids, especially of supercritical carbon dioxide (CO 2 ), can minimize
or even eliminate the use of organic solvents and renders work at moderate temperatures
possible [64] . The term supercritical defi nes the area above the critical
point, which specifi es the fi nal point of the liquid – gas phase transition curve. Beyond
that critical point, isobar/isotherm alterations of pressure or temperature alter the
density of the critical phase but do not lead to a separation into two phases. A
density change is directly associated with a change of the solvent power, and thus
the method features a high variability. Usually CO 2 is used as supercritical fl uid due
to its critical point ( Tc = 31.1 ° C, Pc = 73.8 bars), which can be easily reached. That
allows a moderate working temperature and leaves no toxic residues since it returns
to the gas phase at ambient conditions. Two SC CO 2 - based processes have been
reported for the preparation of drug - loaded polymeric microspheres: fi rst, the rapid
expansion from supercritical solutions (RESS) process, whereby a SC CO 2 solution
of an active agent and a polymeric carrier is rapidly expanded. This quickly transforms
the SC CO 2 into a liquid that is a much poorer solvent, thereby precipitating
the active agent – carrier mixture as small particles [65] . Second is the aerosol solvent
extraction system (ASES), also referred as the gas antisolvent spray precipitation
(GAS) process [66] . Here, a solution of the active agent and the polymeric carrier
is sprayed into a chamber loaded with SC CO 2 . The SC CO 2 extracts the solvent
from the spray droplets and induces coprecipitation of the active agent and the
polymeric carrier in the form of small, solvent - free particles [67, 68] . However, the
use of organic solvents cannot be avoided, which is to be deemed a major disadvantage
of both techniques.
In peptide/protein pharmaceuticals, the GAS process is predominantly applied
for the preparation of microparticulate protein powders as an alternative to common
drying processes. However, Winters et al. [69] reported an increase of . - sheet aggregates
during the precipitation of lysozyme, trypsin, and insulin as a consequence of
stress parameters such as organic solvent, pressure, and shear forces. One reason
why these methods were not credited as encapsulation techniques for protein within
PLGA may be the tendency of several polymers to rapidly precipitate and agglomerate
during the process [70] .
ASES has been compared with conventional spray drying in terms of effects on
the stability of the peptide tetracosactide [71] . Almost no intact peptide was recovered
from spray - dried PLA particles, whereas the tetracosactide was well protected
against oxidation during ASES ( . 94% unmodifi ed peptide). In general, the particle
formation step seems to be less detrimental to proteins than the loading step. For
example, emulsifi cation in an aqueous phase or spray drying of rhEPO/PLGA
emulsions was mild compared to the fi rst emulsifi cation step [72] . Also, variation of
the particle formation step (spray drying or coacervation) had a minor impact on
diphtheria toxoid (DTd) antigenicity when compared to other process variables
[73] .
A serious limitation of GAS, ASES, and RESS for producing microspheres is the
need of polymer types that form discrete crystalline domains upon solidifi cation,
such as l - PLA [74, 75] . The advantages of these methods offer (e.g., over spray
drying) are the low critical temperatures for processing (34 ° C) and the avoidance
of oxygen exposure during atomization, with both parameters being potentially
important to peptide/protein stability.
Ultrasonic Atomization Ultrasonic atomization of w/o dispersions is presently
under investigation for preparing especially protein antigen containing microspheres.
In one setup, the atomized antigen/polymer dispersion was sprayed into a
nonsolvent where the polymer solvent was extracted, resulting in microspheres [76] .
A comparable technique was proposed where the antigen or polymer dispersion
was atomized into a reduced pressure atmosphere and the preformed microspheres
hardened in a collection liquid [77] . Similarly, PLGA solutions were also atomized
by acoustical excitation and the atomized droplets transported by an annular stream
of a nonsolvent phase [aqueous polyvinyl alcohol (PVA)] into a vessel containing
aqueous PVA [78] . Solvent evaporation and microsphere hardening occurred in the
vessel over several hours. The main advantages of these atomization techniques
encompass the possibility of easy particle size control and scale - up, processing at
ambient or reduced temperature, and the suitability for aseptic manufacturing in
a small containment chamber such as an isolator.
In Situ Formed Injectable Microspheres All the encapsulation techniques discussed
so far rely on the preparation of solid microspheres. However, a method for
preparing a stable dispersion of protein containing semisolid PLGA microglobules
has been reported [79] . Here, a protein dissolved in PEG 400 was added to a solution
of PLGA in triacetin or triethyl citrate. This mixture, stabilized by Tween 80,
was added dropwise and under stirring to a solution of Miglyol 812 or soybean oil,
containing Span 80, resulting in a stable dispersion of protein inside semisolid
PLGA microglobules. The microglobules remained in an embryonic state until
mixed with an aqueous medium, so that the water - miscible components were
extracted and protein containing matrix - type microspheres formed. Myoglobin was
encapsulated and found to remain physically unchanged (circular dichroism
analysis) after the process and during storage of the microglobular dispersion (15
days, 4 ° C).
PEPTIDE/PROTEIN-LOADED MICROSPHERE PRODUCTION METHODS 403
404 BIODEGRADABLE POLYMER-BASED MICROSPHERES
Preformed Porous Microspheres A new approach for attaining sustained release
of protein is introduced involving a pore - closing process of preformed porous
PLGA microspheres [80] . Highly porous biodegradable PLGA microspheres were
fabricated by a single w/o emulsion solvent evaporation technique using Pluronic
F127 as an extractable porogen. The rhGH was incorporated into porous microspheres
by a simple solution dipping method. For its controlled release, porous
microspheres containing rhGH were treated with water - miscible solvents in the
aqueous phase for production of pore - closed microspheres. These microspheres
showed sustained - release patterns over an extended period; however, the drug
loading effi ciency was extremely low. To overcome the drug loading problem, the
pore - closing process was performed in an ethanol vapor phase using a fl uidized - bed
reactor. The resultant pore - closed microspheres exhibited high protein loading
amount as well as sustained rhGH release profi les. Also, the released rhGH exhibited
structural integrity after the treatment.
Charged (Anionic and Cationic) PLGA Microspheres PLGA or any other type
of microspheres can be readily decorated with positive or negative surface charges
by simply preparing the particles by a w/o/w solvent evaporation/extraction process
where the second water phase contains a cationic emulsion stabilizer [hexadecyltrimethylammonium
bromide; poly(ethyleneimine); stearylamine] or an anionic
emulsifi er [sodium dioctyl - sulfosuccintate; sodium dodecyl sulfate (SDS)]. Such
compounds attach tightly to PLGA surfaces during preparation and provide the
necessary surface charge for ionic adsorption of counterions. It is known that a
protein ’ s surface charge depends on its isoelectric point (pI) and the pH of the
medium in which it is dispersed. The use of particles with ionic surface charge offers
several advantages over classical microencapsulation, among which the mild conditions
for loading are probably the most attractive. PLGA microspheres with surface -
adsorbed protein antigens and DNA have been highly effi cient in inducing strong
immune responses, as reviewed by Singh et al. [81] and Jilek et al. [82] . Nonetheless,
it remains to be shown whether such particles are also suitable for eliciting long -
term immunity after one or two injections.
Jabbal - Gill et al. [83, 84] noted the tendency for microencapsulated protein antigens
to distribute heavily at the surface of PLGA microspheres and developed
polymeric lamellar substrate particles (PLSP) by precipitating a highly crystalline
poly(l - lactic acid)/organic solvent solution with water, followed by removal of
remaining organic solvent with nitrogen purge. The particles, which can be sterilized
by gamma irradiation and stored as a suspension for several months without changes
to antigen absorption [84] , possessed a large lamellar surface area and highly negative
zeta potential ( .. 35 to . 42 mV) and could adsorb signifi cant amounts of antigen
(up to 50 . g/mg microspheres) depending on pH, ionic strength, antigen – polymer
ratio, and other factors. Release of protein antigen (TT) could be extended to over
1 month with minimal antigenic losses in released antigen, although most of the
antigen was lost to the initial burst or to apparent irreversible adsorption (as indicated
by the absence of reaching 100% release). Elevated antibody responses in
mice were elicited using PLSP similar to one dose of aluminum adjuvant following
subcutaneous administration of OVA at elevated doses (100 or 300 . g). Both immunoglobulin
IgG1 and IgG2a antibody subtypes were of similar magnitude over 28
days in the PLSP/OVA groups, and cellular immunity was also observed following
immunization with a 38 - kDa protein antigen against tuberculosis [85] . Similarly,
Kazzaz et al. [86] created anionic PLGA microparticles by substituting the standard
nonionic emulsifi er PVA with anionic SDS during microsphere preparation. In addition
to eliciting elevated antibody responses in mice relative to the soluble antigen,
the adsorbed antigen elicited a potent cytotoxic T - cell (CTL) response, similar to
that observed after infection from virus expressing the p55 gag and polymerase
proteins. Moreover, the CTLs were formed from the more challenging intramuscular
route but not signifi cantly by the soluble antigen, even at elevated doses. The
SDS - PLGA particles could also be gamma irradiated before adsorption and were
shown to effectively boost antigen in nonhuman primates [87] .
5.2.3 ANALYTICAL CHARACTERIZATION OF PEPTIDE/
PROTEIN - LOADED MICROSPHERES
An area requiring additional efforts is analytical characterization of peptides and
proteins encapsulated in PLGA microspheres. The high complexity of the therapeutic
peptides and proteins requires not only physicochemical methodologies but
also immunochemical and biological techniques for the characterization and quality
control of these substances. In general, the analytical methods can be broadly
viewed from the following study perspectives: methods meant for microsphere
product quality checking, methods used for peptide/protein stability identifi cation
inside the microspheres, and methods called for peptide integrity detection following
liberation from the microspheres immediately upon placement in release
medium either in vitro or in vivo. Therefore, in most cases, a combination of several
analytical methods is necessary for a comprehensive characterization of the
peptide/protein under investigation and for appropriate quality control of the
product concerning identity, purity, and potency. However, some of the analytical
methods have potentially appealing applications to interplay among the mentioned
perspectives. In Table 4 , a selection of widely used analytical methods is given,
showing which technology is applicable for the testing of identity, purity, and
potency of peptides and proteins. In addition, peptide/protein integrity evaluation
is indeed likely to be affected by artefacts during the sample preparation before
analysis and during the analysis itself. Therefore, artefacts might prevent the scientist
from critically ascribing detected protein denaturation to manufacturing
conditions [88] .
In order to measure the extent of peptide/protein degradation within the carriers
and during release, the encapsulated molecule has to be removed from the polymeric
matrix. Moreover, for avoiding artefacts such as underestimation of drug
content, recovery methods need to be tried by an empirical trial - and - error approach
as each peptide/protein is different one from the other. Recovery methods so far
reported include extraction - based method with the help of potentially deleterious
organic solvents, hydrolysis of the polymer matrix with alkaline medium, dissolution
of polymer matrix in an organic solvent, recovery of suspended insoluble protein
by fi ltration [89] , total protein quantifi cation after complete digestion of carriers
followed by amino acid analysis [90, 91] , electrophoretic extraction of the protein
using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS – PAGE) [92,
93] , and direct dissolution of both the polymer and the protein drug in a single liquid
ANALYTICAL CHARACTERIZATION 405
406 BIODEGRADABLE POLYMER-BASED MICROSPHERES
TABLE 4 Analytical Methods for Characterization and Quality Control of
Pharmaceutical Peptides and Proteins
Methods
Indicated Usage/Checking
Identity Purity Potency
Physicochemical
Chromatography
Reversed - phase high - performance liquid chromatography
(HPLC, RP - 1)
+ + .
Ion exchange . + .
Affi nity . + .
Size exclusion chromatography (SEC) . + .
Spectroscopy
Infrared spectroscopy + + .
Raman spectroscopy + + .
Fluorescence spectroscopy . + .
Ultraviolet/visible (UV/VIS) spectroscopy + + .
NMR spectroscopy + + .
Mass spectrometry + + .
Circular dichroism (CD) + + .
Matrix - assisted light desorption ionization – time - of - fl ight
(MALDI - TOF) mass spectrometry
+ + .
Electrophoresis
Capillary electrophoresis . + .
SDS – polyacrylamide gel electrophoresis (PAGE) + + .
Isoelectric focusing . + .
Immunochemical
Radioimmunoassay (RIA) . . +
ELISA . . +
Western blot + . .
Biological
In vivo assays . . +
In vitro (cell culture) assays . . +
Abbreviation: SDS: sodium do decyl sulfate.
phase containing water - miscible organic solvents such as acetonitrile or dimethylsulfoxide
(DMSO) [94, 95] .
Following successful recovery of peptide/protein molecule from the microspheres,
a simple spectrophotometric method does not always allow discrimination between
the monomeric protein form and its aggregates. However, HPLC might separate
these species and thus provides more accurate qualitative data [96] . But HPLC
cannot quantify exclusively the amount of active protein antigen, as is the case with
ELISA techniques [97] . Nowadays, Fourier transform infrared (FTIR) spectroscopy
has become a popular, noninvasive method, as it is able to characterize the secondary
structure of entrapped proteins [26, 95, 98 – 101] . Only recently, the integrity
of their primary structure was evaluated, thanks to a new matrix - assisted laser
desorption ionization – time - of - fl ight (MALDI - TOF) mass spectrometry method [94,
102] . The method was shown to require little sample material and only a simple
dissolution of the carrier was needed prior to the analysis. The MALDI - TOF allowed
elucidation of a new degradation pathway, that is, peptide acylation within PLGA
carriers resulting from a chemical interaction between peptide and degraded
polymer [102] . Moreover, the method was also useful for quantifi cation, and it
should be underlined that no interference from PLGA was detected during the
measurements. For all the reasons cited above, mass spectrometry should be considered
one of the most promising methods for protein analysis inside polymeric
carriers including microspheres. Using erythropoietin as an example, an exploratory
and elaborative discussion was made on the analytical techniques used for the
characterization and quality control of pharmaceutical peptides and proteins [103] .
A similar discussion was also done on the analytical techniques critical to (as a part
of) the quality assurance after process changes of the production of a therapeutic
antibody [104] .
With an increasing level of sophistication in the design of new protein antigens
and adjuvants (including polymer controlled - release systems), efforts both in the
United States and the EU are underway to respond with more appropriate regulations
[105 – 107] . For example, the Committee for Proprietary Medicinal Products
(CPMP), the primary scientifi c body in EU regulatory matters, is currently updating
its “ notes for guidance, ” which guide/direct industry and regulatory authorities on
content and evaluation of marketing authorization applications for vaccines [105] .
Early drafts of these updates include more rigorous guidelines for new non -
aluminum - based adjuvants, including antigen stability requirements (see Sesardic
and Dobbelaer [105] for a discussion). Similar discussions ongoing in the United
States have attempted to standardize requirements of controlled - release parenterals
[106, 107] , including specifi cs regarding in vitro release assays and the need to
account for 80% or more of the encapsulating agent during the release period.
5.2.4 IMMUNE SYSTEM INTERACTION WITH
INJECTABLE MICROSPHERES
Since microspheres are capable of forming a drug depot, the encapsulated peptide
or protein is being slowly released over days or months at the injection site. Interestingly,
the size of microspheres plays an important role in immune response.
Microspheres with sizes smaller than 10 . m can be directly taken up macrophages
(and dendritic cells) through a phagocytosis mechanism while sizes greater than
10 . m need to undergo biodegradation before phagocytosis can occur [108] . It was
shown that within a few days of intramuscular injection PLGA microspheres less
than 10 . m are completely engulfed in a thin layer of connective tissue and thus
evidenced infi ltration by macrophages as a consequence of wound - healing response
to injected particles [109] . It is feasible that the infl ux of these macrophages may
cause degradation of the encapsulated protein and available protein released in the
vicinity of the microspheres. Furthermore, it has been suggested that these macrophages
are capable of producing proteolytic enzymes [110] , which may result in the
release and circulation of altered, inactive, or immunogenic forms of the encapsulated
peptide or protein.
IMMUNE SYSTEM INTERACTION WITH INJECTABLE MICROSPHERES 407
408 BIODEGRADABLE POLYMER-BASED MICROSPHERES
On the other hand, degradation, protein antigen release, location, and antigen
presentation of microspheres larger than 10 . m are expected to be different from
smaller ones. Larger microspheres can provide an extracellular depot for secondary
immune responses by way of B - cell stimulation [111 – 113] . In both cases, upon
administration of microspheres, a foreign - body response occurs resulting in an acute
initial infl ammation despite the excellent tissue compatibility and biodegradability
properties of polymers such as PLGA. This initial infl ammation is followed by the
infi ltration of small foreign - body giant cells and neutrophils [114] . These immune
cells could consume the released peptide or protein and produce an immune
response. However, if released protein is recognized as a self - protein (e.g., homologus),
the probability of an immune response by these cells is reduced. It is therefore
always essential to release the protein in its native conformation. The release of
aggregated or denatured protein from the microspheres may, in fact, result in an
unwanted immune response [115] . It should be added that systematic studies to
explore the effects of tissue response on the bioavailability of incorporated peptide
or protein drug have not appeared extensively in the literature, with a few exceptions
as described below. Using a light microscopic technique, bumps containing
residual amounts of microspheres were observed at the injection site two weeks
after administration of TT - encapsulated PLGA microspheres to mice and guinea
pigs [116] . These bump formations may be due to chronic reactions, long - term
immunogenicity, and immunological priming of mice and guinea pigs against the
injected polymeric microspheres. The immunogenicity of microsphere - encapsulated
vaccines can be varied to some extent by changing the physicochemical properties
of the microspheres, for example, size, surface properties, and release kinetics of
the antigen from the microspheres [111] . An interesting review by Jiang et al. [117]
details the various reports on the relationship between in vitro protein antigen stability
and immunogenicity, modulation of cell - mediated immune responses, and
different formulation approaches to achieve the appropriate immune response with
microencapsulated vaccine antigens. There has been some debate, arising from some
animal experiments, that the antigenicity does not directly correlate with immunogenicity.
However, the stability of protein antigens is considered to play a signifi cant
role in the quality and magnitude of immune response for the controlled - release
single - dose vaccines as degraded or nonantigenic proteins may not be able to
provide a continuous boost for generation of protective levels of high - affi nity
antibodies.
5.2.5 EXCIPIENT INCLUSION: INJECTABLE PEPTIDE/
PROTEIN - LOADED MICROSPHERES
Peptide and protein molecules are highly prone to degradation mechanisms that
can be divided into two classes: physical and chemical [118] . Whereas chemical
degradation leads to the loss of the protein ’ s primary structure through oxidation,
deamidation, peptide bond hydrolysis, isomerization, disulfi de exchange, and covalent
aggregation, physical degradation refers to the changes in the higher order
structure (secondary and above) mainly by noncovalent aggregation and precipitation.
In particular, aggregates formation during the encapsulation process must be
avoided because these aggregates always represent loss of therapeutic effi cacy and
increased immunogenicity which can endanger the patient ’ s health [119, 120] . The
following few examples indicate the fragility of peptide and protein molecules due
to physical or chemical degradation: Aggregation of insulin has been well characterized
and depends on unfolding of the insulin molecules [121] ; aggregation of lyophilized
formulations of BSA, . - galactoglobulin, and glucose oxidase are attributed to
disulfi de interchange [118] ; deamidation contributes to reduction in catalytic activity
of lysozyme [122] and ribonuclease at high temperatures [123] ; and peptide bond
hydrolysis results in loss of activity of lysozyme when heated to 90 – 100 ° C [122] . A
recent introduction to this list is formaldehyde - mediated aggregation pathway
(FMAP) unique to formaldehyde - treated protein antigens such as TT [117, 124,
125] .
The formulator of injectable microspheres for peptide and protein faces multiple
challenges: (i) to maximize physical and chemical stability, (ii) to prolong biological
half - life, (iii) to increase absorption, (iv) to decrease antigenicity, and (v) to minimize
metabolism. Thus, it is quite obvious that the fabrication of peptide - and
protein - loaded microspheres requires several kinds of excipients for effective stabilization
or immobilization of encapsulated therapeutic molecules. Excipients of
choice are included specifi cally for controlling protein degradation in microspheres
due to (a) external and internal environmental changes, (b) manipulating the initial
burst release, (c) preventing protein adsorption onto delivery devices, and (d) neutralizing
the causative acidic microclimate formation due to the acids liberated by
the biodegradable lactic/glycolic - based polymers. Therefore, it is generally best to
fi nd conditions to stabilize the protein before other aspects of the formulation, such
as controlled - release characteristics, are optimized. Typically, the appropriate excipients
for the protein under investigation are experimentally selected among various
substances by screening. This tedious experimental screening is partly necessary due
to the present inability to predict protein stability after addition of such excipients.
Moreover, since individual entrapped peptides and proteins differ in terms of physicochemical
properties and chemical/therapeutic function, each species is expected
to demonstrate a different degree of sensitivity to stress and react differently to the
same stabilization strategy. For example, a sugar, amino acid, or antacid excipient
may be required to stabilize protein, each of which can increase water uptake in the
polymer matrix leading to an increase in release rate. In the scenario in which controlled
- release conditions are optimized before such a stabilizer has been identifi ed,
it is likely that upon addition of the new stabilizer the release kinetics may change
enough to require reformulation. Certainly, there is a sharp contrast between encapsulating
a highly water soluble protein [126] or a poorly soluble zinc – protein complex
[37, 57] . Switching between these two cases would be expected to alter the requirements
in the formulation necessary to attain the controlled - release function (e.g.,
low versus high polymer matrix permeability for the protein, respectively) because
protein solubility in water may be important for any diffusion component of release.
The principal stresses causing instability of encapsulated peptide/proteins in PLGA
microspheres are elaborated in a book chapter [127] and in a jounal publication
[128] . This subject was again reexamined in a review based on new fi ndings since
the previous book chapter by the same author [129] . An interesting review from the
same research group was published on the biodegradable PLGA microparticles for
injectable delivery of vaccine antigens [117] , where they focused on mechanistic
approaches to improve the stability of PLGA - encapsulated protein antigens.
INJECTABLE PEPTIDE/PROTEIN-LOADED MICROSPHERES 409
410 BIODEGRADABLE POLYMER-BASED MICROSPHERES
Another review by Bilati et al. [130] also envisioned the strategic approaches for
overcoming peptide and protein instability within biodegradable nano - and microparticles.
The reader is also referred to related publications edited by Sanders and
Hendren [131] and Senior and Radomsky [132] for information on excipients used
in injectable peptide/protein - loaded formulations including microspheres. This
section will not cover all excipients used in parenteral protein formulations because
the aforementioned publications already do so. Rather this section highlights examples
of synergestic and antagonistic interactions that have been reported mainly
between the excipients and the peptide/protein drugs, especially before microsphere
preparation, followed by a brief discussion of a major instability problem of proteins/
peptides inside the microspheres. The published research paper is being organized
according to major functions of parenteral excipients, namely, solubilization,
stabilization, and preservation (see Figure 2 ) [133] .
5.2.5.1 Solubility - and Stability - Increasing Excipients
The traditional approach is to solubilize directly the peptide/protein in organic solvents.
This can be achieved by different means. Cleland and Jones [61] assumed that
native protein conformation could be maintained by precipitating the protein at its
pI. The molecule is then free of charge and can be readily solubilized in organic
solvents. Conversely, an alternative concept is based on the freeze drying of the
protein at a pH away from its pI value before formulating it. It was thought that
this strategy could increase protein solubility and stability in various polar and
water - miscible organic solvents such as DMSO [134, 135] . It should be noted that
a preformulation procedure consisting of using spray freeze drying with a suitable
excipient was able to stabilize BSA before encapsulation by a nonaqueous method
[35] . Using the dissolution approach, lysozyme was successfully formulated but
incomplete lysozyme release from microspheres was observed and ascribed to
aggregation [136] . Protein solubility can also be increased via an ion - pairing mechanism.
The protein is modifi ed by adding an oppositely charged surfactant that binds
to the protein, so as to obtain a neutral hydrophobic entity and thus reduce direct
contact between the protein and the organic solvent. Positively charged proteins
and negatively charged surfactants should be employed, since cationic surfactants
might have toxic side effects. This technique was shown to improve lysozyme conformational
stability after a hydrophobic complex between lysozyme and oleic acid
[137, 138] . A new interesting concept is to encapsulate an aggregated protein in a
reversibly dissociable form in order to avoid the formation of irreversible aggregates
during processing and to promote the sustained release of the native monomeric
form. Growth hormone was successfully formulated with this approach [139] .
Solubilization Preservation Stabilization
Excipients used
Moisture-induced instability prevention Microclimate pH-induced instability prevention
FIGURE 2 Flow chart of excipients used to prevent/minimize protein instability
problems.
Cyclodextrins (CD) have emerged as very effective additive compounds for
solubilizing hydrophobic drugs. In the parenteral dosage form area, modifi ed cyclodextrins
such as hydroxylpropyl - . - cyclodextrin and sulfobutylether - . - cyclodextrin
have been reported to solubilize and stabilize many injectable drugs, including
dexamethasone, estradiol, interleukin - 2, and other proteins and peptides [140]
without apparent compatibility problems [141] . In addition, CD - containing formulations
(either 0.1 M sulfobutylether - . - cyclodextrin or 0.1 M hydroxylpropyl - . -
cyclodextrin) were shown to cause less damage to venous epithelial cells at the site
of injection compared with formulations containing organic cosolvents [142] . When
CD were coentrapped in the internal aqueous phase, erythropoietin (EPO) covalent
aggregate formation was signifi cantly reduced during microsphere preparation by
the double - emulsion method [72] and lysozyme stability was improved [88] . Although
the precise mechanism is unclear, interactions between amino acids and the hydrophobic
inner cavity of CD may play a role [143] . However, CD showed no protecting
effect on insulinlike growth factor - 1 (IGF - 1) [144] and hepatitis B core antigen
(HBcAg) [145] and even promoted the loss of superoxide dismutase activity at high
CD concentrations [146] . By contrast, carboxymethylcellulose (CMC) did not effi -
ciently stabilize HBcAg and GH against dichloromethane - induced denaturation
[61, 145] . Various types ( . , . , and . ) of CD were examined for encapsulating TT in
PLGA microspheres [147] , with . - hydroxypropyl - cyclodextrin effectively increasing
TT encapsulation. However, CD also showed low effi ciency in retaining spray - dried
TT antigenicity, probably due to antigenic epitopes being buried inside the molecular
CD core [147] .
Surfactants have the ability to lower surface tension of protein solutions and
prevent protein adsorption and/or aggregation at hydrophobic surfaces such as
PLGA. Among them, nonionic surfactants are generally preferred as ionic surfactants
might bind to groups in proteins and cause denaturation. Tween 20 was
shown to greatly reduce the rate of formation of insoluble aggregates of recombinant
human factor XIII caused by both freeze thawing and agitation stresses
[148] . Maximum protection occurs at concentrations close to the critical micelle
concentration of Tween 20, independent of initial protein concentration. In
another report, Tween 20 at a 1% (w/v) concentration caused precipitation of a
relatively hydrophobic protein ( Humicola lanuginosa lipase) by inducing nonnative
aggregates [149] . Similarly, nonionic surfactants such as Tween 20 or 80 were
not good stabilizers for lysozyme and rhGH against the unfolding effect of the
water – dichloromethane interface. It has been assumed that both the hydrophilic
(PEG chains) and hydrophobic (fatty acid chain) parts of the polysorbate molecules
were preferentially partitioned in the dichloromethane phase, leading
to low protection effi cacy [61, 98] . Exchange of Tween 20 for a less hydrophobic
surfactant, PEG 3350, provided almost complete rhGH recovery irrespective of
protein concentration. However, an opposing trend was seen with EPO encapsulation
in PLGA microspheres [72] . Encapsulated protein aggregates increased
(. 15%) with different PEG types codissolved in the w 1 phase. Conversely, when
three nonionic surfactants of different hydrophilic – lipophilic balances (HLBs)
were coencapsulated with insulin by the w/o/w double - emulsion method, only
Tween 20 was able to improve insulin stability within particles and to limit formation
of high - molecular - weight products during the sustained - release period
[150] .
INJECTABLE PEPTIDE/PROTEIN-LOADED MICROSPHERES 411
412 BIODEGRADABLE POLYMER-BASED MICROSPHERES
Tween 80 is well known to protect proteins against surface - induced denaturation
[151] . Tween 80 was demonstrated to reduce hemoglobin aggregation in solution by
preventing the protein from reaching the air – liquid interface or the liquid – surface
interfaces [152] . Polyoxyethylene surfactants such as Tween 80 can form peroxide
impurities after long - term storage. Knepp et al. [153] concluded that Tween 80 and
other nonionic polyether surfactants undergo oxidation during bulk material storage
and subsequent use and the resultant alkyl hydroperoxides formed can contribute
to the degradation of proteins. In such formulations, they further reported that thiols
such as cysteine, glutathione, and thioglycerol were most effective in stabilizing
protein formulations containing peroxide - forming nonionic surfactants.
The Pluronics, also known as poloxamers (e.g., poloxamer 188, British Pharmacopoeia
standard) are a well - studied series of commercially available, nonionic, triblock
copolymers with a central block composed of the relatively hydrophobic
poly(propylene oxide) fl anked on both sides by blocks of the relatively hydrophilic
poly(ethylene oxide) [154, 155] . The Pluronics possess an impressive safety profi le
and are approved selectively by the Food and Drug Administration (FDA) for
pharmaceutical and medical applications, including parenteral administration [156] .
The strong safety profi le, commercial availability, ease of preparation, and well -
studied physical properties make the Pluronics particularly appealing for drug delivery
purposes. They have been used in several patented protein formulations as
stabilizers and sustained - release injectables in development as solubilizing and stabilizing
agents [157] . However, poloxamers, like Tweens, can form peroxide impurities
over time. Poloxamer 188 was successfully used when mixed with PLGA for
prolonged release of active interferon - . (INF - . ) [158] , but such a formulation had
no effect on BSA secondary structure compared to PLGA alone [35] . Poloxamer
188 was not effective in preventing nerve growth factor (NGF) aggregation during
in vitro release from microspheres generated by spray drying [159] . Complex interactions
between poloxamer, BSA, and PLGA were believed to have infl uenced BSA
microencapsulation [160] . The gelling property of the amphiphilic poloxamer 407
was successfully employed for urease encapsulation. The protein was likely protected
during the microsphere preparation by a hydrated gelled structure due to the
hydrophilic polyoxyethylene chains [161] . EPO aggregates in PLGA microspheres
decreased when poloxamer 407 was incorporated at a level of 10% (w/w) [72] .
Interleukin - 1 . (IL - 1 . ) was protected by phosphatidylcholine from damage
during the double - emulsion process but underwent inactivation during microsphere
incubation [162] . Sodium dodecyl sulfate signifi cantly reduced insulin aggregation
at the dichloromethane – water interface, whereas dodecyl maltoside did not, this
surfactant being more effi cient at air – water or solid – water interfaces [163] . It should
be mentioned that surfactants are used along with sugars, proteins, and polymers
effectively for solubilization and stabilization purposes of peptide/protein in microspheres.
Bilati et al. [130] give an overview on various proteins and polymers that
act as stabilizing excipients during the development of peptide/protein - loaded
microspheres.
5.2.5.2 Preservation - Imparting Excipients
Prevention/Minimization of Moisture -Induced Instability Moisture - and microclimate
acid pH – induced instability (typically the aggregation) of the peptide/protein
encapsulated in PLGA microspheres has been monitored. Even several formulation
strategies to inhibit these instability problems are being actively investigated. If the
protein is expected to exist in the solid state within the PLGA polymer, the protein
is remarkably prone to aggregation when formulated under conditions that allow
moisture - and microclimate acid pH – induced instability. The two covalent aggregation
mechanisms commonly described during exposure of the solid protein to moisture
are the disulfi de interchange/exchange [164] and the FMAP, which is operative
for protein antigens that have been detoxifi ed with formaldehyde exposure [124] .
In the former pathway, the reaction is typically initiated by a thiolate ion on the
protein or free thiolate ions that accompany . elimination of an intact disulfi de
[165] . Decreasing the concentration of the reactive species (e.g., lowering pH to
favor the nonionized thiol, covalently blocking the thiol group, or oxidizing free
thiols as they appear with divalent copper ion) has been shown to block this mechanism
[165] . To inhibit the FMAP, strongly formaldehyde - interacting amino acids
such as histidine and lysine [166] have been colyophilized with the formalinized
protein antigen. On exposure to moisture, the amino acids appear to bind with the
reactive Schiff base or equivalent electrophile [167] in the protein before a neighboring
protein nucleophile can react to form an intermolecular cross - link [124] .
Sorbitol has also been identifi ed to inhibit the FMAP of TT at the maximal aggregating
water content of the antigen, about 30 g H 2 O/g protein [168] , although whether
this is a humectant effect [169] or a possible covalent reaction with the highly reactive
electrophile in the antigen has not been determined. Several techniques have
been developed to successfully bypass the destabilizing stress either by altering the
role of water in the solid or immobilizing the protein or, alternatively, by directly
inhibiting the aggregation. Clearly, one of the most signifi cant fi ndings in the fi eld
of peptide/protein stability in polymers is the success of the immobilization strategy
of Zn 2+ precipitation, as performed with human growth hormone [37, 57, 170, 171] .
The 2 : 1 mole ratio Zn – protein complex, which immobilizes the rhGH as a solid
precipitate in a near - native state [99] , has been shown to confer superior stability
on the protein encapsulated in PLGA for a one - month release incubation. Since
then, other proteins such as INF - . [172] and NGF [173] were also stabilized in
PLGA microspheres by this approach. Another interesting approach originating in
the patent literature is the precipitation of erythropoietin with salting - in salt, ammonium
sulfate [174] , which is a technique commonly used in protein processing. Other
methods to alter the role of water in the reaction involve the addition of agents that
alter the amount of water sorbed in the polymer and/or the activity of the water
present. For example, both water - soluble salts (NaCl) and antacid excipients
(Mg(OH)2 ) are known to dramatically increase the amount of water sorbed in
PLGAs, with the former due to osmosis and the latter to a complex effect of neutralizing
acidic degradation products and end groups of the polymer (which also
involves an osmotic component) [126] . In contrast, for a given moisture content,
humectants such as sorbitol, which dissolve in water bound to the protein, reduce
the available free water necessary to mobilize the protein or perform other roles in
deleterious reactions [169] .
The alternative to bypassing the deleterious role of moisture is to inhibit the
aggregation mechanism directly. Several ways to accomplish this have been reported,
particularly in the solid state and in the absence of the polymer. Well - referenced
and useful book chapters by Johnson [175] and Carpenter and Chang [176] are
INJECTABLE PEPTIDE/PROTEIN-LOADED MICROSPHERES 413
414 BIODEGRADABLE POLYMER-BASED MICROSPHERES
available to thoroughly focus on the importance of making a lyophilized powder
before loading the peptide or protein into an injectable microspheres. It has been
stated that, in comparison to protein solution, the protein in the solid state would
be less susceptible to shear forces that occur during an emulsifi cation procedure or
denaturation at oil – water interfaces. However, special precautions should be taken
during freeze drying because the drying process itself will expose the protein to
destabilizing stresses. To circumvent this problem, cryo - and lyoprotectants and
bulking agents are usually included along with a peptide or protein solution while
it undergoes the drying stages of the lyophilization process.
Cryo - and Lyoprotectants and Bulking Agents Various mechanisms are proposed
to explain why excipients serve as cryo - or lyoprotectants. The most widely accepted
mechanism to explain the action of cryoprotection is the preferential exclusion
mechanism [177] . Excipients that will stabilize proteins against the effects of freezing
do so by not associating with the surface of the protein. Such excipients actually
increase the surface tension of water and induce preferential hydration of the
protein. Examples of solutes that serve as cryoprotectants by this mechanism include
amino acids, polyols, sugars, and polyethylene glycol.
For lyoprotection, that is, stabilization of proteins during the drying stages of
freeze drying and during storage in the dry state, two mechanisms are generally
accepted. One is the water substitute hypothesis [178] and the other is the vitrifi cation
hypothesis [179] . Both are legitimate theories, but both also have exceptions;
that is, neither fully explain the stabilization of proteins by excipients during dehydration
and dry storage [180] . The water substitute hypothesis states that a good
stabilizer is one that hydrogen bonds to the protein just as water would do where
it presents and, therefore, serves as a water substitute. Sugars are good water substitutes.
(It may at fi rst appear contradictory that sugars can serve both as cryoprotectants
because of being excluded from the surface of the protein and as
lyoprotectants that hydrogen bond to the protein. However, keep in mind that the
excluded solute concept involves a frozen aqueous system whereas the water substitute
concept occurs in a dry system.) This is why many freeze - dried protein formulations
contain sucrose or trehalose. Nevertheless, during a w/o/w procedure to
prepare peptide/protein - loaded PLGA microspheres, sugars are often added to the
inner aqueous phase. Trehalose was shown to partially improve the BSA secondary -
structure protection within PLGA microspheres and to facilitate BSA monomer
release [26] . Trehalose and mannitol had a signifi cant effect on the recovery of
soluble nonaggregated interferon - . (INF - . ) and rhGH after emulsifi cation and
ultrasonication [61] , whereas no or very little protecting effect on IGF - 1 against
these stress factors was observed [144] . No effect of trehalose, mannitol, and sucrose
was observed against o/w interface - induced degradation of lysozyme, whereas
lactose and lactulose signifi cantly improved its structural stability and activity, mostly
if these additives were also added to the second aqueous phase [88, 100] . Lysozyme
and trypsin activity was not improved by addition of sucrose, which was unable to
protect them from an emulsion - induced denaturation and from sonication [98, 181] .
Mannitol and sucrose dissolved together in the inner aqueous phase had slight effect
on NGF activity [182] and neither mannitol nor lactose improved HBcAg immunogenicity
during dichloromethane/water emulsifi cation [145] . Surprisingly, sucrose
and trehalose even decreased urease bioactivity, showing the opposite effect to that
expected [161] . Coencapsulation of maltose reduced . - chymotrypsin aggregation
[183] . With respect to microspheres generated by spray drying, trehalose was effective
in retaining TT antigenicity [147] and in preventing BSA secondary - structure
degradation [35] . Trehalose protected effi ciently NGF during the processing but did
not prevent its aggregation during in vitro release [159] .
The vitrifi cation hypothesis states that excipients that remain amorphous (glass
formers) form a glassy matrix with the protein with the matrix serving as a stabilizer.
Acceptance of this hypothesis requires formulators to determine glass transition
temperatures of formulations to be freeze dried and to develop freeze - dry cycles
that maintain drying temperatures below the glass transition temperature. Reports
are available to indicate that excipient stabilizers, which are capable of undergoing
crystallization during storage, caused degradation (typically aggregation and loss of
potency) of the protein [176, 184, 185] .
Freeze - dried formulations typically contain one or more of the following bulking
agents: mannitol, lactose, sucrose, trehalose, dextran 40, and povidone. These excipients
may also serve as cryo - and/or lyoprotectants in protein formulations. Fakes
et al. [186] studied these bulking agents for moisture sorption behavior before and
after freeze drying. Moisture uptake certainly can affect drug stability in the freeze -
dried state, particularly with peptides and proteins. When selecting a bulking agent,
these properties, particularly the tendency for moisture uptake, must be considered
by the formulation scientist in developing an optimally stable freeze - dried formulation.
Several excipients can serve as stabilizers for proteins that are unstable during
the drying phases of freeze drying and/or during long - term storage in the dry state.
Typically, additives that will crystallize during lyophilization (e.g., mannitol) or will
remain amorphous but unable to hydrogen bond to the dried protein (e.g., dextran)
are not effective lyoprotectants for proteins. Excipients that will crystallize during
freeze drying will also be relatively ineffective, as was shown with sucrose in
H. lanuginosa lipase formulations [149] . However, these authors also reported that
sucrose crystallization could be inhibited by decreasing the mass ratio of sucrose to
protein and by minimizing the moisture content that serves to decrease the glass
transition temperature during storage. The reverse can also be true for certain small
molecules. For example, excipients (mannitol or sodium bicarbonate) that promoted
the crystallization of cyclophosphamide during freeze drying stabilized the fi nal
product whereas excipients that did not allow crystallization (e.g., lactose) destabilized
the fi nal product [187] . Costantino et al. [188] studied the effects of a variety
of parenteral excipients on stabilizing human growth hormone in the lyophilized
state. Mannitol, sorbitol, methyl a - d - mannopyranoside, lactose, trehalose, and cellobiose
all provided signifi cant protection of the protein against aggregation,
particularly at levels (131 : 1 excipient - to - protein molar ratio) to potentially satisfy
water binding sites on the protein in the dried state. At higher excipient - to - protein
ratios, mannitol and sorbitol crystallized and were not as effective in stabilizing the
protein compared with low levels in which they remained in the amorphous, protein -
containing phase.
Reducing sugars may not be as effective as other bulking agents, cryoprotectants,
or lyoprotectants because they may potentially react with proteins via the Maillard
reaction. For example, glucose will form covalent adducts with side - chain amino
acids lysine and arginine of human relaxin [189] . In addition, a signifi cant amount
of serine cleavage from the C terminal of the B chain of relaxin was formed when
INJECTABLE PEPTIDE/PROTEIN-LOADED MICROSPHERES 415
416 BIODEGRADABLE POLYMER-BASED MICROSPHERES
glucose was used as the excipient. These reactions did not occur if mannitol and
trehalose replaced glucose in the lyophilized formulation. Lactose will react with
primary amines in the well - known Maillard - type condensation reaction to form
brown - colored degradation products [190] . Thus, lactose is known to be incompatible
with amine - containing compounds such as aminophylline, amphetamines, and
amino acids/peptides. This reaction occurs more readily with amorphous lactose
than crystalline lactose.
Hydrophilic additives such as glucose are known to increase the porosity of
microspheres, causing an increase in permeability to mass transport and a higher
burst. However, a signifi cant reduction in initial burst release of a highly water -
soluble model peptide, octreotide acetate, from poly( d , l - lactide - co - glycolide)
microspheres by the coencapsulation of a small amount of glucose (e.g., 0.2% w/w)
was reported [191] . Using the double emulsion – solvent evaporation method of
encapsulation, the effect of glucose on initial burst in an acetate buffer pH 4 was
found to depend on polymer concentration, discontinuous phase/continuous phase
ratio, and glucose content. Extensive characterization studies were performed on
two microsphere batches, ± 0.2% glucose, to elucidate the mechanism of this effect.
However, no signifi cant difference was observed with respect to specifi c surface
area, porosity, internal and external morphology, and drug distribution. Continuous
monitoring of the fi rst 24 - h release of octreotide acetate from these two batches
disclosed that, even though their starting release rates were close, the microspheres
plus glucose exhibited a much lower release rate between 0.2 and 24 h compared to
those without glucose. The microspheres plus glucose showed a denser periphery
and a reduced water uptake at the end of the 24 - h release, indicating decreased
permeability. However, this effect at times was offset as glucose content was further
increased to 1%, causing an increase in surface area and porosity. In summary, these
authors concluded that the effects of glucose on initial burst are determined by two
factors: (1) increased initial burst due to increased osmotic pressure during encapsulation
and drug release and (2) decreased initial burst due to decreased permeability
of microspheres [191] .
Mannitol is probably the most widely used bulking agent in lyophilized formulations
because of its many positive properties with respect to crystallinity, high
eutectic temperature, and matrix properties. However, some lots of mannitol can
contain reducing sugar impurities that were implicated in the oxidative degradation
of a peptide in a lyophilized formulation [192] . Mannitol at or above certain concentrations
and volumes in glass vials is well known to cause vial breakage because
of the unique crystallization properties of mannitol - ice during the primary drying
states of freeze drying [193, 194] .
Other Freeze - Dry Excipients High - molecular - weight carbohydrates such as dextran
have higher glass transition temperatures than peptides/proteins. Therefore, when
mixed with proteins, the overall glass transition temperature presumably can be
increased with resultant increases in protein storage stability. Typically, carbohydrates
(sucrose, trehalose, or dextran) alone do not result in appreciable increases
in the storage stability of proteins. However, combinations of disaccharide and
polymeric carbohydrates do tend to improve protein storage stability [195] . However,
singular carbohydrates (sucrose or trehalose at 60 m M ) were also just as effective
in stabilizing a model recombinant humanized monoclonal antibody as combinations
of sucrose and mannitol or trehalose and mannitol. Interestingly, with this
model monoclonal antibody, mannitol alone at 60 m M provided less protection
during storage than sucrose or trehalose alone. A specifi c sugar/protein molar ratio
was suffi cient to provide storage stability for this particular monoclonal antibody
[196, 197] .
Low - molecular - weight additives such as osmolytes ( N,N - dimethylglycine, trehalose,
and sucrose) or salts (sodium chloride, sodium phosphate, ammonium sulfate,
and sodium citrate) were found to be highly effective in stabilizing keratinocyte
growth factor, both against thermal denaturation and enhancing long - term storage
stability [198] . Nevertheless, the stabilizing properties of osmolytes appear to be
balanced between their binding to (deteriorating effect) and exclusion from (stabilizing
effect) the peptide/protein surface. As binding or exclusion predominantly
results from hydrophobic interactions, hydrogen bonding, and electrostatic interactions,
the sum of the various interaction parameters are dissimilar for different
proteins. Therefore, it becomes crucial to examine the individual nature of the additive
toward each individual protein and to assess whether it will offer a stabilizing
or destabilizing effect [199, 200] .
Polyvinyl pyrrolidone (PVP) and glycine were found to stabilize lyophilized
sodium prasterone sulfate whereas dextran 40 or mannitol did not [201] . PVP and
glycine stabilized the pH of the reconstituted solution by neutralizing the acidic
degradation product, sodium bisulfate, formed by the hydrolysis of prasterone
sulfate. Dextran 40 or mannitol was ineffective because of no buffer capacity. Buffering
agents, such as phosphate – citrate buffer and some neutral and basic amino
acids ( l - arginine, l - lysine, and l - histidine), also stabilized prasterone sulfate. l - Cysteine
is an example of an amino acid that did not stabilize the drug, presumably
because of its weak buffer capacity. Although the effi ciency of proteinic additives
for protein stabilization has been clearly demonstrated in several occasions even
during encapsulation processes [31, 72, 98, 144] , their use in pharmaceuticals is at
present not desirable from a strictly regulatory point of view. Additionally, such
agents might contribute to complicate all subsequent protein characterization within
the formulation. Among these additives, albumins and gelatins are those mainly
used for protection purposes. The protective effect of albumins against protein
unfolding and aggregation has been extensively documented and is likely due to
their surface - active properties (see Bilati et al. [130] for details).
Prevention/Minimization of Microclimate pH -Induced Instability Evidence for
acidifi cation within degrading microspheres is investigated and local pH values
between 1.5 and 4.7 are being reported [202 – 204] . Methods to measure microclimate
pH in PLGA microspheres include (i) ensemble average measurements using electron
paramagnetic resonance (EPR) [203, 204] , nuclear magnetic resonance (NMR)
[205] , and potentiometry and (ii) direct visualization techniques such as confocal
imaging of pH - sensitive dyes [206, 207] . In the EPR method, the constant of hyper-
fi ne splitting, 2 aN , was used to determine an average pH inside PLGA microspheres.
Because the experiments relied on the mobility of spin - labeled protein, with an
increase of the microviscosity in the later hours of the experiments, the spectra of
EPR was changed and the signal - to - noise ratio decreased to prevent the measurement
of pH throughout the release period [203] . The potentiometric measurements
can give rapid values of pH for thin polymer fi lms, and the pH of the thin water fi lm
between the electrode and polymer mimics the microclimate pH of aqueous pores
inside the polymer - based drug delivery system. However, it is diffi cult to mimic
INJECTABLE PEPTIDE/PROTEIN-LOADED MICROSPHERES 417
418 BIODEGRADABLE POLYMER-BASED MICROSPHERES
microclimate pH of a small - scale system, such as microspheres or nanospheres,
which may have unique microstructures, excipient/drug distributions, and transport
characteristics. Overall, the ensemble average measurements described above could
give a general picture of microclimate pH at the macroscopic level. However, the
microscopic level of the detection can only be achieved through direct visualization
techniques, such as microscopic imaging. Shenderova et al. [207] fi rst developed the
confocal microscope imaging method to relate the microclimate pH with the fl uorescent
intensity. Because of the diffi culty of controlling and predicting the fl uorescein
concentration in the aqueous pore inside the microsphere, the method was only
semiquantitative. Fu et al. [206] improved the confocal microscopic imaging method
by coencapsulating two dextran fl uorescent dye (NERF and SNARF - 1) conjugates
inside microspheres and related the ratio of the two dye images with microclimate
pH in order to eliminate the poorly controlled effects of dye concentration and pore
distribution. However, both of the dyes emit in the green range (535 nm for NERF
and 580 nm of SNARF), giving rise to poor resolution without a narrow - bandwidth
detector. Because of the high noise - to - signal ratio from the ratio images, the prediction
of pH is also expected to be semiquantitative. In order to overcome the
aforementioned drawbacks in microclimate pH measurement, a new quantitative
ratiometric method based on laser scanning confocal microscopic imaging was
developed to create a pixel - by - pixel neutral range microclimate pH map inside
PLGA microspheres [208] . This method was then applied to both acid - neutralized
and nonneutralized PLGA microspheres during extended incubation in physiological
buffer. In another study, the PLGA water - soluble acid distribution has been
measured with prederivatization HPLC [209] .
Ongoing acidifi cation of the microsphere interior was shown to induce deamidation
and covalent dimerization of nonreleased insulin [202] . Despite the evidence
of acidifi cation mentioned above, there is controversy on this subject. It has been
pointed out that the sampling scheme has a signifi cant impact on the degree of
acidifi cation; frequent replenishment of the release medium or the use of a dialysis
bag can effectively prevent the acidifi cation of the medium with subsequent
reduced protein degradation [93, 210] . It is unsure, however, whether this also
refl ects the situation in vivo, in which the PLGA microspheres are often surrounded
by a fi brous capsule that may reduce effl ux of acidic degradation products
from the PLGA matrix [93] . On the other hand, studies on rhGH - loaded PLGA
microspheres showed a reasonable in vitro – in vivo correlation (IVIVC) only when
a strong high - capacity buffer [200 m M N - (2 - hydroxyethyl)piperazine - N. - 2 - ethanesulfonic
acid (HEPES), pH 7.4] was used, which effectively minimized the pH drop
[211] .
As indicated by the prevention of acid - induced physical aggregation of BSA in
an abstract [212] , three principal ways have been identifi ed thus far to avoid the
formation of highly acidic microclimate regions in the PLGAs during protein
release:
(i) Increasing the permeability of the polymer to facilitate escape of the water -
soluble hydrolytic products of the PLGA polyester [125]
(ii) Decreasing the degradation rate of the polyester [213]
(iii) Coencapsulating additives to neutralize the weak acids formed by PLGA
hydrolysis [126]
In addition, two more ways that are likely to favor a lowering of microclimate pH
are elevated initial acid content in the polymer [214] and low - frequency release
media exchange [206] .
The concept of controlling polymer permeability is diffi cult because attempts to
increase permeability can spoil the controlled - release function of the polymer and
cause the encapsulated protein to be released too rapidly. For example, Jiang and
Schwendeman [213] increased the permeability of slow - degrading PLA (molecular
weight (MW) 145 kDa) by blending in PEG (MW 10 or 30 kDa) at 0, 10, 20, and
30%. Insoluble BSA aggregation in the PLA microspheres containing 4.5 – 5% w/w
BSA was found in 0 and 10% PEG after a one - month incubation, but not in those
preparations containing 20 or 30% PEG. The structural integrity of BSA was also
intact in the stabilized formulations. However, between 10 and 30% PEG, the
release rate of BSA increased rapidly and by 30% PEG, 60% of the protein encapsulated
was released in only three days [213] . In contrast, an abstract [212] implied
that 5% BSA encapsulated in a more permeable PLA (MW 77 kDa), the BSA
formed < 2% insoluble aggregates over one month. This result suggested strongly
that in some instances the slow degradation rate of the non - glycolic - acid - containing
PLA is suffi cient to inhibit acid formation in the microclimate.
In instances in which it is desirable to increase permeability and/or decrease the
hydrolytic rate of PLGAs, that is, where a highly water - soluble protein requires
release for one month or longer, it becomes necessary to coencapsulate a basic
additive. Antacids such as MgCO 3 , Mg(OH) 2 or ZnCO 3 have been found to be particularly
potent in preventing instabilty of acid - labile proteins [126, 215] . By means
of thin fi lms coating pH glass electrodes to measure directly the microclimate acidic
environment in PLGA microspheres, the stabilization against insoluble acid - induced
noncovalent BSA aggregation afforded by a series of antacid excipients has been
correlated with the ability of the antacid to neutralize acidic pores in fi lms of the
same lot of PLGA coating pH glass electrodes [215] . Though much of the physical
chemistry of microclimate pH adjustment with antacid additives is currently unclear,
the strength of the base, the base solubility, and the association of the divalent
cation with the carboxylate of the degradation products and/or polymer end groups
appear to be important. For instance, Shenderova [216] has shown that from microclimate
pH measurements in PLGA fi lms coating pH glass electrodes, MgCO 3 and
Mg(OH)2 were found to be very similar under conditions which favor homogenous
neutralization (i.e., high protein loading suffi cient to make pores for the base to
diffuse all regions of the polymer matrix), but MgCO 3 was found to increase
microclimate pH higher than Mg(OH) 2 . This result was consistent with the improved
BSA stability in PLGA 50/50 microspheres when MgCO 3 was used in place of
Mg(OH)2 [126] .
5.2.6 PEPTIDE/PROTEIN ENCAPSULATED INTO BIODEGRADABLE
MICROSPHERES: CASE STUDY
Selected examples of therapeutic peptide and protein including vaccines which have
been encapsulated into biodegradable polymer - based microspheres are discussed
in this section. Besides what is mentioned below, many other proteins and
vaccines have been encapsulated in biodegradable polymers, so a glimpse of ongoing
PEPTIDE/PROTEIN ENCAPSULATED INTO BIODEGRADABLE MICROSPHERES 419
420 BIODEGRADABLE POLYMER-BASED MICROSPHERES
research on microsphere delivery systems using biodegradable polymers is shown
in Table 2 .
5.2.6.1 Vaccines
Group B Streptococcus Vaccine Group B streptococcus (GBS) is the leading
bacterial cause of neonatal sepsis and meningitis. Although antibiotic prophylaxis
has decreased the infection rate, the best long - term solution lies in the development
of effective vaccines. The GBS capsular polysaccharide (CPS) is a major target of
antibody - mediated immunity. The feasibility of producing a GBS having the ability
to produce both a local IgA immune response at the mucosal surface and humoral
IgG response having capability of transplacental passive immunization was
investigated [217] . Inactivated GBS antigen was encapsulated in PLGA by a w/o/w
multiple - emulsion technique along with immunostimulatory synthetic oligodeoxynucleotides
containing cytosine phosphate guanosine (CpG) as potent adjuvant
[217] . Immunization of female mice with normal immune systems was done with
these PLGA microspheres containing GBS type III polysaccharide and CpG adjuvant
(PLGA/GBS/CpG) and results indicated a signifi cantly higher GBS antibody
response as compared to nonencapsulated GBS antigen or PLG - encapsulated GBS
PS vaccine without the addition of the CpG.
Diphtheria Toxoid ( DT) Diphtheria is a communicable disease caused by Corynebacterium
diphtheriae which colonizes and forms a pseudomembrane at the
infection site. This pathogen produces a potent protein toxin, diphtheria toxin,
which is responsible for the typical systemic toxemia. DT is required for active
immunization against diphtheria. DT was encapsulated in different types of PLA
and PLGA microspheres by spray drying and coacervation. Immunization of
guinea pigs with DT microspheres made with relatively hydrophilic PLGA 50 : 50
resulted in specifi c and sustained antibody responses to alum adjuvanted toxoid in
contrast to microspheres made with hydrophobic polymers where very low antibody
responses were determined confi rming the feasibility of microsphere vaccines
to induce strong, long - lasting protective antibody responses after single immunization
[218] .
In an endeavor toward development of multivalent vaccines based on biodegradable
microspheres, Peyre et al. [219] tested the immunological performance of
several divalent microsphere formulations against tetanus and diphtheria. Microspheres
were made by separate microencapsulation of tetanus and diphtheria toxoid
in PLGA by either spray drying or coacervation. Guinea pigs were subcutaneously
immunized by a single injection of the divalent vaccines or, for control, an equivalent
dose of a licensed vaccine containing both antigens adsorbed on aluminum hydroxide.
All microsphere formulations were strongly immunogenic, irrespective of particle
size and hydrophobicity. Endpoint titers of ELISA antibodies, mainly of the
IgG1 subtype, were comparable to those obtained after immunization with the
licensed vaccine. The microspheres provided increasing levels of antibodies, during
the 16 weeks of testing, and the antibodies were weakly polarized toward tetanus.
The induced antibodies were also toxin neutralizing, as determined for both diphtheria
(1 – 4 IU/mL) and tetanus (5 – 9 IU/mL) eight weeks after immunization. These
neutralization levels were several orders of magnitude above the level considered
minimum for protection (0.01 IU/mL). When the animals were challenged with
tetanus or diphtheria toxins six weeks after immunization, microsphere vaccines
produced protective immunity that was comparable to or better than that induced
by the licensed divalent vaccine. In conclusion, this study showed that a single
administration of biodegradable microsphere vaccines provided protective immunity
against diphtheria and tetanus and that this immunization approach might be
feasible for multivalent vaccines. In a separate study, the same group have studied
for the fi rst time the fate of immunogenic fl uorescent - labeled PLGA microspheres
loaded with DT in vivo following a subcutaneous injection route [220] .
A unique instability problem of DT is being foreseen when the DT would be
encapsulated in PLGA microspheres along with a preservative such as thiomersal
[221] . Thimerosal (TM) — also known as thiomersal, Merthiolate, or sodium
ethylmercuri - thiosalicylate — is a water - soluble derivative complex of thiosalicylic
acid (TSA) that has been used as bactericide in parenteral vaccines and ophthalmic
products for decades. It has been reported that this preservative can be decomposed
by oxidation to 2,2 - dithiosalicylic acid, ethyl mercuric ion, 2 - sulfenobenzoic acid, 2 -
sulfobenzoic acid, and 2 - sulfi nobenzoic acid [222] . Namura et al. [221] demonstrated
in vitro that the TSA, produced after the reduction of TM by lactic acid, reduces
the S – S bridge of the previously incubated DT. This reduction is immediately followed
by blocking the two SH groups formed by the same TSA molecules. In light
of these conclusions, it is necessary now to reinterpret the in vitro protein degradation
– stabilization data in the presence of PLGA microsphere, mainly for those
proteins which contain S – S. The authors propose that all the PLGA microsphere
microencapsulation studies and protein structural considerations should be done in
the absence of TM as preservative.
Tetanus Toxoid ( TT) Tetanus is considered a major health problem in developing
and underdeveloped countries, with approximately one million new cases occurring
each year. Tetanus is an intoxication manifested primarily by neuromuscular dysfunction.
So vaccination is required for prevention of this disease. TT was encapsulated
using PLGA with different molar compositions (50 : 50, 75 : 25) by the w/o/w
multiple - emulsion technique and protein integrity was evaluated during antigen
release in vitro in comparison to alum - adsorbed TT for in vivo induction of tetanus -
specifi c antibodies [223] . TT microspheres elicited antibody titers as high as conventional
alum - adsorbed TT, which lasted for 29 weeks, leading to the conclusion that
TT microspheres can act as potential candidates for single - shot vaccine delivery
systems.
The study by Determan et al. [224] focuses on the effects of polymer degradation
products on the primary, secondary, and tertiary structure of TT, OVA, and lysozyme
after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic
acid) and anhydride [sebacic acid and 1,6 - bis( p - carboxyphenoxy)hexane] monomers.
The structure and antigenicity or enzymatic activity of each protein in the
presence of each monomer was quantifi ed. SDS - PAGE, circular dichroism, and fl uorescence
spectroscopy were used to assess/evaluate the primary, secondary, and
tertiary structures of the proteins, respectively. ELISA was used to measure changes
in the antigenicity of TT and OVA and a fl uorescence - based assay was used to
determine the enzymatic activity of lysozyme. TT toxoid was found to be the most
stable in the presence of anhydride monomers, while OVA was most stable in the
PEPTIDE/PROTEIN ENCAPSULATED INTO BIODEGRADABLE MICROSPHERES 421
422 BIODEGRADABLE POLYMER-BASED MICROSPHERES
presence of sebacic acid, and lysozyme was stable when incubated with all of the
monomers studied.
Jaganathan et al. [225] compared the effi ciency of microspheres produced from
PLGA and chitosan polymers by using protein stabilizer (trehalose) and acid -
neutralizing base [Mg(OH) 2 ]. The immunogenicity of PLGA - and chitosan microsphere
– based single - dose vaccine was evaluated in guinea pigs and compared with
multiple doses of alum - adsorbed TT. Results indicated that a single injection of
PLGA and chitosan microspheres containing TT could maintain the antibody
response at a level comparable to the booster injections of conventional alum -
adsorbed vaccines. Both the PLGA - and chitosan - based stable vaccine formulations
produced an equal immune response. Hence chitosan can be used to replace the
expensive polymer PLGA. This approach should have potential application in the
fi eld of vaccine delivery.
The study by Kipper et al. [226] focuses on the development of single - dose vaccines
based on biodegradable polyanhydride microspheres that have the unique
capability to modulate the immune response mechanism. The polymer system
employed consists of copolymers of 1,6 - bis( p - carboxyphenoxy)hexane and sebacic
acid. Two copolymer formulations that have been shown to provide extended -
release kinetics and protein stability were investigated. Using TT as a model antigen,
in vivo studies in C3H/HeOuJ mice demonstrated that the encapsulation procedure
preserves the immunogenicity of the TT. The polymer itself exhibited an adjuvant
effect, enhancing the immune response to a small dose of TT. The microspheres
provided a prolonged exposure to TT suffi cient to induce both a primary and a
secondary immune response (i.e., high antibody titers) with high - avidity antibody
production, without requiring an additional administration. Antigen - specifi c proliferation
28 weeks after a single immunization indicated that immunization with the
polyanhydride microspheres generated long - lived memory cells and plasma cells
(antibody - secreting B cells) that generally do not occur without maturation signals
from T helper cells. Furthermore, by altering the vaccine formulation, the overall
strength of the T - helper type 2 immune response was selectively diminished, resulting
in a balanced immune response, without reducing the overall titer. This result is
striking, considering free TT induces a T - helper type 2 immune response and has
important implications for developing vaccines to intracellular pathogens. The
ability to selectively tune the immune response without the administration of additional
cytokines or noxious adjuvants is a unique feature of this delivery vehicle that
may make it an excellent candidate for vaccine development.
Polylactide (PLA) polymer particles entrapping TT were evaluated in terms of
particle size, antigen load, dose, and additional adjuvant for achieving high and
sustained anti - TT antibody titers from single - point intramuscular immunization
[227] . Admixture of polymer - entrapped TT and alum improved the immune response
in comparison to particle - based immunization. High and long - lasting antibody titer
was achieved upon immunization with 2 – 8 - . m size microparticles. Microspheres
within the size range 50 – 150 . m elicited very low serum antibody response. Immunization
with very small particles ( < 2 . m) and with intermediate - size - range particles
(10 – 70 . m) elicited comparable antibody response from single - point immunization
but lower in comparison to that achieved while immunizing with 2 – 8 - . m particles.
Potentiation of antibody response on immunization of admixture of microspheres
and alum was also dependent on particle size. These results indicate the need of
optimal particle sizes in micrometer ranges for improved humoral response from
single - point immunization. Increasing antigen load on polymer particles was found
to have a positive infl uence on the generation of antibody titers from particle - based
immunization. Maximum peak antibody titer of . 300 . g/mL was achieved on day
50 upon immunization with particles having the highest load of antigen (94 . g/mg
of polymer). Increase in dose of polymer - entrapped antigen resulted in concomitant
increase in peak antibody titers, indicating the importance of antigen stability, particle
size, and load on generating a reproducible immune response. Optimization of
particle size, antigen load, dose, and use of additional adjuvant resulted in high and
sustained anti - TT antibody titers over a period of more than 250 days from single -
point immunization. Serum anti - TT antibody titers from single - point immunization
of admixture of PLA particles and alum were comparable with immunization from
two divided doses of alum - adsorbed TT.
Vibrio Cholerae ( VC) Whole -Cell Vaccine Cholera, an acute intestinal infection
caused by the bacterium Vibrio cholerae , produces an enterotoxin that causes a
copious, painless, watery diarrhea that can quickly lead to severe dehydration and
death if treatment is not promptly given. For prevention of cholera, cholera vaccine
is usually given. VC was successfully entrapped in the PLGA microspheres by a
double - emulsion method with trapping effi ciencies up to 98%. The immnunogenic
potential of VC - loaded microspheres physically mixed with or without amphotericin
B was evaluated in adult mice by oral immunization in comparison to VC solution.
The immunogenicity of VC - loaded microparticles mixed with amphotericin B in
evoking Vibrio - specifi c serum IgG and IgM responses was higher than that of VC -
loaded microparticles only [228, 229] . However, VC was loaded in different polymer
compositions (50 : 50 PLGA, 75 : 25 PLGA, and PLA/PEG blended), the higher
antibody responses and serum IgG, IgA, and IgM responses were obtained when
sera from both VC - loaded 75 : 25 PLGA and PLA/PEG - blended microparticles
immunized mice were titrated against VC solution [230] .
Japanese Encephalitis Virus ( JEV) Japanese encephalitis is a disease that is
spread to humans by infected mosquitoes in Asia. It is one of a group of
mosquito - borne viral diseases that can affect the central nervous system and
cause severe complications and even death. Vaccination is one of the ways of
treating it. JEV vaccine was encapsulated in PLGA microspheres by a double -
emulsion technique and infl uences of various process variables such as stirring
rate, types and concentration of emulsifi er, and polymer concentration were
studied on size, size distribution, and biodegradation. The mean size of microspheres
decreased with increasing speed, increasing concentration of emulsifi er,
and decreasing polymer concentration. Rate of biodegradation of nonporous
microspheres was slower than that of porous microspheres, leading to the conclusion
that PLGA microspheres can be used to apply oral vaccination through
Peyers patches across the gastrointestinal tract (GIT) [231] .
Several approaches to develop an improved JEV vaccine are in progress in
various laboratories. Of these, immunization of mice with plasmid DNA encoding
JEV envelope (E) protein has shown great promise. The technology, developed by
Kaur et al. [232] , involved the adsorption of DNA onto cetyltrimethyl - ammonium
bromide (CTAB) containing cationic poly(lactide - co - glycolide) (PLG) microspheres.
PEPTIDE/PROTEIN ENCAPSULATED INTO BIODEGRADABLE MICROSPHERES 423
424 BIODEGRADABLE POLYMER-BASED MICROSPHERES
The microsphere - adsorbed DNA induced a mixed Th1 – Th2 immune response as
opposed to Th1 immune responses elicited by the naked DNA.
JEV - loaded poly(lactide) (PLA) lamellar and PLG microspheres were successfully
prepared with low - molecular - weight PLA by the precipitate method and with
6% w/v PLG in the organic phase, 10% w/v PVP, and 5% w/v NaCl in the continuous
phase by using a w/o/w emulsion/solvent extraction technique, respectively [233] .
The JEV incorporation, physicochemical characterization data, and animal results
obtained in this study may be relevant in optimizing the vaccine incorporation and
delivery properties of these potential vaccine targeting carriers.
Hepatitis B Virus Hepatitis B is one of the most important infectious diseases in
the world. Approximately 350 million people worldwide are chronic carriers of the
hepatitis B virus (HBV), which accounts for approximately one million deaths annually.
PLGA microspheres loaded with recombinant HBsAg were formulated using
a double - emulsion technique. The pharmaceutical characteristics of size, surface
morphology, protein loading effi ciency, antigen integrity, release of HBsAg - loaded
PLGA microspheres, and degradation of the polymer in vitro were evaluated [234 –
237] . Based on these fi ndings in vitro and in vivo, it was concluded that HBsAg was
successfully loaded into the PLGA microspheres, which can autoboost an immune
response, and the HBsAg - loaded PLGA microsphere is a promising candidate for
the controlled delivery of a vaccine.
5.2.6.2 Proteins
Prolidase Defi ciency of this enzyme results in chronic intractable ulcerations of
the skin, particularly of lower limbs, since it is involved in the fi nal stages of protein
catabolism. To counteract the problem, the enzyme was encapsulated in PLGA
microspheres by a double - or multiple - emulsion technique, in vitro and ex vivo
evaluations were done, and the results indicated that microencapsulation stabilizes
the enzymatic activity inside the PLGA microspheres resulting in both in vitro and
ex vivo active enzyme release, hence opening the doors for the possibility of enzyme
replacement therapy through microencapsulation [238] . Further evaluation from
the same research group for prolidase - loaded PLGA microspheres is reported
elsewhere [239, 240] .
Insulin Insulin is the most important regulatory hormone in the control of glucose
homeostasis. The World Health Organization (WHO) has indicated that more than
50 million people around the world suffer from diabetes and require daily parenteral
injections of insulin to stay healthy and live normally. For the treatment of type
I diabetes insulin still is number one, with three subcutaneous injections to be taken
per day. A controlled - release system for a long - term therapy of this disease is the
need of the hour, as this can obviate the need for painful injection given a number
of times to the diabetes patients. Insulin was encapsulated in blends of poly(ethylene
glycol) with PLA homopolymer and PLGA copolymer by a w/o/w multiple -
emulsion technique with entrapment effi ciencies up to 56 and 48% for PLGA/ PEG
and PLA/ PEG, respectively [12] . Insulin - loaded microspheres were capable of
controlling the release of insulin for 28 days with in vitro delivery rates of 0.94 and
0.65 . g insulin/mg per particle per day in the fi rst 4 days and steady release with a
rate of 0.4 and 0.43 . g insulin/mg per particle per day over the following 4 weeks,
respectively, along with the extensive degradation of PLGA/ PEG microspheres as
compared to PLA/ PEG blends which resulted in a stable particle morphology along
with reduced fragmentation and aggregation of associated insulin.
Two types of injectable cationized microspheres were prepared based on a native
gelatin (NGMS) and aminated gelatin with ethylenediamine (CGMS) to prolong
the action of insulin [241] . Release of rhodamin B isothiocyanate insulin from
CGMS was compared with that from NGMS under in vitro and in vivo conditions.
Lower release of insulin from CGMS compared with that from NGMS was caused
by the suppression of initial release. The disappearance of 125 I - insulin from the injection
site after intramuscular administration by NGMS and CGMS had a biphasic
profi le in mice. Almost all the 125 I - insulin had disappeared from the injection site
one day after administration by NGMS. The remaining insulin at the injection site
after administration by CGMS was prolonged, with approximately 59% remaining
after 1 day and 16% after 14 days. The disappearance of CGMS from the injection
site was lower than that of NGMS. However, the difference in these disappearance
rates was not great compared with those of 125 I - insulin from the injection site by
NGMS and CGMS. The time course of disappearance of 125 I - CGMS from the injection
site was similar to that of 125 I - insulin by CGMS. The initial hypoglycemic effect
was observed 1 h after administration of insulin by NGMS, and thereafter its effect
rapidly disappeared. The hypoglycemic effect was observed 2 – 4 h after administration
by CGMS and continued to be exhibited for 7 days. The prolonged hypoglycemic
action by CGMS depended on the time profi les of the disappearance of insulin
from muscular tissues, which occurs due to the enzymatic degradation of CGMS.
A novel controlled - release formulation was developed with PEGylated human
insulin encapsulated in PLGA microspheres that produces multiday release in vivo
[242] . The insulin is specifi cally PEGylated at the amino terminus of the B chain
with a relatively low molecular weight PEG (5000 Da). Insulin with this modifi cation
retains full biological activity but has a limited serum half - life, making microencapsulation
necessary for sustained release beyond a few hours. PEGylated insulin can
be codissolved with PLGA in methylene chloride and microspheres made by a
single o/w emulsion process. Insulin conformation and biological activity are preserved
after PEGylation and PLGA encapsulation. The monolithic microspheres
have inherently low burst release, an important safety feature for an extended -
release injectable insulin product. In PBS at 37 ° C, formulations with a drug content
of approximately 14% show very low ( < 1%) initial release of insulin over one day
and near - zero - order drug release after a lag of three to four days. In animal studies,
PEG - insulin microspheres administered subcutaneously as a single injection produced
< 1% release of insulin in the fi rst day but then lowered the serum glucose
levels of diabetic rats to values < 200 mg/dL for approximately nine days. When the
doses were given at seven - day intervals, steady - state drug levels were achieved after
only two doses. PEG - insulin PLGA microparticles show promise as a once - weekly
dosed, sustained - release insulin formulation.
Shenoy et al. [243] developed an injectable, depot - forming drug delivery system
for insulin based on microparticles technology to maintain constant plasma drug
concentrations over a prolonged period of time for the effective control of blood
sugar levels. Formulations were optimized with two well - characterized biodegradable
polymers, namely PLGA and poly - . - caprolactone, and evaluated in vitro for
PEPTIDE/PROTEIN ENCAPSULATED INTO BIODEGRADABLE MICROSPHERES 425
426 BIODEGRADABLE POLYMER-BASED MICROSPHERES
physicochemical characteristics, drug release in phosphate - buffered saline (pH 7.4),
and evaluated in vivo in streptozotocin - induced hypoglycemic rats. With a large
volume of internal aqueous phase during a w/o/w double - emulsion solvent evaporation
process and high molecular weight of the polymers used, they could not achieve
high drug capture and precise control over subsequent release within the study
period of 60 days. However, this investigation revealed that upon subcutaneous
injection the biodegradable depot - forming polymeric microspheres controlled the
drug release and plasma sugar levels more effi ciently than plain insulin injection.
Preliminary pharmacokinetic evaluation exhibited steady plasma insulin concentration
during the study period. These formulations, with their reduced frequency of
administration and better control over drug disposition, may provide an economic
benefi t to the user compared with products currently available for diabetes
control.
Interferon a2a ( IFN a2a) Interferon .2a is indicated for the treatment of adults
with chronic hepatitis C virus infection who have compensated liver disease and
have not been previously treated with interferon . . To improve the stability and
loading effi ciency of protein drugs, a new microsphere delivery system comprises
calcium alginate cores surrounded by PELA [poly - D,L - lactide - poly - (ethylene
glycol)]. Recombinant IFN .2a as a model drug was entrapped within calcium alginate
cores surrounded by PELA by a w/o/w multiple - emulsion technique [244] .
Core - coated microspheres stabilized the IFN in the PELA matrix. The core - coated
microspheres indicated high encapsulation effi ciency and biological retention as
compared to conventional PLGA microspheres. The extent of burst release reduced
to 14% in core - coated microspheres from 31% in conventional microspheres, indicating
a new approach for water - soluble macromolecular drug delivery.
5.2.7 CONCLUSION
From this chapter, it has become apparent that a number microencapsulation
methods are available today for the preparation of microspheres on an industrial
scale. In fact, parenteral drug delivery systems based upon biodegradable microspheres
are a true success story for the concept of drug delivery. However, the production
of biodegradable microspheres containing a stable therapeutic peptide or
protein still remains a major challenge in terms of technical obstacles. Ideally, peptides/
proteins of therapeutic interest should be studied case by case, so as to bring
to the fore processing steps and stress factors which damage them. Continued efforts
to establish methods for stable protein, especially antigen, delivery from microspheres
may hopefully pave the way for future microsphere - based vaccines. Areas
of further research should focus on the performance of peptide/protein - loaded
microspheres under in vitro and in vivo conditions. Interestingly, the addition of
medium - chain triglycerides (MCT) modifi es/shifts the triphasic release pattern of
leuprolide acetate - loaded PLGA microspheres to a more continuous release in vitro
[245] . Alternatively, BSA - loaded PLGA microspheres were coated with a thermosensitive
gel, Pluronic F127 (PF127) [246] . The results demonstrated that PF127,
which gelled at 37 ° C, inhibited the initial burst release of BSA from microspheres
effectively. It is anticipated that more efforts will be invested in the future to develop
REFERENCES 427
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attain a more continuous release. In addition, an in vitro release model mimicking
the fate of biodegradable microspheres applied through the parenteral route would
be highly desirable. Also, new strategies to stabilize proteins in microspheres during
manufacturing, shelf life, or in vivo could be of general interest. Moreover, the use
of analytical techniques such as FTIR or MALDI - TOF mass spectrometry certainly
constitutes a step forward for protein analysis in more appropriate conditions.
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with PLGA microspheres , J. Pharm. Sci ., 91 , 1020 – 1035 .
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( 1997 ), Controlled release microparticles as a single dose hepatitis B vaccine: Evaluation
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238. Genta , I. , Perugini , P. , Pavanetto , F. , Maculotti , K. , Modena , T. , Casado , B. , Lupi , A. ,
Iadarola , P. , and Conti , B. ( 2001 ), Enzyme loaded biodegradable microspheres in vitro
ex vivo evaluation , J. Controlled Release , 77 , 287 – 295 .
239. Perugini , P. , Genta , I. , Pavanetto , F. , Modena , T. , Maculotti , K. , and Conti , B. ( 2002 ),
Evaluation of enzyme stability during preparation of polylactide -co - glycolide microspheres
, J. Microencapsul ., 19 , 591 – 602 .
240. Lupi , A. , Perugini , P. , Genta , I. , Modena , T. , Conti , B. , Casado , B. , Cetta , G. , Pavanetto ,
F. , and Iadarola , P. ( 2004 ), Biodegradable microspheres for prolidase delivery to human
cultured fi broblasts , J. Pharm. Pharmacol ., 56 , 597 – 603 .
241. Morimoto , K. , Chono , S. , Kosai, T. , Seki, T. , and Tabata, Y. (2005), Design of novel injectable
cationic microspheres based on aminated gelatin for prolonged insulin action ,
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242. Hinds , K. D. , Campbell , K. M. , Holland , K. M. , Lewis , D. H. , Pich e , C. A. , and Schmidt ,
P. G. ( 2005 ), PEGylated insulin in PLGA microparticles. In vivo and in vitro analysis ,
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243. Shenoy , D. B. , D ’ Souza , R. J. , Tiwari , S. B. , and Udupa , N. ( 2003 ), Potential applications
of polymeric microsphere suspension as subcutaneous depot for insulin , Drug Dev. Ind.
Pharm ., 29 , 555 – 563 .
244. Zhou , S. , Deng , X. , He , S. , Li , X. , Jin , W. , Wei , D. , Zhang , Z. , and Ma , J. ( 2002 ), Study on
biodegradable microspheres containing recombinant interferon - alpha - 2a , J. Pharm.
Pharmacol ., 54 , 1287 – 1292 .
245. Luan , X. , and Bodmeier , R. ( 2006 ), Modifi cation of the tri - phasic drug release pattern
of leuprolide acetate - loaded poly(lactide - co - glycolide) microparticles , Eur. J. Pharm.
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246. Wang , Y. , Gao , J. Q. , Chen , H. L. , Zheng , C. H. , and Liang , W. Q. ( 2006 ), Pluronic F127
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61 , 367 – 368 .
443
5.3
LIPOSOMES AND DRUG DELIVERY
Sophia G. Antimisiaris, 1 Paraskevi Kallinteri, 2 and
Dimitrios G. Fatouros 3
1 School of Pharmacy, University of Patras, Rio, Greece
2 Medway School of Pharmacy, Universities of Greenwich and Kent, England
3 School of Pharmacy and Biomedical Sciences, Portsmouth, England
Contents
5.3.1 Introduction
5.3.2 Liposome Structure and Characteristics
5.3.2.1 Phospholipids: Structure Stability and Characterization of Lipid
Membranes
5.3.2.2 Physicochemical Properties of Liposomes
5.3.2.3 Preparation of Liposomes
5.3.2.4 Functionalization of Liposomes
5.3.3 In Vivo Distribution
5.3.3.1 Conventional Liposomes
5.3.3.2 Long - Circulating or PEGylated Liposomes
5.3.3.3 Other Routes of Administration
5.3.4 Applications of Liposomes in Therapeutics
5.3.4.1 Anticancer Drug Delivery
References
5.3.1 INTRODUCTION
Liposomes are vesicles in which an aqueous volume is entirely surrounded by a
phospholipid membrane and their size can range between 30 and 50 nm up to
several micrometers. They can consist of one (unilamellar) or more (multilamellar)
homocentric bilayers of amphipathic lipids (mainly phospholipids). Based on their
lamellarity (number of lamellae) — and size — they are characterized as SUVs/LUVs
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
444 LIPOSOMES AND DRUG DELIVERY
(small or large unilamellar vesicles) or MLVs (multilamellar vesicles). MLV liposomes
are always large (at least cannot be considered small) and aqueous spaces
exist in their center and also between their bilayers.
Liposomes were initially invented by Alec Bangham [1] to serve as a model for
cell membranes in biophysical studies. In the 1970s they started to be investigated
as promising drug carriers [2, 3] . The main advantages of liposomes as a drug delivery
system are the following: (i) They have a very versatile structure which can be
easily tairored in order to bear the properties needed for each specifi c application.
(ii) They can accommodate any type of drug molecules either in their aqueous
compartments (hydrophilic drugs) or in their bilayers (lipophilic drugs) or both
(amphiphilic drugs). (iii) Last, but not least, they are nontoxic, nonimmunogenic,
and fully biodegradable. Early attempts to use liposomes as a drug delivery system
revealed the main limitation of the system and resulted in disappointment due to
the fast nonspecifc clearance of liposomes from circulation by reticuloendothelial
system (RES) cells [4] . However, it was realized that recognition by the RES macrophage
system could be useful in antigen presentation, macrophage activation or
killing, and elimination of parasitic infections (what is called passive targeting).
From then on many different types of liposomes or methods to obtain longer circulation
half - lives have been successfully invented by controlling the physicochemical
characteristics of lipid bilayers and their interaction with the biological
environment. Some of the basic methods to control the properties of liposomes are
presented in Table 1 . Today (Figure 1 ), several different liposome types are available,
mainly conventional, long circulating (or “ stealth, ” sterically stabilized, or PEGylated),
targeted [or ligand bearing or immunoliposomes (when antibodies are used as
targeting ligands)], cationic (for genetic material delivery), and deformable or
elastic (or “ transferosomes ” ) [will be discussed in the paragraph about of skin
delivery in Section 5.3.3.3 ]. As a consequence of these advances in liposome technology,
a very broad range of liposome applications for drug delivery are being explored,
some of which have resulted in life - saving products on the market or under late -
stage clinical testing.
In this chapter we will provide information about the basic characteristics of
liposomes staring from their building blocks, that is, phospholipids. After this, liposome
structure, physicochemical properties, and stability, which are most important
for their in vivo performance, will be discussed as well as methods used for liposome
preparation, characterization, and stabilization. Following this fi rst part which is
more technological, we will move into the biological part and talk about the fate of
conventional liposomes and sterically stabilized liposomes, as well as liposomal
drugs, after in vivo administration by different routes [mainly intravenous (i.v.),
intraperitoneal (i.p.), or subcutaneous (s.c.)] and also give some information about
other possible routes for in vivo administration of liposomes. Finally, specifi c applications
of liposomes in therapeutics will be presented, some in more detail, mainly
for the therapy of different types of cancer.
5.3.2 LIPOSOME STRUCTURE AND CHARACTERISTICS
The main building blocks of most liposomal drug formulations are phospholipids.
This chapter will start with an introduction of phospholipid structure and briefl y
TABLE 1 Methods and Results of Modifying Physicochemical Properties of Liposomes
Properties Method Result
Physicochemical properties
Size distribution Sonication, extrusion,
microfl uidization
Control of circulation time,
Increased extravasation
Membrane
permeability
Lipid composition modifi cation
(cholesterol, thermotropic
transitions)
Increased liposome stability,
proton or temperature - induced
sensitivity
Tendency for
aggregation
or fusion
Lipid composition modifi cation,
addition of cations
Formation of structures (as
cochleates) that can carry
antigens, deoxyribonucleic acid
(DNA), vaccine formulations
Surface
hydrophilicity
Steric stabilization by grafting
hydrophilic molecules on
liposome surface (linear
dextrans [5] , sialic acid –
containing gangliosides [6] ,
lipid derivatives of hydrophilic
polymers [as PEG [7, 8] , poly -
N - vinylpyrrolidones [9] , and
polyvinyl alcohol [10] )
Increase of circulation time,
modifi cation of
pharmacokinetics and tissue
dispotition of liposomes and
encapsulated drugs
Drug
encapsulation
effi ciency
Active or remote loading [11] Stable liposome encapsulation of
drugs at high drug - to - lipid
ratios, Many applications in
drug delivery
Elasticity,
rigidity
Introduction of detergents or
edge activators and “ skin
lipids ” (as ceramides) in
liposome preparations [12]
Increased skin penetration and
retention in skin which results
in increased transdermal
absortption of liposomal drugs
Surface
properties,
liposome
stability, and
other properties
Addition of spefi fi c ligand on
liposome surface, together
with steric stabilization
Increased potential for effi cient
targeting
describe the most important (for liposomes) physichochemical characteristics of
lipids and lipid membranes. Afterward properties of liposomes and fi nally methods
used for the preparation of liposomal drug formulations will be discussed.
5.3.2.1 Phospholipids: Structure Stability and Characterization
of Lipid Membranes
Phospholipids are naturally occurring biomacromolecules that play an important
role in the physiology of humans as they serve as structural components of biological
membranes and support organisms with energy [13] . They are amphiphilic molecules
with poor aqueous solubility and typically consist of two parts: a water - soluble
group, the so called polar head, and an insoluble one, the backbone (Figure 2 ). The
polar head group contains hydroxyl groups which are responsible for the surface
LIPOSOME STRUCTURE AND CHARACTERISTICS 445
446 LIPOSOMES AND DRUG DELIVERY
charge of the lipids that can be positively or negatively charged, zwitterionic, or
noncharged. During liposome formation, these molecules arrange themselves by
exposing their polar parts toward the water phase, while the hydrocarbon moieties
(hydrophobic) adhere together in the bilayer. Two classes of lipids are mainly used
for liposome preparation: double - chain polar lipids and sterols (mainly cholesterol).
Such lipids form bilayers, in contrast to single - chain lipids (e.g., short - chain
phosphatidylcholine) that form micelles, upon their dispersion in water. [14] .
Double - chain lipids are either naturally occurring or synthesized in the laboratory.
They consist of glycerol or sphingosine and a polar head containing a phosphor -
or glyco - group. Glycerophopsholipids or phospholipids are the most popular, among
the other lipids, for the preparation of liposomal dispersions [15] . Phosphatidylcho-
Conventional PEGylated or
Longcirculating
Targeted or
immunoliposomes
Cationic
Phospholipid
PEGlipid
Cationic lipid
Helper lipid
Antibody or
targeting ligand
FIGURE 1 Liposome types. Conventional liposomes are composed of phospholipids that
form bilayers enclosing an aqueous compartment. Cholesterol may be included in the bilayer
to increase membrane rigidity. Hydrophilic drugs can be encapsulated in the aqueous interior
of the vesicles and lipophilic drugs can be included in their membranes. Pegylated or long -
circulating liposomes have a surface coating of polyethylene glycol (PEG) molecules that
permits liposomes to escape opsonization (coating with plasma proteins — opsonins — that
make liposomes visible by RES macrophages). PEG - conjugated lipids are used for the preparation
of this type of vesicle. Targeted liposomes or immunoliposomes are liposomes that in
addition to a PEG coating (in most cases) have targeting moiety on their surface that directs
them to the preferred target. This targeting moiety may be a sugar (i.e., galactose, to target
cells with galactose receptors on their membranes) or other type of molecule or an antibody
(usually monoclonal antibody), in which case the liposomes are characterized as immunoliposomes.
Cationic liposomes are vesicles that consist of positively charged lipids (cationic
lipids) which may form complexes with negatively charged DNA molecules and thus are used
for gene delivery or targeting applications. For their preparation an additional lipid (helper
lipid) is usually required. Cationic liposomes can also have PEG molecules on their surface
(for longer circulation in the bloodstream) and/or targeting moieties. The last type of liposome
is the so called transformable liposome or elastic liposome . ( structure is presented and
explained in Figure 10 ).
line (PC) or lecithin, one of the main components of the liposomal bilayer, belongs
to this group [1] . Phosphatidylcholine is zwitterionic in the range of physiological
pH [16] and can be found in egg yolk. Other naturally occurring glycerophospholipids
are the following: phosphatidylethanolamine (PE), which is isolated from
brain lipids and is zwitterionic; phosphatidylserine (PS), which is found in bovine
brains and posseses a negative charge; cardiolipin (CL), which is isolated from heart
tissue or mitochondrial membranes and is negatively charged; phosphatidylglycerol
(PG), which can be found in mitochondria or chloroplasts of mammalian cells and
is negatively charged; and fi nally phosphatidylinositol (PI), a negatively charged
lipid found in mammalian tissues [13] .
Another group of naturally occurring lipids with applications in liposome technology
is comprised of the sphingophospholipids (mainly sphingomyelin) which are
derivatives of ceramides [17] . Sphingomyelin (SM) is found in the outer leafl et of
plasma membranes [17] and has many similarities with PC since they both have the
same zwitterionic polar group and two hydrophobic acyl chains.
Custom - made lipids can be produced by de - or reacylation of natural lipids. Commonly
used phospholipids with polar heads containing myristoyl (14 : 0), palmitoyl
(16 : 0), stearoyl (18 : 0) fatty acids are all classifi ed by four - letter abbreviations, for
example, DMPC, where DM stands for the number and type of fatty acids (di - myristoyl)
and PC for the type of polar head (phosphatidylglycero - choline), and similarly
DPPC and DSPC (Figure 2 ).
Positively charged lipids are capable of making complexes with deoxyribonucleic
acid (DNA) (since it is negatively charged) and are currently very popular.
Examples of such lipids are: N - [1 - (2,3 - dioleyloxy)propyl] - N , N , N - trimethylammonium
chloride (DOTMA), 1,2 - dioleoyl - 3 - trimethylammoniopropane (DOTAP),
FIGURE 2 Typical phospholipid structure.
Polar head
(hydrophilic)
O– O–
O
O O
O O O
O O
O
–O O –O
O
O P P
Backbone
(lipophilic)
H2C
H2C H2C
H2C CH2 CH2
HC
HC
C C C C
H2C H2C
LIPOSOME STRUCTURE AND CHARACTERISTICS 447
448 LIPOSOMES AND DRUG DELIVERY
and dicetyl phosphate (DCP), and dioctadecyldimethylammonium bromide
(DODAB).
The last group of amphiphiles contains sterols that are present in the membranes
of cells. The most popular among them is cholesterol (Chol), which can be easily
incorporated in lipid bilayers, increasing their rigidity and making them less permeable,
due to the interactions taking place with phospholipids in lipid membranes
which result in modifi cation of the lipid acyl - chain conformation.
Recently polyethyleneglycol (PEG, of varying molecular weight) – lipid conjugates
have become commercially available and are frequently used in liposome
applications. Aditionally, functionalized phospholipids exist for the covalent or noncovalent
attachment of proteins, peptides, or drugs to the liposome surface. Most of
these lipids fall into three major classes of functionality: Conjugation through amide
bond formation, disulfi de or thioether formation, or biotin/streptavidin binding.
Active lipids — mostly with anticancer activity — have also been added in liposome
membrane for production of active liposomes. Examples of such lipids are
ether lipids [18] and arsonolipids [19] .
Several techniques are employed for the physicochemical characterization of
lipid membranes, as summarized in Table 2 . Thermal analysis, mainly differential
scanning calorimetry (DSC), has been used extensively, offering information on the
thermodynamics of various types of liposomes. The phase behavior of lipid components
of membranes determines membrane fl uidity. Each lipid has a characteristic
lipid chain transition temperature, T m . Changes in the structure of lipids occur below
and above this temperature [20] . The temperature at which these changes occur
depends on the head group, the chain length, and the degree and type of unsaturation
of each lipid [21] . Using DSC studies in has been demonstrated that heat capacity
curves are affected by the size of vesicles [20, 22] , and can be modifi ed by
introduction of drugs [23, 24] or peptides [25, 26] in the lipid membranes (due to
interactions between incorporated molecules and lipids).
TABLE 2 Methods for Physicochemical Characterization of Lipid Membranes
Method Information References
Thermal analysis, mainly Membrane fl uidity 20 – 26
Differential scanning calorimetry
(DSC)
Lipid chain transition temperature, T m
Fluorescence spectroscopy Phase transitions 27, 28
Membrane dynamics 29, 30
Nuclear magnetic resonance (NMR) Polymorphism 31, 32
Lamellarity 33
Membrane dynamics 34, 35
Electron paramagnetic resonance
(EPR)
Fluidity of membranes 36
Liposomal internal volume 37
Membrane dynamics 38
Membrane – drug interactions 39
Fluorescence quenching Fusion processes 40, 41
X - ray diffraction Structural information; thickness of
the membrane and water layers
42 – 45
5.3.2.2 Physicochemical Properties of Liposomes
The in vitro and in vivo performance of liposomes is highly dependent on their structural
and surface properties. Liposome size and size distribution, surface charge (zeta
potential), and trapping effi ciency of the drug incorporated in the liposomes are
important parameters that should be measured when developing a liposomal drug
formulation. To obtain optimum performance of a liposomal preparation, parameters
infl uencing both the liposome and the drug need to be carefully considered during
early stages of development. In this chapter the most important physicochemical
properties of liposomes will be discussed in terms of the way they may affect liposomal
drug performance as well as the techniques used for their measurement.
Liposome Size Distribution Liposomes have to be smaller than the vascular pore
cutoff (380 – 780 nm) to extravasate and reach solid tumors [46] . Liposome size also
plays an important role in complement activation and RES clearance of liposomes
[47] and [48] . In general, vesicles that are larger than 100 nm require additional
strategies for preventing surface opsonization and prolonging their circulation half -
life (Table 1 ). Light scattering, fi eld fl ow fractionation, microscopy, size exclusion
chromatography, and turbidity are commonly used techniques for the physicochemical
characterization of liposomal dispersions.
Quasi - elastic light - scattering or photon correlation spectroscopy is the most
popular light - scattering technique. The Brownian motion of the particles causes
fl uctuations in the light intensity versus time. The hydrodynamic radius and the
polydispersity index of liposomes can be easily determined from these studies [49 –
51] . Field fl ow fractionation is another approach to measure the particle size and
the surface charge of liposomes [52 – 54] . The liposomes are exposed to a perpendicular
fi eld under laminal fl ow and their size and mass distribution can be determined.
Size exclusion chromatography is a simple method to determine the size
distribution of liposome dispersions [13, 55] , especially on a routine basis, if other
sophisticated equipment is not available. Samples with high heterogeneity are suitable
to be analyzed with this method [55] . The selection of the proper gel, pretreatment
of the column with sonicated vesicles to avoid any loss of material by adsorption,
and use of isotonic buffer to avoid osmotic shock and frequent column calibration
can secure the reproducibility of the method [13, 55] .
Turbidity is a spectroscopic technique determining the optical density of colloidal
particles. A wavelength between 350 and 500 nm is the fi rst choice for such studies
[56] . Turbidity measurements can offer important information on the kinetics of
membrane – surfactant interactions since membrane solubilization changes refl ect
changes to the optical density of the dispersion [57 – 60] . It is also widely used as a
technique to investigate liposome aggregation and fusion [61] . However the exact
particle size of liposomes cannot be determined using turbidity techniques.
Optical microscopy is applicable for LUVs, MLVs, and especially giant liposomes
[62, 63] . For instance, mechanical properties of the liposomal membrane can be
studied by combining optical microscopy and a micropipette technique [64] . Electron
microscopy can give information about both the morphology and homogeneity
of liposomes. Quantifi cation in terms of number can be carried out measuring at
least 300 particles from different images [65] . Transmission electron microscopy
(TEM) is a powerful technique since magnifi cations up to 200,000 and a resolution
of approximately 1 nm can be achieved. Negative staining is a quite common
LIPOSOME STRUCTURE AND CHARACTERISTICS 449
450 LIPOSOMES AND DRUG DELIVERY
technique for visualization studies. A small amount of the sample is dried on a grid
coated with carbon fi lm. Then the fi lm is coated with an electron - dense solution
(e.g., tungsten molybdate). However the technique suffers from some drawbacks,
such as artifacts due the fi xation process or the extraction of lipid material by
embedments [66, 67] . With freeze - fracture electron microscopy (FFEM) samples are
quickly frozen and fractured. Compared with negative staining technique, FFEM
has the advantage of preserving water - dependent lipid phases because no dehydration
steps are involved. Therefore phase transitions, lipid polymorphism, or fusion
processes can be visualized with this approach [68 – 71] . Recently cryogenic transmission
electron microscopy (cryo - TEM) has been employed for visualization studies.
With this technique very precise morphological assessment of liposome interior
FIGURE 3 Morphology of liposome of liposome by cryo - TEM (transmission electron
microscopy). ( a ) Empty liposomes (liposomes with entrapped unbuffered CuSO 4 in the
absence of drug). ( b ) Topotecan - encapsulating liposomes (drug was added to the empty
liposomes of ( a ) to achieve a fi nal drug - to - lipid ratio of 0.2 mol/mol, and the system was
incubated at 20 ° C). ( Reproduced from ref. 72 with permission of Elsevier .)
(a)
(b)
(Figure 3 ) as well as liposome surface (Figure 4 ) can be carried out as recently
demonstrated for Copper - Topexan encapsulating [72] and transferring - coated liposomes
[73] , respectively. Compared with the previous techniques the main advantage
of cryo - TEM is avoidance of any fi xation of the grid, which can create artifacts
induced by staining and thus keeps the sample close to the original state [74, 75] .
The samples for the cryo - TEM studies are prepared in a controlled environment
vitrifi cation system (CEVS). A small amount of the sample is placed on a grid - supported
fi lm. The grid is quenched in liquid ethane and it is vitrifi ed. Then the samples
are characterized with a TEM microscope. In a manner similar to the previous discussion,
lipid polymorphism or fusion can be investigated [61, 76 – 79] . Finally scanning
probe microscopy (SPM) including atomic force microscopy (AFM) has been
recently applied in the liposome fi eld [73, 80] . Some of the advantages of the technique
are the high resolution in atomic dimensions (Figure 5 [73] ), the production
of three - dimensional images with high resolution, and the versatility of the operation
conditions (vacuum air liquid). Aditionally, sample preparation does not involve
any staining, freezing, embedding, or fracturing procedures.
Surface Charge of Liposomes The electrical properties of liposomal surfaces can
infl uence the physical stability of liposomal dispersions during storage as well as the
behavior of liposomes in the biological milieu and their interaction with cells
[47, 81] .
Microelectrophoresis is used to measure the electrophoretic mobility or, in other
words, the movement of liposomes under the infl uence of an electric fi eld. From the
electrophoretic mobility the electrical potential at the plane of shear or . (zeta)
potential can be determined (by the Helmoholtz – Smoluchowski equation). From
the zeta potential values the surface charge density ( . ) can be calculated.
Aggregation of liposomes both in vitro and in vivo is one of their main stability
problems. According to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory,
or theory of colloidal stability, a colloidal system is stable if the electrostatic repulsion
forces between two particles are larger than the attraction van der Waals forces.
Therefore charged liposomal formulations are highly desirable. Manipulation of
FIGURE 4 Morphological assessment of liposome surface coating using cryo - TEM. Liposomes
that have been conjugated with transferrin display small particles on their surface ( a ),
while control preparations appear to be smooth and undecorated ( b ). ( Reproduced from ref.
73 with permission of Elsevier .)
(a) (b)
LIPOSOME STRUCTURE AND CHARACTERISTICS 451
452 LIPOSOMES AND DRUG DELIVERY
liposome surface renders them stable against self - aggregation or nonspecifi c interactions,
and this can be achieved by introducing other molecules on the surface,
which may be natural molecules such as glycolipids [82, 83] or antibodies [84] or
lectins [85] (that are usually grafted on the liposome surface by chemical linking).
As also mentioned in the introduction, the RES has been the “ Achilles heel ” to
the delivery of liposomes by injection to the blood stream because of their rapid
FIGURE 5 Morphological assessment of liposome surface coating using atomic force
microscopy (AFM). AFM images of liposomes prepared with DSPE – PEG 200 – COOH.
( a ) Plain liposomal formulations. The liposomes show a smooth surface morphology. ( b )
Liposomes covalently modifi ed with transferring. Small globular structures are visible at the
surface. ( Reproduced from ref. 73 with permission of Elsevier .)
(a)
(b)
250 nm
250 nm
uptake by the macrophages of the RES. Grafting of liposome surface with hydrophilic
polymers has been used successfully as a method to protect the liposomes
with a steric barrier which inhibits the adsorption of blood components [7, 86, 87] .
It has been shown that sterically stabilized liposomes have signifi cantly lower zeta
potential values compared with conventional liposomes [87] , indicating that the
presence of PEG might shift the plane of shear away from the phosphate moieties.
Thereby, zeta - potential modifi cation can serve as proof of successful coating of
liposomes by polymers.
Drug Loading Effi ciency and Techniques The amount of drug incorporated (for
amphiphilic or lipophilic drugs) or encapsulated (for hydrophilic drugs) is a very
important parameter that determines largely the selection of liposome type and
components. Terms used to quantitate drug loading are drug – lipid (mol/mol) ratio
(or D/L) and trapping or encapsulation effi ciency (which is the percent D/L in the
fi nal liposomal formulation compared to the initial D/L used for liposome preparation).
In many cases, the percent encapsulation is mentioned (percentage of drug
encapsulated in relation with the amount of drug offered for encapsulation during
liposome preparation); however, this number is practically useless and misdirecting,
since it highly depends on the initial amount of drug offered to the specifi c lipid
quantity, while the amount of lipid is not quantifi ed.
Several parameters infl uence the encapsulation of drugs into liposomes. The
types of vesicle and drug used play signifi cant roles in the percentage of encapsulation.
Hydrophobic drugs have higher loading effi ciency in MLVs since they consist
of high numbers of bilayers and low aqueous volumes [88 – 91] . In contrast, hydrophilic
drugs have higher encapsulation in LUVs [91, 92] . Generally, for hydrophilic
drugs, the percentage of encapsulation increases in the order of SUV . MLV <
LUV [92] .
The method used for liposome preparation has a signifi cant impact on drug
loading as well. Larger surface areas for the formation of thin fi lms are preferable
since they facilitate the hydration process of the bilayer, as demonstrated for doxorubicin
(DOX) using fi ve different hydration protocols [93] . The highest encapsulation
was achieved when a thin fi lm with large surface area was formed. Glass beads
can be used for this purpose. From 8 to 10 times higher values for hydrophobic drug
encapsulation were obtained after the addition of such beads [94] . The type of
hydration (conditions and media used) also infl uences drug loading.
Drug encapsulation and stability of liposomes are also affected by the length of
the acyl chain and the degree of saturation of the lipids used for their formation.
As the acyl chain length of the lipid increases, so does the partitioning of hydrophobic
drugs in the lipid membrane [95] , as demonstrated for atenolol and propranolol
in MLVs and SUVs [96] . The impact of the head group of different lipids used for
the preparation of liposomes on encapsulation of citicoline was investigated by
Puglisi et al. [97] . The fl uidity of the membrane has also been demonstrated to infl uence
encapsulation of different compounds [98 – 100] .
When galactocerebroside was incorporated into liposomes, the percentage of
encapsulation of mintoxantrone increased proportionally to the amount of glycolipid
present in the membrane [101] . Numerous studies have demonstrated the
impact of vesicle surface charge on drug loading effi ciency, which is important for
charge - bearing molecules. Encapsulation of hydroxycobalamin [102] and doxorubicin
[103] was higher in negatively charged liposomes compared to neutral ones. In
LIPOSOME STRUCTURE AND CHARACTERISTICS 453
454 LIPOSOMES AND DRUG DELIVERY
a similar manner, higher amounts of calcitonin were encapsulated in positively
charged compared to neutral liposomes [104] while the highest loading of indomethacin
in liposomes was obtained in the following order: positively charged >
negatively charged > neutral liposomes [105] .
Cholesterol and . - tocopherol are used quite often to increase the rigidity and
stability of liposomal membranes [88, 106 – 108] . In most cases cholesterol appears
to improve the encapsulation of both hydrophilic and hydrophobic compounds.
However, if the drug is lipophilic and partitions in the liposome membrane, there
is a good chance that it might be displaced by adding increasing amounts of cholesterol
in the bilayer (as observed in the case of dexamethasone encapsulating liposomes
in our laboratory).
A novel system for enhancing the drug loading of lipophilic drugs combining
liposomes and cyclodextrin – drug complexes by forming drug - in - cyclodextrins -
in - liposome preparations has been proposed [109 – 111] . Cyclodextrins (CDs) are
hydrophobic, cavity - forming, water - soluble oligosaccharides that can accommodate
water - insoluble drugs in their cavities, increasing their water solubility. The basic
intention is to encapsulate a stable water - soluble drug – cyclodextrin complex in the
aqueous compartments of liposomes (Figure 6 ). As will be discussed below, this
system can also serve as a method to increase the retention of lipophilic drugs in
diluted liposome dispersions.
Finally, remote loading and active loading [11] are other methods used to achieve
high trapping effi ciency in liposome formulations, but unfortunately they can only
be applied to a small number of drugs with specifi c physicochemical properties. This
technique will be discussed below in Section 5.3.2.3 .
= =
Lipid bilayer
Aqueous interior
Drug–CD
complex
(High aqueous solubility)
CD Drug
(Low aqueous solubility)
FIGURE 6 Representation of drug in – cyclodextrin in liposome technique for encapsulation
of lipophilic drugs in aqueous interior of vesicles. Drug molecules have low aqueous
solubility and thus cannot be encapsulated in the aqueous compartment of the vesicle.
However, the drug – cyclodextrin complex has high aqueous solubility and can thus be encapsulated
in high concentrations in the vesicles.
Stability of Liposomes A shelf life of at least two years is requested for pharmaceutical
products. Therefore, chemical stability and physical stability are important
parameters for the overall performance of liposomal formulations. Additionaly,
another very important factor is the retention of encapsulated drug.
Several studies have suffi ciently addressed the chemical degradation of liposomes
during storage [112 – 115] . This is due to the hydrolysis of the phospholipids
to fatty acids and 1 - and 2 - acyl - lysophospholipids. Further hydrolysis leads to the
production of glycerol phospho compounds. Antioxidants ( . -tocopherol), complexing
agents [e.g., ethylenediaminetetraacetic acid (EDTA)], and inert atmosphere
(e.g., nitrogen) are most commonly used to overcome this problem. Moreover, the
presence of . - tocopherol can reduce the auto - oxidation of lipids, which is usually
induced by light, metal ions, and temperature. Prevention of the chemical decomposition
of the lipids by adding . - tocopherol in liposomal dispersions can increase
the shelf life of liposomes [116] . Furthermore, the coexistence of cholesterol and . -
tocopherol in the lipid bilayer can improve the antioxidant activity of tocopherol
[117] .
An alternative to circumvent problems related to the chemical decomposition
of liposomes is their storage in the dry state (freeze dried). However, the protection
against damage by freezing (cryoprotection) [118 – 120] and the protection
against damage by dehydration (lyoprotection) [121, 122] require special attention
for the proper storage of liposomes. Depending on the drug encapsulated in the
liposomes and possible interactions between drug molecules and components of
the lipid membrane, initial studies should be carried out in order to fi nd the proper
cryo - and lyoprotectant which preserves the integrity of the specifi c liposomal
formulation.
The physical stability of liposome dispersions is mainly related with possible
aggregation and leakage of the liposomal membrane. The size and surface charge
play a signifi cant role for the stability of liposomal dispersions, as has been discussed
in the previous paragraph.
In addition to physical stability, the retention of drug in liposomal formulations
is particularly important, not only during storage, but also during in vivo administration.
Especially when targeted or long - circulating liposomes are used, association
of the drug in the liposome carrier until the carrier reaches its biological target is
a prerequisite for achieving any therepeutic benefi t. However, in many cases,
although the amount of drug loaded in liposomes is initially high, for different
reasons most of the liposome - associated drug is rapidly released from the vesicles.
The main causes for such behavior are different according to the physicochemical
properties of the drug. For hydrophilic drugs: (i) The low integrity of liposome
membranes after in vivo administration and contact with blood compomnents,
which results in removal of some lipid molecules and concurrent opening of pores
in the liposomal bialyer through which the loaded drug molecules may leak out,
and (ii) the physical instability of liposomes that results in aggregation and fusion.
and fi nally release of drug from liposomes. For amphiphilic and lipophilic drugs ,
the main problem is caused by the dilution of liposome dispersions, which usually
occurs immediately after in vivo administration [by most routes, especially i.v.
(diluted in 4 L of blood)]. This results in release of drug (since the drug can permeate
membranes) until the drug saturates the full aqueous volume in which the
liposome vesicles are diluted.
LIPOSOME STRUCTURE AND CHARACTERISTICS 455
456 LIPOSOMES AND DRUG DELIVERY
For hydrophilic drugs, the problem can be solved by increasing the rigidity of the
liposome membrane while concurrently decreasing their tendency for aggregation,
after proberly selecting the liposome lipid components. However, for membrane -
permeable drugs, drug leaking upon dilution cannot be easily, if at all, confronted.
Theoretically, one method may be to increase the affi nity of the drug molecule with
the lipid membrane (and at the same time decrease its aqueus solubility) by chemical
modifi cation (conjugation to a lipid or fatty acid). However, this is neither easy
nor generally applicable. The association of liposomes with cyclodextrins has been
recently proposed as a method to ensure high and stable entrapment of lipophilic
drugs in aqueous compartments of liposomes [109 – 111, 123] (Figure 6 ), providing
that the drug has high affi nity for the cyclodextrin molecule and is not displaced
from the cyclodextrin cavity by components of the lipid membrane (mostly cholesterol).
In such cases, this approach may not increase drug retention, but encapsulation
of drug may be substantially improved [111] .
5.3.2.3 Preparation of Liposomes
Liposome Preparation Techniques In most cases, liposomes are named by the
preparation method used for their formation, Such as sonicated, dehydrated – rehydrated
vesicle (DRV), reverse - phase evaporation (REV), one step, and extruded.
Several reviews have summarized available liposome preparation methods [91, 124,
125] . Liposome formation happens spontaneously when phospholipids are dispersed
in water. However, the preparation of drug - encapsulating liposomes with high drug
encapsulation and specifi c size and lamellarity is not always an easy task. The most
important methods are highlighted below.
Thin Film Method It was in 1964 that Alec Bangham introduced the “ thin - fi lm
method ” for liposome preparation [1, 126] . Lipids are dissolved in organic solvents
(chloroform or mixtures with methanol) and the solvent is removed under a high -
vacuum rotor evaporator forming a thin fi lm on the walls of round - bottomed fl ask.
Depending on the phase transitions of the lipids used for the preparation of liposomes,
the aqueous phase for the rehydration should be prewarmed at temperatures
above the phase transition of the lipid. After addition of the aqueous phase the thin
fi lm is detached from the fl ask walls by agitation and a highly heterogeneous population
of MLVs is produced. Depending on the physicochemical properties of drugs,
they can be introduced either in the thin fi lm together with the lipids (lipophilic
compound) or in the rehydration solution (hydrophilic compound).
Sonication This approach uses energy (ultrasound) and can be applied to a dispersion
of MLVs [127] or to solid lipids mixed with aqueous solution. The fl ask with the
liposome dispersion is placed in a bath sonicator or a probe sonicator (tip) is immersed
in the tube containing the liposome dispersion. With the fi rst setup it is diffi cult to
reduce the size to the nanometer level since the energy produced by the bath sonicators
is rather low. However, it has the advantage that there is no contact with the liposome
dispersion. The position of the fl ask in the sonicator is equally important. It is
easy to understand if it is in the right place from the noise produced by ultrasound
waves. For instance, if foams are produced or there is no noise at all, that implies the
sample is misplaced and fi nally the size of vesicles will not be reduced.
In the second approach (probe sonicator), the size of the liposomes can be
reduced to nanometers and SUVs can be produced. As previously, the position of
the probe plays an important role on the ability to minimize vesicle size. Because
the energy produced from the transducer is high, overheating of the system is quite
common; therefore, a water bath fi lled with ice is recommended. After sonication,
fragments of Ti originated from the probe are scattered in the dispersion. Centrifugation
for 4 – 5 min at 10.000 rpm will cause sedimentation of these fragments, giving
clear liposome dispersions.
Injection Methods
Ethanol Injection Small unilamellar vesicles (with diameter of 30 nm) can be
prepared with the ethanol injection technique [128] . Lipids are dissolved in
ethanol and injected rapidly in the aqueous solution under stirring (fi nal concentrations
up to 7.5% (v/v) ethanol can be applied). The method is very easy,
having the advantage of avoiding chemical or physical treatment of lipids.
However, there is an extra step to remove ethanol and the concentration of
vesicles produced is rather low. Also encapsulation of hydrophilic drugs is also
low, due to the high volumes used.
Ether Injection The general principle of this method is the same as ethanol
injection. The only difference is that the lipid is injected slowly in the aqueous
solution that is warm [129] . Furthermore, the concentrations used in this case
are somewhat higher (up to 10 m M ) compared to the ethanol injection
approach.
Extrusion (Extruded Vesicles) The extrusion method, which today is very popular
for the production of homogenous vesicle samples of a predetermined size, was
introduced by the group of D. Papahadjopoulos [65] . Multilamellar vesicles are
extruded through fi lters with well - defi ned pores under pressure. Polycarbonate is
the most commonly used material for these membranes, which have pore sizes from
30 nm up to several micrometers. For lipids with high melting point, the extrusion
should be carried out above their phase transition temperature. The operating
volumes are from 1 to 50 mL with a liposome dispersion concentration up to 150 m M .
Generally, repeated extrusions reduce the number of lamellae and the produced
liposomes are mainly unilamellar. High pressure can cause disruption of membranes.
The reproducibility of the method is good; however, it is quite time consuming
and membrane rupture problems occur quite often.
French Press With this approach MLV liposomes are introduced in a cell and a
piston presses the dispersion [130] . Pressures up to 25,000 psi can be achieved and
SUVs are produced. The main disadvantage of this technique is that it is not applicable
for lipids with phase transition temperatures lower than 20 ° C and the concentration
of liposomes that can be used is relatively low (maximum 20 m M ).
Microfl uidization During microfl uidization, MLVs are circulated with a pneumatic
pump operating under high pressure through a prefi lter and then to the interaction
chamber [131] . From there, they are separated into two streams and they pass
through defi ned microchannels under high velocity to a heat exchanger which is
LIPOSOME STRUCTURE AND CHARACTERISTICS 457
458 LIPOSOMES AND DRUG DELIVERY
connected with a water bath. This is repeated many times until the size of the liposomes
is signifi cantly reduced. They can operate with volumes from 0.1 up to 10 L
of liposomes and with concentrations up to 300 m M , which is by far the highest
capacity from all other methods. Small unilamellar vesicles less than 100 nm can be
produced, but the population is not completely homogeneous.
Reverse - Phase Evaporation The REV method was developed by Szoka and Papahadjopoulos
[132] . Lipids are dissolved in organic solvent and the solvent is removed
with evaporation. The thin fi lm is resuspended in diethyl ether (1 mL solvent/mL
liposomes) followed by the addition of one - third of water and sonication in a bath
sonicator for 1 min. This water - in - oil (w/o) - emulsion is evaporated until a dry gel is
formed, and fi naly the gel is broken by agitation and water addition. Sometimes this
step is quite diffi cult. The remnants of the organic solvent are removed by evacuation
and the resulting dispersion is REV liposomes.
Dehydrated – Rehydrated Vesicles DRV liposomes were developed by Kirby and
Gregoriadis in 1984 [133] and are capable of encapsulating high amounts of aqueous
soluble molecules under mild conditions (conditions that do not cause decomposition
or loss of activity). The high entrapment ability of this type of liposomes is due
to the fact that preformed, “ empty ” SUVs are disrupted during a freeze - drying step
in the presence of the solute destined for entrapment. Subsequently, during controlled
rehydration that is carried out in the presence of concentrated solution of
the solute (to be encapsulated), the vesicles fuse into large oligolamellar vesicles
entrapping high amounts of solute. The produced liposomes are multilamellar and
their size is between 200 and 400 nm up to a few micrometers. Recently, with the
addition of certain amounts of sucrose, DRV liposomes with diameter between 90
and 200 nm were obtained entrapping considerable proportions (up to 87) of the
solute [134] .
Giant Vesicles Large or giant liposomes have been developed by Reeves and
Dowben [135] . Briefl y, lipids are dissolved in chloroform/methanol 2 : 1 and dispersed
on a piece of glass. Water is added for their rehydration; however, their
population is quite heterogeneous. Other types of particle - encapsulating giant liposomes
[136] can be prepared by applying a double - emulsion technique followed by
a freeze – drying step.
Detergent Depletion With this method phospholipid – detergent mixed micelles are
initially produced, and during controlled - rate detergent removal, liposomes are
formed. The rate and method of detergent removal determine the size and size
homogeneity of the liposomes produced. Gel fi ltration and dialysis are the most
popular approaches [137, 138] . Although liposomes are produced under mild conditions
(low temperature and low shear mechanical forces applied), this method
suffers from low encapsulation effi ciency of hydrophilic drugs.
Large Unilamellar Vesicles from Cochleates Large unilamellar vesicles can be produced
with the “ cochleate ” approach [139] . Small unilamellar vesicles consisted
from phosphatidylserine adopt a cochleate shape after addition of calcium. Addition
of EDTA creates complexes with calcium, turning the cochleates to LUVs.
One - Step Method The “ one - step method ” has been introduced by Talsma et al.
[140] . Lipid dispersions are hydrated at high temperatures in the presence of a steam
of N 2 . Liposomes between 200 and 500 nm can be prepared with this approach.
Large - Scale Manufacturing Despite the fact that there are many methods to
prepare liposomal dispersions not all of them are applicable for scaling up. In fact,
scaling up to larger batches could be a monumental task. Among all the methods
reviewed so far, microfl uidization and homogenization are the most powerful
methods to produce large quantities of liposomes. New homogenizers have a capacity
of 1000 L/h and require a minimal sample volume of 2 L [141] . The fact that lipid
concentrations up to 300 m M can be used secures high encapsulation capacity.
Remode and Active Drug Loading Techniques The main advantages of these
approaches are the high encapsulation effi ciency and low leakage of the encapsulated
material.
An in situ method for “ remote ” drug loading based on the development of a pH
gradient across the internal and external water phases of the membrane has been
established. A transmembrane pH gradient induces the uptake of charged drugs
into liposomes. Drug encapsulation is based on its partitioning between the lipid
and aqueous parts. This process is governed mainly by pH and to some extent by
the ionic strength of the medium. Drugs that are weak bases can diffuse through
the lipid membrane as unprotonated species. The presence of a proton gradient
makes them more hydrophilic, allowing them to accumulate in the intraliposomal
aqueous phase. Encapsulations up to 90% have been reported for doxorubicin
[142 – 144] and vincristine [145] .
An alternative but similar technique is based on an ammonium sulfate gradient
used to obtain “ active ” loading of amphipathic weak bases into the aqueous compartment
of liposomes. This has been used for active loading of anthracyclines,
acridine orange, epirubicin, and doxorubicin [11, 146, 147] at very high effi ciency
( > 90%). In the case of doxobubicide most of the intraliposomal drug is present in
the aggregated state. Additionally, antracycline accumulation in liposomes is stabilized
for prolonged periods of storage due to aggregation and gelation of antracycline
sulfate salt.
Active entrapment and loading stability are dependent on liposome lipid composition,
lipid quality, medium composition, and temperature as well as on the p K a and
hydrophobicity of the base. The ammonium sulfate gradient approach differs from
most other chemical approaches used for remote loading of liposomes, since it does
not require liposomes with acidic pH interior or an alkaline extraliposomal phase.
In addition to the remote or active loading techniques mentioned above, metal
complexation reactions have been demonstrated to achieve accumulation of doxorubicin
in liposomes [148, 149] . Furthermore, copper – topotecan complexation has
been recently seen to mediate drug accumulation into liposomes and is proposed
as a methodology to prepare liposomal camptothecin formulations [72] .
5.3.2.4 Functionalization of Liposomes
“ Active targeting ” is used to describe the specifi c liposomal drug localization achieved
by grafting various moieties (antibodies, lectins, polymers, etc.) on the carrier surface.
LIPOSOME STRUCTURE AND CHARACTERISTICS 459
460 LIPOSOMES AND DRUG DELIVERY
There are a number of techniques available to attach the suitable ligand on the liposome
surface, either by covalent or noncovalent coupling [150, 151] .
These techniques should be fast, effi cient, and reproducible, yielding stable nontoxic
bonds, while the conjugated ligands should maintain the ability to recognize
the target site with high binding affi nity. Also, the coupling method should not affect
the blood clearance of the formulation, colloidal stability, drug incorporation, and
release in a negative way. For example, when antibodies were attached on the liposome
surface where PEG molecules were grafted as well to ensure prolonged carrier
retention in the blood, it was shown that the ligand binding effi ciency on the bilayer
was low as well as the binding ability to the target [152, 153] . The latter problem
was opposed by attaching the ligand at the distal end of the PEG molecules already
grafted on the liposomal bilayer [154] .
Covalent Binding of Ligands The majority of ligand coupling is achieved by
covalent reactions with hydrophobic anchors. The procedure could be carried out
in two patterns: Either the hydrophobic anchor is included already in the liposomal
bilayer and the ligand interacts with the anchor on preformed liposomes [155] or
the ligand – hydrophobic anchor conjugate in the form of micelles is mixed with the
liposomes [156] . In the fi rst instance, where a hydrophobic anchor is mediated
between the liposomal surface and ligand, covalent attachment can occur via a
thioether bond [157 – 161] , via a disulfi de bridge, between carboxylic acid and the
primary amine group [162] , via hydrazone, or via cross - linking between two primary
amine groups (Figure 7 ).
The formation of the stable thioether bond is a reaction between thiol moieties
of proteins mainly with maleimide groups. Usually, PE or PEG – PE or PEG – DSPE
(distearoyl - phosphatidylethanolamine) has been functionalized with maleimido
(Mal - ), maleimido - phenylpropionate (MP), or pyridil - dithio - propionylamino
(PDP) groups, which eventually will react with the thiol groups. Also, sometimes
the ligand does not carry enough thiol groups or those are completely absent, so
they have to be introduced using a heterobifunctional cross - linker, such as SPDP
[ N - hydroxysuccinimidyl 3 - (2 - pyridyldithio) propionate] or SATA (succinimidyl -
S - acetylthioacetate), which introduce one amine group. Still, in the case of SATA,
deacetylation using hydroxylamine is necessary to uncover the thiol group, while if
SPDP is used, the produced disulfi de bond has to be reduced to thiol groups with
dithiothreitol (DTT). The former cross - linker is more preferable as only mild conditions
are used to make the thiol group available.
It has been shown that attaching the ligand at the distal end of PEG molecules
combines the advantages of a specifi c drug delivery system with steric stabilization
for higher stability in the blood [163, 164] . However, Longmuir et al. showed that
introducing a peptide from Plasmodium at the free end of PEG 3400 – AP (aminopropane)
was not capable of retaining the liposome stability, so PEG 5000 – PE molecules
were added to the liposomal composition resulting in PC/PE – PEG 5000 /AP – PEG 3400 -
peptide liposomes with molar ratio of 86 : 10 : 4 [165] . The coupling effi ciency of
ligand attached on PEG was higher (60 – 70% or even 100% in some cases) compared
to that achieved using, for example, N - (4 . - 4 . - maleimidophenyl)butyrol) -
dioleoylphosphatidylethanolamine (MPB – DOPE) (only 10%) [164, 166] .
Antibodies, whole or fragments, have been attached successfully on liposomal
surfaces and more commonly at the free end of PEG molecules, as has been reported
FIGURE 7 Schematic of different coupling methods used: ( a ) reaction between meleimide
and thiol functions; ( b ) formation of disulfi de bond; ( c ) reaction between carboxylic acid and
primary amine group; ( d ) reaction between hydrazide and aldehyde functions; ( e ) cross -
linking between two primary amine functions. ( Reproduced from ref. 150 with permission of
Elsevier .)
(a)
R1 R2 R2
R1S
SH
O
O
N N
O
O
+
(b)
R1 R1 R2 R2 SH HS S S +
(c)
CH3
CH3
NH2
CH3
CH3
CH2CH3
CH2CH3 (CH2)
3
(CH2)
3 N
N
N
N HN
HN
C
C
C
N
O
C
C
O
O
O
OH Anchor
Anchor
Anchor
BDAC
Ligand
Ligand
+
+
(d)
R1
R1
R2 R2
R2
NH2
N
H
NH
NH
C C
O
O
O
O
OH
CH
Nalo4
+
(e)
R1
R1
R1
R2
R2
NH2
NH2
CH2 CH2 CH2
CH2
CH2 CH2 CH2 CH2 CH2
CH2 CH2
C
C C N
N N
C H
H
H
H
H
H
O
O
O
+
+
Glutaraldehyde
LIPOSOME STRUCTURE AND CHARACTERISTICS 461
462 LIPOSOMES AND DRUG DELIVERY
in a number of studies since the interaction with the cells is much more favored
[161] . Using Fab . fragments is more advantageous as the Fc part which mediates
MPS activation through a receptor, is omitted [157, 160] . Moreover, the distance of
the antibody fragment from the liposomal surface is another important factor which
determines the drug delivery system uptake by the cells, as reported by Mamot
et al. [167] . Besides the latter has been reported elsewhere even if the coupling
reaction is different from thioether formation [168] .
Examples of peptides attached on liposomal surface via thioether bonds were
TAT - peptide and antagonist - G on Mal – PEG2000 – DSPE and PDP – PEG – DSPE,
respectively [169, 170] . Both ligands exhibited signifi cant increase in cell uptake.
Another possible way for ligand conjugation on liposomes is the formation of a
disulfi de bridge, which is quite unstable in serum and thus it is not used as much
[171] .
However, an amide bond formed between the carboxylic acid group on the liposome
surface (DSPE – PEG – COOH) and the primary amine of the ligand is favored
as the ligand modifi cation is not necessary. According to this method, an acyl amino
ether is produced in the presence of 1 - ethyl - 3 - (dimethylaminopropyl) carbodiimide
(EDAC) and N - hydroxysulfosuccinimide (NHS), which eventually will react with
the primary amine of the ligand [150] . For example, Wartchow et al. improved the
effi cacy of a small integrin antagonist of the extracellular domain of the . . . 3 integrin
by grafting it on dextran - coated liposomes [172] . DPPE - succinate was included
in the liposomal bilayer and a 3 - amino - 2 - hydroxypropyl ether derivative of dextran
was added to preformed liposomes in the presence of EDAC, while unreacted succinyl
groups were converted to amides. The amino groups of dextran were succinylated
and integrin antagonist (IA) was attached on the succinamidodextran liposomes
in the presence of EDAC. The fi nal IA – dextran liposomes had a size of 110 nm,
which is attributed to dextran coating, as the liposomes without dextran were 60 nm
in diameter. The antiangiogenic mechanism of IA – dextran liposomes as well as the
apoptotic potency was proved after a series of studies.
Moreover, Voinea et al. attached antibodies against vascular cell adhesion
molecule - 1 (VCAM - 1) overexpressed on activated human endothelial cells on liposomes
with the intention of using them as drug carriers [162] . N - gluraryl - PE was
used as membrane anchor for the antibody coupling via its free amino groups after
its activation with carbodiimide. There is no necessity of antibody modifi cation
before the coupling reaction.
Also, transferrin was grafted onto liposomes containing N - glutaryl - PE activated
with carboxidiimide with the fi nal plan to use those as carriers for inhalation therapy
for lung cancer [173] . Tfr liposome uptake was signifi cantly higher from immortalized
or cancer cell lines, but to reduce uptake from alveolar macrophages, PEG molecules
are attached on the liposome surface. In general, transferrin is a glycoprotein
that consists of a single chain of amino acids which has been coupled on the liposome
surface for a number of applications because its receptor is overexpressed at malignant
cells so a higher amount of transferrin binds on the cell membrane [174] . Its
covalent attachment on liposomes takes place either by conjugation between transferrin
- lipid and insertion of it on the preformed vesicles [174, 175] or preparation of
liposomes with activated lipids and reaction with activated transferrin [176] .
In addition, Torchilin et al. synthesized pNP ( p - nitrophenylcarbonyl) – PEG –
DOPE to enable protein coupling via its amino group at the distal end of PEG mol
ecules on liposome surface in a quantitative manner at pH around 8.0 [177] . The only
disadvantage mentioned is the hydrolysis of pNP groups from PEG – DOPE at pH
higher than neutral (complete hydrolysis occurs in 1 – 2 h at pH around 8.0). Therefore,
the coupling reaction between protein and pNP has to take place faster or at
least at the same ratio. It was shown that 65% of pNP binds to ligand at pH 9, so the
binding effi ciency and time are adequate at the conditions studied. Also, the amount
of pNP – PEG – DOPE was critical for successful protein binding; it was shown that
1 mol % of pNP – PEG – DOPE was enough to bind approximately 100 protein molecules.
Incorporation of antibodies, lectins, avidin, and nucleosomes did not seem to
alter the activity of those molecules even at high concentrations of pNP – PEG –
DOPE. However, Savva et al. conjugated a genetically modifi ed tumor necrosis
factor (TNF) at the free end of the PE 3500 molecule by reacting the latter with NHS
and DCC ( N , N . - dicyclohexyl carbodiimide) to introduce the succinyl groups which
will react with the phospholipid to produce DOPE – PEG – COOH [178] . Then, the
carboxyl group of the derivatized PEGylated molecule located on the liposomal
bilayer was activated with EDAC and NHS. Consequently, recombinant TNF was
added in the liposome suspension for the fi nal conjugation to occur at 4 ° C where the
overall coupling effi ciency was approximately 55%. However, it was shown that the
biological activity of TNF was reduced when attached on the liposomes as the degree
of PEG modifi cation increased irrespective of the PEG molecular weight (MW).
Also, the formulation did not show the prolonged blood circulation expected due to
the PEG presence. Those results were attributed to a number of reasons, including
either damage of the protein during the coupling reaction or possible dissociation of
the trimeric form of rKRKTNF to a monomeric less active form or cross - linking
between PEG and rKRKTNF during the coupling reaction.
A hydrazone bond is an alternative way of antibody coupling onto liposomal
surface so as to avoid the use of essential (for the recognition) amino groups at the
coupling reaction (by using the maleimido method) or the risk of rapid clearance
due to Fc moiety/segment [150, 179] . According to this method, the carboxylic group
of the heavy chain of the antibody undergoes mild oxidation by sodium periodate
or galactose oxidase to aldehyde groups. The oxidized product can be coupled either
on the hydrazide - hydrophobic anchor inserted in the lipid bilayer [180] or at the
free end of PEG molecules [181, 182] . Hydrazide cross - linkers used are, for example,
S - (2 - thiopyridyl) - l - cysteine hydrazide (TCPH), N - acetylmercaptobutyric hydrazide
(AMBH), and 3 - (2 - pyridyldithio)propionic acid hydrazide (PDPH). TCPH is structurally
closely related to PDPH and could be expected to behave in a similar manner
[183] . Unprotected mercaptohydrazides such as AMBH are unsuitable since the free
thiol function is susceptible to oxidation and may also reduce the disulfi de bonds
in immunoglobulin IgG at the concentrations required for conjugation. The disadvantage
of this method is the low coupling effi ciency (17% only) [166] . According
to Ansell et al. [179] , the drawbacks reported was the possible damage of some
amino acid residues, such as methionine, tyrosine, and tryptophan, due to periodate.
Therefore, antibodies sensitive to periodate treatment would be unsuitable candidates
for the PDPH protocol. Also, the hydrazone bond might undergo hydrolysis
after six weeks of storage, which would be a potential problem if the conjugate
would not be used soon after preparation. However, it is possible to stabilize the
bond using sodium cyanoborohydride to reduce the hydrazone linkage if long - term
storage is required [179] .
LIPOSOME STRUCTURE AND CHARACTERISTICS 463
464 LIPOSOMES AND DRUG DELIVERY
However, the labile nature of the hydrazone bond is used to formulate “ smart ”
drug delivery systems where the basic idea is to introduce in parallel a pH - responsive
ligand spacer in the lipid bilayer (which is PEG 5000 – Hz – PE), a temporarily
shielded biotin or TATp and mAb (monoclonal antibody) attached to the surface
of the drug delivery system via a noncleavable bond (TATp – PEG 2000 – PE) [183] .
Such a system will be able to respond to environment stimuli such as pH changes,
where, for example, at acidic pH (5.0 – 6.0) PEG5000 molecules will be detached from
the carrier surface and biotin or TATp will be available to either bind to avidin or
be internalized by the cells, respectively (Figure 8 ). The monoclonal antibody
and biotin or TAT has been attached on pNP – PEG – PE. The produced mAb DDS
(drug delivery system) demonstrated clear immunoreactivity toward the antigen.
However, some affi nity decrease was observed for the antibodies modifi ed with the
pNP – PEG – PE anchor and incorporated onto the immunoliposomes. Biotin binding
to avidin was pH dependent with higher retention (75%) at pH 5.0, where the
shielding PEG molecules were cleaved away. Signifi cant increase of DDS uptake
by the cells was achieved when TAT was incorporated on the liposome surface at
pH 5.0.
An avidin – biotin system has been used to attach antibodies in the bilayer of
DDSs. Xiao et al. developed a three - step strategy to improve the tumor - to - tissue
ratio of anticancer agents [184] . Two antibodies specifi c for the CA - 125 antigen that
is highly expressed on NIH:OVCAR - 3 cells were used. These cells were prelabeled
with biotinylated anti - CA - 125 antibody and fl uoroscein isothiocyanate (FITC) –
labeled streptavidin (SAv) prior to administration of biotinylated liposomes. Both
antibodies were specifi cally bound to the cell surface of OVCAR - 3 cells but not to
SK - OV - 3 cells, which do not express the specifi c antibody. Antibody biotinylation
did not affect its immunoreactivity.
Schnyder et al. explored the targetability of biotinylated immunoliposomes to
skeletal muscle cell line in vitro [185] . OX26 mAb binds to transferrin receptor and
is covalently attached to streptavidin by introducing thiol groups using 2 - iminothiolane
(Traut ’ s reagent). Immunoliposomes consisted of DSPC (5.2 . mol), cholesterol
a a a a a a a a
a
a
a
a
a
a
a
a
c
b
b
b b
c
Targeting by target specific antibody
and/or long circulation
Incubation at lowered pH
Removal of PEG chains
De-shielding of the “hidden” function
FIGURE 8 Schematic for design of multifunctional drug delivery system (DDS) that
includes pH - cleavable PEG - Hz - PE (a), temporarily “ shielded ” biotin or TATp (b), and
monoclonal antibody (c) attached to surface of DDS via pH - noncleavable spacer. ( Reprinted
with permission from ref. 183 . Copyright 2006 by the Americam Chemical Society .)
(4.5 . mol), PEG – DSPE, (0.3 . mol), and linker lipid (bio - PEG – DSPE; 0.015 . mol).
OX26 mAb – streptavidin was added to preformed liposomes in a 1 : 1 ratio. According
to estimations, the average number of bio - PEG – DSPE molecules was 30, assuming
that one 100 - nm liposome contains 100,000 phospholipid molecules. Uptake
experiments with muscle cell line using the OX26 mAb, fl uorescence - labeled OX26 –
streptavidin, or fl uorescent OX26 – immunoliposomes demonstrated cellular uptake
and accumulation within an intracellular compartment of the OX26 mAb and its
conjugates.
All the methods described earlier consider that coupling of the ligand on the
anchor already existed on the liposome surface. Another option is the ligand –
anchor conjugate in the form of micelles to mix with the liposomes. According to
that, anti - CD19 mAb was thiolated using Traut ’ s reagent and reacted with Mal -
PEG – DSPE in a micellar form with PEG – DSPE and molar ratio 4 : 1 [186] . Antibodies
were coupled at the end of the PEG – DSPE. Consequently, micelles were
incubated with preformed liposomes at molar ratio 0.05 : 1, respectively, for 1 h
at 60 ° C.
In another study, mAb 2C5 with nucleosome - restricted specifi city, which recognizes
specifi cally human brain tumor cells, was tested as a potential ligand candidate
for liposome targeting to brain tumor cells [187] . The mAb was attached to pNP –
PEG – DSPE and the formed micelles incubated with preformed liposomes. The
100 – 200 mAbs bound per single liposome of approximately 200 nm in diameter. A
slight reduction in immunoreactivity was observed for a single antibody molecule
for a number of reasons; the overall evaluation was suffi cient target recognition and
affi nity due to multipoint attachment of immunoliposomes to the target via several
antibody molecules. Indeed, the immunoliposomes showed threefold higher accumulation
in the tumors compared to nonspecifi c carriers.
At this point it has to be emphasized that this method seems to be the most
advantageous one because damaging chemical reactions are excluded as they happen
at a different stage. Also, this method provides the fl exibility of attaching a large
variety of ligands on liposomes of various compositions loaded with different drugs.
Apparently, targeted liposomes produced with this last technique have shown similarities
in the in vitro drug leakage, cell association, and therapeutic effi cacies to
liposomes made by conventional coupling procedures.
Noncovalent Binding of Ligands According to this procedure, the ligand is added
to the lipid mixture during liposome preparation. Small molecules such as sugars
have been attached on the liposome or lipoplex surface in this way. At fi rst galactose,
mannose, and fucose were modifi ed to Gal – C4 – Chol, Man – C4 – Chol, and Fuc – C4 –
Chol and they were added in the lipid mixture of DSPC/Chol/Sugar – C4 – Chol with
a ratio 60 : 35 : 5. Chol was chosen due to the stability in the liposomal membrane
while only one sugar was conjugated so the lipophilicity of the fi nal glycolipid would
not be altered considerably, and thus the stability of the latter in the liposomal
membrane would be more secure [188] . After in vivo administration of 0.5% Gal,
Man, and Fuc liposomes it was found that the ratio of their uptake from parenchymal/
nonparenchymal liver cells was 15.1, 0.6, and 0.2, respectively. Also, in high
doses, 5% Gal liposomes are taken up by nonparenchymal liver cells as well as the
parenchymal ones, while they are capable of inhibiting the uptake of Fuc liposomes
by nonparenchymal cells.
LIPOSOME STRUCTURE AND CHARACTERISTICS 465
466 LIPOSOMES AND DRUG DELIVERY
Even if this is a simple and mild technique, there is always a concern about ligand
orientation, very low attachment effi ciency achieved (4 – 40%), and the liposome
aggregation often observed.
5.3.3 IN VIVO DISTRIBUTION
Successful treatment depends not only on the formulation characteristics but also
on the route of administration. For example, the schistosomicidal drug tartar emetic
incorporated in PEGylated liposomes was delivered either intraperitoneally or
subcutaneously (27 mg Sb/kg) to mice infected with Schisostoma mansoni [189] .
Indeed, 82 and 67% reduction levels of worm were obtained, respectively. However,
the effi cacy of the formulation given by either administrative route was not signifi -
cantly different. The only difference was the slower liposome absorption by the
subcutaneous route.
Also, the therapeutic effect of liposomal adriamycin (PC/Chol, 120 nm) was
enhanced signifi cantly after concurrent i.v. and s.c. administration to rabbits bearing
VX2 tumors in the mammary gland [190] . The i.v. route signifi cantly inhibits breast
tumor and metastasis, while the s.c. route acts on local - regional lymph nodes. That
was proved by slowed growth rates, decreased messenger ribonucleic acid (mRNA)
expression of proliferating cell nuclear antigen, and extensive necrosis and apoptosis
of tumor cells. Even if allergic reactions have not been reported after s.c. injection
of liposomes, there is more to be done on systemic toxicity.
The therapeutic effi cacy of paclitaxel is stronger after drug incorporation in magnetoliposomes
injected either i.v. or s.c to an EMT - 6 breast cancer mouse model
[191] . The carrier manipulation due to the application of a magnetic fi eld led to their
increased accumulation to the tumor site. However, paclitaxel accumulation is
slightly lower after s.c. administration, probably attributed to time delay during the
drug transportation process to the circulation.
In another study by Wang et al., i.v. injection of liposomes carrying rat insulin
promoter (RIK) – thymidine kinase (TK) was found to be less toxic to the liver than
the i.p. injection of the same formulation to severe combined immunodefi cient mice
(SCID) [192] . The direct injection of the liposomes to abdominal cavity probably
leads to higher local absorption and, thus, higher liver toxicity. In contrast, the i.v.
injected volume is smaller (70 . L to 100 . L of i.p. injection), which is diluted fast as
soon as it enters the body.
5.3.3.1 Conventional Liposomes
Conventional liposomes are those that do not carry any sterically stabilizing or targeting
moieties on their surface. Their biodistribution depends strongly on their
physicochemical properties (size, . potential, composition) and physiological and
pathological conditions of the body [193] . Thus, conventional liposomes comprise
the passive targeting of drug molecules.
Intravenous Administration Liposomes administered intravenously face barriers
such as the endothelial lining of the vasculature and the blood – brain barrier. Extravasation
of the liposomes occurs only in organs such as liver, spleen, and bone
marrow (due to leaky fenestrae and loose junctions between the endothelial cells)
and under certain pathological conditions (presence of tumors, infl ammation, infection).
Besides, neutral (uncharged) liposomes of size smaller than 100 nm show slow
blood clearance compared to others of larger size and/or positive or negative charge
(due to presence of opsonins) [194, 195] . Also, lipid exchange between liposomal
carriers and plasma lipoproteins contributes to liposomal membrane rupture and
consequently loss of the therapeutic substance [196] .
Therefore, conventional liposomes are used mostly in treating the RES system or
to mask the toxic side effects of anticancer drugs. Many anticancer drugs entrapped
in liposomes have shown altered biodistribution and reduced toxicity. Plain SUV
liposomes consisting of DSPC/Chol (known as “ Stealth ” ) in a molar ratio 2 : 1 or 1 : 1
have shown particularly promising vehicles as reported in a number of studies of
animal models. They have undergone preclinical and clinical studies due to relatively
low levels of RES uptake and the high level of tumor targeting exhibited [197 – 200] .
Indeed, 111 In - labeled DSPC/Chol liposomes have proven capable of targeting a
number of tumors [201 – 206] . Although the signifi cant RES uptake was a fact, about
45 – 50% of liposomes remained in the blood circulation 4 h after i.v. injection. Positive
images on gamma camera were reported. In patients with recurrent high - grade
gliomas, 1% of the injected liposomal dose was taken up by the tumor [205] .
Moreover, anthracyclines have been formulated in conventional liposomes and
are commercially available. Doxorubicin for i.v. use is commercially available in the
form of Myocet, which consists of egg PC and cholesterol [207] . It is recommended
in combination with cyclophosphamide for metastatic breast cancer. Thus, drug
entrapment in liposomal vesicles may reduce the incidence of cardiotoxicity and
lower the potential for local necrosis, but infusion reactions, sometimes severe, may
occur. Hand – foot syndrome (painful, macular reddening skin eruptions) occurs
commonly with liposomal doxorubicin and may be dose limiting. Daunorubicin also
has general properties similar to those of doxorubicin. A liposomal formulation
(DSPC/Chol, 2 : 1, size 45 nm) for i.v. use is licensed for patients with AIDS - related
Kaposi ’ s sarcoma [207, 208] . The plasma pharmacokinetics of DaunoXome differs
signifi cantly from the results reported for free daunorubicin hydrochloride.
DaunoXome has a small steady - state volume of distribution of 6.4 L (probably
because it is confi ned to vascular fl uid volume) and clearance of 17 mL/min. These
differences in the volume of distribution and clearance result in a higher daunorubicin
exposure [in terms of plasma “ Area on the curve ” or “ bioavailabity ” (AUC )]
from DaunoXome than with free daunorubicin hydrochloride. The apparent elimination
half - life of DaunoXome (daunorubicin citrate liposome injection) is 4.4 h,
far shorter than that of daunorubicin, and probably represents a distribution half -
life. Although preclinical biodistribution data in animals suggest that DaunoXome
crosses the normal blood – brain barrier, it is unknown whether DaunoXome crosses
the blood – brain barrier in humans [207] .
In addition, TAS - 103 (a novel quinoline derivative, topoisomerase inhibitor)
incorporated in DPPC/Chol (2 : 1) liposomes (size < 80 nm) enhanced the survival
time of mice with Lewis lung carcinoma to 42 days in comparison to the 38.6 days
of those treated with free TAS - 103 [209] . The increases in lifetime were 45 and 58%
for the free TAS - 103 and liposomal TAS - 103, respectively.
In another study, the antibiotic cefoxitine was incorporated in DMPC/Chol (2 : 1)
liposomes prepared using the reverse - phase evaporation technique in order to
IN VIVO DISTRIBUTION 467
468 LIPOSOMES AND DRUG DELIVERY
increase the effi cacy of the drug characterized by a short half - life and poor intracellular
diffusion [210] . It was shown that the cefoxitin levels achieved in liver and spleen
5 h postinjection were 6 - and 16 - fold higher than those observed after administration
of free antibiotic. Also, the elimination rate through the kidney was slower.
Intraperitoneal Administration Intraperitoneal administration has the biological
and pharmacological advantage of creating direct exposure of the tumor, infection,
or infl ammation to the therapeutic agent. This drug delivery method increases the
dose intensity within the peritoneal cavity [211 – 213] . Intraperitoneal administration
of liposomal formulations of anticancer drugs is preferred to the i.v. one, due to the
higher drug accumulation in the tumors and lower drug plasma concentration minimizing
drug toxicity [214 – 216] .
Size, liposomal composition, charge, drug density in the liposomal membrane,
and preparation method are some of the important parameters which need to be
considered carefully to design an effi cient DDS. Sadzuka et al. assessed DOX -
incorporating liposomes made by either DMPC or DSPC of a variety of sizes (150,
600, and 4000 nm) and surface charge (positive and negative) on the therapy of solid
tumors and peritoneal dissemination in Ehrilch ascites carcinoma - bearing mice
[214] . When using small negative liposomes, lipid composition did not affect the
clearance or stability of liposomes in the abdominal cavity. However, for neutral
liposomes, DSPC ones were found more effective for the treatment of the solid
tumor due to the higher stability of those liposomes in comparison to DMPC ones.
Thus DSPC exhibited longer plasma circulation. As for the effect of surface charge,
the positive vesicles were cleared faster from the abdominal cavity until 1 h postinjection
and then showed a slower clearance rate until 48 h, in opposition to the
negative ones. Larger particles were found in abundance in the peritoneal cavity
and stayed longer there, inducing toxicity due to liposomal membrane disruption
and release of the anticancer drug. Overall, it was concluded that the larger liposomes
were effective against peritoneal dissemination and the smaller ones against
the solid tumor.
The same author evaluated the method of preparation by using DOX -
encapsulating liposomes on the peritoneal dissemination of tumor in Ehrlich ascites
carcinoma - bearing mice [217] . The liposomal carriers were made either with the
method of Bangham et al. [1] (BLDOX), the pH gradient [144] (PLDOX), or the
gelation method (GLDOX) [147] . It was shown that survival in the BLDOX group
was signifi cantly prolonged compared to that in the DOXsol (DOX as solution)
group, while there was no effect on survival of the GLDOX group. BLDOX liposomes
appeared to be less stable and released DOX in a higher degree than the other
formulations. The latter seems to be of high importance due to increased DOX level
in the abdominal cavity and enhancement of drug effi ciency for the local therapy.
Also, positive outcome was achieved after i.p. administration of the liposomal
formulation of an l - dopa prodrug derivative to rats [218] . It was shown that the
level of dopamine in rat striatum was 2.5 - fold higher to what was obtained after
i.p. administration of l - dopa or the free prodrug itself.
Subcutaneous Administration Liposomes given s.c. aim to target the lymphatic
system for imaging, distribution of therapeutic agents, or vaccination [219, 220] .
According to Oussoren and Storm, the determining factors infl uencing lymphatic
absorption are liposome size and site of injection [219] . Liposome charge, composition,
or PEG coating does not have a signifi cant effect on the fate of the liposome
trip in the lymphatic system.
Liposomes injected s.c. that do not enter the bloodstream either enter the lymphatic
capillaries or stay at the site of injection. In the fi rst case, 1 – 2% of the injected
liposomal formulation is captured by the lymphatic nodes 12 h postinjection.
However, this depends on liposome size. Neutral vesicles smaller than 100 nm pass
through the interstitium and then to the lymphatics a lot easier than the larger particles.
Drug carriers remaining at the site of injection will release the entrapped
molecule. Often, 40% of the injected dose of small liposomes (about 70 nm) is
retained at the injection site. Therefore, liposome surface modifi cation was attempted
using non specifi c human antibodies and saccharides. Only saccharide - modifi ed liposomes
enhanced absorption from the injection site and enhanced lymph node uptake
was in comparison to control liposomes [221] . Also, the specifi c site of injection is
very important and species dependent. Taking the rat as animal model, s.c. injection
in the dorsal foot or the footpad results in higher liposome uptake by the lymph
nodes, in contrast to the fl ank as an injection point.
As mentioned earlier, the s.c. route for delivery of anticancer agents could prevent
the metastatic spread of tumors that occurs often through the lymphatic system.
However, a number of limiting factors, such as incomplete absorption of drug -
loaded liposomes, which would increase, for example , the toxicity of the released
drug at the surrounding tissue and the development of tumors in the regional lymph
nodes could limit the therapeutic potential of liposomes.
In addition, for imaging studies, only liposome - encapsulated gadolinium was used
successfully [222, 223] . In a more recent study, electron spin resonance (ESR) was
applied successfully to investigate the integrity of MLV and the possibility of a depot
effect after the s.c. injection in mice [224] .
Also, Gregoriadis et al. evaluated the type and degree of immune response after
s.c. injection of ovalbumin (OVA) – encoding plasmid DNA (2.5 or 10 . g) either
alone or in liposomes, in mice [220] . Anti - OVA serum antibody titers were detected
in animals immunized with 10 . g of liposomal DNA (after a single dose of antigen)
and with both 2.5 and 10 . g of liposomal DNA (after two doses of antigen) [225] .
However, the anti - OVA response was not detected using the DNA alone.
Similarly, signifi cantly higher humoral responses were obtained after s.c. administration
of either a lipid and/or a nonionic - based vesicle - entrapped plasmid for the
nucleoprotein of H 3 N 2 infl uenza virus in comparison to the naked DNA alone
[226] .
5.3.3.2 Long - Circulating or PEG ylated Liposomes
The liposome biodistribution profi le changes signifi cantly when the vesicle surface
[227 – 229] is coated with polymers, usually PEG. Longer blood circulation, lower
liver uptake, and higher accumulation in tumors have been reported. The presence
of the hydrophilic groups of PEG on the liposome surface provides electrostatic
and steric repulsion between PEG - grafted liposomes. PEG molecules neutralize the
surface charge of vesicles and thus prevent their opsonization. Also, liposome opsonization
is reduced due to inability of opsonins to bind to hydrophilic surfaces.
Moreover, the thickness of the PEG layer infl uences the interaction of the liposomes
IN VIVO DISTRIBUTION 469
470 LIPOSOMES AND DRUG DELIVERY
with the macrophages. The thickness of the PEG layer depends on the PEG molecular
weight and the amount (%) incorporated in the liposomal composition. For
example (Figure 9 ), the fast clearance of positively charged stearylamine liposomes
can be reversed by attachment of 6% mol of PEG with molecular weight of 750 or
5000 kDa [230] . In the case of phosphatidic acid – containing liposomes, only PEG
5000 can prolong blood circulation while phosphatidylserine - containing liposomes
are eliminated fast due to the insuffi cient effect of 6% PEG 750 or 5000.
However, an optimum level of PEGylation (PEG 2000 kDa) was estimated for
PC (1.85 mol %)/Chol (1 mol %) liposomes as to the effect on blood circulation
[231] . As reported, after 5 mol % of PEG incorporation the accumulation in the liver
was signifi cantly decreased, while the minimum uptake by the spleen was achieved
with 9.6 mol % of PEG insertion. The same authors showed that as the PEG amount
grafted increased, the liposome accumulation in the heart increased. But above a
9.6 mol % of PEG, the circulation time was slightly decreased in blood and was
increased in the liver and spleen. The uptake of liposomes by RES was even higher
when 13.7% of PEG was present on the vesicle surface. As shown by another group,
the optimum PEG amount required for liposome stabilization and prolonged half -
life was 5 – 10 mol % DSPE – PEG 2000 [232] . Higher amounts than that led to disruption
of the vesicles.
Intravenous Administration The effect of PEG on liposome biodistribution presented
in the previous paragraph is for i.v. administration of liposomes. Several
recent examples for the effect of PEG coating of liposomal drug formulations on
the biodistribution (and thus pharmacological outcome) for different drugs follow.
DOX concentrations were estimated in various organs after i.v. administration of
DOX - loaded liposomes (PC/Chol/PEG2000, molar ratio 55 : 40 : 5) in xenograft
tumor - bearing mice [233] . Obviously, the encapsulation of DOX in conventional or
PEGylated liposomes reduced the drug concentration in liver, heart, kidney, and
stomach compared to the drug solution and prolonged the circulation half - life to
46.09 h, in contrast to 26.04 and 23.72 of conventional and free DOX, respectively.
However, PEGylated DOX showed higher antitumor activity in comparison to that
entrapped in conventional liposomes. In comparison with free DOX, the inhibition
rate of both liposomal formulations was higher. Doxil is a commercially available
formulation of DOX entrapped in HSPC/Chol/mPEG liposomes [234] . Thereafter,
PEGylated liposomes are in common use in many applications. Covalent attachment
of specifi c molecules (folate) at the free end of PEG molecules results in
liposomes able to be recognized by specifi c receptors (folate receptors on cancer
cells). There are a huge number of research papers on the active targeting of modi-
fi ed PEGylated liposomes [235 – 237] with very promising results.
Intraperitoneal Administration The potential of PEGylated liposomes administered
intraperitoneally has been evaluated for cancers located in the peritoneal
cavity. For example, Syrigos et al. [238] studied the biodistribution of indium ( 111 In) –
labeled PEGylated liposomes [Hydrogenated soya PC (HSPC)/Chol/PEG – DSPE ]
compared to free 111 In via either i.p. or i.v. route to non - tumor bearing mice . The
AUC of In - PEG liposomes was 74 - fold higher than that of free indium. The relative
ratio of the AUCs (RR - AUCs) for i.p. versus i.v. administration for peritoneum was
1.36 [423.6 vs. 311.3% individual dose (ID) g/h]. The blood AUC values for i.p. and
FIGURE 9 ( a ) Clearance from circulation and ( b ) accumulation in liver and spleen of
liposomes composed of lecithin (LS) mixed or not with charged lipids (PS = phosphadityl
serine, PA = phosphatidic acid, SA = stearylamine) and bearing or not, a surface coating with
PEG molecules after IV administration (PEG - 750 and PEG - 5000 = polyethylene glycol with
molecular weights 750 and 5000). ( Reproduced from ref. 230 with permission of Elsevier .)
100
80
60
40
20
0
100
80
60
40
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0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
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0
100
80
60
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0
0 60 120 180 240 300 360
0 60 120 180 240 300 360 0 60 120 180 240 300 360
0 60 120 180 240 300 360 0 60 120 180 240 300 360
0 60 120 180 240 300 360
Time, min Time, min
Time, min Time, min
Time, min Time, min
% injected dose/g
% injected dose/g % injected dose/g
% injected dose/g % injected dose/g
% injected dose/g
Plain LS
Plain LS
LS + PEG-750
LS + SA + PEG-750
LS + SA + PEG-5000
LS + PEG-5000
LS + PS
LS + PA
LS + PA
Plain LS
LS + SA + PEG-750
LS + SA + PEG-5000
LS + PA
Plain LS
LS + PA + PEG-750
LS + PA + PEG-5000
LS + PA
Plain LS
LS + PA + PEG-750
LS + PA + PEG-5000
LS + PA
LS + SA
Plain LS
LS + PEG-750
LS + PEG-5000
LS + PS
LS + PA
LS + SA
(a) (b)
100
80
60
40
20
0
100
80
60
40
20
0
0 60 120 180 240 300 360 0 60 120 180 240 300 360
Time, min Time, min
% injected dose/g
% injected dose/g
Plain LS
LS + PEG-750
LS + PEG-5000
LS + PS
Plain LS
LS + PS + PEG-750
LS + PS + PEG-5000
LS + PS
IN VIVO DISTRIBUTION 471
472 LIPOSOMES AND DRUG DELIVERY
i.v. administration were essentially the same (RR - AUC 1.03; 453.7 vs. 439.2% ID
g/h) 18 h postinjection despite the delayed absorption of the liposomes from the
peritoneal cavity. However, the relevant values for organs in the peritoneum were
higher in case of i.p. administration. An increase in the range 1.2 – 5.1 was seen for
organs such as stomach, pancreas, ileum, colon, gallbladder, ovary, and adrenal
glands. This is an advantage compared to the i.v. administration because the drug
can target both the primary site and any peritoneal deposits. The encapsulation of
doxorubicin and cis - platin (small molecules with high toxicity and short half - lives)
in PEGylated liposomes might increase the retention from the peritoneal cavity and
reduce the drug toxicity.
Another study points out the vesicle size rather than the presence of PEG as a
more determining factor to successfully tackle peritoneal cancers [239] . According
to this study, the synergistic effect of Doxil after coadministration of PEG – SUV –
interleukin - 2 (IL - 2) or MLV – IL - 2 via either the i.p. or i.v. route to mice bearing
M109 lung adenocarcinoma was studied. The cancer was inoculated i.p., resulting in
multiple i.p. masses. Small PEGylated liposomes as vehicles for IL - 2 for systemic
treatment of metastatic lung cancer boosted the antitumor effect of Doxil to the
same level achieved with soluble IL - 2. In contrast, in the regional model, the most
effective combination was Doxil with MLV – IL - 2 liposomes. This is attributed to the
retention and slow release of IL - 2 in the peritoneal cavity due to the inability of
MLVs to enter the circulatory system or the draining lymph vessels, whereas IL - 2
in small liposomes or in soluble form escapes rapidly from the peritoneal cavity.
Another possibility could be the enhanced immunostimulation results from the
uptake of MLV – IL - 2 by peritoneal macrophages as opposed to the stealth properties
of PEGylated SUV – IL - 2.
Subcutaneous Administration As for the use of PEGylated liposomes subcutaneously,
there is not much reported. In one paper the infl uence of the administration
either s.c. or i.m. of mitoxantrone - loaded liposomes was studied. It was reported
that mitoxantrone showed reduced irritation when the formulation was administered
i.m. rather than s.c. However, when PEG was incorporated on the liposome
surface, there was no apparent protective effect of the liposomes [240] .
5.3.3.3 Other Routes of Administration
In general, the administration route plays an important role on the impact of the
therapeutic treatment, and it is chosen according to the kind or purpose of the
treatment (local or systemic), toxicity, and accessibility of the diseased area. In this
section specifi c characteristics of other routes of administration that are currently
receiving attention will be emphasized and the most recent developments with
respect to liposomal drug applications will be presented.
Pulmonary Drug Delivery Pulmonary epithelium offers many advantages for
drug delivery due to easy access and the large surface area provided by alveoli [241] .
Also, macromolecules can penetrate the lungs much easier and faster than other
noninvasive routes avoiding the fi rst - pass meabolism; therefore, many promising
applications are being considered for the delivery of proteins and peptides. For a
drug or drug delivery system to reach the lungs successfully, it has to be aerosolized
with optimum aerodynamic particle diameter between 1 and 3 . m. The latter, in
combination with how the patient inhales determines if the drug particles deposit
primarily in the conducting ways or in the alveoli.
Pulmonary delivery of liposomes has focused on the treatment of asthma,
infectious diseases, genetic diseases (cystic fi brosis), and lung injury and lately on
gene therapy.
Special attention has been paid on the physical characterization of liposome
aerosols [242 – 244] , including dry powder formulations [245] , and cationic liposome
DNA complexes [246, 247] . Corticosteroid therapy using liposomal formulations has
focused on the development of aerosols containing beclomethasone dipropionate,
triamcinolone acetonide, and triamcinolone acetonide phosphates [248 – 251] . Aminoglycosides
have been considered as good candidates for pulmonary delivery
because of their potency and their ability to directly target the lungs. When amikacin
was encapsulated in liposomes [fl uid or rigid state (chol containing)] and administered
in sheep, the drug mean residence time (MRT) increased 5 times compared
to the instilled solution (rigid liposomes gave 2 times higher MRT compared to fl uid
ones) [252] . In a similar study, prolonged retention of liposome - encapsulated tobramycin
was reported after administration in Pseudomonas aeruginosa – infected rat
lungs [253] . Liposomal formulation of antioxidants has also been investigated for
pulmonary delivery [254] . Liposomes containing . - tocopherol prolonged the residence
time of radioactively labeled glutathione [255] . Paraquat poisoning, which
causes extended damage to lung tissues, was attenuated after pretreatment with . -
tocopherol - containing liposomes [256] . Liposomes containing CuZn/superoxide
dismutase and catalase were found to protect the lungs of premature rabbits when
exposed to hyperoxia [257] .
Drugs for the treatment of infections such as aspergillosis, tuberculosis, and anticancer
therapy have been formulated in liposomes and tested in vivo by administration
via the respiratory system [258 – 260] . More specifi cally, rifampicin used to treat
pulmonary tuberculosis associated with AIDS has been incorporated in MLV liposomes
(PC/Chol, 7 : 3 molar ratio) with the aim of increasiing its effi cacy to macrophages
and reduce side effects [258] . The rifampicin retention was found higher in
PC/Chol/DCP (7 : 3 : 0.1) liposomes tested due to electrostatic interaction between
DCP and the drug. Both liposomal formulations showed greater accumulation in
the lungs when compared to the controls. In the case of free drug and after 0.5 h
postadministration, only 39.12% of the administered dose was retained in the lungs
and 29.84% of it was found in the serum. No drug was estimated in the lungs 24 h
later. PC/Chol MLVs demonstrated higher lung accumulation (49.03%) after 0.5 h,
but the overall distribution pattern was not much different to that of the free drug
in solution with no drug estimation at the 24 - h time point. It seems that they were
rapidly passed to the systemic circulation and to the RES organs (liver and spleen).
The negatively charged MLVs showed even higher drug accumulation (53.86% of
the administered dose) while 4.14% of it was still present after 24 h. This is attributed
to the interaction of the negatively charged liposomes with the scavenger receptors
on alveolar macrophages [261] . Also, it has to be mentioned here that the ligands
maleylated bovine serum albumin (MBSA) and O - steroyl amylopectin (O - SAP)
were incorporated on the liposome surface because they are recognized by the
scavenger receptors of the macrophages. The lung accumulation levels were 61.49
and 65.14% of the administered dose, respectively, after 0.5 h, while after 24 h they
were 8.12 and 10.75%, respectively. The relative lung retention of the various
formulations after 6 h of administration was 1.3 times for PC/Chol MLVs, 3.4 times
IN VIVO DISTRIBUTION 473
474 LIPOSOMES AND DRUG DELIVERY
for PC/Chol/DCP MLVs, 4.53 times for MBSA - PC/Chol, and 4.76 for O - SAP – PC/
Chol in comparison to plain drug solution administered by aerosolization.
The same group (Vyas et al.) studied the impact of amphotericin B entrapped in
liposomes (MLVs: PC/Chol, 7 : 3 molar ratio) in the absence or presence of ligands
[ O - palmitoylated mannan (OPM) or O - palmitoylated pullulan (OPP)] on the vesicle
surface as potential use for the treatment of aspergillosis [259] . Optimized formulation
was the one of PC/Chol 7 : 3 molar ratio with the production of spherical MLVs
of approximate mean vesicle size 2.56 . m and maximum entrapment effi ciency
78.2%. Again the lung uptake of the ligand - appended liposomes was higher
compared to the plain liposomes. The lung accumulation levels of OPM - and
OPP - coated liposomes were 58.12 and 55.02% of the administered dose 5 h postadministration,
respectively, while 24 h later the relevant lung retentions were 11.23
and 9.86%, respectively.
Methotrexate (MTX) (a folic acid antagonist) was entrapped into liposomes
(PC/PI/Chol 2 : 1 : 1 molar ratio) so as to reduce nephrotoxicity and investigate the
pharmacokinetics of the liposomal MTX [260] . Indeed, the latter showed increased
MTX retention in the lungs while the biodistribution in spleen and kidney was less
than that obtained with free MTX. Similar results were obtained by liposomalization
of various anticancer drugs elsewhere [262 – 264] . For example, cytosine arabinoside
was administered intratracheally to rats in the free or the liposomal form
[262, 263] . Liposomal drug was effective into the lung but not other tissues, contrary
to the free drug. Moreover, the antitumor properties of the anticancer 9 -
nitrocamptothecin (9NC) after its liposomalization were tested in three different
human cancers xenografted s.c. in mice as well as murine melanoma and human
osteosarcoma pulmonary metastases [264] . The liposomal form of anticancer drug
inhibited the growth of subcutaneous tumors and metastatic pulmonary cancers
given via the respiratory system. Intramuscularly administered liposomal drug
exhibited some anticancer activity, but that achieved using the aerosol was superior.
Thus, the liposomal 9NC aerosol was proved to be of high potential for the treatment
of cancers throughout the body.
Recently the gene therapy of pulmonary diseases using liposomal formulations
has attracted a lot of attention. Transfection of the lungs of animals with aerosolized
cationic liposome – DNA complexes has been attempted [265, 266] . However, the
transfection effi ciency was rather low despite the large amounts of DNA used in
these studies. Cationic liposomes from 2, 3 - dioleyloxy - N - [2(sperminecarboxamido)
ethyl] - N, N - dimethyl - 1 - propanaminium trifl uoracetate (DOSPA), ( ± ) N - (2 - hydroxyethyl)
- N, N - dimethyl - 2, 3 - bis(tetradecyloxy) - 1 - propanaminium bromide (DMRIE)
mixed with DOPE, and DNA at fi xed ratios of DNA/lipid 1 : 4 for DMRIE and 1 : 3
for DOSPA were tested with two different types of jet aerosols (Aerotech II and
Puritan - Bennett 1600) [246] . The decrease in transfection activity was gradual with
Puritan - Bennett 1600, in contrast with Aerotech II, which rapidly lost transfection
effi ciency. That was attributed to the increased throughput of the Aerotech II,
resulting in more frequent cycling and therefore damage of the complex.
The impact of the zeta potential of the formulation was emphasized by Eastman
et al. [247] . The maximal aerosol transfer effi ciency of cationic lipid/DNA complexes
was achieved in the presence of a salt concentration of 25 m M . The authors attributed
that to the fact that the formulation kept its zeta potential between . 40 and
. 50 mV. As a closing remark, one of the major problems and challenges of aerosol
delivery is the duration to deliver therapeutic doses of DNA to the lungs. This possibly
could be overcome with dry powders avoiding volume limitations of aqueous
dispersions.
Oral Delivery The destructive effects of the conditions in the gastrointestinal tract
(GIT), especially due to the presence of bile salts, are known and well established
[267, 268] . The lipid composition of liposomes determines to a large extent the possibility
of remaining intact under such conditions [58] . Quadachi et al. attempted to
provoke IgA response from the M cells of Peyer ’ s patches after oral administration
of OVA - loaded MLV liposomes made by either soya PC or DSPC to a model of
hypersensitivity to OVA Balb/C mouse [269] . Clearly, DSPC MLVs were much more
stable as demonstrated in in vitro stability studies in simulating GIT media. However,
liposome incorporation of OVA did not cause any signifi cant impact on the reduction
of hypersensitivity to that obtained with the free allergen. Surprisingly, the
empty liposomes show some immunoresponse which was attributed to nonspecifi c
stimulation.
The biodistribution of novel liposomal - like spherical carriers called Spherulites,
consisting of PC/Chol/polyoxyethylene alcohol (43 : 4 : 3 w/w/w), was shown to be
more promising [270] . These were prepared by shearing the phospholipid bilayer
and labeling with 111 In. Their integrity was demonstrated by an increase in radioactivity
in the blood 1 h after oral administration to fasted rats, while no increase
was seen for free label.
Skin Delivery Dermal delivery of phospholipid - based vesicles fi rst appeared in
the literature in the early 1980s [271, 272] . Liposomes can play a dual role after their
application to the skin: retention (and perhaps protection) of the active compound
across the stratum corneum and acting as a penetration enhancer. The composition,
size, and vesicle surface charge are parameters that can infl uence the transport rate
of drugs contained in liposomal formulations across the skin [273] . Regarding their
composition, liquid - state liposomes resulted in higher skin permeation rates compared
with gel – liquid liposomes for progesterone and Triamcinolone (TRMA) [274,
275] . Moreover skin lipid liposomes have provided higher drug disposition in the
deeper layers of the skin for corticosteroids and acyclovir [276, 277] . Neverthless, in
another study [278] higher amounts of acyclovir in the skin were delivered from
conventional lipid liposomes compared with liposomes containing skin - based lipids.
These studies emphasize that a careful design is needed to defi ne optimal compositions,
depending also on the specifi c objective which may be either dermal (topical)
delivery of the liposome - encapsulated drug (which is easier since only increased
retention of the drug in the skin is desired) or transdermal delivery (systemic
absorption of the drug), which is more complicated, and in general deeper penetration
of the liposome carrier in the skin is required. What is also important as regards
the fi nal outcome (especially when transdermal delivery is the objective) is the type
of fi nal formulation with respect to the conditions applying, occlusive or
nonocclusive.
Amphotericin B encapsulated in charged liposomes demonstrated 10 - fold higher
transport rates across the skin compared with neutral liposomes [279] . In a similar
manner, the retention of acyclovir from positively charged vesicles was much higher
compared with other formulations [277] . This could be attributed to the attraction
IN VIVO DISTRIBUTION 475
476 LIPOSOMES AND DRUG DELIVERY
between the positively charged liposomes and the negative charge of the skin [277] .
The pore size of the skin is approximately 0.3 nm; however it can be opened up to
40 nm without signifi cant damage to its structure. To some extent, vesicles can transport
across the skin via the follicular and transcellular routes. Therefore the size of
the vesicles used for transdermal delivery is a crucial parameter for the overall
performance of the formulation [280 – 282] . Several studies have also emphasized
the impact of the size of the liposomes on the transport rate of active compounds
across the skin [277, 280, 283, 284] . Biologically active macromolecules, including
superoxide dismutase [285] and interferon - . [286] , have been successfully applied
to skin in liposomal formulations. Generally it is believed that the main pathway
for transdermal delivery of active compounds is either intercellular or paracellular.
However, the appendage transport (e.g., follicular route) has attracted a lot of interest
lately. The pilocebaceous units (hair follicle, hair shaft, sebaceous glands) can be
used for drug targeting. Combinations of liposomes with DNA [287, 288] and monoclonal
antibodies [289] have demonstrated that liposome composition, hair structure,
and hair cycle play signifi cant roles in the transfection of human hair follicles.
Two different mechanisms have been suggested for the incorporation of active
compounds to the hair shaft: (1) direct permeation of the vesicular formulations
with the active compound to the hair shaft and (2) incorporation of the active compounds
in the follicular matrix cells and then into the hair shaft as the matrix cells
develop and differentiate into new hair shafts [290] .
Recently, for the transdermal delivery of drugs using carrier systems, attention
has been focused on the development of transformable [284, 285] or elastic vesicles
[12] . These vesicles are liposomes that contain surfactants or in general “ edge activators
” in addition to phospholipids in their lipid membranes (Figure 10 ), a fact that
Double-chain nonionic surtactant or lipid
Emphiphilic or lipophilic drug
Charged hydrophilic drug
Single-chain surfactant
Hydrophilic drug
FIGURE 10 Conventional liposomes/elastic vesicles. Charged hydrophilic, amphiphilic,
and lipophilic drug molecules can be associated with the bilayers of the vesicles, whereas
hydrophilic substances can also be entrapped in the vesicles. Rigid vesicles consist of double -
chain nonionic surfactants or lipids in the presence or absence of cholesterol (left image).
Elastic vesicles consist of double - chain surfactants or lipids and an edge activator. The edge
is often a single - chain surfactant (right image). ( Reprinted from ref. 273 with permission of
Elsevier .)
increases their elasticity (ability to be deformed, without being disrupted, after
applying pressure on them). Modifi ed liposomes called ethosomes (containing
alcohol) have shown increased skin permeability. Because of their structure, ethosomes
are able to encapsulate and deliver through the skin highly lipophilic molecules
such as cannabinoids, testosterone, and minoxidil as well as cationic drugs such
as propranolol and trihexyphenidil or even plasmids and insulin [291] . Although the
mechanism of the increased transdermal delivery of drug molecules still remains a
controversial issue, mainly with respect to the depth at which intact vesicles can
travel in the skin, many interesting results are being generated. However, due to the
limited permeability of the skin membrane, physical enhancement mechanisms,
including iontophoresis, electroporation, and ultrasound, have been used in combination
with chemical enhancers (liposomes) to increase skin permeability. One of
the methods, application of an electric current to the skin, has been shown to
promote the transdermal transport of drugs by an additional driving force, namely,
an electrical potential gradient across the skin [292] . Transdermal iontophoretic
transport of a liposomal formulation across human cadaver skin was fi rst reported
for [Leu5] enkephalin [293] . Liposomes could penetrate into the skin. Enkephalin,
when delivered iontophoretically from liposomes carrying positive or negative
charge on their surface, was found to be the same or less than that of the controls;
however, the degradation of enkephalin was less in liposome formulations as compared
to controls, demonstrating that liposomes can protect peptides from the proteolytic
environment of skin. However, when enoxacin was encapsulated in different
liposome formulations and was transported (electrically assisted) across the skin,
the drug transport results showed that the permeability of enoxacin released from
liposomes was higher compared to that of free drug [294] . In vitro transdermal
iontophoretic delivery of estradiol from ultradeformable liposomes, saturated
aqueous solution [295] , and conventional liposomes [296] has demonstrated the
superiority of ultradeformable liposomes (Transfersomes) due to their lipid composition.
Liposomal formulations of . blockers were iontophoresced in vivo to hairless
rats [297] . Skin irritation was signifi cantly reduced when a liposomal formulation of
the propranolol base was used rather than the base itself, emphasizing another
important role liposomes could play. When adriamycin was delivered via the hair
follicles using various liposomes and iontophoresis combined with application of
ionic liposomes, higher transport rates were obtained with the latter, emphasizing
their synergistic effect [298] . Moreover skin electroporation was applied to enhance
gene transfer into subcutaneous MC2 murine breast tumor skin in combination with
cationic liposomes demonstrating signifi cant transfection improvement [299] .
However, when electoporation combined with estradiol - loaded liposomes were
applied to skin, the estradiol skin penetration was not affected signifi cantly. That
was attributed to the antienhancer or retardant effect of liposomes [300] . In a
mechanistic study, anionic phospholipids were found to enhance the transdermal
transport of molecules by electroporation compared to cationic or neutral phospholipids,
offering new insights to design better enhancers for transdermal drug and
vaccine delivery [301] . Finally higher transport across the skin obtained after combined
application of ultrasound and liposomal formulations of diclophenac demonstrated
a synergistic effect [302] .
Ocular Delivery Ocular drug delivery has evolved into a great challenge and a
subject of interest for many scientists with different backgrounds, including medical,
IN VIVO DISTRIBUTION 477
478 LIPOSOMES AND DRUG DELIVERY
clinical, pharmaceutical, physical, chemical, biochemical, and toxicological sciences.
For ailment of eye diseases, topical administration is preferred over systemic in
order to avoid systemic toxicity, for rapid onset of action, and for decreasing the
required dose.
The main route for intraocular absorption is across the cornea [303] . In terms of
drug delivery, the cornea presents an effective barrier to the absorption of both
hydrophilic and lipophilic compounds. Actually, the main constraints in topical
ocular delivery are (i) poor ocular retention of conventional dosage forms [304] and
(ii) poor corneal absorption. Various approaches have been developed to increase
the bioavailability and duration of therapeutic action of ocular drugs. One such
approach is based on the use of drug delivery systems [305, 306] , which provide
controlled and continuous delivery of drugs and can also provide improved
(increased) residence time of the drug at the delivery site. Recently, intravitreal drug
injection has evolved into a preferred administration method for therapy of disorders
in the posterior segment of the eye [305] . The procedure is associated with a
high risk of complications, particularly when frequent, repeated injections are
required. Thus, sustained - release technologies are being proposed, and the benefi ts
of using colloidal carriers in intravitreal injections are currently under investigation
for posterior drug delivery.
Between the different types of particulate drug delivery systems, liposomes offer
additional advantages for ophthalmic delivery, since they are completely biodegradable
and relatively nontoxic and thus are well tolerated by the eye [305, 306] . Indeed,
when using other types of colloidal systems, for example , nanoparticles consisting
of polyalkyl cyanoacrylate, infl ammation and damage of the corneal epithelium
have been reported [307 – 309] . Another potential advantage of liposomes is their
ability to come in intimate contact with the corneal and conjunctival surfaces. This
results in increased probability of ocular drug absorption [310, 311] .
The potential of liposomes in topical ocular drug delivery was fi rst exploited in
the 1980s by a number of research groups [303, 310 – 315] . As an example, higher
levels of inulin were found in the cornea when it was encapsulated in liposomes as
compared to its aqueous solution [301, 314] , and this was attributed to the physical
adsorption of lipid vesicles onto the epithelial surface of the membrane [315, 316] .
More recently a number of liposomal applications for ocular delivery have been
under investigation [305, 306] for anterior as well as posterior segment administration,
as outlined in Table 3 . Indeed, a large number of ophthalmic drugs used in
cases of ocular surface disorders (such as dry eye syndrome) [317] , keratitis and
uveitis [318 – 327] , and keratoplasty [328 – 331] have been studied in liposomal form,
and in most cases the results were promising in terms of drug penetration and retention
in the various ocular tissues (cornea, sclera, retina, and choroids), following
subconjunctival administration. In some cases, detectable levels of drugs were found
in ocular tissues up to 7 days after administration [305, 306] .
As mentioned above, the ability to adsorb to the cornea and an optimal drug
release rate have been defi ned as the two liposomal attributes most responsible for
increasing bioavailability after topical ocular administration. A number of factors,
including drug encapsulation effi ciency, liposome size and charge, distribution of the
drug within liposomes, stability of liposomes in the conjunctival sac and ocular
tissues, their retention in the conjunctival sac, and most importantly their affi nity
toward the corneal surface and the rate of release of the encapsulated drug, have
been found to infl uence the effectiveness of liposomes in topical ocular drug delivery
[310, 323, 332 – 335, 337] . Indeed, liposomal manipulation to increase corneal
adherence has met with some success [310] .
Positively charged liposomes seem to be preferentially captured at the negatively
charged corneal surface as compared with neutral or negatively charged liposomes.
Aditionally, cationic vehicles are expected to slow down drug elimination by the
lacrymal fl ow both by increasing solution viscosity and by interacting with the negative
charges of the mucus [334, 335] . Indeed, positively charged phospholipids
yielded superior retention of liposomes at the corneal surface in rabbits [336] .
Schaeffer et al. [310] worked with indoxole and penicillin G and reported that liposome
uptake by the cornea is greatest for positively charged liposomes, less for
negatively charged liposomes, and least for neutral liposomes. Positively charged
unilamellar liposomes enhanced transcorneal fl ux of penicillin G across isolated
rabbit cornea more than fourfold. Similar results were also obtained by others [336,
323] . By observing the morphology of corneal surface treated with liposomes, it was
suggested that positively charged liposomes formed a completely coated layer on
the corneal surface [323] . These liposomes bind intimately on the corneal surface,
TABLE 3 Experimental Liposomal Preparations of Drugs for Anterior and Posterior
Segment Administration
Drug Class Anterior Segment Drugs Posterior Segment Drugs
Antibiotics Gentamicin
Norfl oxacin
Tobramycin
Clindamycin
Gentamicin
Penicillin
Antifungals Amphotericin B Amphotericin B
Antivirals Acyclovir
Idoxuridine
Ganciclovir
Trifl uorothymidine
(Trifl uridine)
Steroids Dexamethasone
Immunosuppressives Cyclosporine
FK506 (Tacrolimus)
Cyclosporine
Antimetabolites 5 - Fluorouracil (5 - FU)
5 - Fluoroorotate
5 - Fluorouracil (5 - FU)
5 - Fluorouridine (5 - FUR)
Bleomycin
Cytosine arabinoside
(Cytarabine)
Daunomycin
Daunorubicin
Etoposide (VP - 16)
Platelet - aggregating
agents
Adenosine diphosphate Adenosine diphosphate
Photosensitive
cytotoxic agents
Verteporfi n (BPD - MA)
Miscellaneous Dichloromethylene
diphosphonate (Clodronate)
Disulfi ram
Immunoglobulins
Dichloromethylene
diphosphonate (Clodronate)
Source : S. Ebrahim, G. Peyman, and P. J. Lee, Survey of Ophthalmology , 50, 167 – 182, 2005.
IN VIVO DISTRIBUTION 479
480 LIPOSOMES AND DRUG DELIVERY
leading to an increase in residence time and therefore to an increase in corneal
absorption time.
With respect to vesicle size, larger particles are more likely to be entrapped
under the eyelids or in the inner canthus and can thus remain in contact with
the corneal and conjunctival epithelia for extended periods. Indeed, larger liposomes
have been found to resist drainage at the inner canthus and are more
bioavailable at the ocular surface [337] . However, for patient comfort, it is considered
that solid particles intended for ophthalmic use should not exceed 5 –
10 . m diameter [338] .
Assessment of ocular irritability of neutral or positively charged liposomes by
the Draize test, histological examination, and the rabbit blinking test has also
been reported in the literature [339] . The mean total score (MTS) of the Draize test
was found to show a slight increase immediately following instillation of liposome
preparations. However, it did not exceed the “ practically nonirritating level, ” and no
corneal histological alteration was observed by optical microscopy. Neutral liposome
preparations were confi rmed to be nonirritating; however, positively charged liposomes
may cause initial pain or unpleasantness following instillation. Thus, althouth
positive charge helps in improving the contact time with the cornea, at the same time
it can lead to irritation. Additionally, the release rate of the drug is found to be more
in neutral liposomes, while increased liposome size restricts solution drainage, thus
prolonging contact time of the drug, but it can be increased within the limits of not
inducing any irritancy. From all the above it is understood that in each case liposome
properties have to be adjusted for best in vivo therapeutics results. As mentioned
above, liposome stability is another important factor. Barber and Shek reported that
increasing the cholesterol content of the liposomal membrane decreased the rate of
tear - induced release of its contents [340] .
The use of bioadhesive polymers (e.g., a polyacrylic acid, chitosan, hyaluronic
acid) to prolong the residence time of an ocular preparation in the precorneal
region, due to increased formulation viscosity, is another approach which can further
improve liposomal drug delivery. In this respect “ collasomes ” (liposomes coupled
to collagen matrices) increased the bioadhesive ability of liposomes, were well tolerated,
and could be instilled safely and effectively by patients in the same fashion as
ointments or drops [341] . It was also demonstrated that liposomes coated with collagen
layer bound to cell monolayer with higher affi nity [342] . Other approaches
were used to increase the contact time of ocular liposomes, such as the case in which
prolonged retention of liposomal suspension of oligonucleotide was achieved
by dispersing liposomes within an in situ gel - forming medium [343 – 345] . Novel
measures to further enhance adsorption of liposomes and increase penetration
of the cornea included application of a natural lectin which promoted binding of
ganglioside - containing liposomes to the cornea [346] .
Liposomes can also be used as promising dosage forms for topical administration
of immunosuppressive compounds for the treatment of ocular immune - mediated
diseases [327] . Indeed, it was found that liposomes containing immunosuppressive
compound FK506 were effective in delivering signifi cantly higher drug concentrations
to all ocular tissues and particularly aqueous humor and vitreous humor as
compared to the oil formulation of the agent. Further, liposomes can be used to
protect drug molecules from attack of metabolic enzymes present at the tear/corneal
epithelium interface, as demonstrated for the O - palmitoyl prodrug of tilisolol
[347] .
As mentioned above, intravitreal injection of drugs should be used in many cases
to achieve therapeutic intravitreal drug levels. This is especially true for cases of
viral retinitis, such as cytomegalovirus (CMV) retinitis and acute retinal necrosis
(ARN) which require intravitreal injection of antivirals, or for the treatment of
bacterial and fungal endophthalmitis or proliferative vitreoretinopathy [305] . It still
remains a controversial issue whether liposomes can reach the retina after intravitreal
injections and which vesicle physicochemical characteristics should be preferred
for such formulations.
In addition to conventional drugs, liposomes were also used for intravitreal
administration of oligonucleotides in order to treat ocular viral infections such as
herpes simplex virus or CMV [344] . Antisense oligonucleotides are poorly stable in
biological fl uids and their intracellular penetration is limited. Hence a system that
is able to permit a protection of oligonucleotides against degradation and their slow
delivery into the vitreous should be favorable for improving the therapeutic outcome
in addition to patient compliance. It was found that lipid vesicles are able to protect
oligonucleotides against degradation by nucleases [344, 345] . Furthermore, they
increase the retention time of many drugs in the vitreous. Thereby, the use of liposomes
for intravitreal administration is a very promising approach.
Nasal Delivery Liposomes are able to decrease mucociliary clearance in the case
of nasal administration due to their low viscosity. This is attributed to the incorporation
of liposomal lipids in the membranes of the nasal epithelial cells, which results
in the opening of pores in the paracellular tight junctions [348] . MLVs containing
nifedipine administered via the nasal route could attain a constant plasma level
[349] . Liposomal formulations of levonorgestrel containing carbopol or chitosan
demonstrated prolonged contact time with the absorptive surfaces, resulting in
increased bioavailability of the intranasally administered drug [350] .
The nasal route may be highly promising for candidate vaccines against potential
pathogens (and infection - related diseases such as cancers) that utilize this route of
infection [351, 352] . In addition to the likelihood of increased patient compliance,
immune responses elicited by nasal administration may be more predictable when
compared with the vaginal route due to immunological changes in the female reproductive
tract during the menstrual cycle [352] . Previous work with liposome -
containing vaccines for nasal delivery demonstrated that liposomes can confer
adjuvancy to the subunit infl uenza vaccine, but also empty liposomes administered
48 h prior to immunization resulted in immune stimulation, emphasizing that the
properties and composition of the liposomes play a signifi cant role facilitating the
transport of the antigen across the membrane [353] . More recently, several promising
liposome - based vaccines are being designed and investigated for delivery by the nasal
route, such as the liposome - encapsulated plasmid DNA - encoding infl uenza virus
hemagglutinin, which has been reported to elicit mucosal, cellular, and humoral
immune responses after intranasal administration in Balb/C mice [354] . Additionally,
nasal immunization studies using liposomes loaded with tetanus toxoid were perfomed,
and it was found that intranasal administration of liposome - encapsulated
vaccines can be an effective way for inducing mucosal immune responses [355] . Furthermore,
intranasal immunization studies have been carried out with liposomes
containing recombinant meningococcal opacity proteins [356] and with anthrax -
protective antigen protein incorporated in liposome – protamine particles [357] , both
with promising results.
IN VIVO DISTRIBUTION 481
482 LIPOSOMES AND DRUG DELIVERY
Vaginal Delivery The vaginal route has been under investigation in the last years,
especially for the topical delivery of drugs that are intended to act in the vagina, as
contraceptives, microbicides and antibiotics, as recently reviewed [358] . In such cases
the main advantage of using liposomes would be the controlled and sustained
release of the drug at the site, which would result in a less frequent drug administration
and improved patient compliance.
However, the major limitation of using liposomes topically and vaginally is the
liquid nature of preparation. Nevertheless, several formulation characteristics should
be optimized in order to achieve the needed rheological and mucoadhesice properties
for maximum retention of the delivery system in the vagina. Research for the
development of liposomal gels (gels that contain liposomes) with the required
properties is currently ongoing [358 – 360] .
It has been demonstrated that, by their incorporation in an adequate vehicle, such
as carbopol resins, the original structure of vesicles is preserved [359] , while liposomes
are fairly compatible with gels made from polymers derived from such resins.
A previous study has suggested application of liposomes containing antimicrobial
drugs for the local therapy of vaginitis [359] , while recently the design and in vitro
evaluation of bioadhesive liposome gels containing clotrimazole and metronidazole,
or acyclovir, were carried out [359, 360] .
Some other applications are arising lately concerning liposomes and vaginal
administration. As mentioned above (in the nasal administration section), mucosal
surfaces serve as a gateway to disease. It was recently demonstrated that RNA
interference can be used to manipulate mucosal gene expression in vivo. Using
a murine model, it was shown by Zhang et al. [361] that direct application of
liposome - complexed small interfering RNA (siRNA) mediates gene - specifi c
silencing in cervicovaginal and rectal mucosa. A single vaginal or rectal administration
of siRNA targeting hematopoietic or somatic cell gene products reduced
corresponding messenger RNA (mRNA) levels by up to 90%. Additionaly, liposomal
siRNA formulations proved nontoxic, did not elicit a nonspecifi c interferon
response, and provided a means for genetic engineering of mucosal surfaces in
vivo.
In addition, it was recently found that when human immunodefi ciency virus
type 2 (HIV - 2) DNA vaccine were formulated with cationic liposomes [362] ,
stronger immune responses in mice were observed compared with naked DNA
alone. Using this knowledge, very recently a vaccine consisting of some HIV - 2
genes ( tat, nef, gag , and env ) was formulated within cationic liposomes by Lochera
et al. [363] . Baboons ( Papio cynocephalus hamadryas ) that were immunized by
the intramuscular, intradermal, and intranasal routes with these expression constructs
were challenged with HIV - 2 UC2 by the intravaginal route, and the results
of this study demonstrate that partial protection against HIV - 2 vaginal challenge,
as measured by reduced viral load, can be achieved using only a DNA vaccine
formulation.
5.3.4 APPLICATIONS OF LIPOSOMES IN THERAPEUTICS
It is well known that liposomes have many applications in drug delivery. Initially,
after the limitations of conventional liposomes were noticed, great effort was given
toward the therapy of parasitic diseases, due to the fact that the targeting of RES
macrophages, the place were parasites are mainly located, was considered to be very
easy and fast (usually mentioned as passive targeting). Indeed, even now, research
is ongoing, such as the treatment of drug - resistant visceral leishmaniasis with liposomal
amphotericin - B [364] or with sterically stabilized liposomes containing camptothecin
[371] . Nevertheless, possibly due to the high cost of liposome manufacturing,
in relation to other drug formulations, and the fact that the main need for such
medicaments would be for third world countries, such products have not been marketed
for these diseases, despite the fact that they offer therapeutics advantages. In
addition, again due to the same etiology, a very small part of recent research efforts
and money are devoted to such diseases.
A list of the marketed liposomal products is presented in Table 4 [364 – 366] . In
addition, liposomes are currently being investigated for a variety of conventional
and novel drugs: therapeutic agents, including antibiotics (as amikacin [367] , vancomycin,
and ciprofl oxacin [368] ); anticancer agents (e.g., paclitaxel [369] and cisplatin
[370] ; camptothecin and analogs [371 – 373] ), biologics such as antisense oligonucleotide
[374] , DNA, and siRNA [375] ; and muramyl tripeptide [376] . A list of most
liposome - based products currently under clinical investigation is presented in
Table 5 . Additionally many products are currently being evaluated in preclinical
studies. In many of the latest studies, the liposomes used have been surface modifi ed
with active targeting ligands to improve delivery of therapeutics to target cells [238,
377 – 379] .
After investigating the recent literature, we have seen that most recent efforts
connected with the use of liposome in therapeutics are mainly connected with the
treatment of cancer. This is the reason why here we deal with the most common
cancer types (brain, breast, lung, and ovarian cancer), and ongoing research and
clinical treatments are discussed in more detail. However, we do not want to
imply that the future of liposomes in drug delivery is limited to cancer therapy.
Indeed, liposome structure, characteristics, and versatility are sure to fi nd, in
TABLE 4 Currently Marketed Liposome - Based Products
Active Agent a Application
Daunorubicin (DaunoXome, Gilead Sciences, Inc.) Kaposi ’ s sarcoma
Doxorubicin (Doxil/Caelyx, Ortho Biotech
ProductsLP/Sequus Pharmaceuticals)
Kaposi ’ s sarcoma
Amphotericin B (Ambisome/Abelcet, Fujisawa
Healthcare, Wyeth Pharmaceuticals)
Fungal infections in
immunocompromised patients
Doxorubicin (Myocet/Evacet, Sopherion/
Liposome Company)
Metastatic breast cancer
Hepatitis A virus envelope proteins (Epaxal,
Berna Biotech)
Hepatitis A
Infl uenza virus (Infl exal V, Berna Biotech) Infl uenza
Verteporfi n (Visudyne, Novartis Ophthalmics) Age - related macular degeneration
Source : D. Felnerova, J. F. Viret, R. Gluck, and C. Moser, Current Opinion in Biotechnology , 15, 518 – 529,
2004.
a Product names and companies given in parentheses.
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 483
484 LIPOSOMES AND DRUG DELIVERY
TABLE 5 Liposome - Based Products Currently under Clinical Testing
Active Agent or Product a Application
Company
and Trial Phase/Reference
Drug delivery
Caelyx Bladder cancer Schering Plough; approved EU
Doxil Multiple myeloma Schering Plough; III ALZA
Pharm; III
Bladder, liver cancer ALZA Pharm; II, III
Pancreatic cancer ALZA Pharm; II, Sequuz; II
Doxorubicin combined
with ATB
Prostate cancer Pharmacia; III, Neopharm; III
Bladder cancer Neopharm; II, III Pharmacia;
II, III
Myocet combined with
ATB
Bladder cancer Liposome; III
Liposomal ether lipid Bladder cancer Liposome; I
(TLC ELL12) Lung, prostate, skin cancer Elan Pharm; I
Platinum Cervical, ovarian, kidney Aronex Pharm; II
(Aroplatin) Lung, pancreatic cancer Aronex Pharm; I, II
Annamycin Leukemia
Paclitaxel Head and neck cancer Pharmacia; II, III
Lung cancer Neopharm; II, III
Vincristine (Onco - TCS) Lung cancer Inex Pharm; II
Topoisomerase inhibitor
(OSI 211)
Lung, ovarian cancer OSI Pharm; II
All - trans retinoic acid
(ATRA - IV)
Lung cancer Antigenics; II
Mitoxanthrone Other cancers Neopharm; II
Nystatin (Nyotran) Leukemia (antifungal) Pharmacia; II
Lung (antifungal) Aronex Pharm; II
Prostate cancer (antifungal) Abbott Laboratory; II
DNA delivery
Human leucocyte
antigen (HLA) B and
. 2 microglobulin
plasmid DNA
(Allovectin)
Head and neck cancer Vical; II
Interleukin - 2 plasmid
DNA
Kidney, prostate cancer Vical; II
Antisense toraf - 1
(LerafON)
Leukemia Neopharm; I
Antigen delivery:
MUC - 1 peptide: BLP25
(human epithelial
mucin peptide)
Lung cancer Biomira; II
Merck; III
Source : D. Felnerova, J. F. Viret, R. Gluck, and C. Moser, Current Opinion in Biotechnology , 15, 518 – 529,
2004.
a Commercial names are given in parentheses. ATB, antibiotika.
addition to those existing already, numberous applications in drug delivery in the
future.
Recently, a multicomponent liposomal drug delivery system consisting of doxorubicin
and antisense oligonucleotides targeted to MRP1 mRNA and BCL2 mRNA
to suppress pump resistance and non – pump resistance, respectively, has been developed
[379] . This liposomal system successfully delivered the antisense oligonucleotides
and doxorubicin to cell nuclei, inhibited MRP1 and BCL2 protein synthesis,
and substantially potentiated the anticancer action of doxorubicin by stimulating
the caspase - dependent pathway of apoptosis in multidrug resistant human lung
cancer cells.
5.3.4.1 Anticancer Drug Delivery
Brain Tumors Brain tumors are classifi ed as gliomas (astrocytomas, oligodendrogliomas,
ependymomas) and primitive neuroectodermal tumors (PNET) (medulloblastoma
and supratentorial PNETs). Approximately half of all primary brain
tumors are gliomas, while 80% of those are astrocytomas and glioblastomas. Most
chemotherapeutic drugs are toxic to the healthy tissue and have damaging side
effects due to their nonspecifi c nature. Incorporation of those drugs in liposomes
can enhance the therapeutic effi cacy and reduce the toxicity. Drug delivery to the
brain via the intravenous route has been a very challenging task due to the strict
selectivity of the blood – brain barrier (BBB) as to the number and kind of molecules
able to pass through.
The BBB is the tight junction formed between the cerebral endothelial cells
(Figure 11 ). These cells are in close contact with astrocytes and pericytes connected
over a basal membrane. Only small (MW < 400 – 600) lipophilic molecules can diffuse
FIGURE 11 Schematic of neurovascular unit/cell forming BBB (brain – blood barrier).
( Reproduced from D. J. Begley, Pharmacology & Therapeutics , 104, 29 – 45, 2004, with
permission by Elsevier .)
Axonal
ending
Pericyte
Endothelium
Extraccllular matrix
Astrocytic
foot process
Microglial
cell
Tight
junction
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 485
486 LIPOSOMES AND DRUG DELIVERY
through the BBB, while the majority of the circulating drugs cannot access the brain.
Nutrients and peptides pass through the BBB via receptor - mediated or carrier -
mediated transport systems. These mechanisms are exploited in an attempt to deliver
drug - loaded liposomes into the central nervous system (CNS) [380] . The most
common are low - density lipoprotein (LDL) receptors, insulin receptors, and transferrin
receptors.
Thus, mAb against the transferrin receptor OX26 mAb has been conjugated via
a stable thioether bond to the end of the PEG chain inserted on the liposome
surface. Tritiated daunomycin was incorporated in OX26 mAb – PEG liposomes and
the formulation was given intravenously to rats. The brain volume of distribution
of daunomycin increased with time and exceeded 200 . L/g 24 h after injection [381] .
In contrast, the pharmacokinetics of the free drug and the drug loaded in conventional
liposomes was much lower [382] .
In another study, the OX26 mAb has been grafted on the PEG chains by the
biotin streptavidin coupling [383] . Brain tissue distribution obtained using biotinylated
immunoliposomes was the same with that reported in the previous work where
the mAb was chemically linked on the distal end of PEG. Therefore, the coupling
method has not had a great impact on the brain accumulation of immunoliposomes.
However, accumulation of the biotinylated PEG immunoliposomes was quite high
in tissues such as liver, spleen, heart, muscle, and kidney. The latter was attributed
to either the fact that the OX26 – biotinylated PEG immunoliposomes could pass
through the BBB by an active transport system or the biotinidase activity, which
could mediate cleavage of the targeting antibody from PEG and interfere with the
tissue distribution of the formulation.
An increase in therapeutic effi cacy and lower toxicity was reported with liposomes
where the bradykinin analogue RMP - 7 was chemically attached at the end
of PEG molecules of PEGylated liposomes (approximate size 70 nm) [384] . RMP - 7
exhibits high selectivity for the B2 receptor of the BBB endothelial cells, which
“ shrunk ” and let the RMP - 7 – PEG liposomes to pass into the brain. Actually the
mechanism used in that study was based on opening the tight junctions of the BBB.
Liposome - incorporated nerve growth factor (NGF) concentration increased 10
times in comparison to free NGF, while they accumulated mainly in striatum,
hippocampus, and cortex.
A different type of immunoliposome was developed using antinuclear autoantibodies
with nucleosome (NS) – restricted specifi city [187] . Anti - NS mAb 2C5
specifi cally recognizes human brain tumor cells. Evaluation of immunoliposomes
2C5 – PEG – PC/Chol was carried out in nude mice bearing subcutaneous brain tumor
(U - 87 astrocytoma) and exhibited a threefold higher accumulation in the tumor in
comparison to control (IgG – PEG liposomes).
Moreover, disialoganglioside (GD 2 ) is expressed in abundance by neuroectodermal
cancer cells and in low levels by normal cells located mainly in cerebellum and
peripheral nerves. The Fab . fragment of the monoclonal antibody anti - GD 2 was
grafted on PEG chains of sterically stabilized liposomes loaded with doxorubicin
and their potential in treating neuroblastoma was assessed in nude mice with
HTLA - 230 xenografts [160] . Mice receiving intravenously the immunoliposomes
showed signifi cant improvement in long - term survival compared with other mice
that received free DOX, freeGD 2 Fab . , Fab . – PEG liposomes, PEG liposomes – DOX.
The control mice died from metastatic disease, while the immunoliposome - treated
group lived 4 months longer. The same ligand, anti - GD 2 – Fab . , was used with liposomes
carrying the antisense oligonucleotide c - myb [385] . After intravenous injection
of the targeted liposomes into nude mice with HTLA - 230 xenografts, signifi cant
prolonged survival times were obtained in comparison to controls (Figure 12 ). The
suggested mechanism was downregulation of c - myb proto - oncogene expression.
Although all the active targeting liposomes mentioned earlier have not left the
laboratory, nonspecifi c sterically stabilized liposomes are being tested in clinical
trials. Doxorubicin is the anticancer agent which is used as standard therapy, and it
has the most serious side effects (mucositis, cardiotoxicity), so its incorporation in
liposomes and bioavailability enhancement are under scrutiny [386 – 388] .
It has also been demonstrated that PEGylated liposomal doxorubicin, Caelyx,
can cross the BBB with a consequent accumulation in primary and secondary brain
lesions [389] . In 10 patients with metastatic brain tumors treated with radiolabeled
Caelyx concurrent with radiotherapy, the accumulation of the liposomal doxorubicin
was 7 – 13 times higher in the metastatic lesions compared to the normal
brain.
Liposomal formulations, type of tumor, anticancer agent, delivery pathway, day
of treatment, and general conclusions are given in Table 6 . DaunoXome (liposomal
daunorubicin) and Doxil (liposomal doxorubicin) have been proved to have good
response in clinical trials, in the range of approximately 40%.
Also, according to Arnold et al. [390] , the dose scheme of doxorubicin - loaded
liposomes affects the drug accumulation in various tissues as well as the tumor. The
authors reported that after repetitive doses of sterically stabilized liposomes (SSL) -
DOX every week, the plasma half - life of the drug increased, the deposition in liver
and spleen decreased, and peak concentrations of DOX in the heart were threefold
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Control
Free- CpG-myb-as
CCL-CPG-myb-scr
CCL-CpG-myb-as
Targeted-CCL-CpG-myb-scr
Targeted-CCL-CpG-myb-as
100
75
50
25
0
0 40 80 120 160
Time (days)
Survival (%)
FIGURE 12 Survival of NB - bearing nude mice after injection of oligonucleotides
(free or encapsulated within liposomal formulations). Nude mice were injected intravenously
with 3.5 . 10 6 HTLA - 230 neuroblastoma cells. After 4 h each mouse received 50 . g of
oligonucleotides either free or encapsulated in targeted or nontargeted liposomes.
Control mice received HEPES - buffered saline. ( Reprinted from ref. 385 with permission of
Elsevier .)
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 487
488 LIPOSOMES AND DRUG DELIVERY
TABLE 6 Recently Completed and Ongoing Clinical Trials of Low - Molecular - Weight Drug -
Carrying Liposomes for Brain Tumors
Tumor
Number
of
Patients Treatment Delivery
Day of
Treatment General Conclusions
Recurrent
tumor
14 Daunorubicin IV Once every 4
weeks
6/14: Patients showed
positive response
GBM 8 Daunorubicin IV 24 h before
surgery
Concentration similar
in tumor mass and
peripheral regions
Pediatric
glioma
7 Daunorubicin
+ free
carboplatin
and
etoposide
IV Daunorubicin:
day 1 and 2;
carboplatin
and etoposide:
day 1
5/7: Showed positive
response with
monthly treatment
GBM 15 Doxorubicin +
radiation
IV Doxorubicin:
days 1, 21;
radiation: days
1 – 21, 21 – 23
4/10: Patients
completely
responded
Solid tumor 22 Doxorubicin IV Once every 4
weeks
Phase I: dose - limiting
toxicity: 70 mg/m 2
Glioma 40 Daunorubicin
+ free
tamoxifen
IV PEG - dox: day 4
every 14 days
Phase II: response
(including
stabilization) 40%;
tamoxifen: day 1
every 4 days
Glioma 8 111 In labeled IV Contrast only Tumor uptake: 1.1%
max tumor/brain
contrast 7:5
Glioma 3 Bleomycin IT Twice weekly
for up to 6
weeks
All patients
deteriorated, no
toxicity
Recurrent
meningeal
malignancies
15 Cytarabine IT Once every 2
weeks for 2
courses; in
positive
response,
second
induction in 2
weeks after
fi rst dose
Meningeal
neoplasms
100 Cytarabine
against free
methotrexate
IT Once every 2
weeks
Ongoing
Source : From G. H. Huynh, D. F. Deen, and F. C. Szoka, Jr., Journal of Controlled Release , 110, 236 – 259, 2006.
Reprinted with permission of Elsevier.
Note : GBM: glioblastoma; IV, intravenous; IT: intratumor; PEG: poly(ethylene glycol). See http://www.
clinicaltrials.gov .
lower. These results were not obtained using free DOX. In addition, a signifi cant
increase in survival was achieved in animals treated weekly with SSL - DOX, while
animals treated with free drug did not survive longer than the untreated controls.
In phase I clinical trials children with recurrent or refractory tumors previously
treated with free doxorubicin were administered i.v. Doxil in various doses (40 –
70 mg/m 2 ) in order to determine the best tolerated one [391] . It was concluded that
the maximum tolerated dose was 60 mg/m 2 because at the highest, 70 mg/m 2 , some
patients developed mucositis, so dose adjustment was necessary.
Administration of a combination of liposomal anthracyclines in parallel with
other anticancer agents has been found of great advantage as they prolong
the patients ’ survival. Caraglia et al. evaluated in a phase II study the use of
combination Doxil with temozolomide in the treatment of brain metastases from
brain tumors [392] .
It is worth mentioning that temozolomide accumulates signifi cantly in the brain
after oral administration. It is well tolerated and therefore is considered a potential
candidate for combination chemotherapy. Administration of 200 mg/m 2 of temozolomide
for 5 days and liposomal doxorubicin 35 mg/m 2 on day 1 was performed
on 19 patients. The overall response rate was 36.8% and the median overall survival
was 10.0 months.
Moreover, Fiorillo et al. [393] studied the effect of a combination of liposomal
daunorubicin, etoposide, and carboplatin administered to seven children with recurrent
malignant supratentorial brain tumors as a second - line therapy. Chemotherapy
consisted of infusion of liposomal daunorubicin on days 1 and 2 and infusion of
etoposide and carboplatin on day 1 whereas courses were repeated every 3 – 4 weeks.
After a total of eight courses, fi ve of seven children evaluated were alive 12 – 64
months after diagnosis and 8 – 29 months from the start of the second - line chemotherapy.
Of the seven children, three showed complete response, two partial
responses, one stable disease, and one progressive disease. The time to the best
response was 3 – 10 months, while the median time to progression was 23 months.
The toxicity observed was minimum.
Boron neutron capture therapy (BNCT) is also of high interest in treating brain
tumors, especially glioblastoma multiforme, due to the high degree of normal brain
infi ltration, the high histological complexity, and the heterogeneity of the cellular
composition of the latter. This method is based on the nuclear reaction which
occurs when boron - 10 is irradiated with low - energy thermal neutrons, producing
high linear energy transfer of . particles and lithium - 7 nuclei [394] . Development
of BNCT has been ongoing over the last 50 years and the greatest challenge is to
achieve selective tumor targeting at a suffi cient therapeutic dose with minimal
toxicity. Various compounds are currently being used or have been used in BNCT
(shown in Figure 1 of [394] ), such as BPA [( l ) - 4 - dihydroxy - borylphenylalanine]
and BSH (sodium mercaptoundecahydro - closo - dodecaborate), which are the fi rst
most successfully used chemical compounds (so - called second - generation compounds)
due to low toxicity, longer retention to the tumor site and tumor/brain,
and tumor/blood ratios higher than 1. However, these drugs are not ideal, but they
are safe after i.v. administration, so they are being used in clinical trials in Europe,
the United States, Japan, and Argentina. The next group of advanced boron molecules
(third generation) consists of stable boron or a cluster attached to a tumor -
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 489
490 LIPOSOMES AND DRUG DELIVERY
targeting moiety, such as monoclonal antibodies or low - MW biomolecules with
amphiphilic properties. Other anionic compounds show little specifi city, so their
potency increases when they are incorporated in either targeted or nontargeted
liposomes [395, 396] .
According to Feakes et al. [396] , boron - loaded DSPC/Chol liposomes of 40 nm
average size were prepared and injected i.v. in murine mice carrying EMT6 tumors.
Those liposomes showed high tumor retention, while boron amount was at therapeutic
levels through the entire course of the experiment (more than 15 . g B/g
tumor). However, targeted liposomes would, in theory, assure higher boron accumulation
than the nontargeted ones [397] .
Due to the overexpression by glioma cells, the most potent ligands for glioma
treatment are endothelial growth factor receptor (EGFR) [398] , the vIII mutation
of EGFR [399] , platelet - derived growth factor (PDGFR) [400] , and tenascin epitopes
[401] .
Liposomes are also used as carriers for gene delivery to gliomas while the cationic
ones have demonstrated better interaction with cells in comparison to other types
of liposomes [402, 403] . However, cationic liposomes suffer from toxicity, which
varies according to cell type, duration of exposure, and density of cell culture. Antisense
genes have been incorporated into liposomal carriers. For example, the EGFR
antisense gene was packaged in PEGylated immunoliposomes [carrying human
insulin receptor antibody (HIR)] [404] . It was reported [405] that the liposomes
could cause 70 – 80% inhibition in human glioma cell growth. The same authors
reported a 100% increase in survival time of mice with intracranial human brain
cancer with weekly i.v. injections of antisense gene therapy directed at the human
EGFR [405] .
Moreover, double immunoliposomes were developed in order to treat intracranial
human brain cancer in mice [406] . The mAbs used were the rat 8D3 mAb to
the mouse transferrin receptor and the mAb against the HIR. RNAi (intereference
RNA) is a new antisense gene therapy, where an expression plasmid encodes for a
shRNA (short hairpin RNA). The shRNA is processed in the cell to an RNA duplex.
The latter mediated RNAi. Indeed, weekly i.v. RNAi gene therapy reduced tumor
expression of immunoreactive EGFR and caused an 88% increase in survival time
of mice with advanced intracranial brain cancer.
In another study, the herpes simplex virus thymidine kinase (HSVtk) gene was
evaluated as to its potency to increase the sensitization of ganciclovir (GCV) to
glioma cell lines when that gene was incorporated in liposomes [407] .
Although the effi ciency of transfection was 18.6% in vivo after intratumoral
injection of DNA liposomal complexes, the sensitivity to ganciclovir was improved
as tumor weight induction was observed. In 2001, the FDA approved a clinical protocol
relevant to liposomal gene therapy with the HSVtk/GCV system for the treatment
of glioblastoma multiforme [408] .
Hybrid vectors consisting of adeno - associated virus (AAV) vectors enclosed in
liposomes lead to a10 - fold increase in transduction effi ciency compared to liposomes
containing plasmid DNA and 6 - fold increase compared to AAV vector alone
[409] .
However, alternative routes are currently used in the clinic according to the
site/location and type of tumor to achieve higher therapeutic effi ciency [410] because
systemically administred drugs are not able to pass to cerebrospinal fl uid (CSF),
whereas there is direct tumor exposure to the drug, characterized with increased
drug concentration and half - life [410] . Thus, drugs can be administered in CNS via
intrathecal, intraventricular, and intraparenchymal routes. Intrathecal and intraventricular
routes result in a high drug concentration in the bulk CSF but with limited
penetration to parenchyma. Thus, they are suited for treating meningeal and ventricular
diseases. The intraparenchymal route assures the delivery in a local region
in the parenchyma and so is useful for solid tumors and degenerative diseases that
are surgically accessible.
DepoCyt, a slow - release liposomal cytosine – arabinoside, undergoes clinical trials,
as in some previous preclinical and clinical tests it was shown of good potency for
the treatment of meningeal malignancies. For example, DepoCyt was administered
to children with refractory neoplastic meningitis via lumbar puncture using an
Ommaya reservoir or intraventricularly [411] . That study demonstrated the safety
and feasibility of using liposomal cytarabine in children older than three years at
the recommended dose of 35 mg with concomitant administration of dexamethasone.
The latter is given to reduce the side effects of liposomal cytarabine, which is
mild headache and back or neck pain. In a relevant study on adults with lymphomatous
meningitis, DepoCyt given once every two weeks at a dose of 50 mg yielded
a response rate of 71%, whereas free cytarabine at a dose of 50 mg twice a week
produced a response rate of only 15% [412] . In patients with solid tumor neoplastic
meningitis, 50 mg DepoCyt given once every two weeks yielded a response rate in
evaluable patients of 36%, whereas free methotrexate 10 mg given twice a week
produced a response rate of 21%. However, a problem accompanies the direct
delivery to the brain parenchyma: the limited diffusion coeffi cient of particles in
general (liposomes, nanoparticles, viral vectors) from the injection site in the brain
tissue [410] . Thus, intraparenchymal delivery of liposomal carriers is facilitated using
convection - enhanced delivery (CED) to distribute the drug through a larger region
in the tissue. CED utilizes a bulk fl ow mechanism to deliver and distribute macromolecules
to clinically signifi cant volumes of solid tissues providing a larger volume
of distribution. A range of parameters affecting CED are connected with the physicochemical
properties of liposomes, that is, size, surface charge, and steric stabilization
[413] .
According to investigations by MacKay et al. [413] , the ideal liposome (or in
general nanoparticle) for CED will be PEGylated and of less than 100 nm in diameter,
have neutral or negative surface charge, and need a targeting ligand to direct
the particle to the target cell. For example, mannosylated liposomes containing clodronate
infused directly to the fourth ventricle of the rat brain were selectively
taken up by macrophages but not from microglia [414] . Also, the lipid dose is important
as it was shown that infusing a high total lipid concentration reduced the fraction
of the dose taken up by perivascular cells in the brain.
However, MacKay and co - workers [413] carried out the previously mentioned
study using healthy animals. Mamot et al. administered liposomes of either 40 or
90 nm with 25% mannitol in U - 87 glioma xenograft animals [415] . In contrast to
MacKay et al. [413] , the small 40 - nm liposomes infused via CED without mannitol
achieved distribution over nearly all the tumor tissue. The addition of mannitol to
CED further enhanced distribution, as liposomes accumulated throughout all sections
of tumor and further penetrated beyond the tumor boundary into adjacent
normal brain tissue. For the 90 - nm liposomes, CED with mannitol resulted in more
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 491
492 LIPOSOMES AND DRUG DELIVERY
than 50% tumor penetration of the total area evaluated , which also included extension
into surrounding normal brain. Distribution was somewhat less extensive than
with the 40 - nm liposomes, though.
The effi cacy of liposome administration with CED was demonstrated using real -
time magnetic resonance imaging (MRI) of rat brain tumors [416] . Two types of
liposomes were used: doxorubicin - loaded liposomes and gadolinium (Gd) – loaded
(MRI contrast agent) liposomes. According to the authors, MRI facilitated the distinction
of distribution between different tumor models, such as C6 gliomas and
9L - 2 gliomas. Also, after CED of Doxil, the drug presence was identifi ed in the tissue
several weeks after a single administration, while the therapeutic response achieved
was greater compared to systemic administration of Doxil. The fact that therapeutic
liposomes can be coinfused with liposomal Gd to successfully monitor the distribution
of the drug carrier in the nonhuman primate brain was shown by Saito and
co - workers [417] . A 2 : 1 ratio between the volume of distribution and the volume
of liposomes infused was found while liposomal Gd was still detectable 48 h postinfusion,
confi rming the previous fi nding of prolonged retention of liposomal Gd in
the tissues and negligible toxicity in rat. With this study, the authors showed that
CED is a technique enabling safe and extensive liposome distribution. Also, real -
time MRI is a potentially useful tool to estimate the concentration and tissue half -
lives of liposome - loaded drugs within target tissues.
However, CED might not always be successful as good technical skills are
required (if the catheter is not placed properly, the liposomes will escape within the
CSF). Also CED is an invasive technique and could cause infl ammation and neurotoxicity
and it is determined by many formulation characteristics and diffusional
properties of the latter in brain tissue [418] .
Breast Cancer Breast cancer is the second leading cause of cancer deaths in
women today (after lung cancer) and is the most common cancer among women,
excluding nonmelanoma skin cancers [419] . According to the World Health Organization,
more than 1.2 million people will be diagnosed with breast cancer this year
worldwide. Breast cancer stages range from stage 0 (very early form of cancer) to
stage IV (advanced, metastatic breast cancer). Each patient ’ s individual tumor
characteristics, state of health, genetic background, and so on, will impact her survival.
In addition, levels of stress, immune function, will to live, and other unmeasurable
factors also play a signifi cant role in a patient ’ s survival. The majority of women
with breast cancer will undergo surgery as part of their cancer treatment (lumpectomy
and mastectomy). In addition to surgery, some women will receive adjuvant
(additional) treatment (chemotherapy, radiation therapy, and drug treatments) to
stop cancer growth, spread, or recurrence. Occasionally women may be treated with
chemotherapy, radiation, or drugs without having breast surgery.
From a clinical point of view and with relevance to liposomes, administration of
either PEGylated (Doxil) or non - PEGylated liposomal doxorubicin (Myocet) has
improved the safety and tolerance of patients with breast cancer by signifi cantly
reducing the main side effect of those drugs, cardiotoxicity. However, drug effi cacy
remains the same either incorporated or not in the liposomes as this is reported
after conducting various clinical studies [420, 421] . For example, Myocet (M) received
European approval for use in patients with metastatic breast cancer at a dose of
60 mg/m 2 in combination with cyclophosphamide (C) having shown equivalent effi -
cacy at a phase III study [422] . Also, doxorubicin is given at a dose of 60 or 75 mg/m 2
[423] . Chan et al. [424] studied the effi cacy and tolerability of Myocet in a dose of
75 mg/m 2 in combination with cyclophosphamide in 160 patients. A high incidence
of neutropenia was obtained. Thus, that group confi rmed that the use of 60 mg/m 2
is an appropriate choice. However, administering a combination of either Myocet
and cyclophosphamide or epirubicin and cyclophosphamide to patients with metastatic
breast cancer, the response rate was not signifi cantly different, which showed
that the use of the liposomal formulation just reduced the cardiotoxicity of
doxorubicin.
In a phase I study, Myocet was administered in combination with docetaxel in 21
metastatic breast cancer patients with the aim of estimating the safety and maximum
tolerated dose of Myocet in parallel with docetaxel [425] . The latter is proved highly
active in metastatic breast cancer as well as doxorubicin. The maximum tolerated
dose was 50 mg/m 2 of Myocet and 25 mg/m 2 of docetaxel. As reported in the previous
study, neutropenia was the most common toxicity effect while some incidents of
congestive heart failure were observed after a total doxorubicin dose of 540 mg/m 2 .
Moreover, a combination of Myocet (75 mg/m 2 ) with gemcitabine (350 mg/m 2 ) and
docetaxel (75 mg/m 2 ) was injected intravenously in 44 patients with early breast
cancer every three weeks for six cycles [426] . The overall clinical response rate was
80% and the pathological complete response was 17.5%. The toxicity of the regimen
was moderate and, as expected, neutropenia and leukopenia were the most prominent
side effects. The latter effects were well managed and treatment discontinuation
was not required.
Using PEGylated liposomal doxorubicin (Caelyx), Keller et al. [427] compared
the effi cacy of the liposomal formulation with that of a common regimen in patients
with taxane - refractory advanced breast cancer. The regimen scheme was Caelyx
(50 mg/m 2 every 28 weeks) or vinorelbine (30 mg/m 2 ) or mitomycin C (10 mg/m 2
every 28 days) plus vinblastine (5 mg/m 2 at days 1, 14, 28, 42). Finally, progression
free survival and overall survival were similar for Caelyx and the comparative
regimen.
The effi cacy and toxicity of Caelyx in combination with paclitaxel (Taxol) were
investigated as a fi rst - line therapy in 34 patients with advanced breast cancer in a
multicentric phase II study [428] . Paclitaxel at a dose of 175 mg/m 2 and Caelyx
(30 mg/m 2 ) were administered intravenously every 3 weeks. It was shown that the
response rates of the combination were over 70% while the median time to treatment
failure was 45 weeks. No signifi cant clinical cardiotoxicity was observed and
the usual side effects (mucositis, stomatitis, hand – foot syndrome) were treated
accordingly.
In another phase II study, Caelyx was used in combination with cyclophosphamide
(CP) as a fi rst - line therapy in patients with metastatic or recurrent breast
cancer [429] . Three different schemes were given in groups of patients: Caelyx
50 mg/m 2 intavenously on day 1 and CP 100 mg/m 2 (orally on days 1 – 14) every
28 days, 30 mg/m 2 Caelyx and 600 mg/m 2 i.v. on day 1 every 21 days, and 35 mg/m 2
Caelyx plus 600 mg/m 2 CP i.v on day 1 every 21 days. The responses were similar
among the different groups (51%) of patients, but less side effects were observed
in group 2, making that regimen the one of choice as a fi rst - line therapy for patients
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 493
494 LIPOSOMES AND DRUG DELIVERY
with metastatic or recurrent breast cancer. The median duration of response was
35.1 weeks.
Coleman et al. [430] demonstrated that using Caelyx in a dose format of
50 mg/m 2 every four weeks in patients with metastatic breast cancer is quite effective
(objective partial responses 31%) while the main side effect was hand – foot
syndrome.
From a research point of view, liposomes have been used in order to facilitate
or alter the pharmacokinetics/bioavailability of various molecules. For example,
ceramide is an antiproliferative and proapoptotic molecule produced after sphingomyelin
metabolism [431] . Ceramide C6 – loaded PEG(750) - C8 liposomes were
injected intravenously in mice bearing syngeneic or human xenografts of breast
adenocarcinoma. Administration of 36 mg/kg of liposomal C6 over a three - week
period caused a sixfold decrease of tumor size in the case of syngeneic tumor –
bearing mice. Liposomal C6 accumulated in caveolae and mitochondria, while a
marked increase of apoptotic cells was observed. The PEGylated liposomal ceramide
followed fi rst - order kinetics in the blood and a steady - state concentration was
achieved in tumor tissue (Figure 13 ). Also, a decrease in tumor size was obtained
in the human xenograft model. Minimal toxicities were observed in tumor - bearing
mice, suggesting that the bioactive concentration of C6 achieved in the tumor tissue
is not active in normal tissues. Besides, contortrostatin (CN), a molecule isolated
from snake venom, has been proved to interact with integrins on tumor cells and
with newly growing vascular endothelial cells via its two Arg – Gly – Asp sites [432] .
Thus, interactions between CN and integrins disrupt several steps critical to tumor
growth: angiogenesis and metastasis. CN was incorporated in PEGylated liposomes
with an encapsulation effi ciency of approximately 80% while retaining full biological
activity. Intravenous injection of approximately 100 . g of liposomal CN twice a
week caused a signifi cant reduction of 94% of microvascular density and hindered
the tumor growth in the MDA - MB - 435 xenograft model. Also, liposomal CN demonstrated
prolonged circulation ( t 1/2 values were 19 and 0.5 h for liposomal and
nonencapsulated CN, respectively).
In another study, the authors modifi ed the liposome composition by including
polyoxy - ethylenedodecyl ether [C 12 (EO) n ] in the DMPC - made liposomes [433] .
These vesicles caused activation of caspases 3, 8, 9 and eventually induction of
apoptosis in MDA - MB - 453 cells. Another idea is based on using methods to
direct the drug loading carriers to the cancer site. More specifi cally, the paclitaxel
is successfully used for the treatment of breast cancer among other types of
cancers but has side effects such as neutropenia, peripheral neuropathy, and
hypersensitivity reactions. Also, it is formulated with Cremophor EL due to its
high lipophilicity. In order to decrease the drug toxicity and enhance the therapeutic
potential of that drug, paclitaxel was incorporated in negatively charged
magnetic liposomes and its effi cacy was evaluated in vivo [191] . A magnetic fi eld
of suitable strength was used to direct the magnetoliposomes to the desired site.
Indeed, after intravenous injection the AUC obtained for the magnetic carriers
and the Cremophor EL/ethanol were 20.7 and 6.8 h · . g/mL, respectively. The liposomal
paclitaxel concentration was much higher in the tumor in comparison to
other organs while the concentration peak was reached sooner (19.85 . g/g at
0.25 h). Even after 8 h, paclitaxel concentration in the plasma was 2.71 – 0.33 . g/mL
for the Cremophor EL formulation. The antitumor effi cacy of magnetoliposomes
FIGURE 13 Blood and tumor concentrations of bioactive ceramide – lipid C6 in tumor -
bearing mice were maintained over a 48 - h period: ( a ) 10 - and 40 - mg/kg doses of liposomal - C6
followed fi rst - order kinetics with blood concentration exceeding in vitro IC 50 sustaibed at
48 h; ( b ) at these doses steady - state concentration of C6 in tumor tissue achieved at . 60 min.
The 40 - mg/kg dose maintained a concentration above the desired IC 50 up to 48 h. ( Reprinted
from ref. 431 with permission of American Association for Cancer Research .)
40 mg/kg
10 mg/kg
IC50 (–5.M)
40 mg/kg
10 mg/kg
IC50 (–5.M)
(a)
(b)
40
30
20
10
0
0
5
10
15
20
0.0 0.5 1.0 1.5 2 12 22 32 42 52
0.0 0.5 1.0 1.5 2 12 22 32 42 52
Hours
Hours
.g C6 / MI Blood Ng C6 / mg Tumor Tissue
(expressed as change rate of tumor weight) after subcutaneous injection of 10 mg/
kg drug was equivalent to that achieved with Cremophor EL 50.4 and 51.9%.
When the administered dose was 20 mg/kg, the change rate of tumor weight was
the highest, 60.5%.
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 495
496 LIPOSOMES AND DRUG DELIVERY
Pulsed high - intensity focused ultrasound (HIFU) is another suggested method
to enhance the delivery and therapeutic effect of Doxil in a murine breast cancer
tumor model with the fi nal aim of reducing the Doxil dose given during therapy
[434] . However, the results were not particularly encouraging as there was not any
signifi cant difference between free and liposomal Doxil distribution in the tumor
after HIFU exposure. The latter was possibly attributed to the ability of liposomes
to extravasate easily through the leaky vasculature of the tumor, so the use of HIFU
did not add any therapeutic or other advantage.
In an attempt to achieve active targeting, sigma receptor ligands were incorporated
on to the liposomal surface. Sigma receptors are overexpressed in various
human tumors, including breast cancer cells [435] . Haloperidol has shown high affi nity
for sigma receptors; thus it has been attached at the end of PEG molecules, which
are protruding from the surface of cationic lipoplexes. Indeed, haloperidol – PEG
lipoplexes showed more than 10 - fold higher gene expression in MCF - 7 (breast
carcinoma) cells than control lipoplexes, while the presence of haloperidol or 1,3 -
ditolylguanidine suppressed the expression of the reporter gene [435] .
Transferrin is another ligand attached on the lipoplex surface so to cause higher
transfection effi ciency, as has been reported [436] . Thus, Basma et al. [437] studied
the effect of cis - diaminedichloro platinum (CDDP) in combination with bcl - 2 antisense
treatment on p53(+)MCF - 7 and p53( . )MCF - 7/E6 breast cancer cells using
transferrin bcl - 2 lipoplexes. The median inhibitory concentration (IC 50 ) for bcl - 2
antisense delivered with lipoplexes plus transferrin was approximately 1.4 . M for
MCF - 7 and 1.2 . M for MCF - 7/E6 cells (Figure 2 of ref. 437 ). In CDDP - treated cells,
the IC 50 was approximately 5 . M for both cell lines. In general, bcl - 2 antisense
delivered in the form of transferrin bcl - 2 lipoplexes in combination with cisplatin
induced cell death and apoptosis in a higher degree in MCF - 7/E6 cells rather than
MCF - 7 cells. Cisplatin demonstrated higher caspase - 8 activation compared to the
targeted lipoplexes, which suggested that possibly caspase - 8 is the major pathway
for cancer cell death. G3139 is another oligonucleotide (ODN) capable of downregulating
bcl - 2 and, its effi cient delivery to breast cancer cells would potentially
make it a successful candidate for antisense therapy [438] . Thus, the effi ciency of
different liposomal formulations for the lipid composition (DOTAP, DC - Chol,
CCS) 1 with or without helper lipids DOPC, DOPE, and Chol and liposomal size
[approximately 100 nm LUV or unsized heterolamellar vesicles (UHV)] on MCF - 7
breast cancer cells was examined [438] . Out of 18 tested formulations, only the CCS -
bcl - 2 lipoplexes (UHV derived) have been effective, causing a larger than 50%
decrease of cell growth in comparison to free ODN. The possible mechanism of
action of the particular formulation is attributed to the presence of one primary and
two secondary amines on the spermine moiety of the CCS molecule. Because of
this, CCS lipoplexes are taken up by cells via adsorptive endocytosis and the secondary
amines cause a “ proton sponge effect ” due to which ODN escapes from the
endosomes and so has the ability to interact with the bcl - 2 neutralizing it. Of course,
the effect of the L + /DNA . ratio is a determining factor along with the lipid composition
and size.
1 DOTAP: N - (1 - (2,3 - dioleoyloxy)propyl) - N , N , N - trimethylammonium chloride; DC - Chol: 3 . [ N - ( N . N . - di
methylaminoethane)carbamoyl] - cholesterol; CCS: ceramide carbomoyl spermine.
Diagnosis is always the fi rst aim in effectively treating breast cancer, so the need
to develop or reveal more tumor markers at an early stage of the cancer is absolutely
critical [439] . Such are the circulating epithelial cells, the cyclins, and the urokinase -
type plasminogen activator and plasminogen activator inhibitor, which indicate
breast cancer or metastatic spread apart from the already existing markers estrogen
receptor, progesterone receptor, and human epidermal growth factor receptor - 2
(HER - 2). Liposomal formulations have facilitated anticancer activity of highly toxic
anticancer drugs as well as altered their biodistribution, while targeted drug - loaded
liposomes have shown some promising results in the laboratory. Development of
new drugs and use of particulate carriers to increase bioavailability could be one
way forward in the battle for improvement of quality of life for patients suffering
from breast cancer.
Lung Cancer Lung cancer might be the most common form of cancer and the
most common cause of death in both men and women, although it affects more men
than women [440] . There are three main types of lung cancer, based on their appearance
when examined by a pathologist: small cell carcinoma, squamous cell carcinoma,
and adenocarcinoma. The latter two consist of non – small cell lung cancer. It
is important to know which type of cancer a patient has because small cell cancers
respond best to chemotherapy (anticancer medicines) whereas the other types
(often referred to collectively as non – small cell cancer) are better treated with
surgery or radiotherapy (X - ray treatment).
Surgery can cure lung cancer, but only one in fi ve patients are suitable for this
treatment. If the tumor has not spread outside the chest and does not involve vital
structures such as the liver, then surgical removal may be possible, but only if the
patient does not also have severe bronchitis, heart disease, or other illnesses. Small
cell lung cancer is treated with chemotherapy. Non – small cell cancer may be treated
with radiotherapy and chemotherapy (as part of a research trial) or with supportive
care. Radiotherapy is either “ radical ” or “ palliative. ”
Regarding drug treatment of lung cancer, Merck KGaA and Biomira will soon
start phase III trial on their BLP - 25 liposomal vaccine for patients with non – small
cell lung carcinoma [441] . L - BLP25 is a synthetic MUC1 peptide vaccine. MUC1 is
a mucinous protein expressed on the apical borders of normal epithelial cells.
It is overexpressed and glycosylated on tumor cells, where it appears to be antigenically
distinct from the normal protein. The liposomal vaccine can induce a MUC1 -
specifi c T - cell response. Median survival for patients with stage IIIB locoregional
non – small cell lung cancer who received L - BLP25 in a phase IIb study was 30.6
months in the vaccinated group compared with 13.3 months for the unvaccinated
group.
Caelyx is liposomal doxorubicin very well used as a treatment of choice for a
number of cancers with good tolerability and antitumor activity, as has been demonstrated
in many phase I or II clinical trials. One such example is the conduct of
phase I study of Caelyx (PEGylated liposomal doxorubicin, 25 – 40 mg/m 2 ) in combination
with cyclophosphamide (750 – 1000 mg/m 2 ) and vincristine (1.2 mg/m 2 ) every
21 days in patients with relapsed or refractory small cell lung cancer [442] . The suggested
doses were CaelyxTM 35 mg/m 2 , cyclophosphamide 750 mg/m 2 , and vincristine
1.2 mg/m 2 intravenously every 21 days. This combination was well tolerated
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 497
498 LIPOSOMES AND DRUG DELIVERY
while antitumor activity was observed for patients with relapsed small cell lung
carcinoma with survival duration of 5 months. The latter is similar to what is achieved
using only camptothecin analogues taxanes or vinorelbine (survival duration . 6
months).
Also, paclitaxel is the most widely used anticancer agent for non – small cell
lung cancer [420] . Liposomal encapsulated paclitaxel faces the problem of formulation
due to the drug ’ s high hydrophobicity. However, a phase I trial with liposomal
paclitaxel reported dose - limiting toxicity at the dose of 150 mg/m 2 /week. Besides,
the whole blood clearance of paclitaxel was similar for liposomal and free
paclitaxel.
Promising results that were referred to liposomal encapsulated paclitaxel easy to
use (LEP - ETU; NeoPharm) consisted of DSPC/Chol/cardiolipin molar ratio 90 : 5 : 5,
lipid/drug molar ratio 33 : 1, and paclitaxel concentration 2 mg/mL (mean liposome
size 150 nm). In a phase I study of increasing doses of liposomal paclitaxel (135 –
375 mg/m 2 ) in 25 patients, the enhanced effectiveness was proved with much better
tolerability and with 3 partial remissions and 11 patients with stable disease. Lurtotecan,
a camptothecin analogue, was incorporated in PC/Chol (2 : 1 molar ratio)
liposomes (size 50 – 100 nm) with a lipid/drug molar ratio 20 : 1. A remarkable 1500 -
fold AUC increase was obtained after administration in nude mice and several
xenograft models.
9 - Nitro - 20( S ) - camptothecin (9NC), another lipophilic camptothecin analogue,
showed antitumor effects in mice and milder effect in humans after oral administration.
Thus, the potency of 9NC - loaded di-lauryl-PC (DLPC) liposomes was investigated
in patients with primary or metastatic lung cancer after aerosol administration
(aerosol droplet size 1 – 3 . m) for fi ve consecutive days per week [373] . Indeed, the
most serious side effect of 9NC, hematological toxicity, did not appear in any of the
patients, while the dose - limiting toxicity, chemical pharyngitis, was observed at a
dose of 26.6 . g/kg/day. 9NC plasma levels, after aerosol administration (13.3 . g/kg/
day), were similar to that given orally (2 mg/m 2 ), despite the lower dose administered
in the fi rst case. More specifi cally, C max and AUC were 76.7 ± 39.1 ng/mL and 275 ±
149 ng - h/mL, respectively, after aerosol administration and 111 ng/mL and 194.4 ±
108.4 ng - h/mL, respectively, after oral administration. The recommended dose for
phase II studies is 13.3 . g/kg/day on a 1 - h exposure for fi ve consecutive days per
week and for eight weeks, with 0.4 mg/mL 9NC concentration in the nebulizer.
Moreover, targeting moieties have been grafted on the vesicle surface, exploiting
the fact that antigen/receptor overexpression on cancer cell membranes increases
the specifi city of the active substance. For example, antagonist - G is a hexapeptide
which blocks the action of multiple mitogenic neuropeptides by interacting with
their receptors [170] . Antagonist - G was chemically attached at the distal end of
PEG molecules of stealth liposomes (HSPC/Chol/mPEG – DSPE/PDP – PEG – DSPE,
2 : 1 : 0.08 : 0.02 molar ratio) (SLG) loaded with doxorubicin. The antiproliferative
activity of doxorubicin was estimated on the human variant small cell lung carcinoma
(SCLC) H82 cell line. Indeed, 20 - to 30 - fold increase of both, binding, and
internalization took place after SLG incubation on cells in comparison to stealth
liposomes only, or PEGylated liposomes without the antagonist - G. The 0.03 . g of
antagonist - G on the liposome surface was enough to cause 50% cell liposome association.
Doxorubicin accumulation in the whole cell was 20 - fold higher with SLG.
Eventually, there was the suggestion that the main antagonist - G mechanism of
action is to bind to vasopressin receptor, which is expressed abundantly on the
SCLC cells.
Hyaluronan (HA) is another potential candidate for tumor targeting because it
has been proven that hyaluronan receptors CD44 and RHAMM are overexpressed
on several tumor types [443] . In this case, hyaluronan is used not only as a targeting
moiety but also to prolong the circulation half - life of the vesicles in question; in
other words, it replaces PEG molecules. HA was chemically attached on the vehicle
surface (57 . g/ . mol lipid), which was comprised of PC/PE/Chol (3 : 1 : 1) (HA – LIP).
Doxorubicin was entrapped in 78 ± 5% encapsulation effi ciency, while the . potential
and size of the targeted liposomes were . 13.1 ± 3.9 mV and 81 ± 13. Pharmacokinetic/
biodistribution studies were carried out with Doxil (PEGylated liposomal
doxorubicin) as a comparison to the new hyaluronan liposomes. Those studies plus
therapeutic responses were tested on three mice models, one of which was C57Bl/6 -
bearing B16F10.9 lung metastasis. Clearly, ha-liposomal (HA–LIP)–DXR exhibited
prolonged blood circulation similar to Doxil (approved formulation in the market),
which indicated that the amount of attached HA on the vehicle surface is enough
to stabilize and offer the steric stabilization required (Figure 14 ). Also, accumulation
of HA – LIP – DXR was much more reduced in liver of C57Bl/6 - bearing B16F10.9
lung metastasis mice, while DXR accumulation in the tumor site was threefold
higher to that achieved using Doxil. Signifi cant improvement was obtained in both
metastatic burden and survival with Doxil and HA – LIP – DXR (Figure 15 ). Besides,
the HA – LIP – DXR had positive results on all tumor types tested.
Manipulating the genetic material by injecting tumor - suppressing genes has been
an alternative way in cancer treatment. The 3p FUS1 gene is a tumor suppressor
FIGURE 14 Doxorubicin (DXR) plasma concentration ( . g/mL) as function of time from
dosing: ( a ) C57BL/6 mice inoculated (by intraveneous injection) with B16F10.9 cells;
( b ) healthy C57BL/6 mice; ( c ) BALB/c mice inoculated with C - 26 cells (injected into right -
hind footpad). A single dose of the selected formulation was injected into the tail vein. DXR
formulations and doses are specifi ed. ( Reprinted from ref. 443 with permission of Neoplasia
Press, Inc .)
Free DXR (10 mg/kg)
nt-LIP-DXR (10 mg/kg)
Doxil (10 mg/kg)
tHA-LIP-DXR (10 mg/kg)
C57BL/6
B16F10.9
Time (h)
DXR plasma concentration (.g/mL)
100
10
1
0.1
0.01
0.001
0 20 40 60 80 100
(a)
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 499
500 LIPOSOMES AND DRUG DELIVERY
gene which belongs to a 120 - kb region of the 3p chromosome on the region 3p21.3.
That 120 - kb region is missing so injection of DOTAP/Chol lipoplexes with the FUS1
gene could induce apoptosis and inhibit tumor growth [444] . A signifi cant inhibition
of lung metastatic tumor was obtained in animals (bearing subcutaneous lung tumor
xenografts) treated with liposome – FUS1 DNA complex (total of six doses). Also,
intravenous treatment of lung tumor - bearing animals with DOTAP/Chol – FUS1
complex prolonged the animal survival; 40% of the animals were still alive after
approximately 100 days. Also, the antitumor potency of FUS1 was demonstrated to
be superior to the one obtained with p53 as the same therapeutic effect was achieved
by using less amount of FUS1 gene in the lipoplexes in comparison to p53. This
would be an advantage because lowering the DNA dose would result in much lower
toxicity of DNA due to infl ammatory response.
FIGURE 15 Therapeutic responses of mice bearing B16F10.9 - originating lung metastatic
disease. Doxobubicin (DXR) formulations and doses are specifi ed. Treatments were on days
1, 5, and 9 by injection of selected formulation to the tail vein. ( a ) Lung metastatic burden.
Light - shaded bars are data for increase in lung weight; dark - shaded bars are data for number
of lung metastasis: ( * * * ) P < 0.001 compared with free drug. ( b ) Survival ( n = 5). Each line
connects the symbols representing the daily survival state of the group. ( Reprinted from
ref. 443 with permission of Neoplasia Press, Inc .)
400
300
200
100
0
100
80
60
40
20
0
Increase in lung weight (%)
Number of lung metastases
160
120
80
40
0
Saline Free DXR nt-LIP-DXR Doxil tHA-LIP-DXR
Saline
Free DXR(10
mg/kg)
nt-LIP-DXR
(10 mg/kg)
Doxil (10
mg/kg)
tHA-LIP-DXR
(10 mg/kg)
Survival (%)
0 20 40 60 80 100
Time from tumor inoculation (days)
(a)
(b)
Moreover, the effi ciency of gene therapy depends very much on the vector used
to achieve the gene delivery, the properties and stability of the fi nal lipoplexes in
the presence or absence of serum, and the pharmacokinetics and biodistribution.
On this basis, Li et al. studied the effect of lipoplex size on the lipofection effi ciency
of TFL - 3/pDNA (plasmid - encoding luciferase) liposomes in the absence and presence
of serum and investigated the correlation between in vitro and in vivo results
using either B16BL6 murine melanoma cell line or the same cells injected i.v. in
C57BL/6 mice in order to produce pulmonary metastases [445] . The authors incubated
a range of different ratios of pDNA/TFL - 3 (P/L) ranging from 8 to 120 P/L
on the previously mentioned cell line in the absence and presence of serum. Serum
had a dramatic effect on lipoplex size; in the absence of serum, the size increased
as the pDNA amount increased and it reached a maximum value at the ratio 80 g/
mol P/L, while the . potential decreased as the P/L ratio increased and at 80 g/mol
P/L was approximately +2 mV. Maximum luciferase activity was observed with
lipoplexes of 80 g/mol. In contrast, in the presence of serum, the size increased from
8 to 20 g/mol and decreased from 40 to 120 g/mol as the P/L increased. Maximum
luciferase activity was obtained with 40 g/mol in the presence of serum. However,
the in vitro transfection effi ciency of the lipoplexes in the presence of serum at the
highest point of 40 g/mol was twofold less than the one achieved at the peak of
transfection effi ciency at 80 g/mol in the absence of serum. However, the plasmid
expression was higher with 8 and 80 g/mol lipoplexes in tumor - bearing animals in
contrast to the in vitro fi ndings in the presence of serum. This was attributed to less
aggregate formation in the blood due to the lipoplex/serum ratio (50 – 100 . L serum/
200 . L lipoplex dispersion) so there is no complete interaction between lipoplexes
and serum proteins. Also, pulmonary gene expression was dependent on the time
after cell inoculation. It was shown that gene expression takes place at different
parts of the lung; for example, 8 g/mol P/L lipoplexes expressed luciferase in the
cells surrounding the tumor, while 80 g/mol lipoplexes expressed the gene in
the entire lung without any specifi city. Possible explanations for the latter are the
extended lung capillary bed and increased lipoplex uptake by tumor cells compared
to the normal cells. Of course many other factors could contribute to the different
patterns of gene expression, such as endosomal release, nuclear uptake, increased
translation, and transcription.
All - trans retinoic acid (ATRA) has shown anticancer activity in a number of
types of cancer cells [446] . However, some non – small cell lung carcinoma is resistant
to ATRA probably due to the high lipophilicity of the molecule, which makes it
unable to pass through the cellular membrane. Therefore, cationic liposomes
(DOTAP/Chol 1 : 1 molar ratio) were used to incorporate and facilitate ATRA ’ s
action in the resistant NSCLC in A549 human lung cancer cells. The produced
ATRA – DOTAP/Chol lipoplexes were 125 nm in diameter with 50 mV of . potential,
while for the DSPC/Chol liposomes used as control these were 110 nm and . 3 mV,
respectively. The former lipoplexes exhibited higher uptake by the cells in comparison
to DSPC/Chol due probably to the positive surface charge, which interacts to a
higher degree with the negatively charged cell membrane. Thus, the apoptosis
induced on these cells in the presence of ATRA lipoplexes was much higher than
the one caused by ATRA only or ATRA – DSPC/Chol (Figure 16 ).
Lastly, although antisense therapy is considered a simple and effi cient procedure,
its products have never reached the market. This is due to the instability of phos-
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 501
502 LIPOSOMES AND DRUG DELIVERY
phodiester oligonucleotides (PO - ONs) in the cytoplasm, while the phosphothioate
ONs demonstrate unwanted side effects [447] . In order to design better delivery
systems for PO - ONs, the intracellular fate of the PO - ON and PS - ON lipoplexes
with DOTAP/DOPE was investigated after their application on A549 cells. After
endocytosis, endosomal localization, and endosomal escape of the lipoplexes, the
ONs were localized in the nuclei. However, PO - ONs were degraded in the nucleus
(degradation products diffuse out of the cells after 2 h) whereas PS - ONs were still
intact and remained in the nucleus for 8 h. It is worth mentioning that PS - ONs were
eliminated from the nucleus and were found in cytoplasmic granules, which indicated
that the cell has a mechanism of elimination of intact PS - ONs. Also, it seemed
that the amount of PO - ONs was quite important for the ONs ’ stability as the
degradation was reduced after injecting 22 . M , in contrast to complete degradation
with injection of 2 . M . From the study it was concluded that ON degradation
happened after their release from the lipoplexes. Thus, for the successful delivery
of ONs, they have to remain complexed with their carrier. Polyethylenimine (PEI)
was suggested as a possible carrier due to its proton sponge effect, leading to
endosomal rupture without releasing the ONs as well as graft - pDMAEMA – PEG
[poly(2 - dimethylamino)ethyl methacrylate - co - aminoethyl methacrylate – bearing
polyethylene glycol chains]. According to the authors, probably cationic polymers
are more effi cient than cationic lipids, but lots of work still needs to be done on the
matter.
Ovarian Cancer Ovarian cancers start at the ovaries [448] . They can be either
benign, and so never spread from the ovary, or malignant, in which case they can
Percent of cells
Viable cells
Apoptosis cells
Necrotic cells
Control ATRA Bare
DOTAP/
cholesterol
liposome
ATRA/
DOTAR/
cholesterol
liposome
H2O2
0
20
40
60
80
100
120
FIGURE 16 Flow cytometric analysis of A549 cells treated with 1.0 . M ATRA, bare
DOTAP/cholesterol liposomes, or ATRA incorporated in DOTAP/cholesterol liposomes for
48 h in A549 cells. As a control. Cells were incubated with 1% DMSO and 400 . M H 2 O 2 .
Signifi cant differences: ( * ) P < 0.05 vs. control; (#) P < 0.05 vs. ATRA; ( ‡ ) P < 0.05 vs. DOTAP/
cholesterol liposomes. ( Reprinted from ref. 446 after permission of Elsevier .)
metastasize to other parts of the body. Malignant cancers are classifi ed into germ
cell, stromal, and epithelial tumors. Germ cells produce eggs, stromal cells produce
progesterone and estrogen, and epithelial cells cover the ovary. The majority of the
malignant cases are epithelial cancer (almost 90%). As with most tumors, the usual
treatment is surgery, chemotherapy, and radiotherapy.
Chemotherapy refers to drug administration with highly serious side effects, such
as nausea, hand and foot rashes, mouth sores, and increased risk of infection, easy
bruising, and so on. Therefore, liposomal carriers have been used in order to improve
the drug ’ s biodistribution and protect the patient from those side effects. The
main anticancer drugs used to treat ovarian cancer are carboplatin and cisplatin,
paclitaxel, topotecan, and lurtotecan. PEGylated liposomal doxorubicin has been
approved as a regimen for patients with metastatic ovarian cancer refractory to both
paclitaxel and platinum based - therapy [449] .
A number of clinical trials have been reported and many refer to a combined
therapeutic regimen of liposomes with other anticancer drugs. PEGylated liposomal
doxorubicin was used in a phase II trial on patients with platinum/paclitaxel pretreated
ovarian cancer with or without topotecan. Approximately 36% of patients
showed stable disease [420] . A phase III study followed up the one already mentioned
in 474 patients with pretreated epithelial ovarian cancer that failed or
recurred upon platinum - based combination chemotherapy. The patients received
either PEGylated liposomal doxorubicin (50 mg/m 2 for 1 day every 4 weeks) or
topotecan (1.5 mg/m 2 for 1 – 5 days every 3 weeks). The response rates and the overall
progression - free survival (PFS) were similar for the two formulations. The platinum -
sensitive patients showed longer PFS and overall survival of 108 weeks against the
72 weeks obtained using topotecan. The interesting result of the study was the
reduced cytotoxicity recorded for PEG – liposomal DXR compared to topotecan,
proving that the former treatment improves patient quality of life.
A phase III study was conducted to compare PEG – liposomal DXR (50 mg/m 2
every 4 weeks) with paclitaxel (175 mg/m 2 every 3 weeks) using 214 patients
with relapse after fi rst - line platinum - based chemotherapy [420] . As previously,
the response rates and PFS were not signifi cantly different, but again the liposomal
formulation was notably less toxic as fewer patients recorded with grade
4 adverse effects (17% compared to 71% for topotecan) and thus was more
tolerable.
The most common toxic effects associated with liposomal DXR treatment were
hand - and - foot syndrome and stomatitis, which can be handled by modifying the
dose so there is no need for the regimen to cease. For example, administration of
40 mg/m 2 liposomal DXR every 4 weeks reduces the incidences of hand - and - foot
syndrome and stomatitis compared to 50 mg/m 2 liposomal DXR every 4 weeks
without loss of drug potency [450] . A series of clinical trials using patients with a
variety of ovarian cancers in terms of characteristics (relapsed, refractory, platinum,
paclitaxel resistant) were carried out. The optimized dose regimen for PEGylated
liposomal doxorubicin was 10 – 12.5 mg/m 2 per week when it is used as a single
therapy. Combining liposomal DXR with other anticancer drugs is an equally or
more effective strategy due to the lower dosage regimen of the two formulations
avoiding relevant toxicities.
While platinum compounds and paclitaxel comprise the fi rst - line treatment in
combination with PEGylated liposomal doxorubicin for patients with ovarian
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 503
504 LIPOSOMES AND DRUG DELIVERY
cancer, vinorelbine has been used in combination with liposomes as a pallia -
tive second - line therapy for patients with refractory/resistant ovarian cancer to
platinum – paclitaxel [451] . The best MTD (mean therapeutic dose) was 25 mg/m 2
at days 1 and 8 for vinorelbine and 30 mg/m 2 at day 1 for liposomal DXR of every
21 days, which was well tolerated with moderate hematologic and mild nonhematologic
toxicities. A phase II study relevant to the clinical effi cacy, toxicity, and
pharmacokinetics of that combined therapeutic regimen was carried out by
Katsaros et al. in 30 patients with platinum – paclitaxel pretreated recurrent ovarian
cancer [452] . Caelyx (30 mg/m 2 ) and vinorelbine (30 mg/m 2 ) were administered every
3 weeks for six cycles. The regimen was proved of signifi cant activity for patients
pretreated with paclitaxel – platinum fi rst - line therapy. The overall response rate was
37% with 10% of patients demonstrating stable disease. Vinorelbine bioavailability
was higher under the current regimen. The overall survival was 9 months while the
toxicity was mild and reversible. There were no treatment - related deaths and there
were only 2 patients, one reported with grade 4 and the other with grade 3 hand – foot
syndrome. Also, the toxicity due to liposomal formulation was much lower compared
to that reported from a phase III study with the drug given as a single agent
[453] . In another phase II study, the combination of liposomal doxorubicin and
infused topotecan was studied in 27 patients with platinum - resistant ovarian cancer
in two cohorts [454] . Liposomal DXR (30 mg/m 2 at day 1) and topotecan (1 mg/m 2
for 5 days) were infused and the cycle was repeated every 21 days. The overall
response rate of the regimen was 28% and the median overall survival was 40 weeks.
However, neutropenia and thrombopenia were observed at 70 and 41% of the
patients. Therefore, the topotecan dose was reduced to 0.75 mg/m 2 and liposomal
DXR was increased to 40 mg/m 2 in order to reduce the toxicity. After that, the cytotoxic
effect due to liposomal doxorubicin increased and the bone marrow cytotoxicity
remained the same despite the effectiveness of the regimen.
Lurtotecan is a more advanced camptothecin analogue with probably greater
potency with regard to topotecan and therefore was encapsulated in liposomes to
investigate the toxicity and pharmacokinetics [455] . In a multi - institutional open -
label phase II study, 22 patients with topotecan - resistant ovarian cancer were administered
liposomal lurtotecan in a dose of 2.4 mg/m 2 on days 1 and 8 every 21 days.
Although the toxicity profi le of the drug was lower, no response was observed.
Others used a regimen of liposomal delivery at days 1 and 3 with more promising
therapeutic results, but higher toxicity, too [456] .
A variety of new molecules either in combination with liposomal doxorubicin or
not are in development at the moment [457] . For example, a phase III study will be
conducted to test the effi cacy and safety of pattupilone versus PEG – liposomal DXR
in taxane/platinum refractory/resistant patients with recurrent epithelial ovarian,
primary fallopian, or primary peritoneal cancer. A phase III randomized study of
Telcyta with Doxil/Caelyx versus Doxil/Caelyx has been planned in patients with
platinum - refractory or platinum - resistant ovarian cancer. A phase II study relevant
to side effects and best dose of ixabepilone combined with liposomal DXR will be
assessed in patients with advanced ovarian epithelial, peritoneal cavity, or fallopian
tube cancer or metastatic breast cancer.
Gemcitabine is a clinically active antineoplastic drug in platinum - refractory
ovarian cancer. The effi cacy and tolerability of the particular drug in combination
with liposomal DXR were investigated in athymic mice bearing cisplatin - resistant
human ovarian carcinoma [458] .
Using two therapeutic regimens, either 80 mg/kg of gemcitabine and 15 mg/kg of
liposomes or 20 mg/kg of gemcitabine and 6 mg/kg of liposomes, the same trend of
response was observed, with some of the animals having complete tumor regression
at the end of the study. The lack of toxicity and therapeutic effi cacy observed makes
that regimen promising for clinical trials.
In terms of drug development, a novel analogue of vitamin E assembled into
liposomes was evaluated as a potent anticancer agent in combination with cisplatin
in mice bearing human ovarian cancer xenografts [459] . The analogue is the 2,5,7,8 -
tetramethyl - 2 R - (4 R , 8 R , 12 - trimethyltridecyl)chimoran - 6 - yloxyacetic acid ( . - TEA),
which has apoptotic properties to cancer and not the normal cells in a dose - and
time - dependent manner. Liposomes were administered in the form of aerosol while
cisplatin was injected intraperitoneally to mice with either small or large tumor
volume. It was shown that the combined therapeutic scheme demonstrated the
highest antitumor activity compared to . - TEA itself in both cases. Also, the current
regimen signifi cantly reduced micrometastases observed in lungs and lymph nodes,
while the ratio of proliferating to apoptotic cells in the tumor was decreased due to
induction of apoptosis. However, the specifi c apoptotic mechanism of the particular
molecule needs to be elucidated.
By grafting folate molecules on the liposome surface, their accumulation is highly
increased in macrophages located in tumor ascites fl uid but not in solid tumors,
which have been tested and the existence of functional folate receptors has been
confi rmed [460] . Folate receptors are overexpressed on epithelial tumor cells, and
thus folate was attached on PEG molecules via cysteine (folate – cysteine – PEG3400 –
PE), which, consequently, was one of the components of the liposomal bilayer (PC/
Chol/PEG – PE). The ligand - bearing liposomes (diameter of 65 – 90 nm) were injected
i.p. in tumor - bearing mice. Signifi cant folate - bearing liposome accumulation was
obtained in the ascites fl uid and more specifi cally by macrophages, which indicates
macrophage activation. A reduced targeted liposome accumulation in the tumor
could occur for a number of reasons, such as poor liposome penetration into solid
tumor mass. Activated macrophages secrete immunosuppressive cytokines and
angiogenic factors, so liposome targeting could possibly eliminate them during
malignancy therapy.
Gene delivery is another approach trying to tackle the problem of cancer. Mutations
of p53 tumor suppressor gene contribute to genetic abnormalities in ovarian
cancer. Kim et al. developed a nonviral vector for delivery of p53 in ovarian cancer
cells (OVCAR - 3) [461] . The nonviral (liposomal) vector consisted of DOTAP,
DOPE, and Chol in the molar ratio 1 : 0.7 : 0.3. High expression of p53 mRNA and
proteins in OVCAR - 3 cells indicated successful transfection of the lipoplexes to the
cells used. The latter was indicated with the inhibition of cell growth obtained in
OVCAR - 3 cells due to apoptosis caused by that protein. Intratumoral injection of
DDC/pp53 - GFAP lipoplexes into mice bearing the OVCAR - 3 cells clearly showed
the tumor growth inhibition, which suggests the therapeutic effi ciency of the particular
lipoplexes.
The neutral 1,2 - dioleoyl - sn - glycerol - 3 - phosphatidylcholine (DOPC) was used to
make complexes with the siRNA targeting the oncoprotein EphA2 and then injected
i.v. into ovarian tumor - bearing mice [462] . According to the authors, the signifi cance
of the particular study is the formulation of siRNA in liposomes with successful
outcome after delivery of the loaded carrier, which showed signifi cant reduced
protein expression and tumor growth (Figure 17 ).
APPLICATIONS OF LIPOSOMES IN THERAPEUTICS 505
506 LIPOSOMES AND DRUG DELIVERY
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FIGURE 17 Therapeutic effi cacy of siRNA - mediated EphA2 down regulation. A and B
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and randomly allocated to one of fi ve groups, with therapy beginning 1 week after cell injection.
(a) Empty DOPC liposomes, (b) control siRNA in DOPC, (c) EphA2 - targeting siRNA
in DOPC, (d) paclitaxel + control siRNA in DOPC, or (e) paclitaxel + EphA2 siRNA in
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(100 . g) or vehicle (fi rst three groups) was injected i.p. once weekly. ( Reprinted from ref. 462
and with permission of the American Association for Cancer Research .)
*
+++++++++
+++
§
§
†
*
*
*
*
†
† *
(a)
(b)
HeyA8: Mean weights Individual weights
SKOV3ip1: Mean weights Individual weights
Tumor weight (g)
Empty liposomes
Control siRNA
EphA2 siRNA
Paclitaxel +control siRNA
Paclitaxel +EphA2 siRNA
Empty liposomes
Control siRNA
EphA2 siRNA
Paclitaxel +control siRNA
Paclitaxel +EphA2 siRNA
4.0
3.0
2.0
1.0
0.0
5.0
4.0
3.0
2.0
1.0
0.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
p<0.05 compared to control siRNA
p<0.05 compared to empty liposomes
p<0.05 compared to control siRNA plus paclitaxel
2.40
0.81 0.35
0.70 0.22
0.04
1.51
0.98 0.84
0.21
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530 LIPOSOMES AND DRUG DELIVERY
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535
5.4
BIODEGRADABLE NANOPARTICLES
Sudhir S. Chakravarthi and Dennis H. Robinson
University of Nebraska Medical Center, College of Pharmacy, Omaha, Nebraska
Contents
5.4.1 Introduction
5.4.1.1 Classifi cation of Nanoparticles
5.4.2 Natural Biodegradable Polymeric Nanoparticles
5.4.2.1 Physical Properties of Natural Polymers and Methods Used to Prepare
Nanoparticles
5.4.2.2 Drug Delivery Applications and Biological Fate of Natural Polymeric
Nanoparticles
5.4.3 Synthetic Biodegradable Polymeric Nanoparticles
5.4.3.1 Synthetic Polymers: Physical Properties and Methods of Preparation of
Nanoparticles
5.4.3.2 Drug Delivery Applications and Biological Fate of Synthetic Biodegradable
Polymers
5.4.4 Thermosensitive and pH - Sensitive Nanoparticles
5.4.4.1 Physical Properties and Methods of Preparation
5.4.4.2 Drug Delivery Applications and Biological Fate of Thermosensitive and
pH – Sensitive Nanoparticles
5.4.5 Applications of Biodegradable Nanoparticles Other Than Drug Delivery
5.4.6 Physicochemical Characterization of Polymeric Nanoparticles
5.4.6.1 Molecular Weight
5.4.6.2 Hydrophobicity
5.4.6.3 Glass Transition Temperature
5.4.6.4 Particle Size and Particle Size Distribution
5.4.6.5 Surface Charge and Zeta Potential
5.4.6.6 Surface Hydrophilicity
5.4.6.7 Drug Loading and Encapsulation Effi ciency
5.4.6.8 Drug Release
5.4.6.9 Physical Stability of Polymeric Nanoparticles
5.4.7 Targeting Nanoparticles by Surface Conjugation with Ligands
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
536 BIODEGRADABLE NANOPARTICLES
5.4.8 Cellular Traffi cking of Biodegradable Nanoparticles
5.4.9 Conclusions
References
5.4.1 INTRODUCTION
Interest in nanotechnology has increased exponentially in many scientifi c areas,
including drug delivery, nanoimaging, and other medical - related applications.
Nanoparticles can be fabricated in many different shapes and sizes using a wide
range of organic and inorganic materials. However, by defi nition, these particles
must be within the size range of 1 – 1000 nm. Because the use of nanoparticles in drug
delivery and nanomedicine invariably requires parenteral administration, there has
been, and continues to be, a major need for the use of polymeric carriers that are
both biocompatible and biodegradable. This review will focus on the application of
nanotechnology to deliver therapeutic or diagnostic agents using biodegradable
polymeric nanoparticles for systemic, localized, or targeted delivery.
5.4.1.1 Classifi cation of Nanoparticles
Depending on the method of preparation and resulting structure, nanoparticles are
broadly classifi ed as either matrix - type or encapsulated particles. Hence, a drug is
either homogenously dispersed in the polymeric matrix or encapsulated within the
core of the particle. In drug delivery applications, biodegradable polymers used to
prepare nanoparticles are either natural or synthetic in origin (Table 1 ). Natural
polymers, or biopolymers, include alginates, chitosan, cellulose, gelatin, gliadin, and
pullulan. A recent review describes the applications of these polymers in gene delivery
and tissue engineering [1] . Natural polymers may vary widely in their composition
and therefore physicochemical properties. Such variability in properties may
result in poor reproducibility in delivery characteristics, such as drug loading and
release kinetics. Further, their purifi cation from natural sources may be diffi cult. In
contrast, synthetic polymers can be prepared with relatively precise properties such
as molecular weight, solubility, and permeability characteristics. Examples of synthetic
polymers used to make biodegradable nanoparticles include polylactide,
poly(lactide -co - glycolide) (PLGA), polyanhydride, polycyanoacrylate, poly( . -
caprolactone), and polyphosphoester. As liposomes are more commonly prepared
in the micrometer - size range, they were considered out of the scope of this review.
Because there are numerous reviews of these polymers in the literature, their physicochemical
properties, methods of synthesis, applications, and biological fate of each
of these polymers are only briefl y described in this chapter.
5.4.2 NATURAL BIODEGRADABLE POLYMERIC NANOPARTICLES
Natural polymers extracted and purifi ed from plant and animal sources often vary
signifi cantly in their purity. For example, alginate is available in over 200 different
TABLE 1 Classifi cation of Natural and Synthetic Polymers and Their Methods of
Purifi cation or Synthesis
Polymer Source
Method of
Purifi cation or
Synthesis Solubility
Sodium alginate Natural
(seaweed)
Alkali - based
extraction
Water soluble (pH >
3), insoluble in
organic solvents
Chitosan Natural
(crab
shells)
Deacetylation of
chitin
Soluble in aqueous
solutions (low pH),
insoluble in organic
solvents
Gelatin Natural
(collagen)
Hydrolysis Soluble in hot water
(> 34 ° C), acetic acid,
forms insoluble gel
with water at room
temperature,
insoluble in organic
solvents
Polysaccharides Natural Enzymatic reactions Pullulans (soluble in
water), dextrans
(soluble in water)
Albumin Natural
(plants,
animals)
Separation
techniques
(chromatography)
Soluble in water
Gliadin Natural
(wheat)
Alcohol extraction Insoluble in water,
soluble in ethanol
Poly(lactide) and
Poly(lactide- co -glycolide)
Synthetic Ring - opening
polymerization
Insoluble in water,
soluble in organic
solvents
Poly( . - caprolactone) Synthetic Anionic, cationic,
free - radical, ring -
opening
polymerization
Soluble in select
organic solvents
such as chloroform,
dichloromethane
Polyanhydrides Synthetic Melt condensation,
ring - opening
polymerization
Most polyanhydrides
soluble in organic
solvents, insoluble in
water
Poly - alkylcyanoacrylates Synthetic Emulsion and
interfacial
polymerization
Soluble in organic
solvents
Polyphosphoesters Synthetic Polyaddition, ring -
opening
polymerization
Available as water -
soluble and water -
insoluble types
grades and is extracted from various sources that differ in molecular weight and the
percentage and arrangement of guluronic and mannuronic acid blocks. Further,
chitosan, poly - . (1 - 4 - d - glucosamine), is available in grades varying in molecular
weight, degree of deacetylation (from parent compound, chitin), and viscosity [2] .
NATURAL BIODEGRADABLE POLYMERIC NANOPARTICLES 537
538 BIODEGRADABLE NANOPARTICLES
Some natural polymers may be chemically modifi ed to tailor solubility properties.
An example is the reaction of the free amino groups of chitosan to form the more
water soluble derivative, methoxy - polyethylene glycol (PEG) chitosan [3] . Collagen
is marketed as six different types, I – VI, depending on its source and physiological
applicability. Similarly, the properties of gelatin are dependent on the method of
preparation using acid - or base - catalyzed hydrolysis from collagen.
5.4.2.1 Physical Properties of Natural Polymers and Methods Used to
Prepare Nanoparticles
Sodium Alginate Alginates are primarily derived from the algae Macrocystis pyrifera
and Laminaria hyperborea . These are linear, unbranched polymers containing
. - (1 – 4) - linked mannuronic acid and . - (1 – 4) - linked guluronic acid residues that are
either arranged in blocks, commonly called G blocks and M blocks, or alternate with
each other. Alginates are hydrophilic, anionic polymers that vary in molecular
weight, depending primarily on the G and M blocks. They are characterized by the
ratio of guluronic and mannuronic acids, which can be quantifi ed by ultraviolet
(UV) spectrophotometry, gas chromatography, and high - performance liquid chromatography
(HPLC) [4] . For example, the polymer obtained from M. pyrifera has
an M/G ratio of 1.6. Alginate obtained from seaweed must be purifi ed by one of
several applicable alkali and acid treatment protocols [5, 6] .
Alginate nanoparticles can be prepared using ionotropic gelation, emulsifi cation/
internal gelation, and emulsifi cation/solidifi cation methods. Ionotropic gelation
results when the anionic alginate reacts with cationic ions or molecules such as
calcium or poly - l - lysine. Gelation occurs when cations chelate the guluronic and
mannuronic acid groups to produce an “ egg - box ” structure that encapsulates the
drug. The size of the alginate particles is determined by the molar concentration of
calcium or poly - l - lysine and the method of addition of these counterions to alginate
[7] . In the emulsifi cation/internal gelation method, the sodium alginate and an
insoluble calcium salt are dispersed in a vegetable oil and the calcium ions are liberated
to form an alginate gel when the pH of the dispersion is lowered [8, 9] . An
advantage of the use of alginate polymers to deliver drugs is that nanoparticles are
prepared in aqueous media and may be more suitable to formulate compounds that
are unstable in organic solvents such as proteins and peptides. However, since the
chelation to form the gel is reversible, a disadvantage of unmodifi ed, alginate - based
delivery systems is rapid drug release due to the collapse of the egg - box structure
when exposed to monovalent ions in physiological media.
Chitosan Chitosan is a nontoxic, biodegradable polymer obtained by hydrolysis
of chitin, a natural polysaccharide that is a chief component of the crustacean exoskeleton.
Unmodifi ed chitosan is soluble in acidic media and has signifi cant mucoadhesive
properties.
Chitosan nanoparticles may be prepared using various methods, including emulsion
cross - linking, coacervation – precipitation, spray drying, emulsion droplet
coalescence, ionic gelation, reverse - micellar method, and sieving. A relatively recent
review describes the methods and applications of chitosan nanoparticles in drug
delivery [10] . Chitosan nanoparticles have also been prepared using water - soluble
cross - linking agents such as carbodiimide with the size being controlled by changing
pH [11] . More monodisperse nanoparticles may be prepared using fractionated and
deacetylated chitosan [12] . In general, the size of nanoparticles will depend on the
molecular weight of chitosan, its concentration, and its surface charge [13] . The
physicochemical properties of chitosan are determined by the solution pH and ionic
strength [14] .
Gelatin Gelatin is obtained by either alkaline or acidic hydrolysis of collagen. It
has a triple helical structure with a high content of glycine, proline, and hydroxyproline
residues. Gelatin that is formed from alkaline treatment of collagen has
more carboxyl groups and a lower isoelectric point than that derived from acidic
hydrolysis [15] . The physicochemical properties of gelatin depend on the method
of extraction and the extent of thermal denaturation that occurs during the
purifi cation.
Gelatin nanoparticles can be prepared by various methods, including chemical
cross - linking, water - in - oil (w/o) emulsifi cation, and desolvation. Gelatin is cross -
linked with agents such as glutaraldehyde. Effi cient cross - linking usually results in
decreased rate of drug release. The w/o emulsifi cation involves extruding a preheated,
aqueous solution of gelatin into vegetable oils, such as corn or olive oils
[15] . The two - step desolvation method involves the dropwise addition of a water -
miscible nonsolvent such as acetone and ethanol [16] . While the use of collagen,
the parent compound of gelatin, in drug delivery is rare, collagen nanoparticles
have been used to deliver genes by exploiting the electrostatic interaction between
the positively charged polymer and negatively charged deoxyribonucleic acid
(DNA) [17] .
Polysaccharides The macromolecular polysaccharides that include pullulan,
mannan, and dextran are the main constituents of the cellular glycocalyx and play
an important role in cell – cell adhesion and the cell – cell recognition process [18] .
Pullulan is a nonimmunogenic, nontoxic, water - soluble, linear, nonionic polysaccharide
with . (1 – 4) and . (1 – 6) linkages with free hydroxyl groups for drug conjugation
[19] . Pullulans are intracellularly synthesized and secreted by a fungus,
Aureobasidium pullulans [20] . On the other hand, dextrans are anionic glucose
polymers derived from sucrose with . (1 – 6) glucosidic linkage. A class of enzymes,
glucansucrases, produced by two genera of lactic acid bacteria, namely, Leuconostoc
and Streptococcus, catalyze the synthesis of dextrans from sucrose. The extraction
of dextrans as well as their physical properties and drug delivery aspects has been
reviewed [21, 22] . When coated with mannans, the biological response of both
natural and synthetic polymeric nanoparticles may be changed [23] .
To make pullulan nanoparticles, the polymer must fi rst be made hydrophobic,
typically by conjugating alkyl groups, cholesterol groups, or succinyl groups. Hydrophobic
pullulans self - assemble to form stable hydrogel nanoparticles [24] . Alternately,
pullulan nanoparticles can be formed by cross - linking reverse micelles of the
polymer with glutaraldehyde [25] . Nanoparticles form when a solution of pullulan
acetate in N,N - dimethyl acetamide is used and dialyzed with borate buffer [26] .
Dextran is commonly conjugated to other polymers, such as PLGA, PEG,
polystyrene, and poly(methyl methacrylate) for preparation of nanoparticles.
Complex coacervation has been used to prepare dextran nanoparticles using
oppositely charged polymers such as polyethyleneimine [27] . Although nanoparticle
NATURAL BIODEGRADABLE POLYMERIC NANOPARTICLES 539
540 BIODEGRADABLE NANOPARTICLES
formulation using mannans has not been reported, mannan - coated nanoparticles
increased cell binding and macrophage uptake [28] .
Albumin Human serum albumin, the most abundant plasma protein, is a positively
charged, multifunctional protein and is involved in transport, ligand binding, and
enzymatic activities. Albumin is a globular protein containing approximately 585
amino acids in an . - helical tertiary structure. Several exogenous and endogenous
compounds can covalently or reversibly bind to albumin, and because of the
excellent adsorptive properties of human serum albumin, this polypeptide can be
adsorbed onto the surface of polymeric nanoparticles [29] . In addition, as it is
amphoteric, albumin can be used as a surfactant during the preparation of
nanoparticles where it is irreversibly adsorbed onto the surface of biodegradable
polymers such as poly(lactic acid) (PLA) and PLGA [30] . These protein – particle
interactions are mainly driven by electrostatic forces further stabilized by hydrophobic
forces [31] .
Albumin nanoparticles can be prepared by controlled desolvation, pH - induced
coacervation, and/or chemical cross - linking with glutaraldehyde. Briefl y, the pH of
an aqueous solution of albumin is raised to about 9.0 and nanoparticles are precipitated
by adding a miscible cosolvent such as acetone [32] . A study reports attempts
to optimize the desolvation method to prepare albumin nanoparticles of a more
controlled particle size and narrower particle size distribution [33] .
Gliadin Gliadin, a glycoprotein derived from gluten, is extracted from wheat and
separated by capillary electrophoresis [34, 35] . Gliadin is classifi ed as . - 5, . - 1,2, . ,
and . - type based on its structure and electrophoretic mobility [36] . The glycoprotein
is water insoluble due to the presence of interpolypeptide disulfi de bonds and
hydrophobic interactions. A limitation on the use of gliadins is that patient sensitivity
causes an autoimmune disorder called celiac disease.
Gliadin nanoparticles are prepared by the desolvation method by fi rst pouring
an organic solution of polymer into an aqueous phase such as physiological saline
containing a surfactant stabilizer (e.g., Pluronic). The nanoparticles are formed by
evaporating the organic solvent.
5.4.2.2 Drug Delivery Applications and Biological Fate of Natural
Polymeric Nanoparticles
Alginate Nanoparticles Alginate nanoparticles have been used to formulate a
wide range of drugs. Because they are prepared in an aqueous environment under
mild conditions, alginate nanoparticles are particularly suitable for formulating
proteins, peptides, and oligonucleotides [37] . Further, in addition to being biodegradable,
alginates are nonimmunogenic. To decrease the rate of exchange of cations
such as Ca 2+ with monovalent ions in the dissolution medium, the anionic alginates
are often treated with cationic molecules such as chitosan, poly - l - lysine, or tripolyphosphate.
Some examples of the wide range of applications of alginate - based
nanoparticles are described. Alginate nanoparticles prepared with tripolyphosphate
were used in oral delivery [38] . A study of physical properties demonstrated that
alginate – chitosan nanoparticles are suitable for the delivery of DNA [39] . Alginate -
coated chitosan nanoparticles increased stability and decreased the burst release of
ovalbumin [40] . A study reported that chitosan - stabilized alginate nanoparticles
increased bioavailability and sustained release of antifungal drugs compared to
PLGA nanoparticles [41] . Although predominantly used for oral administration,
inhaled alginate nanoparticles improved the bioavailability of antitubercular drugs
[42] . In vivo, alginate nanoparticles accumulate in the Kupffer cells, parenchymal
cells of liver, and phagocytes of spleen and lungs [43, 44] . Alginate nanoparticles
have also been reported to be absorbed into Peyer ’ s patches, suggesting that they
may enhance targeting to the intestinal mucosa [45] . In the body, the alginates
degrade by acidic hydrolysis of the guluronic and mannuronic segments [46] .
Chitosan Nanoparticles In addition to low - molecular - weight drugs and nutraceuticals,
chitosan nanoparticles are widely used in the delivery of macromolecules such
as DNA and small interfering ribonucleic acid (siRNA) [47] . Apart from sustaining
the release of macromolecules, chitosan nanoparticles protect them from nucleases.
Placebo chitosan nanoparticles exhibited antibacterial activity against several
microbes, including Escherichia coli [48] . The surface of chitosan nanoparticles was
hydrophobically modifi ed with linoleic acid for delivery of trypsin [49] . Other applications
of chitosan nanoparticles include lung [50] and ocular delivery [51] . The
primary amine group at the 2 position can be modifi ed to tailor chitosan for specifi c
applications. For example, chemical conjugation of these amine groups to methoxy -
PEG groups increased water solubility [52] . Thiolation of chitosan enhanced the
mucosal permeation of the nanoparticles [53] . Hydrophobically modifi ed glycol
chitosans that self - assemble into nanoparticles have been used to deliver doxorubicin
[54] . Targeting chitosan nanoparticles to folate receptors on the surface of cells
enhanced the transfection effi ciency of DNA [55] . N - Succinyl chitosan nanoparticles
containing 5 - fl uorouracil demonstrated excellent activity against sarcoma tumors
[56] . Self - assembled N - acetyl histidine – conjugated glycol chitosan nanoparticles
were effi ciently internalized into cells by adsorptive endocytosis [57] . No toxic
effects have been observed with chitosan nanoparticles. Upon intravenous (i.v.)
administration, chitosan nanoparticles accumulated in the liver with minimal concentrations
in the heart and lung [54] .
Gelatin Nanoparticles Gelatin nanoparticles have been used as a delivery system
for several drugs, including pilocarpine, hydrocortisone [58] , methotrexate [59] ,
paclitaxel [60] , and chloroquine [61] . High protein loading and sustained release
were achieved using composite gelatin and PLGA nanoparticles [62] . Surprisingly,
placebo gelatin nanoparticles exhibited antimelanoma activity in vivo [63] . Primary
amine groups of gelatin molecule can be chemically conjugated or cross - linked using
bifunctional cross - linkers. This is demonstrated in the delivery of biotinylated
peptide nucleic acid using avidin - cross - linked gelatin nanoparticles [64] . PEGylation
of gelatin nanoparticles containing hydrophilic drugs prolonged circulation time in
the body [65] . Thiolated gelatin nanoparticles produced effective transfection of
plasmid DNA encoding the green fl uorescent protein [66] . Following endocytic
uptake by the cells, gelatin nanoparticles concentrate in the perinuclear region [65] .
In vitro, the gelatin nanoparticles are effi ciently internalized into macrophages and
monocytes [67] . In tumor - bearing mice, PEGylated gelatin nanoparticles predominantly
accumulated in the liver and tumor [68] while in dendritic cells they are primarily
localized in the lysosomes [69] . In vivo, gelatin is degraded by proteases to
NATURAL BIODEGRADABLE POLYMERIC NANOPARTICLES 541
542 BIODEGRADABLE NANOPARTICLES
amino acids. Although cardiotoxicity and mild immunogenicity were reported with
gelatin nanoparticles covalently coupled to doxorubicin, this was attributed to the
coupling reagent glutaraldehyde [70] .
Pullulan and Dextran Nanoparticles Pullulan nanoparticles successfully delivered
HER2 oncoprotein to induce humoral and cellular immune responses against
HER2 - expressing murine sarcomas [71] . Hydrophobic polysaccharides such as pullulan
and mannan enable soluble proteins to induce cellular immunity and therefore
may be a potential delivery vehicle for vaccines [72] . pH - sensitive pullunan nanoparticles
prepared by conjugating sulfonamides are stable at physiological pH but
aggregate and release the encapsulated drug when exposed to the lower tumor pH
[73] . Coating of magnetic nanoparticles with pullulan enhanced their cellular uptake
by endocytosis [74] . Amphotericin - loaded, dextran – polyethyleneimine nanoparticles
were active against Candida albicans [27] . Similarly, insulin - containing dextran –
polyethyleneimine nanoparticles prolonged the hypoglycemic effect in diabetic
rats [75] . Hydrogels prepared from blends of polyvinyl alcohol and dextrans have
been used as matrices to entrap PLGA nanoparticles [76] . In vitro, immuno-
fl uorescent staining illustrated that pullulan nanoparticles are internalized by active
endocytosis [25] . However, other studies suggest that both absorption and
internalization of pullulan nanoparticles are inhibited by coating them with dextran
[77] .
Albumin Nanoparticles A signifi cant development in the drug delivery of albumin
nanoparticles has been the recent marketing of the commercial product Abraxane ®
for chemotherapy of breast cancer. This delivery system is prepared by nab TM
technology, which involves noncovalent complexation of albumin with paclitaxel.
Albumin nanoparticles have been used to deliver antisense oligonucleotides, interferon
. , and anticytomegaloviral drugs [78, 79] . Intravitreal injection of gancicyclovir
- loaded albumin nanoparticles was attempted to prolong residence time in the
eye [79] . PLA nanoparticles coated with albumin degrade more rapidly in the gastrointestinal
(GI) region, resulting in effi cient delivery of water - soluble drugs across
the GI tract [80] . In addition, intra - arterial chemotherapy with paclitaxel - containing
albumin nanoparticles effectively treated squamous cell carcinoma [81] . Human
serum albumin – polyethyleneimine nanoparticles optimized transfection of the
luciferase gene in human, embryonic, and epithelial kidney cells [82] . Conjugation
of the cellular targeting agent, folic acid, resulted in increased cellular uptake of
albumin nanoparticles compared to unmodifi ed particles [83, 84] . Transferrin - conjugated
PEGylated albumin nanoparticles demonstrated enhanced uptake into the
brain tissues [85] . Glycyrrhizin was conjugated to the amine groups of albumin
nanoparticles targeting hepatocytes [86] . After i.v. administration, albumin - coated
PLA nanoparticles were distributed in the liver, bone marrow, lymph nodes, spleen,
and peritoneal macrophages [87] . In the body, albumin nanoparticles are actively
taken up by macrophages.
Gliadin Nanoparticles The hydrophobic nature of gliadin makes this polymer
ideal for delivery of hydrophobic compounds such as all - trans retinoic acid and
vitamin E. Gliadin nanoparticles adhered to the stomach mucosa and signifi cantly
increased the bioavailability of carbazole [88] . Typically, encapsulation effi ciency
and drug loading of gliadin nanoparticles are higher for hydrophobic drugs [89] .
Gliadin nanoparticles were targeted to Helicobacter pylori by chemical conjugation
of lectin glycoproteins to their surface, resulting in a twofold increase in inhibition
of bacterial activity compared to unmodifi ed nanoparticles [90] . Although gliadins
adhere to the mucosa, internalization of gliadin nanoparticles into cells has not been
reported. More defi nitive studies are required to fully understand the biological fate
of these particles.
5.4.3 SYNTHETIC BIODEGRADABLE POLYMERIC NANOPARTICLES
A detailed description of the methods of polymerization and variables employed in
polymer synthesis are beyond the scope of this review and can be found in many
texts and review articles. The focus of this section is to provide an overview of the
properties and methods used to prepare nanoparticles from each class of synthetic
polymer.
5.4.3.1 Synthetic Polymers: Physical Properties and Methods of Preparation
of Nanoparticles
Poly(lactic acid) and Poly(lactide -co-glycolide) These poly - hydroxy acids are
approved for human use by the Food and Drug Administration (FDA) and have
been widely used to prepare biodegradable nanoparticles. PLA exists in optically
active and inactive forms and is a semicrystalline, hydrophobic molecule that
degrades in the body over a period of months. Conversely, poly(glycolic acid) is
amorphous and hydrophilic and degrades more rapidly than PLA. In aqueous
media, these polymers degrade by random hydrolysis of ester bonds that is autocatalyzed
in acidic media to form lactic and glycolic acids [91] . The factors that affect
the rate of hydrolytic degradation include type and composition of the polymer
backbone, nature of pendent groups, molecular weight, pH, enzymes, and geometry
of the delivery device.
The preparation and characterization of PLA and PLGA nanoparticles have
been extensively reviewed elsewhere [92, 93] . Various techniques may be used to
prepare PLA and PLGA nanoparticles, including simple and multiple emulsions,
nanoprecipitation, gas antisolvent method, supercritical fl uid technology, coacervation/
phase separation, and spray drying [91] . Briefl y, in the single - emulsion method,
an organic solution of the polymer and drug is emulsifi ed with an aqueous solution
of surfactant such as polyvinyl alcohol (PVA). While PLA and PLGA nanoparticles
containing hydrophobic drugs are prepared by the two - phase emulsion method, a
w/o/w multiple - emulsion method is needed to encapsulate hydrophilic drugs. In the
phase separation method, the addition of a nonsolvent precipitates or coacervates
the polymer from solution to encapsulate the drug. The experimental variables for
each protocol can be altered to infl uence the physicochemical properties, such as
particle size, particle size distribution, morphology, and zeta potential [93] . The
release of encapsulated drug from PLA and PLGA nanoparticles may occur by a
combination of diffusion and polymer degradation at a rate that is infl uenced by
properties of the polymer and nanoparticles and the environment. The surface of
SYNTHETIC BIODEGRADABLE POLYMERIC NANOPARTICLES 543
544 BIODEGRADABLE NANOPARTICLES
both PLGA and PLA nanoparticles can be modifi ed to target cells and organs by
conjugation with ligands such as folates, transferrin, HIV - TAT, aptamers, heparin,
and lectins. The negative zeta potential of PLA and PLGA nanoparticles can be
altered by coating with cationic polymers such as chitosan and polyethyleneimine,
which promote nanoparticle – cell interaction. As with liposomes, PEGylation of
PLGA nanoparticles prolongs circulation times in the body.
Poly( e-caprolactone) Poly( . - caprolactone) is a semicrystalline polymer synthesized
by anionic, cationic, free - radical, or ring - opening polymerization [94] . It is available
in a range of molecular weights and degrades by bulk hydrolysis autocatalyzed
by the carboxylic acid end groups. The presence of enzymes such as protease, amylase,
and pancreatic lipase accelerates polymer degradation [95] . The various methods of
preparation of poly( . - caprolactone) nanoparticles include emulsion polymerization,
interfacial deposition, emulsion – solvent evaporation, desolvation, and dialysis. These
methods and various applications are extensively reviewed [94] .
Polyanhydrides Polyanhydrides have a hydrophobic backbone with a hydrolytically
labile anhydride linkage. These polymers widely vary in chemical composition
and include aliphatic, aromatic, and fatty acid – based polyanhydrides. The rate of
degradation depends on the chemical composition of the polymer. In general, aliphatic
polyanhydrides degrade more rapidly than the aromatic polymer. Hence,
copolymer blends with varying ratios of aliphatic - to - aromatic polyanhydrides can
be synthesized to suit specifi c applications.
The synthesis and physical properties of polyanhydrides have been reviewed [96,
97] . Polyanhydride nanospheres are commonly prepared by the emulsion – solvent
evaporation method using PVA as a stabilizer. However, as polyanhydrides are
hydrolabile, they need to be fl ash frozen in liquid nitrogen and lyophilized immediately
[98] . An example of their use to deliver drugs is entrapment of bovine zinc
insulin by phase inversion nanoencapsulation [99] . Although not used to formulate
nanoparticles, polyanhydride microspheres have been prepared using alternate
techniques involving nonaqueous solvents, such as solid/oil/oil double emulsion and
cryogenic atomization techniques [100] . The surface of polymeric nanoparticles can
be modifi ed for targeted delivery by reaction of the anhydride with an amino group
to form an amide linkage with a ligand [101] . However, application of the ligand -
conjugated nanoparticles for drug delivery is yet to be explored extensively.
Poly(alkyl -cyanoacrylates) As poly(alkyl - cyanoacrylates) form strong bonds
with polar substrates including the skin and living tissues, they exhibit bioadhesive
properties. These polymers are synthesized by free - radical, anionic, or zwitterionic
polymerization. As detailed in a recent review, poly(alkyl - cyanoacrylate) nanoparticles
are prepared by emulsion polymerization, interfacial polymerization, nanoprecipitation,
and emulsion – solvent evaporation methods [102] .
Solid–lipid Nanoparticles Solid – lipid nanoparticles (SLNs) are obtained by high -
pressure homogenization of molten lipids in the presence of surfactant. The major
advantage of using SLN is the ability to have high drug loading and prolonged stability
with lipophilic compounds. In addition to drug delivery, SLNs have been used
in dermatological and cosmetic preparations. The most common carriers used to
prepare SLNs are triglycerides, glycerides, fatty acids, steroids, and waxes and may
contain a wide range of emulsifi ers. Methods of preparation of SLNs include high
speed hot and cold homogenization, ultrasound, emulsion – solvent evaporation, and
microemulsion. The various parameters involved in the preparation of SLNs have
been optimized and thoroughly reviewed and their physicochemical properties
elucidated [103, 104] .
Other Synthetic Biodegradable Polymers Although well investigated for drug
delivery, polyorthoesters, polyurethanes, and polyamides have found limited application
as nanoparticles. A report documents the synthesis and characterization of
polyorthoester nanoparticles [105] .
5.4.3.2 Drug Delivery Applications and Biological Fate of Synthetic
Biodegradable Polymers
PLA / PLGA Nanoparticles A wide range of hydrophilic and hydrophobic drugs,
including low - and high - molecular - weight compounds, have been encapsulated into
PLGA/PLA nanoparticles for a wide range of therapeutic applications and routes
of administration, including oral, intravenous, intra - arterial, nasal, and inhalation
delivery [92, 106] . Extensive reviews describing the application of PLGA nanoparticles
in drug therapy are available [92, 107, 108] .
After i.v. administration, the PLGA nanoparticles are removed from systemic
circulation by the mononuclear phagocytic system in the liver [109] . PLGA nanoparticles
enter cells by absorptive endocytosis and may escape the lysosomes to accumulate
in cytoplasm [110, 111] . In the body, PLA and PLGA degrade into the
monomers lactic and glycolic acids, which enter the citric acid cycle, where they are
metabolized and eliminated as CO 2 and H 2 O. Glycolic acid may also be excreted
through the kidney [91] . Humoral response to these results in mild, acute, and
chronic infl ammation [112] .
Poly( e-caprolactone) Nanoparticles As important applications of poly( . -
caprolactone) nanoparticles have been reviewed previously, only representative
examples will be given [94] . Decreased cardiovascular adverse effects of cartelol
was observed upon ophthalmic administration of poly( . - caprolactone) nanocapsules
[113] . Poly( . - caprolactone) nanoparticles, nanocapsules, or nanoemulsions
increased the ocular uptake of indomethacin [114] . The cytotoxicity of retinoic acid
was enhanced when delivered in core - shell - type nanoparticles formed from poly( . -
caprolactone – polyethylene glycol) blends [115] . Alternately, these nanoparticles
were also chemically modifi ed with folic acid to target the folate receptors for
enhanced cellular uptake [116] . Coating poly( . - caprolactone) nanoparticles with
polysaccharides such as galactose resulted in lectin - dependent aggregation, demonstrating
the potential as a targeted delivery system to hepatocytes [117] . Stable
complexes were formed between anionic DNA and chitosan - modifi ed poly( . -
caprolactone) nanoparticles, demonstrating high transfection effi ciency [118] . After
i.v. administration, these particles are eliminated by macrophages of the reticuloendothelial
system and biodegradation occurs by bulk scission of polymer chains
[94] . However, dextran - coated poly( . - caprolactone) nanoparticles lowered their
uptake into macrophages [119] .
SYNTHETIC BIODEGRADABLE POLYMERIC NANOPARTICLES 545
546 BIODEGRADABLE NANOPARTICLES
Polyanhydride Nanoparticles Polyanhydrides have been more commonly used
to prepare microparticles than nanoparticles. However, the technology is adaptable
for nanoparticles. The transfection effi ciency of fi refl y luciferase DNA was
enhanced when delivered in nanoparticles prepared from polyanhydride – lactic
acid blends, demonstrating the potential application in gene delivery [120] . The
degradation and elimination of polyanhydrides have been reviewed [97] . In vivo,
the anhydride bond degrades to form diacid monomers that are eliminated from
the body.
Poly(alkyl -cyanoacrylate) Nanoparticles The applications of poly(alkyl -
cyanoacrylate) nanoparticles have been reviewed elsewhere and therefore only
representative examples are described [102] . Because of their adhesive properties,
nanoparticles have the potential to prophylactically treat candidiasis of the oral
cavity [121] . Not surprisingly, poly(alkyl - cyanoacrylate) nanoparticles have been
used to deliver drugs to tumors [122] . Enhanced absorption and prolonged
hypoglycemic effect were observed when insulin was delivered in poly(alkyl -
cyanoacrylate) nanoparticles [121] . Nuclear accumulation of antisense oligonucleotides
into vascular smooth muscle cells was increased when delivered using
poly(alkyl - cyanoacrylate) nanoparticles [123] . Dextran - coated poly(alkyl -
cyanoacrylate) nanoparticles lowered protein adsorption in the blood [124] .
Poly(alkyl - cyanoacrylates) degrade by hydrolysis of the ester bond of the alkyl
side chains to form water - soluble alkyl alcohol and poly(cyanoacrylic acid). After
in vivo administration, poly(alkyl - cyanoacrylate) nanoparticles are predominantly
distributed in the liver, spleen, and bone marrow where they are endocytosed into
the cells to become localized in the lysosomes. However, the mechanisms of lysosomal
escape have not been identifi ed [102] .
Solid–Lipid Nanoparticles SLNs have been used to deliver small molecules and
macromolecules such as DNA and peptides. The in vitro and in vivo applications of
SLNs are reviewed elsewhere [125, 126] . The stability and oral bioavailability of
insulin were enhanced when administered in wheat germ agglutinin – conjugated
nanoparticles [127] . A polyoxyethylene stearate coat on the SLN confers stealth
properties [128] .
5.4.4 THERMOSENSITIVE AND p H - SENSITIVE NANOPARTICLES
5.4.4.1 Physical Properties and Methods of Preparation
Thermosensitive Polymeric Nanoparticles Thermoresponsive “ smart ” polymers
that change their physical characteristics, such as shape, surface properties, or solubility,
in response to changes in temperature have been developed to target drugs
[129] . The encapsulated drug is released when the nanoparticles are exposed to
changes in temperature, such as body temperature or external heat source. For
example, poly( N - isopropyl acrylamide), or poly(NIPAAm), is water soluble at room
temperature but aggregates and is insoluble above its lower critical solution temperature
(LCST), which typically ranges between 37 and 42 ° C [129] . The methods
used to prepare thermosensitive nanoparticles have been thoroughly reviewed [127,
129 – 131] . Interestingly, when heated above the LCST, poly(NIPAAm) – PEG block
copolymers spontaneously self - assemble into nanoparticles whose size is controlled
by the rate of heating [132] .
pH-Sensitive Polymeric Nanoparticles Enteric - coated polymers have long been
used to protect drugs from the acidic pH in the stomach. Chitosan is a pH - sensitive
polymer that is soluble only in acidic media. Similarly, the solubility of sulfonamide -
modifi ed pullulans is dependent on pH [133] . Chitosan – insulin nanoparticles are
stable at low pH but dissociate at physiological pH, releasing insulin [134] . The pH
of tumor interstitium is lower than the normal tissue. The pH - sensitive polyethylene
oxide – poly( . - amino ester) microparticles containing paclitaxel signifi cantly reduced
tumor burden [135, 136] . Polyketals, a new generation of acid - sensitive polymers,
degrade by acidic hydrolysis [137] . Low pH inside the endosomes facilitated the
escape of pH - responsive plasmid – lipid nanoparticles, resulting in enhanced transfection
effi ciency [138] .
5.4.4.2 Drug Delivery Applications and Biological Fate of Thermosensitive and
p H - Sensitive Nanoparticles
Thermosensitive block copolymer nanoparticles containing doxorubicin increased
cytotoxicity against Lewis lung carcinoma cells when activated by heating above
the LCST [139] . Chitosan was chemically conjugated to NIPAAm/vinyl laurate
copolymer to enhance gene transfection in mouse myoblast cells [140] . Upon i.v.
administration, poly(NIPAAm) nanoparticles are taken up by the reticuloendothelial
cells of the liver and mild infl ammatory and fi brotic responses are observed
[141] .
After internalization into SKOV - 3 (ovarian adenocarcinoma) cells, polyethylene
oxide – modifi ed poly( . - amino ester) nanoparticles rapidly disintegrated and released
the drug in the low pH of the endosomes [142] . Intravenous administration of polyethylene
oxide – modifi ed poly( . - amino ester) nanoparticles containing paclitaxel
signifi cantly reduced tumor burden in mice with ovarian cancer [142] . N - Acetyl
histidine – conjugated glycol chitosan nanoparticles were used to deliver drugs into
the cytoplasm. These pH - responsive nanoparticles are endocytosed where their
structural integrity is lost due to protonation of imidazoles, resulting in their endo -
lysosomal escape [57] . As most of the pH - sensitive biodegradable polymers are
blends of natural and synthetic polymers, they are degraded by mechanisms specifi c
to individual polymers.
5.4.5 APPLICATIONS OF BIODEGRADABLE NANOPARTICLES
OTHER THAN DRUG DELIVERY
Diagnosis and imaging are important applications of nanoparticles that are briefl y
described. The ability to encapsulate or conjugate fl uorescent compounds into or
onto biodegradable nanoparticles has been used extensively in imaging. Compounds
that have been encapsulated into nanoparticles for imaging include gadolinium,
fl uorescein isothiocyanate (FITC) – dextrans, Bodipy, and the autofl uorescent anticancer
drug doxorubicin. Nanoparticles encapsulating radioactive ligands, such as
APPLICATIONS OF BIODEGRADABLE NANOPARTICLES 547
548 BIODEGRADABLE NANOPARTICLES
99m Tc - labeled colloids and 111 In, have been used in scintigraphic imaging [143] . In
addition, fl uorescent or radioactive moieties can be targeted by noncovalently or
covalently tagging the nanoparticles through avidin – biotin conjugation and thiol
formation [144] . In vitro imaging enables the dynamics of cellular internalization and
localization of nanoparticles to be studied. For example, Bodipy — loaded PLGA
nanoparticles have been used to study their cellular disposition in vitro [145] as well
as the effect of storage temperature on their physical properties [146] . The biotinylated
antibody, specifi c to the CD3 antigen on lymphocytes, was chemically conjugated
to nanoparticles and their binding to leukemic and primary T lymphocytes investigated
[143] . The instrumentation that facilitates imaging of nanoparticles includes
confocal laser scanning microscopy, liquid scintigraphy, and fl ow cytometry.
5.4.6 PHYSICOCHEMICAL CHARACTERIZATION OF
POLYMERIC NANOPARTICLES
The selection of polymer is critical to the performance, properties, and application
of nanoparticles. Further, the physicochemical properties of the polymer will determine
the surface properties of nanoparticles with polymer molecular weight, hydrophobicity,
and glass transition temperature being particularly important. The surface
properties that infl uence their biodistribution and cellular response include particle
size, zeta potential, and surface hydrophilicity.
5.4.6.1 Molecular Weight
Many reviews and textbooks document the experimental methods available to
determine number - average, weight - average, viscosity - average, and z - average molecular
weights. The molecular weight of the polymer will infl uence many parameters
during the preparation of nanoparticles as well as their properties such as drug
loading and rate and extent of drug release.
5.4.6.2 Hydrophobicity
The experimental methods of determining hydrophobicity include interaction chromatography
and two - phase partition using fl uorescent or radiolabeled hydrophobic
probes [147] . Generally, a more hydrophobic polymer degrades more slowly and
releases drugs at a decreased rate. Hence, a blend of hydrophobic and hydrophilic
polymers can be used to tailor drug release kinetics. Depending on the conditions
of polymerization, mixtures of two or more monomers of different types yield block,
alternate, or random cocopolymers, each of which will possess different hydrophobicities
and consequently drug release kinetics. Additionally, the degree of hydrophobicity
can change the mechanism of degradation. For example, by inserting
hydrophilic ethylene glycol moieties within the hydrophobic backbone, polyanhydrides
have been tailored to specifi cally degrade by bulk or surface erosion [148] .
Incorporation of aromatic side chains generally increases hydrophobicity. A difference
in hydrophobicity of individual polymers results in microphase separation of
copolymers followed by a thermodynamic partition and altered release profi le of
encapsulated drugs [149] .
5.4.6.3 Glass Transition Temperature
The morphology and physical properties of nanoparticles are affected by the glass
transition temperature and physical state of the polymer or polymer blends. The
glass transition temperature ( Tg ) is the temperature at which polymers undergo
a change in heat capacity and transform their physical arrangement. The crystallinity
or amorphous nature of the polymers can be altered by synthesizing polymer
blends of varying ratios. The Tg of a polymer is experimentally determined by
differential scanning calorimetry (DSC). The Tg is mathematically calculated using
multidimensional lattice representations and statistical methods [150] . The various
factors that infl uence the Tg of polymers include molecular weight, composition
and stereochemistry of the polymer backbone, type and length of pendent
groups, additives such as copolymers, and plasticizers. Additives and copolymers
may also be used to alter the Tg . For example, incorporation of mPEG signifi -
cantly lowered the Tg of PLA, resulting in rapid release of drug from the nanoparticles
[151] .
5.4.6.4 Particle Size and Particle Size Distribution Instrumentation
Particle size is a critical characteristic of nanoparticles and by defi nition differentiates
them from microparticles. Particle size and particle size distribution play an
important role in the biological performance of the nanoparticles. Important techniques
for measuring particle size are photon correlation spectroscopy (PCS), electron
microscopy, and atomic force microscopy, which have been comprehensively
described [147] . Particle size and particle size distribution are determined by the
method of preparation and experimental variables during manufacture. For example,
in the emulsion – solvent evaporation method, the particle size is determined by
controlling the energy of emulsifi cation and the resulting droplet size of the internal
phase. Control of particle size is also possible by altering experimental variables
such as the volume and phase ratio of the internal and external phases and the
concentration, type, and viscosity of the emulsifying agent. As an example, the
experimental parameters that can infl uence the particle size and size distribution of
PLGA nanoparticles include the method used, polymer concentration, surfactant
concentration, stirring speed, ratio of aqueous and organic phases, and concentration
of the emulsifi er [152] . Methods of separation such as fi ltration and centrifugation
can also infl uence particle size distribution. When chitosan nanoparticles are
prepared using ionic gelation, the critical parameters for a narrow particle size distribution
are molecular weight, degree of deacetylation, concentration and molar
ratio of chitosan, and the presence and concentrations of counterions (e.g., tripolyphosphate)
[12] .
5.4.6.5 Surface Charge and Zeta Potential
The surface charge of nanoparticles is important because it determines the nature
and extent of aggregation of colloids and their interaction with cells and other biological
components within the body. The zeta potential is the potential at the solid –
liquid interface and is commonly determined using light scattering [153] . Decreasing
the zeta potential of nanoparticles below a critical value increases the rate and
PHYSICOCHEMICAL CHARACTERIZATION OF POLYMERIC NANOPARTICLES 549
550 BIODEGRADABLE NANOPARTICLES
extent of their aggregation, resulting in conglomerates with different physical properties.
The surface charge can also be altered by chemical conjugation of ligands to
nanoparticles. As the following examples demonstrate, surface charge may be
modifi ed to facilitate targeting. The degree of positive charge on chitosan – tripolyphosphate
nanoparticles was increased by controlling the processing parameters,
resulting in a stronger electrostatic interaction with negatively charged cell surfaces
[13] . An increase in surface charge increased the adsorption of nanoparticles to
plasma proteins [154] . Positively charged ligands such as chitosan as well as peptides
neutralize the surface charge of negatively charged particles made using polymers
such as PLGA [155] . Positively charged tripalmitin nanoparticles increased circulation
time and higher blood concentrations of etoposide compared to negatively
charged particles [156] . On the other hand, neutrally charged particles may be protected
from opsonization [157] .
5.4.6.6 Surface Hydrophilicity
The surface hydrophilicity of nanoparticles also infl uences the nature and extent
of their interaction with cells and their behavior in the biological environment. The
techniques for determining surface hydrophilicity have been described previously
[147] . As hydrophobic nanoparticles are opsonized and eliminated by the mononuclear
phagocytic system, surface hydrophilicity is an important parameter to
ensure longer circulation times. The hydrophilicity to the nanoparticles may be
modifi ed using several methods, including adsorption of nonionic surfactants
such as Poloxamer as well as adsorption or conjugation of hydrophilic polymers
such as polysaccharides, polyacrylamides, PVA, poly( N - vinyl - 2 - pyrrolidone), PEG,
polyoxamines, and polysorbates [157] . Stealth nanoparticles can be prepared by
adsorbing or conjugating PEG to the particle surface, which protects them from
opsonization [157] . The biological properties of stealth nanoparticles have been
reviewed elsewhere [158] .
5.4.6.7 Drug Loading and Encapsulation Effi ciency
Drug loading or the weight of the drug encapsulated in the polymeric carrier is
expressed as a percentage (w/w) of the delivery system. Encapsulation effi ciency is
the difference between the amount of the drug encapsulated into nanoparticles
compared to the total amount added during preparation. The encapsulation effi -
ciency is dependent on the properties of the polymer and excipients and the method
of preparation of nanoparticles. For example, increased miscibility between the
nanoparticle and water at lower temperatures and high rate of solvent evaporation
at high temperatures result in the formation of an outer sphere wall, increasing
encapsulation effi ciency [159] . Effi cient encapsulation depends on (1) physicochemical
properties of the drug, such as solubility, hydrophilicity, and crystallinity;
(2) physicochemical properties of the polymer, including molecular weight, hydrophobicity,
drug – polymer interactions, and solubility parameter; and (3) variables
involved in the preparation of the nanoparticles, such as drug – polymer weight ratio,
solvents, method of preparation of particles, solvent evaporation rate, type as well
as volume, and concentration of the surface - active agent used.
5.4.6.8 Drug Release
Many cumulative and differential dissolution methods are used to monitor the rate
and extent of drug release. The rate of drug release from nanoparticles depends on
the chemical properties of the polymer, properties of particles such as hydrophobicity
and surface area, and environmental factors such as pH. Drug release may occur
by one or a combination of the following: diffusion, dissolution, degradation, or
swelling. Hence, drug release normally follows fi rst - rather than zero - order kinetics
[160 – 162] . A disadvantage of nanoparticles is that they may have a signifi cant burst
release ( . 40%) due to their high surface area – mass ratio. Surface treatment of
nanoparticles can reduce the burst release of encapsulated drug [163, 164] .
5.4.6.9 Physical Stability of Polymeric Nanoparticles
The main indication of physical instability of nanoparticles is irreversible aggregation.
The principal factors that affect the extent of aggregation are type of polymer,
zeta potential, duration and temperature of storage, and presence of electrolytes.
Generally, homopolymers such as PLA and poly( . - caprolactone) are more stable
than copolymers. However, the stability of PLGA copolymers can be prolonged
by appropriate storage [165] . The duration of storage can also be important. For
example, poly( . - caprolactone) nanoparticles aggregated after four months [166] .
Another study compared the physical stability of PLGA nano - and microspheres
after incubation at different temperatures and demonstrated that, while microspheres
did not aggregate, the extent of aggregation of nanospheres increased as
storage temperature increased [146] . Ideally, PLGA nanoparticles should be stored
desiccated at 4 ° C. Aqueous dispersions of SLN are stable for three years [167] . It
is important to note that electrolytes can accelerate the aggregation and instability
of nanoparticles. For example, multivalent ions caused solid – lipid nanoparticles to
gel [168] . Physical stability can be improved by coating the surface of nanoparticles
with hydrophilic polymers or surfactants [169] . As discussed previously, a reduction
in the zeta potential below critical values causes fl occulation of colloidal systems.
For example, neutralization of the surface charge of colloidal systems by addition
of cationic oligonucleotides resulted in aggregation [170] .
5.4.7 TARGETING NANOPARTICLES BY SURFACE CONJUGATION
WITH LIGANDS
Nanoparticles can be targeted to cells using specifi c or nonspecifi c ligands. Specifi c
ligands can be covalently conjugated to the nanoparticles to target them to a
selected site of action. Some examples of targeting ligands are folic acid [171] ,
transferrin [172] , lectin [173] , and epidermal growth factor [174] . Nonspecifi c targeting
is achieved by attaching ligands that alter the biodistribution of the nanoparticles.
Examples of nonspecifi c targeting include the use of PEG for imparting stealth
properties or hydrolytically cleavable linkers that protect the drug from degradation.
Common methods of conjugation of ligands include carbodiimide coupling,
glutaraldehyde conjugation, peptide bond formation, disulfi de, and thiol linkages
[175 – 178] .
TARGETING NANOPARTICLES BY SURFACE CONJUGATION WITH LIGANDS 551
552 BIODEGRADABLE NANOPARTICLES
5.4.8 CELLULAR TRAFFICKING OF
BIODEGRADABLE NANOPARTICLES
Biodegradable nanoparticles are internalized by one or more of the following
mechanisms: phagocytosis, macropinocytosis, and clathrin - and caveolin - mediated
endocytosis (Figure 1 ). While phagocytosis by macrophages eliminates nanoparticles
from the body, effi cient cellular uptake occurs when high - affi nity receptors
capture the nanoparticles through receptor - mediated endocytosis [179] . On the cell
surface, nanoparticles activate caveolin, a dimeric protein, resulting in their internalization
through caveolae. Clathrin - mediated endocytosis occurs when nanoparticles
accumulate on the plasma membrane and clathrin - coated pits are formed to
transport the nanoparticles into the cell, resulting in the formation of endosomes.
Macropinocytosis is restricted to larger particles, such as nanoparticles typically
greater than 800 nm. The mechanism by which particles enter cells depends on the
composition of the nanoparticles, type of the cell, and particle size. For example, the
uptake of chitosan nanoparticles into lung epithelial and Caco - 2 cells was mediated,
in part, by the clathrin - mediated pathway [180, 181] . However, PLGA nanoparticles
of size 100 nm are internalized by the clathrin - and caveolin - independent pathway
[110] . There are comprehensive reviews of the mechanisms responsible for the
uptake of nanoparticles [182 – 184] .
Nanoparticles that are internalized into cells by these mechanisms fi rst enter the
primary endosomes of the cell and are then transported into sorting endosomes.
While some nanoparticles in the sorting endosomes are transported out of the cell
by recycling endosomes, the remaining nanoparticles are transported into secondary
endosomes that fuse with the lysosomes [107] . The surface charge of PLGA nanoparticles
is reversed in the acidic lysosome, resulting in their escape into the cytoplasm
[111] . A high external concentration of nanoparticles outside the cell prolongs their
intracellular concentration within the cytoplasm [107] .
Ligand - mediated endocytosis targets specifi c cell surface receptors and these
nanoparticles are internalized by a receptor - mediated endocytic pathway. Examples
of targeting transferrin, folate, lectins, and epidermal growth factor receptors are
contained in the literature [185 – 187] .
FIGURE 1 Mechanisms of cellular internalization of biodegradable nanoparticles.
Mechanisms of
internalization
of
nanoparticles
Phagocytosis
Pinocytosis/
fluid-phase
endocytosis
Receptormediated
endocytosis
Caveolinmediated
endocytosis
Clathrin- &
caveolinindependent
endocytosis
Macropinocytosis
Clathrinmediated
endocytosis
Transferrin,
folate,
asialoglycoprotein,
Epidermal growth
factor, etc.
5.4.9 CONCLUSIONS
Biodegradable nanoparticles are a very active area of research in drug delivery,
imaging, and diagnostics. This review has primarily focused on drug delivery applications.
Natural and synthetic polymers used to prepare nanoparticles were discussed
as well as their physicochemical properties that infl uence the biological performance
of particles. Methods used to prepare and characterize the properties of
nanoparticles have also been reviewed. Specifi c and nonspecifi c methods used to
target nanoparticles to cells were mentioned as well as the mechanism for cellular
and intracellular transport. As research into the various uses of biodegradable
nanoparticles increases, so will our knowledge to further optimize their preparation
and formulation and hence improve drug therapy and diagnosis.
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565
5.5
RECOMBINANT SACCHAROMYCES
CEREVISIAE AS NEW DRUG
DELIVERY SYSTEM TO GUT: IN VITRO
VALIDATION AND ORAL
FORMULATION
St ephanie Blanquet and Monique Alric
Universit e d ’ Auvergne, Clermont - Ferrand, France
Contents
5.5.1 Enginereed Microorganisms as Delivery Vectors to Human Gastrointestinal Tract
5.5.1.1 What is the “ Biodrug ” Concept?
5.5.1.2 Medical Applications
5.5.1.3 Choice of Candidate Host Microorganisms
5.5.2 Evaluation of Scientifi c Feasibility of Biodrug Concept Using Yeast as Vector
5.5.2.1 Approach
5.5.2.2 Yeast Survival Rate in Simulated Gastrointestinal Conditions
5.5.2.3 Yeast Heterologous Activity in Simulated Gastrointestinal Conditions
5.5.2.4 Conclusion
5.5.3 Oral Formulation of Recombinant Yeasts
5.5.3.1 Freeze Drying of Recombinant Model Yeasts
5.5.3.2 Immobilization of Recombinant Model Yeasts in Whey Protein Beads
5.5.4 General Conclusion and Future Developments
References
5.5.1 ENGINEREED MICROORGANISMS AS DELIVERY VECTORS TO
HUMAN GASTROINTESTINAL TRACT
5.5.1.1 What Is the “ Biodrug ” Concept?
The development of recombinant deoxyribonucleic acid (DNA) technology has
allowed the emergence of novel applications such as drug production directly in the
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
566 IN VITRO VALIDATION AND ORAL FORMULATION
human digestive environment ( “ in situ ” ) by ingested living recombinant microorganisms
[1 – 3] .
This new kind of vector offers several advantages over classical dosage forms.
First, the microorganisms, by protecting the active compounds, can allow the administration
of drugs known to be sensitive to digestive conditions when given in classical
pharmaceutical formulations. Second, the regulation of gene expression (e.g.,
using an inducible promoter) makes it possible to target specifi c sites throughout
the digestive tract (i.e., the absorption or reaction site of the drug) and to control
drug release. Thus, similar therapeutic effects can potentially be obtained at lower
doses [4, 5] and the degradation of the active compound by acid or proteases should
be avoided upstream from its absorption or reaction site.
In the digestive tract, genetically modifi ed microorganisms can either carry out
a reaction of bioconversion or produce compounds of interest. The bioconversion
reaction can lead either to the production of an active product or to the removal of
undesirable compounds. The active compound produced in situ can be secreted in
the digestive medium [5] , be bound to the cells [6, 7] , or accumulate inside the cells
and be released in the digestive environment by cell lysis [8] .
5.5.1.2 Medical Applications
The biodrug concept involves the use of orally administered recombinant microorganisms
as a new drug delivery route to prevent or treat various diseases. The
potential medical applications are numerous and can be classifi ed in terms of bioconversion
or production of active compounds. Validation studies have yet been
conducted in animals (and even sometimes in human being), as described below
and summarized in Table 1 .
Bioconversion Three main types of medical applications have been considered.
First, recombinant microorganisms could be administered to perform “ biodetoxication
” in the gut [9] . Here, the objective is to increase the body ’ s protection against
environmental xeniobiotics, mainly those ingested with food (e.g., pesticides, procarcinogens,
or chemical additives), by ingesting microorganisms expressing enzymes
that play a major role in the human detoxication system [e.g., phase I xenobiotic
metabolizing enzymes such as cytochrome P450 or phase II – like glutathione S -
transferase (GST)]. Therefore, recombinant microorganisms could be used to
prevent multifactorial diseases that have been associated with anomalies in human
detoxication processes. For instance, a defi ciency in GST - M1 has been correlated
with an increased susceptibility to different cancers, endometriosis, and chronic
bronchitis [10] .
Second, modifi ed microorganisms could correct errors of metabolism resulting
from either gastric or intestinal enzyme defi ciencies (e.g., lipase or lactase) [11] or
organ failure (by removing urea in the case of kidney failure or ammonia in the
case of liver failure) [12, 13] . This could constitute an alternative to current therapy
such as renal dialysis, which is time consuming and uncomfortable for the patient.
The third potential application is the use of recombinant cells to control the
activation of prodrug into drug directly in the digestive tract. This is of interest when
the drug, but not the prodrug, is either toxic at high concentrations or damaged by
digestive secretions [1] .
TABLE 1 Potential Medical Applications of Biodrug Concept and Their Validation in Animals
Biodrug
Concept Applications
Examples
Validation
Recombinant
Microorganisms
(Heterologous Gene) Experimental Models
Effect
Bioconversion Biodetoxication Removal of
benzo( a )pyrene [9]
Escherichia coli
(P450
1A1 and glutathione
S - transferase)
Stomach and duodenum
of a mouse combined
with mutagenesis assay
(Ames assay)
Decrease of the mutagenic
potential of B(a)P
Correction of
errors of
metabolism
Correction of lipase
defi
ciency [11]
Lactococcus lactis
(lipase)
Pig with pancreatic
defi ciency, oral
administration
Increase of lipid
digestibility
Correction of urease
defi
ciency [13]
Escherichia coli
DH5
(urease)
Rat with renal failure,
oral administration
Decrease of plasma uric
acid
In situ
production
of active
compounds
Synthesis of
biological
mediators
Secretion of
Interleukine [5]
Lactococcus lactis
(IL - 10)
Mouse treated with
dextran sulfate sodium,
oral administration
Reduction of colitis
symptoms
Oral vaccines Vaccination against
bacteria [17]
Lactobacillus plantarum
( Helicobacter pylori
urease B subunit)
Mouse,
intragastric
immunization
Induction of immune
response (anti - UreB Ig),
partial protection against
H. felis
Vaccination against
virus [19]
Attenuated
Salmonella
typhimurium
(SIV
capsid antigen p27)
Macaques,
oral
administration
Induction of immune
response (anti - p27 Ig)
Control of food
allergy [20]
Lactococcus lactis
(bovine
. - lactoglobulin)
Model of mouse allergy,
oral administration
Induction of BLG - specifi c
Th1 response
567
568 IN VITRO VALIDATION AND ORAL FORMULATION
In Situ Production of Active Compounds The fi rst (and main) medical application
derived from the biodrug concept is the development of oral vaccines [14] . In that
case, the microorganisms will locally deliver the antigens to the digestive mucosa in
order to stimulate an immune response (production of immunoglobulins) and
ensure a protection against bacterial [7, 15 – 17] or viral [6, 18, 19] diseases or being
used in the management of food allergy [4, 20] . For several immunological and
practical reasons, these new vaccines represent a promising alternative to the traditional
injectable ones [21] . In particular, oral immunization is the most effi cient way
to induce a protective local immune response at the site of pathogen contact.
Recently, clinical trials have shown the vaccinal effi ciency of different recombinant
strains of attenuated Salmonella typhi in humans, but the patients were presenting
undesirable side effects (diarrhea) [22] .
Other medical applications involve the direct production in the digestive medium
of various biological mediators, such as insulin, cytokines [23] , or growth factors. In
particular, new approaches to treating infl ammatory bowel diseases (IBD) such as
Crohn ’ s disease, celiac disease, or ulcerative colitis have been considered [24] . Anti -
infl ammatory and immunosuppressive therapies are commonly used for the treatment
of IBD. However, patients are often subject to unpleasant side effects owing
to the high level of the drug in the body (systemic administration) and some of them
remain refractory to such treatments. Steidler et al. [5] have investigated the potential
of alternative therapeutics and shown the interest of delivering interleukin - 10
(IL - 10), a strong anti - infl ammatory cytokine, in a localized manner by the action of
recombinant Lactococcus lactis . Oral administration of the strain producing mouse
IL - 10 led to 50% curing of dextran sulfate sodium – induced colitis and prevented
the onset of the pathology in mice [5] . The amount of IL - 10 required to achieve the
healing effect was 10,000 - fold lower when the cytokine was in situ delivered by
L. lactis compared to systemic treatment with anti - infl ammatory drugs (e.g., dexamethasone).
A recent study of the same author has reported that the treatment of
Crohn ’ s disease patients with L. lactis secreting human IL - 10 was safe and allowed
a decrease in disease activity [25] .
5.5.1.3 Choice of Candidate Host Microorganisms
Recombinant bacteria, particularly lactic acid bacteria, have been mostly suggested
as potential hosts for this new kind of drug delivery system [2, 3] . However,
yeasts can offer advantages, especially when a eukaryotic environment is required
for the functional expression of human genes. Moreover, the absence of bacterial
sequences liable to promote gene transfer to host bacteria can be ensured using
the effi cient site - targeted homologous recombination machinery of yeasts for
introduction of the heterologous gene into the yeast genome. Lastly, yeasts are
not sensitive to antibacterial agents, allowing the simultaneous administration of
the recombinant microorganisms and antibiotics. In this study, the common
baker ’ s yeast Saccharomyces cerevisiae was chosen owing to its “ generally recognized
as safe ” (GRAS) status and its easy culture and genetic engineering. Saccharomyces
spp. have already been used in humans, mainly in the treatment of
intestinal functional disorders such as colitis [26] or antibiotic - associated
diarrhea [27] .
5.5.2 EVALUATION OF SCIENTIFIC FEASIBILITY OF BIODRUG
CONCEPT USING YEAST AS VECTOR
5.5.2.1 Approach
The scientifi c feasibility of our approach was recently evaluated using recombinant
S. cerevisiae expressing model genes and an original artifi cial digestive system simulating
human gastrointestinal conditions. The survival rate and heterologous activity
of the recombinant model yeasts were followed in this in vitro system.
Recombinant Model Yeasts Three different yeast strains, all derived from the
haploid strain W303 - 1B, were used to evaluate the scientifi c feasibility of the biodrug
concept using yeast as vector.
The fi rst one, WRP45073A1 (provided by Denis Pompon, CNRS, Gif - sur - Yvette,
France), expresses the plant P45073A1 when grown in the presence of galactose
[28] . P45073A1 was chosen as a model for a reaction catalyzed by a P450 owing to
the nontoxicity and easy quantifi cation of both substrate and product [29] . It
catalyzes the 4 - hydroxylation of trans - cinnamic acid into p - coumaric acid [cinnamate
4 - hydroxylase (CA4H) activity]. In this model, the heterologous protein is
an intracellular enzyme, and it was synthesized (induction of CYP73A1 by galactose
during the last 12 h of culture) before yeast introduction into the artifi cial digestive
system. The in situ CA4H activity of yeasts was quantifi ed following the simultaneous
introduction of recombinant yeasts and trans - cinnamic acid into the in vitro
system.
The two other strains — WppGSTV 5 H 6 and WppV 5 H 6 — were genetically engineered
[30] to secrete (i) a model protein derived from the commonly used reporter
protein GST, named GST – V 5 H 6 [molecular weight (MW) 31.5 kDa], and (ii) a model
peptide, peptide – V 5 H 6 (MW 5.6 kDa). The recombinant protein and peptide were
expressed in fusion with the V 5 epitope (V 5 ) and the polyhistidine (H 6 ) tag to allow
their immunological detection and make easier their purifi cation, respectively. A
leader sequence derived from that of the . - factor precursor was used to direct the
secretion of the heterologous protein compounds into the extracellular medium
[30] . In that case, recombinant yeasts and galactose, the inductor of the heterologous
genes, were simultaneously introduced into the artifi cial digestive system to evaluate
the yeast ability to initiate the synthesis and secrete protein compounds of various
sizes, directly in the digestive environment.
Artifi cial Digestive System TIM : Powerful In Vitro Tool The system TIM (TNO
gastrointestinal tract model) is the in vitro model that at present time allows the
closest simulation of in vivo dynamic physiological processes occurring within the
lumen of the stomach and small intestine of humans [31] . It is composed of four
successive compartments reproducing the stomach and the three parts of the small
intestine: the duodenum, jejunum, and ileum (Figure 1 ). This dynamic, computer -
controlled system has been designed to accept parameters and data from in vivo
studies on human volunteers. The main parameters of digestion, such as pH, body
temperature, peristaltic mixing and transport, gastric, biliary, and pancreatic secretions,
and passive absorption of small molecules (e.g., nutrients, drugs) and water,
BIODRUG CONCEPT USING YEAST AS VECTOR 569
570 IN VITRO VALIDATION AND ORAL FORMULATION
are reproduced as accurately as possible (Table 2 ). This system has been previously
described in detail [1, 29 – 31] .
Compared with animal experiments, this in vitro system offers accuracy, reproducibility,
easy manipulation, and the possibility of collecting samples at any level
of the digestive tract and at any time during digestion with no ethical constraint. It
has been validated by microbial, nutritional, and pharmaceutical studies. For instance,
validation experiments demonstrate the predictive value of the TIM with regard to
the survival rate of probiotic bacteria [32, 33] , the digestibility of nutrients [31, 34] ,
and the availability for absorption of minerals [35] , vitamins [36] , food mutagens
[37] , and drugs such as paracetamol [33, 38] .
In the present study, the TIM was programmed to reproduce gastrointestinal
conditions of the adult after the intake of a liquid meal, according to in vivo data
[31, 39 – 41] . The initial “ meal ” (introduced into the artifi cial stomach at the beginning
of digestion) was composed of (i) 10 10 WRP45073A1 cells and 200 . mol of trans -
cinnamic acid or (ii) 3 . 10 10 WppGSTV 5 H 6 or WppV 5 H 6 cells and galactose (40 g/L)
FIGURE 1 Gastric and small intestinal system TIM.
Jejunal
absorption
Pancreatic juice
Hollow fibers
Electrolytes
pH electrode Stomach
Sodium bicarbonate
/electrolytes
Pepsine, lipase
Sodium
bicarbonate
Flexible wall
Bile salts
Hydrochloric
acid/water
37°C 37°C
Dialysates
Pression sensor
Ileal absorption
Meal
Pump
Sodium
bicarbonate
Peristaltic valves
Ileum
Ileal delivery
Duodenum
Jejunum
TABLE 2 Digestive Parameters Reproduced in Gastrointestinal Model TIM and Their
Simulation
pH The pH is computer monitored and continuously controlled in each
digestive compartment.
The fall of gastric pH is reproduced by adding hydrochloric acid.
The pH is kept to 6.5, 6.8, and 7.2 in the duodenum, jejunum, and ileum,
respectively, by secreting sodium bicarbonate.
Temperature The compartments are surrounded by water at body temperature (37 ° C).
Peristaltic
mixing
Peristaltic mixing is mimicked by alternate compression and relaxation of
the fl exible walls containing the chyme, following changes in the water
pressure.
Dynamic of
chyme
transit
A mathematical model using power exponential equations [39] is used to
reproduce gastric and ileal deliveries ( f = 1 . 2 . ( t / t 1/2) . , where f represents
the fraction of meal delivered, t the time of delivery, t 1/2 the half - time of
delivery, and . a coeffi cient describing the shape of the curve).
Chyme transit is regulated by opening or closing the peristaltic valves that
connect the compartments.
Volume The volume in each compartment is monitored with a pressure sensor
connected to the computer.
Digestive
secretions
Simulated gastric, biliary, and pancreatic secretions are introduced into the
corresponding compartments by computer - controlled pumps.
Absorption
of small
molecules
and water
Semipermeable membrane units are connected to the jejunum and ileum
to remove the products of digestion as well as water.
in suspension in 300 mL of yeast culture medium. The parameters of in vitro digestion
are summarized in Table 3 .
5.5.2.2 Yeast Survival Rate in Simulated Gastrointestinal Conditions
At the end of in vitro digestion, yeast survival rate was evaluated by comparing the
total ingested yeasts with the living yeasts recovered in both the ileal effl uents of
the TIM and the residual digestive content. After 240 or 270 min digestion (depending
on the strain), 79.5 ± 12.1% ( n = 3), 63.9 ± 2.4% ( n = 3) and 75.5 ± 25.3% ( n =
4) of the ingested WRP45073A1, WppV 5 H 6 , and WppGSTV 5 H 6 , respectively, were
recovered in the ileal effl uents (Figure 2 ). When the yeasts remaining in the residual
chyme were added ( t = Tf), 95.6 ± 10.1% ( n = 3), 83.1 ± 9.6% ( n = 3), and 95.3 ±
22.7% ( n = 4) survival percentages, respectively, were found, showing the high resistance
of recombinant yeasts to gastric (pepsin and lipase) and small intestinal (bile
salts and pancreatic juice) secretions and low gastric pH [29, 30] . This high survival
rate was confi rmed (Figure 2 ), no signifi cant difference ( p < 0.05) being observed
during digestion between the ileal recovery profi les of recombinant yeasts (except
for WppV 5 H 6 ) and that of a nonabsorbable marker, blue dextran, added in the
artifi cial stomach at the beginning of digestion, as previously described [31] .
The survival rate of other microorganisms, such as lactic acid bacteria, has also
been studied in the TIM. At the end of digestion, Marteau et al. [32] found a bacte-
BIODRUG CONCEPT USING YEAST AS VECTOR 571
572 IN VITRO VALIDATION AND ORAL FORMULATION
TABLE 3 Parameters of In Vitro Digestion in TIM When Simulating Gastrointestinal
Conditions of Adult After Intake of Liquid Meal
Gastric
compartment
Initial volume 300 mL
Time (min)/pH 0/6
20/4.2
40/2.8
60/2.1
90/1.8
120/1.7
Secretions 0.25 mL/min pepsin (590 IU/mL)
0.25 mL/min lipase (37.5 IU/mL)
0.25 mL/min HCl 0.5 M if necessary
Time of half emptying t 1/2 30 min
. coeffi cient 1
Duodenal
compartment
Volume 30 mL
pH Maintained at 6.5
Secretion 0.5 mL/min bile salts (4% during fi rst
30 min of digestion, then 2%)
0.25 mL/min pancreatic juice (10 3 USP/mL)
0.25 mL/min intestinal electrolyte solution
0.25 mL/min NaHCO 3 1 M if necessary
Jejunal
compartment
Volume 70 mL
pH Maintained at 6.8
Secretion 0.25 mL/min NaHCO 3 1 M if necessary
Dialysis 10 mL/min of jejunal fl uid solution
Ileal
compartment
Volume 70 mL
pH Maintained at 7.2
Secretion 0.25 mL/min NaHCO 3 1 M if necessary
Dialysis 10 mL/min ileal fl uid solution
Time of half emptying t 1/2 160 min
. coeffi cient 1.6
rial cumulative delivery from the ileum between 0 and 25% (depending on the
tested strain). In this work, under similar experimental conditions, about 75% of
ingested yeasts were recovered. Until now, the feasibility of the biodrug concept
had been mainly evaluated with lactic acid bacteria (see Section 5.5.1.2 ). The high
viability of S. cerevisiae in the digestive tract might favor the choice of yeasts over
lactic acid bacteria as hosts for the development of biodrugs, particularly if the viability
of the microorganisms is required for their in situ activity.
5.5.2.3 Yeast Heterologous Activity in Simulated Gastrointestinal Conditions
Bioconversion The CA4H activity of WRP45073A1 yeasts was quantifi ed measuring
p - coumaric acid production by high - performance liquid chromatography.
Control experiments showed that both trans - cinnamic and p - coumaric acids were
stable under digestive conditions when no yeast was introduced into the TIM. In
the presence of yeasts with no CA4H gene in their plasmid, no p - coumaric acid was
produced, showing the specifi city of the enzymatic reaction catalyzed by the recombinant
model yeasts.
FIGURE 2 Survival rate of three recombinant model yeasts in TIM. The cumulative ileal
deliveries of viable yeasts and that of a nonabsorbable marker, bleu dextran, are represented.
At the end of digestion, the percentages obtained in the cumulative ileal effl uents (0 – 240 min
or 0 – 270 min depending on strain) and in the residual digestive content are added ( t = Tf).
Results are expressed as mean percentages ± SD ( n = 3 for WRP45073A1 and WppV 5 H 6 ,
n = 4 for WppGSTV 5 H 6 ) of intake.
0
20
40
60
80
100
120
0 60 120 180 240 300 360
Time of digestion (min)
Cumulative ileal delivery of viable yeasts
(% of intake)
WRP45073A1
WppGSTV5H6
WppV5H6
marker
Tf
At the end of digestion (240 min), 41.0 ± 5.8% ( n = 3) of initial trans - cinnamic
acid was converted into p - coumaric acid (Figure 3 a ) [29] . By means of a computer
simulation [29] , in each compartment of the in vitro system, the amount of p -
coumaric acid resulting from the CA4H activity of yeasts could be dissociated from
that delivered by the previous compartment. After calculation, trans - cinnamic acid
conversion rates of 8.9 ± 1.6%, 13.8 ± 3.3%, 11.8 ± 3.4%, and 6.5 ± 1.0% ( n = 3)
were found in the stomach, duodenum, jejunum, and ileum, respectively (Figure 3 b ).
The enzymatic reaction occurred throughout the artifi cial gastrointestinal tract, but
mostly in the duodenum and jejunum. This could be explained by the fact that yeasts
were no longer stressed by the acid pH of the stomach and could metabolize the
trans - cinnamic acid that had previously easily entered the cells, owing to the low
pH ( trans - cinnamic acid is essentially in a cationic form which easily diffuses through
the yeast membrane [42] ). Also, previous studies have demonstrated that bile salts
can favor enzymatic reactions [43] . The lower activity in the ileum might result from
a decrease in the availability of trans - cinnamic acid owing to its previous conversion
into p - coumaric acid in the upper digestive compartments. The computer simulation
that was developed here should prove useful in future stages of the development
of biodrugs, especially if a specifi c level of the digestive tract has to be targeted for
drug action.
Further calculations were performed to quantify the specifi c enzymatic activity
of the WRP45073A1 yeasts. Yeast specifi c activity in the TIM (from 0.05 ± 0.04 .
10 . 10 to 3.36 ± 0.86 . 10 . 10 . mol/cell/min, depending on the digestive compartment
and the sampling time [29] ) was close to that observed in classical batch cultures,
BIODRUG CONCEPT USING YEAST AS VECTOR 573
574 IN VITRO VALIDATION AND ORAL FORMULATION
FIGURE 3 CA4H activity of WRP45073A1 in TIM. The trans - cinnamic acid conversion
rate was evaluated in ( a ) overall TIM and ( b ) each compartment of TIM. Results are
expressed as mean percentages ± SD ( n = 3) of ingested trans - cinnamic acid converted into
p - coumaric acid. (Reprinted with permission from Blanquet et al., Applied and Environmental
Microbiology , 69, 2889.)
0
10
20
30
40
50
0 60 120 180 240 300
0 60 120 180 240 300
Time of digestion (min)
Trans-cinnamic acid conversion (%)
0
2
4
6
8
10
12
14
16
18
Time of digestion (min)
Trans-cinnamic acid conversion (%)
Stomach Duodenum Jejunum Ileum
(a)
(b)
which is very encouraging for a potential use of S. cerevisiae as a biodetoxication
system to the gut.
Secretion of Peptides or Proteins The production of the GST – V 5 H 6 and peptide –
V 5 H 6 in the TIM was examined by Western blotting (data not shown). No signal was
detectable during control digestions without yeast or with yeasts with no heterologous
gene in their plasmid. The model protein and peptide were detected as early
as 90 min after the yeast intake/gene induction in each compartment of the in vitro
system and remained until 270 min of digestion in the lower part of the small intes
FIGURE 4 Immunoenzymatic (ELISA) measurement of GST – V 5 H 6 produced by WppGSTV
5 H 6 in ( a ) different compartments of TIM (ng/mL) and ( b ) the overall TIM ( . g). Error
bars represent standard deviations ( n = 4). (Reprinted with permission from 110, Blanquet
et al., Journal of Biotechnology , 45, 2006. Copyright 2006 by Elsevier.)
0
2
4
6
8
10
12
14
16
18
20
Time of digestion (min)
GST-V5H6 (ng/mL)
Stomach
Duodenum
Jejunum
Ileum
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
Time of digestion (min)
GST-V5H6 (.g)
(a)
(b)
0 60 120 180 240 300
0 60 120 180 240 300
tine [30] . No signal was detectable in the “ meal ” before introduction into the artifi -
cial stomach, showing the effi cient initiation of protein compound synthesis and
secretion by galactose in the digestive environment.
The amount of GST – V 5 H 6 produced in each compartment of the TIM was quanti-
fi ed by enzyme - linked immunosorbent assay (ELISA). The protein concentrations
in the digestive medium reached 15 ng/mL, the highest values being found in the
jejunum and ileum from 150 min to the end of digestion (Figure 4 a ). The GST – V 5 H 6
concentrations in the digestive environment were close to those measured in stan-
BIODRUG CONCEPT USING YEAST AS VECTOR 575
576 IN VITRO VALIDATION AND ORAL FORMULATION
dard batch cultures. Therefore, the low secretion levels of GST – V 5 H 6 may be only
imputed to the large size of the protein and/or the genetic construction, but not to
the particular digestive conditions. This hypothesis is consistent with the results of
Zsebo et al. [44] , who showed that S. cerevisiae is an effi cient host for small polypeptide
secretion, but not for larger proteins, which accumulate in the periplasmic
place and cell wall of the yeast. Improved secretion levels might be obtained with
another heterologous protein or a different expression vector. In the overall in vitro
system, the total amount of GST – V 5 H 6 regularly increased during digestion to reach
3.3 ± 0.7 . g ( n = 4) after 270 min digestion (Figure 4 b ).
To check that the GST – V 5 H 6 recovered in the TIM was truly secreted by living
recombinant yeasts and did not result from cell lysis, a control strain producing an
intracellular form of the model protein was contructed (without the leader sequence)
and tested in similar experimental conditions. Some GST – V 5 H 6 was found in the
ileum showing that cell lysis occurred in this compartment. At the end of digestion,
the total amount of GST – V 5 H 6 released by the control strain represented 30% (1
± 0.2 . g, n = 2) of the protein produced by WppGSTV 5 H 6 , showing a signifi cant
contribution of cell lysis to the release of the protein in the TIM.
For the fi rst time, the amount of heterologous proteins secreted by recombinant
S. cerevisiae was evaluated throughout the upper digestive tract. Until now, the
secretion effi ciency of recombinant microorganisms had never been directly quanti-
fi ed throughout the digestive tract. The ability of recombinant strains to produce
heterologous proteins in the digestive environment had been mainly demonstrated
indirectly, following the biological effect of the protein: immune response (antibody
production) [7, 15 – 17, 19, 20] , growth improvement [45] , or reduction in colitis
symptoms [5] . Nevertheless, the ability of recombinant bacteria to initiate protein
synthesis in situ has been reported in a few studies. Oozeer et al. [46] have shown
that engineered Lactobacillus casei was able to initiate the synthesis of luciferase
during its transit in the digestive tract of a human fl ora - associated mouse model.
Steidler et al. [5] have demonstrated in mice the in situ synthesis of mouse IL - 10 by
recombinant L. lactis , the viability of these microorganisms being required to achieve
their therapeutic effect (see Section 5.5.1.2 ). They have further documented this
result by showing the de novo synthesis of IL - 10 in the colon of IL - 10 . / . mice.
Moreover, these authors have quantifi ed the amount of IL - 10 produced by the
engineered L. lactis in two animal models, but only in a limited part of their digestive
tract : (i) 7 ng of mouse IL - 10 was recovered in the colon of IL - 10 . / . mice, but
the interleukin was not detectable in other areas of the gastrointestinal tract [5] and
(ii) about 470 pg/mL of human IL - 10 was found in an ileal loop of a pig 4 h after
injection of the recombinant bacteria [47] . Unlike what was previously obtained in
the TIM with GST – V 5 H 6 , the concentrations of IL - 10 found in the digestive tract
of the animals were much lower than that recovered in batch cultures [5, 47] .
5.5.2.4 Conclusion
For the fi rst time, the ability of engineered S. cerevisiae to carry out a bioconversion
reaction [29] and initiate the synthesis and secrete protein compounds of various
sizes [30] was shown throughout the upper gastrointestinal tract in human simulated
digestive conditions. The CA4H specifi c activity of WRP45073A1 and the secretion
level of GST – V 5 H 6 were surprisingly similar to that obtained in classical batch cul
ORAL FORMULATION OF RECOMBINANT YEASTS 577
tures. This is particularly remarkable as the expression strategy of the model genes
had not yet been adapted to the particular constraints of the digestive environment
and promising for a future use of recombinant S. cerevisiae as host for biodrug
development.
5.5.3 ORAL FORMULATION OF RECOMBINANT YEASTS
Once the scientifi c feasibility of the new drug delivery system was established, the
development of pharmaceutical formulations allowing the oral administration of
the genetically modifi ed S. cerevisiae was considered. Ideally, these oral drug dosage
forms would improve both the survival and the heterologous activity of yeasts in
the digestive environment. The following works were carried out only with the strain
expressing the model P450. In a preliminary step, the effect of a preservation technique
(lyophilization) and an immobilization procedure (entrapment in whey
protein beads) on the survival rate and heterologous activity of the model strain
WRP45073A1 was assessed in simulated digestive conditions.
5.5.3.1 Freeze Drying of Recombinant Model Yeasts
Freeze - Drying Conditions Freeze drying is a technique of dehydration commonly
used for the formulation of drugs containing nonrecombinant Saccharomyces spp.
[48 – 50] . Standard freeze - drying conditions derived from the literature [51 – 54] and
our own experiments were applied for the lyophilization of the genetically modifi ed
model yeasts. The effect of cryoprotectants was further investigated because it
appears as one of the most important parameters during lyophilization [51, 52] .
Following galactose induction of the heterologous CYP73A1, yeasts in the beginning
of their stationary growth phase (10 9 cells/mL) were lyophilized in suspensions
of trehalose 10% w/v, maltose 10% w/v, lactose 10% w/v, or a mixture of 5% w/v
milk proteins and 10% w/v trehalose. The parameters of lyophilization are summarized
in Table 4 [55] .
Saccharomyces cerevisiae WRP45073A1 survives freeze drying and yeast survival
rates were dependent on the nature of the cryoprotectants: 13.1 ± 1.8%, 9.5 ± 6.0%,
TABLE 4 Parameters of Lyophilization Used for Recombinant Model Yeast
WRP45073A1
Recombinant yeasts Growth phase: beginning of stationnary growth phase
Cell concentration in freeze - drying fl asks: 10 9 cells/mL
Cryoprotectants 10% w/v trehalose
10% w/v maltose
10% w/v lactose
5% w/v milk proteins and 10% w/v trehalose
Control: physiological water
Freeze - drying conditions Volume of sample: 5 mL
Cooling rate: 1 ° C/min
Condenser plate temperature: . 40 ° C
Time of lyophilization: 24 h
Heating temperature (secondary drying): 23 ° C
578 IN VITRO VALIDATION AND ORAL FORMULATION
7.7 ± 4.6%, and 7.1 ± 4.0% ( n = 5) for the milk protein – trehalose mix, lactose,
maltose, and trehalose, respectively [55] . The protective effect of trehalose [51 – 53]
and maltose [52] (but not that of lactose) compared to physiological water (survival
rate 0.3 ± 0.2%, n = 5) had already been shown in non genetically modifi ed S. cerevisiae
. Several mechanisms have been proposed to explain this protective effect.
One hypothesis is related to the ability of these carbohydrates to form a glassy
structure during drying, responsible for the long stability of biological materials [56] .
The milk protein – trehalose mix led to a higher survival compared with trehalose
alone ( p < 0.05). A similar result had already been observed by Abadias et al. [54] ,
who showed an improvement in the viability of another yeast, Candida sake , from
7 to 29% when 5% skim milk is used in combination with 10% trehalose.
Infl uence of Cryoprotectants on Viability and Heterologous Activity of
Lyophilized Yeasts in Simulated Gastrointestinal Conditions To evaluate the
infl uence of cryoprotectants on both the survival rate and CA4H activity of
WRP45073A1 in simulated gastrointestinal conditions, 10 10 viable freeze - dried
yeasts and 200 . mol of trans - cinnamic acid were simultaneously introduced into the
TIM. Yeast cells were lyophilized in the presence of the milk protein – trehalose mix,
trehalose, lactose, or maltose, as previously explained (see above). The freeze - dried
samples were introduced into the artifi cial stomach suspended in 300 mL of yeast
culture medium without any storage period. The number of viable cells introduced
into the TIM was determined from previously obtained survival rates (cf. Section
5.5.3.1 ).
Viability of Freeze - Dried Yeasts in TIM Freeze - dried yeasts showed a high tolerance
to gastric and small intestinal conditions. At the end of digestion (240 min),
61.5 ± 0.7%, 59.9 ± 3.8%, 56.3 ± 5.4%, and 55.6 ± 6.0% ( n = 3) of the ingested
cells were recovered in the ileal effl uents of the TIM, following freeze drying in
the presence of the milk protein – trehalose mix, maltose, lactose, and trehalose,
respectively (Figure 5 ). When the yeasts remaining in the residual chyme were
added ( t = Tf), 84.7 ± 3.5%, 87.0 ± 6.4%, 83.7 ± 6.2%, and 70.7 ± 9.2% ( n = 3)
survival percentages were found, respectively. Nevertheless, whatever the protective
agent, the cumulative ileal delivery of freeze - dried yeasts remained signifi -
cantly lower ( p < 0.05) than that of the nonabsorbable marker (see Section 5.5.2.2 )
and that of nondried ( “ native ” ) yeasts. The higher sensitivity to digestive conditions
of freeze - dried cells compared with native ones may be linked to the damage
caused to cells during drying and rehydration, resulting in increased membrane
permeability [57] .
In addition, no signifi cant difference was observed between the ileal recovery
profi les of yeasts with the various cryoprotectants, showing their lack of infl uence
on the survival of freeze - dried WRP45073A1 in the TIM.
A few studies have evaluated the survival rate of freeze - dried S. cerevisiae spp.
in human volunteers following their oral administration. Nevertheless, comparison
between in vitro results in the TIM and these in vivo data is hampered by the fact
that yeast survival had been evaluated only in feces (and not at the end of the ileum)
after a single or multiple oral administration of the microorganisms. Klein et al. [49]
found a fecal recovery of 0.12 ± 0.04% ( n = 8) after a single dose of 1 g of S. boulardii
[10 10.4 colony - forming units (CFU)] to healthy volunteers, and Blehaut et al. [50]
measured a steady - state fecal recovery of 0.36 ± 0.31% ( n = 8) after oral administra
ORAL FORMULATION OF RECOMBINANT YEASTS 579
tion of 1 g of S. boulardii for 15 days. These survival rates in feces are much lower
than those obtained in the ileal effl uents of the TIM. However, a recent work [29]
has shown that native WRP45073A1 yeasts are very sensitive to colonic conditions:
Yeast viability in an artifi cial digestive system reproducing the human large intestine
[58] was only 1.2 ± 0.4% ( n = 3) after 12 h and no more yeast could be detected
following 24 h fermentation.
Heterologous Activity of Freeze - Dried Yeasts in TIM The ability of freeze - dried
recombinant yeasts to catalyze the bioconversion of trans - cinnamic acid into p - coumaric
acid in the in vitro system was shown whatever the tested protectant. At the
end of the experiment, conversion rates of 24.2 ± 1.0%, 17.7 ± 2.2%, 15.1 ± 3.3%,
and 16.5 ± 0.7% ( n = 3) were found for the yeasts lyophilized in the presence of
milk proteins plus trehalose, maltose, lactose, and trehalose, respectively (Figure 6 ).
During all the in vitro digestion, the CA4H activity of freeze dried cells remained
signifi cantly lower ( p < 0.01) than that of native ones. This lower activity could result
from the P45073A1 damage during freeze drying, probably aggravated by the membranous
location of the enzyme. Among those tested, the cryoprotectant that allowed
the highest CA4H activity was the milk protein – trehalose mix ( p < 0.05).
Conclusion Although the impact of freeze drying on both the survival rate and
heterologous activity of yeasts in the artifi cial digestive system was found to be
adverse, lyophilization appears to be a convenient technique for the dehydration of
recombinant S. cerevisiae . Among the tested cryoprotectants, the association of milk
proteins and trehalose was the most effi cient to maintain the CA4H activity of
FIGURE 5 Effect of cryoprotectants on survival rate of WRP45073A1 in TIM. The cumulative
ileal deliveries of viable freeze - dried and native yeasts and that of the nonabsorbable
marker are represented. At the end of digestion, the percentages obtained in the cumulative
ileal effl uents (0 – 240 min) and the residual digestive content are added ( t = Tf). Results are
expressed as mean percentages ± SD ( n = 3) of intake. Signifi cantly different from the marker
( t = Tf) at p < 0.05 ( * ), p < 0.01 ( * * ) and p < 0.001 ( * * * ). (Reprinted with permission from
Blanquet et al., European Journal of Pharmaceutics and Biopharmaceutics , 61, 37, 2006.
Copyright 2006 by Elsevier.)
0
20
40
60
80
100
120
0 60 120 180 240
Time of digestion (min)
Cumulative ileal delivery of viable yeasts
(% of intake)
Marker Native yeasts
Milk prot + treh Lactose
Maltose Trehalose
Tf
*** **
**
580 IN VITRO VALIDATION AND ORAL FORMULATION
recombinant model yeasts in the stringent digestive conditions. This study also gives
an example of the usefulness of the TIM in the prescreening of pharmaceutical
excipients, such as cryoprotectants.
5.5.3.2 Immobilization of Recombinant Model Yeasts in Whey Protein Beads
Cell Immobilization Among the available techniques, the entrapment in gel beads
is frequently used for the immobilization of living cells in food sciences [59, 60]
because of its simplicity and low cost. This technique has been recently extended to
microorganisms with probiotic activity with the aim of increasing their survival in
the human digestive environment and particularly in the stomach [61, 62] .
Whey proteins have been recently considered a potential alternative to the commonly
used alginate [59, 61 – 63] for the production of gel beads. They have been
used to entrap drugs such as retinol [64] and living microorganisms such as bifi dobacteria
[65] , but until now not yeasts. A new immobilization system using whey
proteins was then developed for entrapping the recombinant model yeasts
WRP45073A1 in order to ensure their oral administration [66] .
The formation of beads is a two - step process based on the cold gelation of whey
proteins in the presence of divalent cations, such as Ca 2+ [67] . Briefl y, the whey
protein isolate (WPI) solution (10% w/v in deionized water) was (i) adjusted at pH
7 to favor the apparition of negative charges implied in ionic bounds with Ca 2+ ions
and (ii) heated (80 ° C, 45 min) to denaturate the proteins. Recombinant cells in the
beginning of their stationnary growth phase were suspended in a sterile solution of
FIGURE 6 Effect of cryoprotectants on CA4H activity of WRP45073A1 in TIM. The
CA4H activity of freeze - dried and native yeasts was evaluated in the overall TIM. Values are
expressed as mean percentages ± SD ( n = 3) of initial trans - cinnamic acid converted into
p - coumaric acid. Signifi cantly different from native yeasts ( t = 240 min) at p < 0.01 ( * * ). Signifi
cantly different from milk proteins/trehalose group ( t = 240 min) at p < 0.05 ( + ) and p <
0.01 ( ++ ). (Reprinted with permission from Blanquet et al., European Journal of Pharmaceutics
and Biopharmaceutics , 61, 38, 2006. Copyright 2006, by Elsevier.)
0
5
10
15
20
25
30
35
40
45
50
Time of digestion (min)
Trans-cinnamic acid conversion (%)
Native yeasts Milk prot + treh Maltose Lactose Trehalose
** ++
+
+
**
**
**
0 60 120 180 240 300
ORAL FORMULATION OF RECOMBINANT YEASTS 581
10% w/v lactose (fi nal concentration 10 9 cells/mL) and added to denaturated WPI
solution (7% v/v). The extrusion of the mixture through a needle led to the production
of droplets forming gel beads in a calcium bath (0.1 M CaCl 2 ). This protocol
allows the obtaining of spherical beads (diameter 2 605 ± 18 . m, n = 3) with an
homogeneous distribution of yeasts through the matrix (1.15 . 10 6 viable cells per
bead) [66] . The lack of infl uence of the immobilizing procedure on the viability of
yeasts was also shown [66] .
Gastric Digestion Protocol A most sought - after property of gel beads is their
potential resistance to gastric conditions. Authors have already shown in vitro
that beads resulting from the cold - induced gelation of a whey protein – oil emulsion
[64] or a whey protein – polysaccharide mix [65] were gastroresistant. To further
investigate the involvement of whey proteins in the gastroresistance of beads, the
behavior of entrapped WRP45073A1 yeasts was followed in simulated gastric
conditions.
The human gastric environment was reproduced using a simple in vitro model
adapted from that initially developped by Yvon et al. [68] . The main parameters of
gastric digestion are reproduced according to in vivo data: decrease of pH, pepsin
supply, body temperature, mixing, and gastric emptying (Figure 7 ). This system was
validated by studies on the digestability of milk proteins (unpublished data). In the
present work, it was programmed to reproduce gastric conditions of the adult after
the intake of a glass of milk. Initially, 10 10 viable entrapped WRP45073A1 cells and
200 . mol of trans - cinnamic acid were simultaneously introduced into the artifi cial
stomach, suspended in 300 mL of yeast culture medium. Table 5 summarizes the
parameters of in vitro gastric digestion.
Release of Yeasts from Beads in Simulated Gastric Conditions The release of
entrapped WRP45073A1 cells from whey protein beads was followed in the artifi cial
FIGURE 7 Gastric digestive system.
pH
temps
pH meter Two-way
valve
37°C
Computer
system
Artificial stomach
in water bath
Gastric
effluents
Pump H2O HCl 0.2 M Pepsin 3804 IU/mL
582 IN VITRO VALIDATION AND ORAL FORMULATION
TABLE 5 Parameters of In Vitro Digestion in Artifi cial Stomach When Simulating
Gastric Conditions of Adult after Intake of Glass of Milk
Initial “ meal ”
Volume 300 mL (constant during all digestion)
Pepsin 75 IU/mL
Acidifi cation
Time (min)/pH 0/6.5, 60/2.1
15/4.4, 75/1.9
30/3.2, 90/1.7
45/2.5, 120/1.6
Flow rate 2 mL/min HCl 0.2 M if necessary
Exponential base e = 1.04
Pepsin supply
Time (min)/pepsine (IU/mL) 0/77.6, 60/139.2
15/99.7, 75/146.7
30/117.1, 90/152.8
45/129.7, 120/163.0
Flow rate From 13 ( t = 0) to 1 ( t = 120 min) mL/min pepsin
3804 IU/mL
Exponential base e = 1.03
Gastric emptying
Flow rate From 10 ( t = 0) to 3 ( t = 120 min) mL/min
Exponential base e = 1.03
Time of digestion 120 min
stomach. During the fi rst 60 min of gastric digestion, a few percentage points (2.2 ±
0.9%, n = 3) of initial entrapped yeasts was recovered in the gastric medium (Figure
8 ). This low percentage cannot be explained by cell death since control experiments
showed the high survival rate of free yeasts (about 90%) during all the digestion
[66] . These results are in agreement with those of Beaulieu et al. [64] , who have
observed that only 5 – 10% of the incorporated retinol was released from the whey
protein – oil matrix following 30 min incubation in HCl 0.1 M and pepsin 24 mg/L.
The low release of yeasts in the fi rst hour of digestion indicates that the beads are
resistant to acidifi cation until pH 2 (cf. Table 5 ) and pepsin attack, which implies
that they might cross the gastric barrier in humans.
From 60 min digestion, the percentage of released yeasts increased regularly to
reach 39 ± 5% ( n = 3) at 120 min. This phenomenon might be explained by two
hypotheses: (i) a swelling of beads due to an increase in the acidity of the medium
or (ii) a degradation of the matrix resulting from a raise in pepsin concentration.
Complementary studies conducted to further evaluate the effect of pH and pepsin
on beads have shown that pepsin has no effect on the protein matrix whatever the
tested pH [66] . As already suggested by other authors [69] , this resistance to enzyme
attack might be due to the formation of hydrophobic interactions between aromatic
amino acids of . - lactoglobulin, the major whey protein. On the contrary, incubation
at pH 2 led to an increase in the diameter of beads, certainly due to high electrostatic
repulsive forces (between positive charges of protoned amino acids and Ca 2+ ), which
induced a raise in the pore size. In conclusion, the release of yeasts observed from
ORAL FORMULATION OF RECOMBINANT YEASTS 583
60 min digestion is provoked by acidic conditions rather than by enzymatic degradation
of beads.
Infl uence of Entrapment on Heterologous Activity of Yeasts in Simulated Gastric
Conditions In order to evaluate the infl uence of the entrapment process of the
CA4H activity of WRP45073A1, the heterologous activity of free and entrapped
yeasts was followed in the gastric system under similar experimental conditions. In
both cases, p - coumaric acid was detected in the gastric medium as soon as 15 min
after the beginning of the experiment (Figure 9 ). This implies that both trans -
cinnamic and p - coumaric acid could diffuse through bead pores.
During all the digestion, the CA4H activity of entrapped yeasts was signifi cantly
( p < 0.05) higher than that of free ones (expected for t = 15 min and t = 75 min). At
120 min, 63.4 ± 1.6% ( n = 3) of initial trans - cinnamic acid was converted into
p - coumaric acid for immobilized yeasts versus 51.5 ± 1.8% ( n = 3) for control yeasts.
This phenomenon was particularly marked from 30 to 60 min of digestion when a
very low amount of recombinant yeasts was released from beads. As suggested by
Bienaim e et al. [70] , beads might create a microenvironment (e.g., a buffer effect
toward low pH or enzyme attack) favoring the heterologous activity of yeasts. In a
similar way, the microenvironment resulting from the presence of an alginate matrix
improves the invertase activity of recombinant S. cerevisiae in batch cultures [71] .
Conclusion This preliminary work reveals whey proteins as a convenient material
for immobilizing recombinant yeasts. Gel beads were resistant to acidifi cation until
pH 2 and pepsin attack, suggesting that they should cross the gastric barrier in
humans. Moreover, the presence of the protein matrix seemed to create “ microconditions
” that favor the heterologous activity of entrapped yeasts.
FIGURE 8 Release of WRP45073A1 cells from whey protein beads in simulated gastric
conditions. Results are expressed as mean percentages ± SD ( n = 3) of initial entrapped yeasts.
(Reprinted with permission from Hebrard et al., Journal of Biotechnology , in press. Copyright
2006 by Elsevier.)
0
0
10
20
30
40
50
Time of digestion (min)
Viable yeasts released (% of initially entrapped)
20 40 60 80 100 120 140
584 IN VITRO VALIDATION AND ORAL FORMULATION
5.5.4 GENERAL CONCLUSION AND FUTURE DEVELOPMENTS
Using genetically engineered microorganisms as new delivery vehicles to the gut is
an important challenge for the development of innovative drugs. A potential application
directly issued from the present work is the development of drug delivery
systems based on orally administered yeasts carrying out a bioconversion reaction
or secreting compounds directly in the human digestive tract.
Soon, the choice of candidate genes as well as the most appropriate dosage forms
will be made according to the therapeutic target. Oral formulations will be optimized
in order to (i) control the release of yeasts according to their action site in
the gastrointestinal tract, (ii) maximize the heterologous activity of yeasts (by addition
of the appropriate substrate and/or inductor), and (iii) ensure a stability of both
yeasts and pharmaceutical dosage forms before administration to the patient. Of
course, heterologous gene expression strategies have to be tailored for a safe use in
humans, the presence of mobilizable vectors, antibiotic selection markers, and bacterial
sequences liable to promote gene transfer to host microfl ora being prohibited.
In addition, environmental confi nement of recombinant cells has to be achieved by
introducing a suicide process that triggers the elimination of the microorganisms
upon leaving the digestive tract. Two types of biological containment systems may
be considered [1, 3] : (i) the active system, which should provide control of the
recombinant microorganism dissemination through the conditional production of a
toxic protein [72 – 74] , and (ii) the passive system, which could render the cell growth
dependent on the complementation of an auxotrophy or other gene defects [75, 76] .
FIGURE 9 Infl uence of immobilization on CA4H activity of WRP45073A1 in simulated
gastric conditions. The CA4H activity of entrapped and free yeasts was evaluated in the arti-
fi cial gastric system. Results are expressed as mean percentages ± SD ( n = 3) of trans - cinnamic
acid converted into p - coumaric acid. Signifi cantly different from free yeasts at p < 0.05 ( * ).
(Reprinted with permission from Hebrard et al., Journal of Biotechnology , in press. Copyright
2006 by Elsevier.)
0
10
20
30
40
50
60
70
Time of digestion (min)
Trans-cinnamic acid conversion (%)
Entrapped yeasts
Free yeasts
*
*
*
*
*
0 20 40 60 80 100 120 140
Steidler et al. [47] have already developed and validated in pigs a passive containment
system for the L. lactis expressing human IL - 10 by deleting the thymidylate
synthase gene which is essential for their growth (the resulting strain being dependent
on thymidine or thymine).
The present study also shows the particular interest of the TIM in drug development
and testing. This artifi cial system will constitute a powerful alternative to
animal experimentation during all preclinical phases of biodrug development. The
in vitro model can aid in the selection of pharmaceutical formulations, ensuring both
the release of yeasts directly at their action site and their optimal activity. The effi -
ciency of newly developed molecular tools (e.g., promoters, selection markers, and
vectors) can also be evaluated to optimize the functionality of recombinant strains
in the digestive environment. As the mucosal layer is not involved in the actual
confi guration of the TIM, this system may be used in combination with intestinal
cells in culture (e.g., Caco - 2) to study the mucosal transport and metabolism of the
active compounds produced by recombinant yeasts. Moreover, experiments in a
large intestinal model [58] , complementary of the gastric and small intestinal system,
could provide necessary data on the biological safety of engineered microorganisms.
For example, the potential gene transfer to the human fl ora can be studied and the
cell death outside of the digestive tract can be checked to ensure there is no dissemination
in the environment.
This study opens up new opportunities in the development of new drug delivery
vectors based on engineered living yeasts for the prevention or treatment of various
diseases in human.
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591
5.6
NASAL DELIVERY OF PEPTIDE AND
NONPEPTIDE DRUGS
Chandan Thomas and Fakhrul Ahsan
Texas Tech University Health Sciences Center, Amarillo, Texas
Contents
5.6.1 Introduction
5.6.2 Nasal Anatomy and Physiology
5.6.2.1 Structure of Nasal and Olfactory Mucosa
5.6.2.2 Nasal Vasculature
5.6.2.3 Enzymes and pH
5.6.3 Factors Infl uencing Nasal Drug Absorption
5.6.3.1 Physiological Factors
5.6.3.2 Nasal Mucociliary Clearance
5.6.3.3 Pathological Condition of Nose
5.6.3.4 Dose Volume and Site of Deposition
5.6.3.5 Physicochemical Properties of Drugs
5.6.3.6 Type of Delivery Device
5.6.4 Animal Models for Nasal Absorption Studies
5.6.5 Enhancement of Intranasal Drug Absorption
5.6.6 Nasal Delivery of Peptide and High - Molecular Weight Drugs
5.6.6.1 Insulin
5.6.6.2 Calcitonin
5.6.6.3 Low - Molecular - Weight Heparins
5.6.6.4 Azetirelin
5.6.6.5 Growth Hormones
5.6.7 Nasal Delivery of Nonpeptide Molecules
5.6.7.1 Morphine
5.6.7.2 Benzodiazepines
5.6.7.3 Buprenorphine
5.6.7.4 Hydralazine
5.6.7.5 Nitroglycerin
5.6.7.6 Propranolol and Other . - Adrenergic Blocking Agents
5.6.7.7 Sex hormones
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
592 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
5.6.7.8 17 . - Estradiol (E 2 )
5.6.7.9 Testosterone
5.6.8 Nose: Option for Delivery of Drugs to Central Nervous System
5.6.9 Nasal Delivery of Vaccines
5.6.9.1 Nasal Vaccines: Ideal Noninvasive Route
5.6.9.2 Immunity after Intranasal Immunization
5.6.9.3 Need for Adjuvants
References
5.6.1 INTRODUCTION
The delivery of drugs via the nasal route has been practiced since ancient times; for
example, psychotropic and hallucinogenic agents have been used as snuff in many
parts of the world for hundreds and possibly thousands of years. More recently,
especially over the past two decades, intranasal drug delivery has shown great
promise in various fi elds of medical practice. At present, a number of conditions
(e.g., rhinitis, migraine, nasal congestion, and osteoporosis) are being treated successfully
with nasal formulations. In addition, an ever - increasing number of nasally
delivered, systemically acting drugs are in the pipeline. Recently this form of therapy
received encouragement with the approval of FluMist (MedImmuneVaccines,
Gaithersburg, MD), an intranasal vaccine against Haemophilus infl uenzae, the infl uenza
virus. This vaccine is the fi rst to be given by the nasal route as a mist rather
than by injection. With its approval, many of the pharmaceutical companies, including
some giants of the pharmaceutical industry, are increasingly looking toward the
area of nasal drug delivery. The market for such therapy in 2005 is reported to have
reached $ 2.4 billion. With 16 of the 20 major pharmaceutical companies conducting
active programs in this area, the fi eld of nasal drug delivery is expected to grow at
an estimated 33% annually [1] . As reported by Koch [2] , the U.S. drug delivery
market in 2005 was somewhere in excess of $ 50 billion and has been predicted to
be around $ 67 billion by the year 2009, whereas the nasal drug delivery market is
expected to be valued at $ 9 billion by 2008 [1 – 3] . Nasal administration — as compared
with injection and oral administration — is more feasible and convenient,
especially in view of the rising number of peptide and protein therapeutics that are
rapidly being developed. In particular, the possibility of delivering drugs to the brain
by the nasal route is eliciting increased interest, especially due to the possibility of
accessing or targeting the local receptors and also of circumventing the blood – brain
barrier.
Drug delivery via the nasal route offers a number of advantages, the most important
of which is the possibility of needle - free treatment. It also means that — in
addition to the newly developed peptide - and protein - based drugs — this method is
also suitable for a wide variety and perhaps most of the drugs that are currently in
use. However, it is not only convenience that sets nasal drug delivery apart: This
method also provides a rapid onset of action and high bioavailability.
Because of its rich vasculature and highly permeable structure, the nasal route
can be used as an alternative to parenteral routes of delivery. It circumvents hepatic
fi rst - pass metabolism and gut - wall enzyme - mediated degradation. It is also easily
accessible for self - administration without the help of a health professional and there
are no associated needle - stick hazards. Other advantages of nasal drug delivery
systems include a rapid onset of action, reduced risk of overdose, and improved
patient compliance. However, there are also several disadvantages, including the
impermeability of the nasal mucosa to lipophilic and high - molecular - weight drugs,
mucotoxicity associated with long - term use of some formulations, the requirement
for an expensive delivery device, and possible dose inaccuracy.
In order to understand the delivery and absorption of drugs by the nasal route
and appreciate the factors that may affect it, one must begin with a clear picture of
the anatomy and physiology of the nose.
5.6.2 NASAL ANATOMY AND PHYSIOLOGY
5.6.2.1 Structure of Nasal and Olfactory Mucosa
The human nasal passage is about 12 cm long and runs from the nostrils to the
nasopharynx (Figure 1 ). The nasal cavity is divided into right and left halves by a
midline septum, or cartilaginous wall, that extends posteriorly into the nasopharynx
(E). Each half of the nasal cavity consists of three well - separated regions: (A) the
vestibule, (B) the olfactory region, and (C) the respiratory region. The vestibule
is the most anterior part of the nasal cavity, which opens to the face through the
FIGURE 1 Sagittal section of nasal cavity showing nasal vestibule (A), atrium (B), respiratory
area and inferior turbinate (C1), middle turbinate (C2) and superior turbinate (C3),
olfactory region (D), and nasopharynx (E). ( Reproduced from ref. 5 with permission of
Pharmaceutical Press. )
D
B
A
C1
C2
C3
E
NASAL ANATOMY AND PHYSIOLOGY 593
594 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
nostrils. The respiratory region and the turbinates make up most of the nasal cavity;
this region has lateral walls that divide it into three chambers, including the superior
nasal turbinate (C3) at the top, the middle nasal turbinate (C2) below, and the
inferior nasal turbinate (C1) at the bottom (Figure 1 ). The total volume of nasal
cavity is 15 mL; its surface area is 150 cm 2 [4, 5] . Air generally travels into the nose
in an approximately parabolic pattern from the vestibule to the nasopharynx. The
hard palatine bone and soft palate form the fl oor of the nose and the roof of the
mouth. The human nasal epithelial surface is covered mainly by stratifi ed squamous,
olfactory, and respiratory epithelia. The stratifi ed epithelium lies in the anterior part
of the nose and becomes pesudostratifi ed columnar epithelium at the posterior part,
which constitutes the respiratory epithelium (Figure 2 ). The olfactory epithelium
covers the olfactory region in the upper part of the nasal cavity. The nasal epithelial
surface is covered by a continuous layer of mucus secreted by various mucosal and
submucosal glands. The mucous layer comprises the gel layer (D) and sol layer (E)
as shown in Figure 2 . The sol layer is a low - viscosity fl uid surrounding the cilia, and
the viscous gel layer covers the tips of the cilia — fi ne, hairlike structures that move
in an organized fashion to ease the fl ow of mucus across the epithelial surface. There
are also microvilli, and every ciliated cell carries some 100 cilia. Each ciliated and
nonciliated cell has about 300 microvilli. Figure 2 also shows both nonciliated cells
and basal cells [4 – 6] .
The olfactory mucosa is discussed further on in relation to the delivery of drugs
via the nose to the brain.
5.6.2.2 Nasal Vasculature
The nasal surface is supplied with a dense network of blood vessels by the external
and internal carotid arteries. Blood from the anterior part of the nose is drained
through the facial vein, but the nose ’ s main blood supply drains through the sphenopalatine
foramen into the pterygoid plexus or via the superior ophthalmic vein
FIGURE 2 Cell types of nasal epithelium showing ciliated cell (A), nonciliated cell (B),
goblet cell (C), gel mucous layer (D), sol layer (E), basal cell (F), and basement membrane
(G). ( Reproduced from ref. 5 with permission of Pharmaceutical Press. )
F G
A B E D C
[6] . The nasal blood vessels can be greatly dilated with blood to facilitate warming
and humidifi cation of inspired air in response to prevailing conditions. Nasal blood
fl ow is very sensitive to a variety of agents applied topically and systemically.
5.6.2.3 Enzymes and p H
Nasal secretions contain a mixture of secretory materials from the goblet cells, nasal
glands, and lacrimal glands. The main constituents of nasal secretions are water, with
2 – 3% mucin and 1 – 2% electrolytes. Nasal secretions also contain several enzymes,
including lysozyme, cytochrome P450 – dependent monooxygenases, steroid hydroxylases,
proteases such as neutral endopeptidase, leucine aminopeptidase, aminopeptidase
peroxidase, carboxypeptidase N, and protease inhibitors [7] . However,
because most studies of peptide and protein degradation are carried out in homogenates
of nasal tissue, the peptides and proteins can be exposed to both intracellular
and extracellular enzymes; therefore, the data regarding peptide stability must be
interpreted with caution. The normal pH of nasal secretions in adults ranges from
about 5.5 to 6.5; in young children, it ranges from 5.0 to 6.7. The nasal pH can vary
depending on pathological conditions such as allergic rhinitis and environmental
conditions such as cold and heat [8] .
5.6.3 FACTORS INFLUENCING NASAL DRUG ABSORPTION
The nasal absorption of drugs is infl uenced by a multitude of factors, including nasal
physiology, nasal pathology, physicochemical properties of drugs, dosage forms, and
delivery method. The presence of pathological conditions such as allergic rhinitis
and the common cold further complicates nasal drug delivery. Moreover, the intimate
contact between the nasal mucosa and the atmosphere leads to variability in
absorption with changes in temperature and humidity.
5.6.3.1 Physiological Factors
Nasal Blood Flow The nasal vasculature differs from the tracheobronchial tree
due to the presence of (a) venous sinusoids, (b) arteriovenous anastosomes, and
(c) the nasal vasculature, which shows cyclical changes of congestion, hence giving
rise to the nasal cycle. In the nasal vasculature, the arterioles lack an internal elastic
membrane, making the endothelial basement membrane continuous with the basement
membrane of the smooth muscle cells. Also present are the fenestrated type
of capillaries lying just below the surface epithelium and surrounding the glands.
Because of this, the capillaries facilitate rapid movement of fl uid through the vascular
wall, allowing water to escape into the airway lumen. The conditioning of the
inhaled air is greatly infl uenced by the nasal blood vessels. In essence, air is heated
and humidifi ed by the fl ow of nasal blood in the opposite direction to the incoming
airfl ow. The nasal blood fl ow also controls the size of the nasal passage ’ s lumen.
Changes in ambient temperature and humidity, nasal administration of vasoactive
drugs, nasal trauma, and compression of large veins in the neck may adversely affect
blood fl ow in the nose [9, 10] . Other factors such as mood changes, hyperventilation,
and even exercise can have an effect on the nasal blood fl ow and hence the nasal
FACTORS INFLUENCING NASAL DRUG ABSORPTION 595
596 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
passages [10 – 12] . In conclusion, any change in blood fl ow can alter the absorption
of a nasally administered drug.
Nasal Enzymes The enzymes present in the nasal cavity may be involved in the
extensive enzymatic degradation of drugs administered nasally. The presence of the
enzymes in the nasal epithelium acts as a defensive mechanism or a barrier against
the entry of xenobiotics [9] . Examples include nasal decongestants, essences, anesthetics,
nicotine, and cocaine, which are metabolized by the nose ’ s P450 - dependent
monooxygenase system [13, 14] . The nasal mucosa also includes oxidative phase I
and conjugative phase II enzymes. The phase I enzymes include aldehyde dehydrogenase,
carboxyl esterase, and carbonic anhydrases; phase II enzymes include glucuronyl,
sulfate, and glutathione transferases. 17 . - Estradiol has shown signifi cantly
more conjugation when administered via the nasal route as compared to the intravenous
route. A variety of other drugs have been shown to be metabolized by nasal
enzymes, including progesterone, testosterone, and insulin [15, 16] .
5.6.3.2 Nasal Mucociliary Clearance
Nasal mucociliary clearance is the transport of the mucous layer covering the nasal
epithelium toward the nasopharynx by ciliary beating for its eventual discharge into
the gastrointestinal tract. Nasal mucociliary clearance plays a very important role
in the upper respiratory tract in preventing various noxious agents such as allergens,
bacteria, viruses, and toxins from reaching the lungs. The ciliated cells of the nasal
mucosa drive the movement of the mucus, and hence the physiological control of
the ciliated cells and the rheological properties of the mucus determine the effi -
ciency of the nasal mucociliary clearance system. In humans the normal mucociliary
transit time is reported to be 12 – 15 min, and transit times of 30 min or more are
likely to be an indication of impaired mucociliary clearance. Impairment of mucociliary
clearance has been associated with longer contact times of various noxious
agents as well as drugs with the nasal mucosa. On the other hand, increases in the
mucociliary clearance rate decrease the contact between drug and the epithelium
and consequently reduce drug absorption. Therefore nasal drug absorption can be
augmented by the use of bioadhesive polymers or microspheres or by increasing
the viscosity of the drug formulation [5, 17] . Hydroxypropyl methylcellulose, polyacrylic
acid, and hyaluronan all enhance absorption by increasing nasal residence
time [18–20] . The effect of mucociliary clearance may vary depending on the site of
drug deposition. Ciliated epithelium is present in the middle and posterior parts of
the turbinates, but there is little or no ciliary epithelium in the anterior regions
of the nasal cavity [21, 22] . This is one of the reasons why a drug deposited in the
posterior part of the nose is washed away more quickly than a drug deposited in
the anterior site of the nasal cavity [23] .
5.6.3.3 Pathological Condition of Nose
As mentioned earlier, the presence of nasal pathological conditions — such as allergic
rhinitis, polyposis, and common colds — infl uences nasal drug absorption to a
great extent. The majority of pathological conditions of the nose show bleeding,
excessive secretion of mucus, nasal blockage, and crusting. It has been reported that
excessive nasal secretion may wash away a nasally administered drug before it can
be absorbed [24] . Nasal drug absorption and distribution are also infl uenced by the
presence of nasal polyps and blockage. Several studies however have suggested that
the presence of nasal pathological conditions do not affect nasally administered
peptide drugs. For example, buserelin and desmopressin absorption studies have
shown similar nasal absorption profi les in normal subjects and in those suffering
from colds or rhinitis [25, 26] .
5.6.3.4 Dose Volume and Site of Deposition
A dose of 25 – 200 . L per nostril is what can be maximally accommodated by the
human nose. A dose higher than this will be drained off and hence shows lower
absorption. Some studies have reported that a 100 - . L volume resulted in a larger
deposition area. Hence, taking into account the volume of administration becomes
very important for manufacturers of nasal drug delivery systems. The site of deposition
of the nasal formulation may also affect the nasal absorption of drugs since the
anterior part of the nose provides greater contact between the nasal epithelium and
drug, but the mucociliary clearance mechanism of the posterior tends to remove
drug more rapidly [27] . It has been found that the permeability of the posterior area
is greater than that of the anterior portion, and hence, based on the formulation,
drugs may be administered in either the anterior or posterior parts of the nose. The
nasal adapter ’ s spray - cone angle defi nes the width of the nasal spray pattern and
thus plays an important role in determining the site of deposition in the nasal cavity.
Changes in the cone angle of the adapter from 60 ° to 35 ° or 30 ° can produce a larger
and more posterior deposition and therefore higher drug deposition in the ciliated
area [10] .
5.6.3.5 Physicochemical Properties of Drugs
The absorption of a drug across the nasal mucosa is a function of its physicochemical
properties, such as molecular weight, lipophilicity, and water solubility, as seen with
most of the mucosal routes of delivery. The majority of studies on the effects of drug
lipophilicity on nasal absorption are rather confl icting. The effect of lipophilicity on
the nasal absorption of barbituric acids has been investigated. It was found that drug
absorption through the nasal mucosa increases with an increase in the partition
coeffi cient. Interestingly, there was only a fourfold increase in absorption between
phenobarbital and barbital despite the fact that the partition coeffi cient of phenobarbital
was 40 - fold higher than that of barbital [28] . Similarly, increases in nasal
absorption have been seen for hydrocortisone, testosterone, and progesterone with
increases in the partition coeffi cient. However, a hyperbolic — rather than a linear —
relationship was observed between the in vivo nasal bioavailability of a series of
progesterone derivatives and their octanol – water partition coeffi cients [29] . In contrast
to this, Kimura et al. [30] showed that, for a series of quaternary ammonium
compounds structurally related to tetraethylammonium chloride, nasal absorption
was inversely related to the partition coeffi cient. All these studies suggest that a
drug ’ s lipophilicity may not be an appropriate indicator of the extent of its nasal
absorption. Besides the drug lipophilicity another important factor most studied for
its infl uence on nasal absorption is the aqueous solubility of a drug. This is because
FACTORS INFLUENCING NASAL DRUG ABSORPTION 597
598 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
the nasal mucosa is constantly kept moist by the nasal secretions and is well perfused
with blood vessels. In addition to limiting drug absorption, it also is a limiting factor
for the formulation of the drug. Further it is also important to understand the relation
between the saturation solubility of the drug and drug absorption. However,
the infl uence of drug solubility on absorption of drugs via the nasal route has not
been signifi cantly explored and needs much more attention. The relative effectiveness
of nasal atropine and hyoscine was compared by Tonndorf et al. [31] by measuring
each drug ’ s capacity to arrest salivary secretion; they found that 0.65 mg of
hyoscine, 40 times more soluble in water than atropine, is equivalent to 2 mg of
atropine. Some authors have suggested that the aqueous pores in the nasal mucosa
play a major role in absorption of hydrophilic drugs [32] . When nasal formulations
are administered as inhaled powders or suspensions, drug dissolution rate becomes
an important factor. Formulations deposited in the nostrils require proper dissolution
for better absorption. Nasal mucociliary clearance may remove the drug if it
remains undissolved in the nostrils. The size and shape of a drug molecule can affect
its nasal absorption. Molecules with an average molecular weight less than 1000 Da
are better absorbed nasally than higher molecular weight drugs whereas linear
molecules are less effectively absorbed than compact ones [33] . The nasal and oral
absorption of polyethylene glycol 600, 1000, and 2000 have been studied by Donovan
et al. [34] in relation to molecular weight; they found that the greater the molecular
weight, the less effective the absorption. This pattern of absorption was seen in the
case of both the nasal and oral routes. The effect of water - soluble compounds such
as 4 - oxo - 4 H - 1 - benzopyran - 2 - carboxylic acid, p - aminohippuric acid, sodium cromoglycate,
inulin, and dextran showing different molecular weights on the nasal absorption
was studied by Fisher et al. [35] . A 43 - fold decrease in the nasal absorption of
the these compounds was observed with a 368 - fold increase in the molecular weight
[9] . Similarly, studies with 13 di - iodo - l - tyrosine - labeled dextran showed an inverse
relationship between the percentage absorbed after nasal administration and the
weight of the molecule; in fact, a 36 - fold increase in molecular weight produced an
88 - fold reduction in nasal absorption [36] . The effect of molecular weight on the
nasal absorption of fl uorescein isothiocyanate and diethylaminoethyl dextrans has
been studied by Maitani et al. [37] . It was found that an inverse relationship between
absorption and molecular weight existed for these compounds. However, since the
nasal absorption of these compounds was low, enhancers were used; hence it is dif-
fi cult to rule out the infl uence of the enhancers used on the extent of absorption
obtained. The absorption of a drug after nasal administration is also infl uenced by
the pH of a drug formulation as well as that of the nasal cavity — along with the p Ka
of the drug substance. Biological membranes form a major barrier to the transport
of drugs into the bloodstream. There are a number of transport mechanisms by
which drugs are transported across the biological membranes. These include transcellular,
paracellular, and carrier - mediated transport mechanisms. The most important
factors that infl uence the above - mentioned mechanisms are the pH, p Ka , and
partition coeffi cient of the drug. The pH of a nasal formulation should be in the
range of 4.5 – 6.5 in order to minimize nasal irritation. However, the drug ’ s p Ka must
also be taken into account so as to maximize the drug ’ s concentration in un - ionized
form. The effect of pH on the nasal absorption of benzoic acid was studied by
Hussain et al. [28] , who showed that the absorption of benzoic acid is pH
dependent.
5.6.3.6 Type of Delivery Device
Both the type of drug delivery system and the specifi c type of delivery device can
affect drug absorption via the nasal route. The choice of delivery system depends
mainly on the physiochemical properties of the drug, its desired site of action, and,
more importantly, patient compliance and marketing aspects. The formulations most
commonly used in nasal delivery are solutions, suspensions, gels, dry powders, and,
most recently, nanoparticulate formulations.
Solutions are most commonly used for intranasal drug delivery. Such solutions
may be used when the active ingredient is soluble in water or in some other vehicle
approved by the Food and Drug Administration. At present, nasal solutions are
available in the form of drops and sprays. Drops are the simplest and the most
convenient nasal dosage form, and they are also easy to manufacture. However,
their major drawback is that exact dosages cannot be administered with them.
Another disadvantage is that they — like most solution - based medications — are vulnerable
to microbial contamination; therefore, preservatives must often be added.
These, in turn, have further disadvantages, as they may both cause irritation and
hamper mucociliary clearance, thus decreasing compliance. Chemical stability is also
often an issue with nasal drops.
Since the introduction of metered - dose inhalers, nasal solutions have increasingly
been formulated as nasal sprays. Initially, aerosol - based systems containing chloro-
fl uorocarbons were employed; however, the Montreal Protocol put an end to this.
Thereafter, mechanical pumps or actuators were employed to deliver nasal formulations
as sprays. These devices, using actuators, can precisely deliver as little as 25 . L
and as much as 200 . L of a formulation. However, various factors must be considered
in formulating the spray; these include viscosity, particle size, and surface
tension, all of which may affect the accuracy of the dose administered.
Suspensions may also be used to deliver nasal formulations, though only rarely,
since a number of complicating factors (e.g., particle size and morphology) must be
considered. Suspensions offer the advantage of increasing residence time in the
nasal cavity, thus possibly augmenting nasal bioavailability.
Gels are thickened solutions that may sometimes be used to deliver drugs via the
nose, since they offer a number of advantages, such as reducing postnasal drip into
the back of the throat and hence reducing the loss of the drug from the nasal cavity,
anterior leakage, and the associated irritation. The use of gels is also reported to
improve absorption and to mask the irritation associated with some ingredients by
the addition of soothing agents and emollients. A vitamin B 12 (cyanocobalamin) nasal
gel, Nascobal (Nastech Pharmaceutical, Kirkland, WA), is available in a metered -
dose formulation. Several other drugs, such as insulin, are being studied with a view
to formulating them as nasal gels [9, 27, 38] . Although nasal powders are more stable
than other formulations, they are rarely used because they tend to irritate the nasal
tissue. However, a powder form may be useful when the active ingredient cannot be
formulated as a solution or suspension. With the development of refi nements in
technology, many researchers are exploring the use of nanoparticle - based formulations
to deliver drugs nasally. The main advantage of these state - of - the - art formulations
is that they ensure increased absorption as well as better compliance.
Microsphere - and liposome - based formulations are being increasingly tested. Some
of these studies are discussed in Sections 5.6.6 and 5.6.7.
FACTORS INFLUENCING NASAL DRUG ABSORPTION 599
600 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
The type of nasal device employed in delivering a drug formulation plays a major
role in the effi cacy of the treatment. In general, two types of delivery systems are
used: mechanical pumps and pressurized aerosol containers. The properties of the
drug to be used infl uence the selection of the system. The various types of delivery
devices are described in the following sections.
Unit-Dose Containers The unit - dose container offers a number of advantages. It
is easy and convenient to carry and also does away with the need for preservatives,
thus greatly increasing patient compliance. The unit - dose container is more accurate
than the multidose container; metered - dose nasal sprays are still more accurate. The
volume of drug held by such a container is usually determined by its fi lling volume,
which greatly infl uences its accuracy. Another type of unit - dose container is based
on an actuator and consists of a nasal adapter, or a small chamber that contains a
piston [9] . Unit - dose containers are used mainly in emergencies (e.g., to manage
pain), although they are not restricted to such use and may also be employed
in instances where a single administration is required (e.g., vaccination). An
example of a product that employs unit - dose containers is Imitirex (sumatriptan)
(GlaxoSmithKline, Research Triangle Park, NC).
Squeeze Bottles This is a smooth plastic bottle with a jet outlet. When the bottle
is pressed, a certain volume of its contents is atomized as the air inside the bottle
is pushed out. This type of device is vulnerable to contamination as ambient air
rushes into it following the release of pressure. The squeeze bottle is used mainly
to deliver decongestants and not vasoconstrictors, as in the latter case the dose
administered would be diffi cult to control [9] .
Metered-Dose Nasal Pump Sprays Metered - dose pumps are the most widely used
devices for the delivery of formulations via the nose. A number of commercially
available products use this technology. The accuracy of the delivered dose is fairly
high and makes it possible to administer dose volumes ranging from 25 to 200 . L.
A metered - dose pump is made up of a container as well as a pump, valve, and actuator.
The characteristics of the spray delivered will differ depending on the properties
of the drug, the precompression mechanism, and the valve and pump selected. The
length of the actuator is an important factor determining the deposition of the drug
in the nose; the collection of residual drops on its tip will affect correct dosing.
Airless and Preservative -Free Sprays There are now pumps that prevent the entry
of air into a dispensing device after use, thus increasing the stability of numerous
compounds that are vulnerable to oxidation; this innovation has also minimized the
use of preservatives. The working principle of these pumps is operation against a
vacuum using a collapsible bag and a sliding piston. This is possible because the
vacuum created when a dose is dispensed is accompanied by a reduction in the
volume of the container, either by deforming the container itself or by dragging
the sliding piston out of it. These maneuvers have no infl uence on the system ’ s effi -
ciency and, in fact, provide an advantage in that the container can be held in any
position without signifi cantly compromising the accuracy of the dose dispensed. This
system is particularly suitable for use with children and bedridden hospitalized
patients.
Whenever systems without preservatives are used for single or double doses, they
pose little risk of contamination. However, multidose systems are generally used
over a longer period of time; therefore, unless their formulations also include preservatives,
the chances of contamination are increased. Scientists from Erich Pfeiffer
and Qualis Laboratorium (both in Germany) have reviewed the latest trends in
preservative - free nasal sprays and report that it is possible to prevent microbial
contamination via the orifi ce in two ways. The fi rst is by introducing a chemical
additive, such as a bacteriostatic agent, into the nasal actuator so that it comes into
contact with both the medication and the environment. However, in the case of an
open system, the formulation within the actuator can still be contaminated. The
second approach is the use of a mechanism whereby the system is sealed behind
the orifi ce, thus preventing microorganisms gaining access [9, 39] .
Some innovative technologies are being developed by a variety of pharmaceutical
fi rms. The following section touches briefl y on the latest of these.
Kurve Technology Kurve Technology has developed a unique system of controlled
particle dispersion (CPD) by which it is possible to deliver drugs to the entire nasal
cavity as well as the olfactory region and the paranasal sinuses. It uses the principle
of vortical fl ow, by which inherent airfl ows of the nasal cavity are disrupted (Figure
3 a and b ). Its advantages include optimization of the size and trajectory of droplets,
which makes it possible to saturate the nasal cavity. CPD also increases nasal residence
time and reduces the deposition of compounds in the lungs and stomach, thus
making the treatment more effective and effi cient. ViaNase ID (Kurve Technology,
Bothell, WA) is a CPD - powered electronic atomizer also developed by Kurve. Its
advantages include generation of narrow droplet distribution between 3 and 50 . m
and control of the atomization rate (i.e., the rate at which the droplets are generated
and how rapidly they exit the device). CPD technology can be used to deliver both
solutions and suspensions; currently, work is in progress to apply this principle to
dry powders. Testing is also under way for the delivery of small and large molecules
as well as peptides and proteins. Finally, CPD technology makes it possible to
provide preservative - free packaging; unit - dose ampules; targeted deposition, as
mentioned earlier; and monitoring of doses and compliance [40 – 42] .
OptiNose OptiNose AS (Oslo, Norway) has introduced the novel idea of bidirectional
intranasal drug delivery, which delivers a drug while the patient exhales and
thus is said to prevent lung deposition. It utilizes the concept that exhalation against
resistance leads to closure of the soft palate, thus separating the nasal cavity from
the mouth as well as cutting off communication between the cranial surface of the
soft palate and the posterior margin of the nasal septum. When this occurs, the air
can enter one nostril through the sealing nozzle, turn 180 ° , and fi nally exit through
the other nostril in the reverse direction. This concept is utilized in breath - actuated
bidirectional delivery; that is, the air is blown out of the container and the sealing
nozzle is used to direct its fl ow of air into the nose. When this approach was compared
with conventional nasal drug delivery in 16 healthy subjects using 99m Tc -
labeled nebulized particles, it was found that bidirectional nasal delivery did, in fact,
prevent deposition in the lungs. The single - use device is already developed and
undergoing clinical testing for the delivery of a variety of compounds; a multidose
liquid reservoir and powder delivery device are also being developed. The technol-
FACTORS INFLUENCING NASAL DRUG ABSORPTION 601
602 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
FIGURE 3 ( a ) Vertical droplet fl ow created by controlled particle dispersion used in
ViaNase ID (Kurve Technology, Bothell, WA). ( b ) Deposition pattern produced by controlled
particle dispersion. ( Reproduced from ref. 42 with permission from Drug Delivery
Technology. )
Controlled particle dispersion
Transistion from aperture to
nasal cavity
Vestibule
Nasal cavity
Delivery path of the droplet
Velocity profile of air steam
a: Nasal vestibule
b: Palate
c: Inferior turbinate
d: Middle turbinate
e: Superior turbinate
f: Nasopharynx
(a)
(b)
ogy is also being tested in connection with vaccination by the nasal route, and vaccines
against diphtheria and infl uenza have already been shown to improve local
and systemic immune responses. Another area being explored is the delivery of
drugs to the brain via the nose. A phase I clinical trial of midazolam has been carried
out and has shown an onset and level of sedation comparable to that of intravenous
administration. The duration of sedation was found to be longer with the OptiNose
technology, but the bioavailability of the drug was found to be only 68%, as
compared with 100% with intravenous administration. Figures 4 a and b show the
Optinose multidose liquid device and multiuse powder device being developed
respectively [43, 44] .
DirectHaler The DirectHaler (Direct - Haler A/S, Copenhagen, Denmark) nasal
delivery device takes advantage of the nasal anatomy and an innovation in device
technology in order to improve nasal drug delivery and patient compliance. Similar
to OptiNose, this novel drug delivery device avoids lung deposition. It takes advantage
of the fact that, when air is blown out of the mouth against a particular resistance,
the oral and nasal cavity airway passage closes on its own. Hence when a
patient blows air into the DirectHaler Nasal device, the nasal dry powder dose is
delivered into the nostril (Figure 5 ). The DirectHaler is reported to solve most of
the problems that are associated with existing drug delivery devices, including dripping
of the liquid dose out of the nostril following its delivery, swallowing of the
dose after the administration and hence low absorption, and other problems of
contamination associated with the liquid and multidose formulations. The device is
quite easy and cost effective to manufacture, fi ll, and assemble using the latest high -
speed technology. The tube of the device is made by using extrusion and roll forming
while the device cap is manufactured by injection molding. A modifi ed high - speed
capsule - fi lling machine is used to carry out the powder dose fi lling. DirectHaler has
also been developed for the combination of oral and nasal drug delivery. For
pulmonary delivery DirectHaler Pulmonary has been developed. Further, for the
FIGURE 4 ( a ) Optinose multidose liquid device and ( b ) Optinose multiuse powder device.
( Reproduced with permission of Per Gisle Djupesland by personal communication. )
(a) (b)
FIGURE 5 DirectHaler Nasal delivery device. ( Reproduced with permission of Troels
Keldmann by personal communication. )
Pharma blister pack
avaiable, extra
protection barrier
Flow valve
for control
PowderWhirl chamber
for turbulent dispersion
of the dose
Inhaler cap
for moisture
protection
Mouth piece
for generation
of air flow
Transparency of
device for visibility
of dose
FACTORS INFLUENCING NASAL DRUG ABSORPTION 603
604 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
complete targeting of the whole respiratory system, Novel DirectHaler Compliance
system has been designed. The device has successfully undergone clinical trials for
its use in nasal drug delivery and has shown patient compliance [45] .
5.6.4 ANIMAL MODELS FOR NASAL ABSORPTION STUDIES
Nasal absorption studies can be carried out in mainly two types of animal models:
the whole - animal model and the isolated organ perfusion model.
In vivo nasal absorption has been studied in a variety of mammals, including the
rat, rabbit, dog, sheep, and monkey. Rats are the most widely used animals for testing
intranasal drug delivery. They are easy to handle and simple and inexpensive to
maintain. Use of the rat model for the nasal delivery of insulin was fi rst described
by Hirai and co - workers more than 25 years ago [46, 47] . The rat has been used in
two basic models, in vivo and in situ — the latter also referred to as the ex vivo nasal
perfusion model. Both models require anesthesia, though an in vivo model not
requiring surgery has also been reported and is discussed later. In early studies of
nasal absorption in rats, the animals were anesthetized and the passage of the nasopalatine
tract was then sealed with acrylic glue to prevent the drainage of drug
solution from the nasal cavity. The trachea was cannulated with a polyethylene tube,
with another tube being inserted through the esophagus toward the posterior part
of the nasal cavity. The drug solution was then delivered to the nose through either
the nasal cavity or the esophageal tubing [8, 47] . Blood samples were then collected
from the femoral vein.
However, a less traumatic and much more feasible rat model has recently been
proposed by Pillion et al. and other groups [48, 49] . In this model, the rats are anesthetized
and kept on their backs; then the drug solution is administered directly into
the nose by inserting a pipette about 3 – 5 mm into each nostril. In some cases the
drug is administered into only one nostril so as to prevent blockage. The rats then
remain on their backs long enough for the formulation to come into contact with
the nasal mucosa. Blood samples are then collected from the tail vein.
In support of the earlier models, it was argued that, by sealing the nasopalatine
tract, the drug would necessarily be fully absorbed and transported into the circulation
via permeation through the nasal mucosa. However, such nasal absorption with
blockage of drainage from the nose to the mouth is not the normal physiological
condition. Although the rat model has been used extensively by investigators
throughout the world, application of the results of such studies to humans is very
limited because of the small body size of these animals and signifi cant interspecies
differences. In fact, signifi cant variability between the rat and the human was
observed in studies of the bioavailability of insulin administered intranasally [50] .
Furthermore, the use of anesthesia has raised concerns because of its potential to
confound the test results.
In the rabbit model, drug solution is delivered by spray instillation into a nostril,
keeping the rabbit ’ s head in an upright position and allowing the rabbit to breathe
normally. Blood samples are then collected from the ear vein. New Zealand White
and Japanese White rabbits are most commonly used in such research. One of the
advantages of the rabbit model is that the blood volume of these animals is large
enough for multiple sampling and pharmacokinetic analysis [51] .
Dogs, sheep, and monkeys can be kept conscious during nasal delivery to mimic
the human [51] . Sheep, because of their large nostrils and docile nature, serve as
excellent models for studies of this kind.
5.6.5 ENHANCEMENT OF INTRANASAL DRUG ABSORPTION
Lipophilic drugs or compounds have consistently been shown to be completely
absorbed across the nasal mucosa; frequently, nasal absorption of these compounds
is identical to that obtained with intravenous administration. In some reports, bioavailability
after nasal administration reached almost 100% and Tmax was similar to
that obtained with intravenous administration. For example, lipophilic drugs such
as propranolol, naloxone, buprenorphine, testosterone, and 17 . - ethynylestradiol
have been reported to be completely or almost completely absorbed after nasal
administration in animal models. However, the same is not true of polar molecules
and certain low - molecular - weight drugs and also high - molecular - weight peptides
and proteins. In these instances absorption enhancers have been employed, and this
is the method most widely used to improve nasal drug absorption [52 – 54] . Such
absorption enhancers belong to a variety of different chemical groups and may have
one or multiple ways of enhancing the absorption. Absorption enhancers work by
(1) altering the mucous layer by decreasing its viscosity or disrupting it; (2) altering
the tight junctions by sequestering extracellular calcium ions, which are reported to
be essential in maintaining the integrity of these junctions; (3) inhibiting mucosal
enzymatic degradation; (4) reverse - phase micelle formation — in certain cases,
reverse - phase micelles may be formed within the cell membranes, thus creating an
aqueous pore through which the drug can pass; and (5) altering membrane fl uidity,
which can be achieved when there is disorder in the membrane phospholipid component
or leaching of proteins from the membrane or by a combination of these
mechanisms [52 – 56] . Table 1 lists some of the selected absorption enhancers based
on the mechanisms mentioned above.
5.6.6 NASAL DELIVERY OF PEPTIDE AND
HIGH - MOLECULAR - WEIGHT DRUGS
Protein and peptide delivery by means other than injection is currently receiving
enormous attention due to the increasing number of biotechnology - based products
being developed. There have been numerous attempts to design systems for oral
peptide, protein, and gene delivery, but these have unfortunately met with limited
success, thus providing an impetus for exploring alternative noninvasive delivery
methods. As mentioned earlier, more and more research has been directed to nasal
drug delivery because of the numerous advantages it offers. The following section
deals with the delivery of peptide and protein drugs.
5.6.6.1 Insulin
Insulin is produced by the . cells of the islets of Langerhans in the pancreas.
It is made up of two peptide chains, which have 21 and 30 amino acid residues,
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 605
606 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
TABLE 1 Enhancement of Nasal Absorption
Type of
Compound
(Absorption
Enhancers ) Examples
Mechanism of
Absorption
Enhancement
Bile salts and
derivatives
Sodium deoxycholate, sodium glycocholate,
sodium taurocholate, sodium
taurodihydrofusidate, sodium
glycodihydrofusidate
Alteration of tight
junctions, membrane
disruption, inhibition
of mucosal enzymatic
degradation, mucolytic
activity
Chelating
agents
Citric acid, EDTA, enamines, N - acyl
derivatives of collagen, salicylates
Alteration of tight
junctions
Enzyme
Inhibitors
Amastatin, bestatin, camostat mesylate,
boroleucine
Enzyme inhibition
Fatty acids and
derivatives
Acylcarnitines, acylcholine, caprylic acid,
capric acid, oleic acid, phospholipids,
mono - and diglycerides, sodium laurate
Membrane disruption
Surfactants Sodium lauryl sulfate, saponin,
polyoxyethylene - 9 - lauryl ether,
polyoxyethylene - 20 - lauryl ether,
alkylmaltosides such as tetradecylmaltoside,
dodecylmaltoside, and decylmaltoside
Membrane disruption
respectively. These two chains are held together by disulfi de linkages between cysteine
residues. Insulin is an anabolic and anticatabolic peptide hormone with a
molecular weight of about 6000. It was fi rst used in the successful treatment of diabetes
mellitus in 1922. Today insulin is widely used in the treatment of both insulin -
dependent and non - insulin - dependent diabetes mellitus [57 – 59] . A nonparenteral
formulation of insulin has been approved only recently; before this development,
insulin treatment required the use of painful injections. Exubera (Pfi zer Labs, New
York, NY) is the fi rst insulin and fi rst biotechnology - based medicine for the treatment
of a systemic disorder that can be administered without an injection. It was
developed by Nektar (San Carlos, CA) and is now a registered trademark of Pfi zer
(New York, NY). It still poses a few concerns, especially its effect on the lungs of
patients with asthma or chronic obstructive pulmonary disease. Because of the
problems associated with the parenteral injection of insulin, many diabetic patients
fl atly refuse to accept insulin therapy. As to subcutaneous injections, these fail to
attain a physiological pattern of insulin owing to their adverse pharmacokinetics,
and normoglycemia is often not achieved [47] . Of all the routes so far studied apart
from delivery via the lungs, the nasal route would appear to be the most advantageous
for the delivery of insulin.
The nasal delivery of insulin was demonstrated as early as 1922 by Woodyatt [59] .
Since then, numerous studies have focused on this methodology. Some of the early
studies included absorption of insulin from the nasal mucosa in human diabetics,
the use of an insulin sprayer that contained saponin, and insulin in ethylene glycol
or trimethylene glycol applied to the nose in the form of drops or sprays; the last
of these methods demonstrated a signifi cant fall in blood glucose levels in normal
rabbits, dogs, and diabetic humans [60 – 62] .
The enhancement of nasal absorption of insulin by hydrophobic bile salts has
also been investigated. It was found that minor differences in the number, position,
and orientation of the nuclear hydroxyl groups as well as alterations to side - chain
conjugation can improve the adjuvant potency of bile salts. Moreover, the absorption
of insulin positively correlated with an increase in the hydrophilicity of the
steroid nucleus of the bile salts. In the presence of bile salts, nasal absorption of
insulin reached peak levels within about 10 min, and some 10 – 20% of the dose was
found to have been absorbed into the circulation. Marked increases in serum insulin
levels were seen with sodium deoxycholate, the most lipophilic of the bile salts,
whereas the least elevation — as well as least lowering of blood glucose levels — was
seen with the most hydrophobic bile salt, sodium ursodeoxycholate [63] .
Morimoto et al. [64] studied the nasal absorption of insulin using polyacrylic acid
gel. When insulin was formulated with 0.1% w/v polyacrylic acid gel base (pH 6.5),
the maximum hypoglycemic effect was seen 30 min following intranasal administration;
in 1% w/v gel base, however, it took 1 h to reach the maximum effect. There
was no effect of the pH (4.5, 6.5, and 7.5) of 0.1% w/v polyacrylic acid gel on the
extent of nasal absorption.
Pillion and his group studied alkylmaltosides differing in chain length for their
abilities to lower blood glucose levels when formulated with insulin [65] . Tetradecylmaltoside
(TDM) was the most effective agent in producing the hypoglycemic
effect, followed by dodecylmaltoside (DDM) and decylmaltoside (DM), all at concentrations
of 0.060%. The onset of hypoglycemic action using these nasal drops
was seen within 30 min and the maximum effect was obtained within 60 – 120 min. It
was also demonstrated that insulin plus TDM at concentrations as low as 0.03%
induced a hypoglycemic effect; however, insulin plus octylmaltoside (OM) failed to
produce any hypoglycemic effect even at OM concentrations as high as 0.50%.
Dodecylsucrose, which differs from DDM by only one carbohydrate residue, had a
similar effect on blood glucose; however, decylsucrose was found to be less potent,
and nonglucosides were able to enhance the nasal absorption of insulin only at
concentrations . 0.50% [65] .
Insulin formulated with 0.06 or 0.125% hexadecylmaltoside produced a pronounced
and rapid dose - dependent decrease in blood glucose levels after nasal
administration. The effects of seven different alkylmaltosides were studied, and all
the reagents (Figure 6 ) showed a similar maximal enhancement of insulin uptake
when a concentration of 0.125% was employed. The fi gure demonstrates that TDM
showed the greatest effect when concentrations of 0.03 and 0.06% were used.
Similar experiments were carried out using sucrose esters in nasal insulin formulations
(Figure 7 ). It was observed that tetradecanoylsucrose and tridecanoylsucrose
were more effective in stimulating insulin absorption as compared with decanoylsucrose
and dodecanoylsucrose. But — compared with TDM at concentrations of
0.03% — the sucrose esters were less effective in promoting nasal absorption [66] .
Sucrose cocoate (SL - 40) is produced by the chemical esterifi cation of coconut oil
with sucrose; it has frequently been used in cosmetic and dental preparations as an
excipient. When this excipient was formulated with insulin at 0.125, 0.25, and 0.5%
concentrations, the associated plasma levels of insulin increased rapidly; whereas
there was no enhancement of insulin plasma levels when insulin in saline was admin-
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 607
608 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
FIGURE 6 Changes in area under the curve (AUC 0 – 120 ) for blood glucose values in rats
that received 2 U insulin in presence of alkylmaltosides. Data represent mean change in
AUC 0 – 120 in arbitrary units (AU) ± standard error of the mean (SEM) compared with rats
that received insulin formulated without alkylmaltoside ( n = 3, 4). ( Reproduced from ref. 66
with permission of John Wiley & Sons. )
Octyl
Decyl
Dodecyl
Tridecyl
Tetradecyl
Pentadecyl
Hexadecyl
Concentration (%)
Alkylmaltosides
AUC Change (AU)
10
9
8
7
6
5
4
3
2
1
0
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
istered. The levels of insulin in plasma increased from a baseline value of 10 . U/mL
to a level of 200 . U/mL after the nasal administration of sucrose cocoate - containing
formulations; the T max was found to be 10 min (Figure 8 ) [67] .
Chitosan is a linear cationic polysaccharide made up of copolymers of glucosamine
and N - acetylglucosamine. It is commercially obtained by alkaline deacetylation
of chitin [53, 68] and has been used for the nasal delivery of a number of
drugs. The usefulness of chitosan in the enhancement of nasal absorption was
reported fi rst by Illum [69] . Later, Illum and his group also published experimental
results indicating that solution formulations with 0.5% chitosan promoted the
absorption of nasally administered insulin in rat and sheep [70] .
The use of chitosan nanoparticles in the enhancement of the nasal absorption
of insulin has also been investigated in rabbits. Chitosan nanoparticles were prepared
by ionotropic gelation of chitosan and pentasodium tripolyphosphate (TPP).
Two types of chitosan were used in the hydrochloride salt form (Seacure ® 210 Cl
and Protasan ® 110 Cl). Insulin loaded in chitosan 210 Cl produced a signifi cant
increase in systemic absorption and also the greatest decline in the level of blood
glucose, as much as 60% of basal levels; this result was found to be signifi cantly
different from that obtained with the insulin control solution as well as the insulin –
chitosan solution [71] . A novel chitosan nanoparticle formulation was prepared by
again employing ionotropic gelation of TPP and chitosan glutamate (A1), and
postloaded insulin – chitosan nanoparticles (A2) were also prepared. Both these
FIGURE 7 Changes in area under the curve (AUC 0 – 120 ) for blood glucose values in rats
that received 2 U insulin in presence of alkanoylsucroses. Data represent mean change in
AUC 0 – 120 in arbitrary units (AU) ± SEM compared to rats that received insulin formulated
without alkanoylsucroses ( n = 3, 4). ( Reproduced from ref. 66 with permission of John Wiley
& Sons. )
Decyl
Dodecyl
Tridecyl
Tetradecyl
Concentration (%)
AUC Change (AU)
10
9
8
7
6
5
4
3
2
1
0
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Alkanoylsucroses
novel formulations were tested in rat and sheep models and also compared with
the insulin – chitosan solution formulation (A3) and subcutaneous injection of
insulin. In the rat model, it was found that A3 performed better than either A1 or
A2. A1 showed a minimum blood glucose concentration ( C min ) and time to reach
C min ( T min ) of 40% and 90 min, respectively. The F dyn — calculated as individual area
over the curve (AOC IN or SC . dose SC /mean AOC SC . dose IN or SC ) . 100 — was found to
be around 48%, whereas the insulin – chitosan nanoparticles showed F dyn values of
38 and 37 for the A1 and A2 formulations, respectively.
Since the concentrations of insulin to be administered in the sheep model would
have been large, the insulin - loaded chitosan nanoparticles were not investigated in
that model. However, the pharmacodynamics and pharmacokinetics of various
insulin – chitosan preparations were compared with postloaded insulin – chitosan
nanoparticles. It was found that chitosan solution and chitosan powder formulations
were far better, with the chitosan powder formulation showing a bioavailability of
17% as against 1.3 and 3.6% for the chitosan nanoparticles and chitosan solution
[72] . The effects of the concentration and osmolarity of chitosan and the presence
of absorption enhancers in the chitosan solution on the permeation of insulin
across the rabbit nasal mucosa in vitro and in vivo were investigated, and the same
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 609
610 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
investigation was also carried out in rats. The results suggested that, by increasing
the concentrations of chitosan from 0 to 1.5%, insulin permeation in vitro could be
increased. As compared with insulin without chitosan, there was a 25 - fold increase
in the permeability coeffi cient of insulin with 1.5% chitosan. A similar increase in
permeability was seen in the case of a hyperosmotic solution, and there was also an
FIGURE 8 Changes in ( a ) plasma insulin levels and ( b ) blood glucose levels after nasal
administration of 0.5 U insulin formulated in saline ( ) or 0.125% ( • ), 0.25% ( ), and 0.5%
(%) sucrose cocoate. Blood glucose concentrations at time 0 (250 – 350 mg/dL) were normalized
to a value of 100% in each animal. Data represent mean ± SEM, n = 3. Inserts represent
changes in plasma insulin AUC 0 – 60 ( a ) and changes in blood glucose AUC 0 – 120 ( b ).
( Reproduced from ref. 67 with permission of Elsevier. )
250
200
150
100
160
140
120
100
80
60
40
0 20 40 60 80 100 120
50
0
0 10 20 30 40 50 60
10
8
6
4
2
0
0 0.1 0.2 0.3 0.4 0.5 0.6
12
10
8
6
4
2
0
0.0 0.10.2 0.3 0.4 0.50.6
Plasma insulin (.U/mL)
Time (minutes)
AUC change (AU) AUC change (AU)
Concentration (%)
Concentration (%)
Initial blood glucose (%)
Time (minutes)
(b)
(a)
increase in permeability when insulin was formulated in deionized water as compared
with phosphate buffer 7.4 (Figure 9 ). An increase in permeability was also
seen when 5% hydroxypropyl . - cyclodextrin (HP . - CD) plus 1% chitosan was
included with insulin as compared with chitosan alone. However, there was no
statistical difference in permeability when insulin was formulated with 0.1%
ethylenediaminetetraacetic acid (EDTA) and 1% chitosan. In the in vivo studies
FIGURE 9 Effect of ( a ) concentrations, ( b ) osmolarity, and ( c ) medium of chitosan solution
on mean serum glucose concentrations after nasal administration of 10 IU/kg insulin to rats.
Bars represent the standard deviation (SD) of fi ve experiment. ( Reproduced from ref. 73
with permission of Elsevier. )
0% chitosan
0.5% chitosan
1.0% chitosan
1.5% chitosan
Control: plain
saline
SC
Hypo-osmolarity
Iso-osmolarity
Hyperosmolarity
Time (h)
Time (h)
Time (h)
Water
pH7.4PBS
Serum glucose
(% of initial value)
Serum glucose
(% of initial value)
Serum glucose
(% of initial value)
140
140
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0 1 2 3 4 5
0 1 2 3 4 5
120
100
80
60
40
20
0
0 1 2 3 4 5
(a)
(b)
(c)
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 611
612 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
in rats, insulin without chitosan did not show any reduction in blood glucose levels,
but as the concentrations of chitosan were increased, a hypoglycemic effect was
seen. With a formulation of 0.5% chitosan, the nadir in glucose levels was observed
within 1 h after administration; with 1 and 1.5% chitosan – insulin formulations, the
lowest levels were reached in about 2 h. In the case of isoosmotic formulations, the
lowering of blood glucose levels was weaker as compared with the hypo - or hyperosmolar
solutions; with deionized water and EDTA, effects similar to those of the
permeability studies were seen in terms of lower serum glucose levels. When insulin
was formulated with 1% chitosan, there was also a decrease in serum glucose levels,
and a similar effect was seen with both 1% chitosan and 0.1% EDTA. A similar
effect was also seen in the case of 5% Tween 80. Insulin formulated with 5% HP . -
CD and 1% chitosan was more effective in reducing serum glucose levels than when
5% HP . - CD or 1% chitosan was used alone. These studies suggested that the concentrations,
osmolarity, medium, and inclusion of absorption enhancers in chitosan
solution infl uence absorption following the nasal delivery of insulin [73] .
Varshosaz and co - workers recently explored the use of chitosan microspheres
and chitosan gels in the nasal delivery of insulin [74] . They prepared microspheres
of chitosan, and insulin was loaded into the microspheres. The formulations were
administered through the nasal cavity by the method described earlier by Hussain
et al. [47] . Varshosaz and colleagues found marked differences in the AUC of blood
glucose reduction and the AUC of insulin concentration between the untreated
controls and those animals that were treated either by intravenous administration
and insulin - loaded chitosan (using ascorbyl palmitate as the cross - linking agent) or
with microspheres. Serum glucose reduction in diabetic rats with nasal insulin –
chitosan microspheres was around 67% in the intravenous group, and absolute
bioavailability ( F abs ) was around 44% [74] . Furthermore, the same researchers prepared
insulin – chitosan gels containing different enhancers and investigated their
nasal absorption. As seen in Figures 10 and 11 , when EDTA was employed as an
absorption enhancer in the chitosan gels and administered nasally, a signifi cant
increase in insulin absorption and a decrease in serum glucose levels by as much as
FIGURE 10 Serum glucose level in four groups of diabetic rats ( n = 6): A , untreated control
group; B , intravenous administration of 4 IU/kg insulin; C , nasal administration of blank gel
base; D , nasal administration of 100 . L/kg chitosan gel containing 4000 IU/dL insulin.
( Reproduced from ref. 75 with permission of Taylor & Francis. )
450
400
350
300
250
200
150
100
50
200 150 100 50 0
0
250 300
Time (min) Serum glucose (mg/dL)
A
B
C
D
***
** *
*** ***
*** ***
*
***
46% compared to the intravenous route of administration were obtained. The
authors suggested that this formulation would be benefi cial in the controlled delivery
of insulin by the nasal route [75] .
5.6.6.2 Calcitonin
Calcitonin is a 32 – amino - acid peptide with a molecular weight of 3418 that is
cleaved from a larger prohormone. It has a single disulfi de bond, which causes the
amino terminus to assume a ring shape. Calcitonin is a hormone that participates
in calcium and phosphorus metabolism. The major source of calcitonin in mammals
is the parafollicular or C cells in the thyroid gland; it is also synthesized in other
tissues, including the lungs and intestinal tract. When serum calcium levels are elevated,
calcitonin is released from the thyroid gland. Salmon calcitonin (sCT) is more
potent and longer lasting than the mammalian form and hence is used clinically.
Calcitonin ’ s main action is to reduce the plasma concentration of calcium. At pharmacological
doses, calcitonin brings about reduction in bone resorption. It is indicated
for the treatment of postmenopausal osteoporosis in women with low bone
mass relative to healthy premenopausal women. The marketed version of intranasal
salmon calcitonin is Miacalcin (calcitonin - salmon) Nasal Spray (Novartis Pharma
AS, Huningue, France). Up to now, this calcitonin treatment has been approved only
for treatment in women. It has been reserved as a second - line treatment, since it
reduces fracture risk less than do other available treatments for osteoporosis.
Miacalcin Nasal Spray, fi rst manufactured by Sandoz Pharmaceuticals, was approved
by the FDA in 1995 and is now distributed by Novartis Pharmaceuticals in the
United States.
Pontiroli et al. [76] looked at the intranasal absorption of calcitonin in normal
subjects. Their study included six healthy volunteers who had no family history of
endocrine or metabolic diseases. Human calcitonin (Cibacalcin; Ciba - Geigy) was
administered intravenously or mixed with sodium glycocholate, a surfactant, in distilled
water and instilled as nose drops. Plasma concentrations of calcitonin were
found to be consistently higher when compared with intranasal administration of
FIGURE 11 Serum insulin levels of four groups of diabetic rats ( n = 6): A , untreated control
group; B , intravenous administration of 4 IU/kg insulin; C , nasal administration of blank gel
base; D , nasal administration of 100 . L/kg chitosan gel containing 4000 . g/dL insulin.
( Reproduced from ref. 75 with permission of Taylor & Francis. )
31,600
31,200
30,800
4,000
3,600
3,200
2,800
2,400
2,000
1,600
1,200
800
400
0
Serum insulin (pmol/L)
0 50 100 150 200 250
AB
C
D
Time (min)
*
***
***
*** ***
** *
* *
* *
**
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 613
614 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
the same dose of calcitonin. However, it was found that intranasal administration
effected a reduction in the plasma concentrations of calcium to a similar extent as
that seen when a similar dose of intravenous calcitonin was given. The data also
showed that although a higher dose of calcitonin by the intranasal route brought
about a higher plasma calcitonin concentration, there was no difference in the
decrease in plasma concentrations when calcitonin was given at doses of 500 and
1000 . g.
Polyacrylic acid aqueous gel enhances the absorption of calcitonin after nasal as
well as rectal administration. When [Asu1,7] - eel calcitonin (10 U/kg) was administered
nasally in polyacrylic acid gel at a concentration of 0.1% w/v, a prominent
hypocalcemic effect was seen in the fi rst 30 min. Nasal administration of [Asu1,7] - eel
calcitonin in saline had no hypocalcemic effect at the same dose when given by the
nasal route. In addition to this, the effect of [Asu1,7] - eel calcitonin in the dose range
of 1 – 10 U/kg has also been studied. The resulting data showed that a rapid reduction
in plasma calcium concentrations can be achieved at doses of 5 and 10 U/kg; however,
at doses of 1 U/kg only a small reduction in the plasma calcium concentration was
observed, suggesting that polyacrylic acid gel can be used for the intranasal administration
of peptides such as calcitonin. The possible side effects, however, were not
known at the time the study was performed [76 – 78] .
Commercially available sCT has been used mainly in the treatment of bone -
related diseases such as Paget ’ s disease, hypercalcemia, and osteoporosis. It can
be used to alleviate pain due to its analgesic properties; hence there may be a
need for its frequent administration. As reported in the literature, owing to chemical
and enzymatic degradation, the polypeptide has a short half - life of only about
14 min. Hence its use calls for measures to improve its in vivo stability and overcoming
the problem of rapid clearance. Polyethylene glycol (PEG) has been
extensively used for this purpose in association with a variety of agents. Lee at
al. [79] have prepared and characterized PEG - modifi ed sCT and studied how
blood clearance is affected with and without PEGylation. Succinimidyl carbonate
mono - methoxy - polyethylene glycol (SC - mPEG) was prepared using mPEG, which
has a molecular weight of 12,000, and bis( N - succinimidyl) carbonate as per the
procedure of Miron and Wilcheck [80] . There are three main reactive sites for
the activated PEG in sCT: the N - terminal amino group (Cys 1 ) and the . - amino
groups of two lysine residues (Lys 11 and Lys 18 ), as shown in Figure 12 . The site
of conjugation of the PEG on sCT determines the stability of the sCT in the face
of enzymatic attacks; hence the three possible mono - PEG - sCTs — Cys 1 - PEG - sCT,
Lys 11 - PEG - sCT, and Lys 18 - PEG - sCT — can withstand the effects of proteolytic
enzymes. It was found that, on PEGylation, the plasma half - life improved to 11.2
and 54.0 min for the mono - PEG - sCT and the di - PEG - sCT as compared with
the non - PEGylated sCT, which showed a plasma half - life of about 4.7 min
(Figure 13 ).
FIGURE 12 Primary structure of salmon calcitonin. Possible PEGylated sites are Cys 1 ,
Lys 11 , and Lys 18 . ( Reproduced from ref. 79 with permission of Taylor & Francis. )
Cys1-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys11-Leu-Ser-Gln-Glu-Leu-His-Lys18-Leu-Gln-Thr-Tyr-
Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2
The same group — Shin et al. [81] — did nasal absorption studies of a low -
molecular - weight PEG (2000) instead of the 5000 and 12,000 they tried previously.
They also used commercially available succinimidyl - propionated monomethoxy -
poly(ethylene glycol) - 2000 (SP - mPEG) for the chemical modifi cation instead of
synthesizing it, as in their previous studies. The PEGylation of sCT was done by
mixing SC - mPEG and sCT, and this mixture was shaken at 25 o C. The reaction
mixture was stopped by using an excess of 1.0 M glycine solution. This conjugated
mixture was then subjected to size exclusion chromatography. Radioiodination of
the sCT and the PEG - sCT were carried out and the radiolabeled 125 I - sCT and
125 I - mono - PEG2000 - sCT were then used for the nasal absorption studies.
Tissue distribution studies were also done in rats after nasal administration. As
seen in Figure 14 and Table 2 , it was found that the elimination half - life of the
unmodifi ed sCT was 199 min, whereas the SP - mPEG2000 - modifi ed sCT showed an
increased terminal elimination with a half - life of 923 min. It was also found that the
SP - mPEG2000 - modifi ed sCT took a signifi cantly longer time to reach its maximum
concentration, 520 min, as compared with the 77 min for the unmodifi ed sCT, and
the AUC was found to be 20,638 . g/min/mL, which is much higher than the 3650 . g/
min/mL for the unmodifi ed sCT. The authors reported that the increase in the terminal
half - life observed could be due to a fl ip - fl op phenomenon. Also, when the
tissue distribution of the formulation was examined 12 h after administration, the
highest radioactivity was found in the liver. The details of the biodistribution studies
are as shown in Table 3 .
The same group [82] further studied the stability of these mono - PEG2000 - sCT
and the unmodifi ed sCT in the rat nasal mucosa. It was found that PEGylated sCT
exhibited signifi cant resistance against trypticlike and nonspecifi c enzymatic degradation.
Ahsan et al. [49] showed that when sCT was formulated with alkylglycosides,
bioavailability was enhanced following both nasal and ocular administration. Miacalcin
(Novartis Pharma AS, Huningue, France) was used to prepare the formulation
FIGURE 13 Blood clearance of PEGylated salmon calcitonins in rat. ( Reproduced from
ref. 79 with permission of Taylor & Francis. )
100
80
60
40
20
0
0 10 20 30 40 50
Time (min)
60
Remaining radioactivity %
Native sCT
Mono-PEG-sCT
Di- PEG-sCT
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 615
616 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
FIGURE 14 Average serum concentration of intact sCT – time curves following nasal
administration of unmodifi ed sCT ( • ) and Mono - PEG2K - sCT ( ) in rats ( n = 9 each).
( Reproduced from ref. 81 with permission of The Pharmaceutical Society of Japan. )
10
1
0 500 1000 1500 2000 2500
Time (min)
Serum concentration (ng/mL)
TABLE 2 Pharmacokinetic Parameters (Mean ± SD) of Unmodifi ed sCT and
Mono - PEG2k - sCT obtained after nasal administration to rats ( n = 9 each)
Parameter Unmodifi ed sCT Mono - PEG 2k - sCT
C max (ng/mL) 10.5 ± 4.7 12.9 ± 3.0
t max (min) 77 ± 22 520 ± 167 a
t 1/2, . z (min) 199 ± 97 923 ± 389 a
CI/F (mL/min) 7.4 ± 5.2 1.3 ± 1.0 a
V ss / F (mL) 1802 ± 811 1392 ± 450
AUC (ng · min/mL) 3650 ± 1894 20638 ± 9486 a
AUC/D (ng · min/mL/ng) 0.18 ± 0.09 1.03 ± 0.48 a
MRT . a (min) 314 ± 131 1505 ± 560 a
Source : From ref. 74 .
a Signifi cantly different from unmodifi ed sCT ( p < 0.05).
TABLE 3 Extent of Total Radioactivity (Mean ± SD)
in Various Body Organs after Nasal Administration
of Unmodifi ed sCT and Mono - PEG2k - sCT to Rats
( n = 9 each)
Tissue
Radioactivity (%) in Whole Organ a
Unmodifi ed SCT Mono - PEG 2k - sCT
Liver 0.80 ± 0.41 1.03 ± 0.65
Kidney 0.30 ± 0.14 0.52 ± 0.24
Lung 0.18 ± 0.12 0.20 ± 0.12
Heart 0.06 ± 0.03 0.13 ± 0.05 b
Spleen 0.04 ± 0.02 0.10 ± 0.05
Thyroid 0.04 ± 0.03 0.05 ± 0.02
Source : From ref. 74 .
a Determined 12 h after administration.
b Signifi cantly different from unmodifi ed sCT ( p < 0.05).
in solutions containing different concentrations of tetradecylmaltoside and octylmaltodise.
These formulations were administered as described by Ahsan et al. [49] .
It was found that when calcitonin was formulated in saline, absorption after administration
by the nasal route was negligible; a similar result was seen with 0.125%
OM. However, when calcitonin was formulated with 0.125% TDM, absorption was
found to be increased, and maximal absorption ( Tmax ) was achieved after 10 min.
The AUC 0 – 40 was found to be fourfold higher than with saline and OM formulations.
The bioavailability was found to be 53% as compared with intravenously administered
calcitonin at the same dose of 2.2 U. The AUC 0 – 40 was found to be 6250 pg/
mL · min as compared with 3500 pg/mL · min when the concentration of TDM was
increased to 0.25% from 0.125%. It was also found that in the absence of the absorption
enhancer increasing the amount of calcitonin from 2.2 to 22 U did not increase
absorption by the nasal route. However, when formulated in the presence of 0.25%
TDM, there was a 23 - fold increase in the relative bioavailability of calcitonin. Also,
when calcitonin was formulated with 0.25% TDM and given as nose drops, there
was a signifi cant reduction in the plasma calcium concentration as compared with
the saline formulation.
5.6.6.3 Low - Molecular - Weight Heparins
Low - molecular - weight heparins (LMWHs) are fragments of natural heparin that
are obtained by either enzymatic degradation or chemical depolymerization of
unfractionated heparins (UFHs). The molecular weight of LMWHs, a heterogeneous
mixture of sulfated glycosaminoglycans, is about one - third that of UFHs.
Owing to the variations in the distribution of molecular weights, they show differences
in their affi nity for plasma proteins, activity against factor Xa, and thrombin,
as well as duration of activity. They have been proven to be useful in the treatment
and prevention of deep vein thrombosis. Of late, LMWHs have been preferred to
therapy with conventional heparin. However, one of the main disadvantages of the
use of LMWHs on a regular, noninstitutional basis is that they must be delivered
by the subcutaneous or intravenous route. There have been concerns regarding
patient compliance, longer hospital stays, and the need for skilled health professionals
for therapeutic drug monitoring and administration. This has prompted a number
of researchers to seek alternative forms of delivery.
The nasal administration of LMWHs was investigated in Sprague - Dawley rats
using TDM as an absorption enhancer [83] . TDM was used at concentrations of
0.125 and 0.25%. Lovenox (Aventis Pharmaceuticals, Bridgewater, NJ) (enoxaparin
sodium injection), a commercially available LMWH, was prepared with 0.25% TDM
and the nasal absorption was compared with and without TDM. It had been previously
reported that a plasma anti – factor Xa level of 0.20 U/mL or higher is required
for an antithrombotic effect to be considered therapeutic [83] . It was found that
enoxaparin when formulated only in saline did not produce a therapeutic anti – factor
Xa level. However, when enoxaparin was formulated with 0.25% TDM, a signifi cant
increase in the AUC and Cmax for the anti – factor Xa level was observed. Dalterparin,
another commercially available LMWH, showed a similar response. When UFH was
formulated with TDM, it produced an increase in the anti – factor Xa levels as compared
with saline, but it was not in the therapeutic range (Figure 15 ).
The bioavailability ( Fabs ) of enoxaparin achieved by the subcutaneous route was
found to be 83% of that achieved by the intravenous route. In the absence of TDM,
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 617
618 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
the nasal F abs was 4.0%, but in the presence of 0.25% TDM, the F abs was 19% compared
with the intravenous route. The relative bioavailability ( F rel ) of nasal enoxaparin
plus 0.25% TDM was found to be 23% compared with the subcutaneous
route.
The use of alkanoylsucroses in the enhancement of nasal absorption of LMWHs
was investigated by our group [84] . As seen in Figure 16 a , enoxaparin plus 0.125%
octanoylsucrose showed no signifi cant increase in anti – factor Xa levels; even when
the concentration was increased to 0.5%, it barely reached the therapeutic level of
0.2 U/mL. Similar results were reported for 0.125% decanoylsucrose; however, when
the concentration was increased to 0.25 or 0.5%, there was an appreciable and rapid
rise in anti – factor Xa levels (Figure 16 b ). The inability of octanoylsucroses and
0.125% decanoylsucrose to increase anti – factor Xa levels is attributed to the critical
micellar concentration as reported by the authors [84] . In the case of dodecanoylsucroses,
all the concentrations produced anti – factor Xa levels well above the therapeutic
level required for an antithrombotic effect (Figure 16 c ).
Our group also compared the effi cacy and potency of alkanoylsucroses with those
of the well - known absorption enhancer 1% sodium glycocholate (Figure 16 d ); the
results were similar to those seen with 0.5% dodecanoylsucrose. Also, 0.5%
dodecanoylsucrose showed the highest increase in C max , and it was found that an
increase in the concentration of alkanoylsucroses led to a subsequent increase in
the C max . When the absolute and relative bioavailabilities of nasal LMWH plus 0.5%
dodecanoylsucrose were compared with those of 1% sodium glycocholate, similar
profi les were found [84] .
FIGURE 15 ( a ) Nasal administration of 100 U of ( A ) enoxaparin, ( B ) dalteparin, or
( C ) UFH formulated with ( • ) and without ( ) 0.25% tetradecylmaltoside. Data represent
mean ± SEM, n = 3. Asterisks indicate results that are signifi cantly different from those
obtained with the drug formulated with saline, P < 0.05. ( b ) Administration of 100 U of
enoxaparin via the subcutaneous ( ), intravenous ( ), and nasal ( ) routes. Nasal administration
was performed with a formulation that included 0.25% TDM. Data represent mean
± SEM, n = 3. ( Reproduced from ref. 83 with permission of John Wiley & Sons. )
A. Enoxaparln B. Dalteparin C. UFH
Anti–factor Xa activity (U/mL)
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
0 0 0 120 120 120 240 240 240 360 360 360 480 480 480
Time (min)
Anti–factor Xa activity (U/mL)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 60 120 180 240 300 360 420 480
Time (min)
(a) (b)
FIGURE 16 Changes in anti – factor Xa activity after nasal administration of enoxaparin
formulated in saline or in presence of different concentrations of the following: ( a ) octanoylsucrose;
( b ) decanoylsucrose; ( c ) dodecanoylsucrose; ( d ) sodium glycocholate and dodecanoylsucrose
to anesthetized rats (enoxaparin dose, 330 U/kg). Data represent mean ± SEM,
n = 3, 5. ( Reproduced from ref. 84 with permission of Pharmaceutical Press. )
(a)
(c)
(b)
(d)
0 100 200 300 400
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Anti–Factor Xa activity (U/mL)
Time (min)
0.5% Decanoylsucrose
0.25% Decanoylsucrose
0.125% Decanoylsucrose
Saline
0 100 200 300 400
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Anti–factor Xa activity (U/mL)
Time (min)
0.5% Octanoylsucrose
0.25% Octanoylsucrose
0.125% Octanoylsucrose
Saline
0 100 200 300 400
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Anti–Factor Xa activity (U/mL)
Time (min)
0.5% Dodecanoylsucrose
0.25% Dodecanoylsucrose
0.125% Dodecanoylsucrose
Saline
0 100 200 300 400
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Anti–Factor Xa activity (U/mL)
Time (min)
1% Sodium glycocholate
0.5% Dodecanoylsucrose
Saline
The authors also studied the infl uence of the chain length of alkylmaltosides on
the nasal absorption of enoxaparin. The results indicated that increases in the concentration
of alkylmaltosides increased the AUC for plasma anti – factor Xa; it was
found that the absolute and relative bioavailabilities of enoxaparin increased by
twofold with an increase in alkyl chain length from 8 to 14 carbons. Of all the alkylmaltosides,
TDM was found to be the most potent absorption enhancer [85] .
Furthermore, we have also shown the effi cacy of cyclodextrins in enhancing
absorption following the nasal delivery of LMWHs. Three different cyclodextrins
were employed: . - cyclodextrins ( . - CD), hydroxypropyl . - CD (HP . - CD), and
dimethyl . - CD (DM . - CD). . - CD showed therapeutic levels of anti – factor Xa only
at 2.5 and 5% . - CD, but there was no signifi cant difference between the two concentrations,
which was attributed to their solubility limit. In the case of HP . - CD,
neither 1.25 nor 2.5% produced an appreciable increase in anti – factor Xa levels;
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 619
620 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
only 5% HP . - CD showed levels above 0.2 U/mL, which, however, was not signifi -
cant. Unlike the other two cyclodextrins, 1.25 and 2.5% DM . - CD showed a fourfold
increase in AUC profi les. The studies reported that 5% DM . - CD produced the
greatest increase in the bioavailability of LMWHs, with an eightfold increase in the
AUC profi le. It was also found that the reduction in transepithelial electrical resistance
(TEER) and changes in tight junction protein ZO - 1 distribution facilitated
by 5% DM . - CD were much greater than with . - CD or HP . - CD [86] . Recently our
group has shown that positively charged polyethylenimines (PEI) increases the
nasal absorption of LMWHs by reducing the surface negative charge of the drug.
When PEI 1000 kDa was employed at a concentration of 0.25%, a four - fold increase
in the absolute and relative bioavailabilities was observed in comparison with the
control formulation of LMWH plus saline [87] .
5.6.6.4 Azetirelin
Azetirelin is a novel analog of the tripeptide thyrotropin - releasing hormone
(TRH). It was discovered in 1969 when two different groups of researchers, led by
Guillemin and Schally, showed that the hypothalamic substance that causes the
anterior pituitary gland to release thyrotropin (thyroid - stimulating hormone, or
TSH) is l - pyroglutamyl - l - histidyl - l - prolineamide ( l - pGlu - l - His - l - ProNH2), now
called TRH. In azetirelin the pyroglutamyl moiety of the TRH is replaced by an
(oxo - azetidinyl) carbonyl moiety. It has been reported that the inhibition of
pentobarbital - induced sleep and reserpine - induced hypothermia due to azetirelin
in mice, as opposed to TRH, are about 10 – 100 times more effective as well as 8 – 36
times longer lasting. Azetirelin is stable in plasma and degrades much more slowly
than TRH in brain homogenates, thus showing improved pharmacological potency
as well as effi cacy over TRH. It is highly potent when given intravenously; however,
when administered by the oral route, it shows very low bioavailability of only 2%
[88 – 91] .
Kagatani et al. [90] studied the effect of acylcarnitines as drug absorption enhancers
for the nasal delivery of azetirelin in a rat model. A buffered azetirelin sample
solution was administered intranasally, as described previously [47] . The nasal and
oral absorptions of azetirelin were then compared. The Fabs after nasal absorption
was found to be 17.1%, which was 21 times greater than the 0.8% after oral administration.
As reported above, a pilot study of oral azetirelin showed a bioavailability
of about 2%. A bioavailability of about 20% was seen in the case of nasally administered
TRH in humans as well as rats. The authors predicted that since azetirelin
is an analog of TRH, its pharmacokinetic properties after nasal delivery in humans
could also be about 20% [90, 91] .
5.6.6.5 Growth Hormones
Recombinant human growth hormone (hGH) is a 22 - kDa protein drug having 191
amino acids. It has been used to treat a number of conditions, including short stature
in children, Turner syndrome, and chronic renal failure. It is said to play an important
role in the metabolism of proteins, carbohydrates, and fats as well as electrolytes
and hence infl uences weight and height. It has been reported that hGH secretion
in humans is pulsatile, showing low basal serum levels in between peaks. It has been
suggested that this secretory pattern of hGH can be mimicked by the nasal route
as opposed to painful subcutaneous injections [8, 92, 93] .
In a pharmacokinetic - based study by Hedin et al. [94] , hGH was administered
with a nasal permeation enhancer, sodium tauro - 24, 25 - dihydrofusidate (STDHF),
in patients defi cient in growth hormone (GH) using a reprocessed lyophilized form
of hGH. The lyophilized material was formulated with STDHF and all the subjects
received the formulation by both the nasal and subcutaneous routes. The dose given
by the subcutaneous route was a standard dose of 0.1 IU/kg body weight (BW),
whereas three different doses (of 0.2, 0.4, and 0.8 IU/kg BW) of the nasal formulation
were given. As compared with the subcutaneous route, all three nasal formulations
showed a rapid increase in the plasma levels of hGH, with Tmax being reached
15 – 25 min after administration, as compared with 3 – 4 h in the case of the subcutaneous
route. However, the Cmax was higher in the case of the latter route, and the nasal
formulations touched baseline after 3 – 4 h, as compared with 14 – 18 h after subcutaneous
delivery.
Several studies have shown that frequent doses of hGH are more benefi cial than
the total amount given as a single dose and that higher peaks of plasma hGH with
low troughs are found in taller children; hence the nasal therapy for defi ciencies in
hGH would not only be more convenient but also offer advantages, including a rapid
decrease of the peaks to zero levels and a mimicking of the pulsatile pattern of the
endogenous hormone [94 – 97] . Nasal irritation studies also were carried out, indicating
that the nasal formulations showed only local short - term irritation.
Didecanoylphosphatidylcholine (DDPC) and . - cyclodextrin ( . - CD) were used
as enhancers and reversibility studies were carried out in vivo in rabbits. Three different
combinations were used: DDPC, . - CD, and DDPC plus . - CD for the nasal
administration of hGH. Vermehren et al. [98] used intravenous hGH as the reference.
When hGH was administered with . - CD as a powder, 23.6% bioavailability
was seen, as compared with 18.1% Frel when given at the same time as two powders.
When hGH only was given, a bioavailability of 8.3% was attained. DDPC plus hGH
together showed a Frel of 22.3 and 21.5% when given as two powders, while simultaneous
administration of DDPC plus . - CD and hGH as two powders showed a
Frel of 14.3%. When dosed as one single powder, it showed a Frel of 31.9%. Reversibility
of the enhancer effect was seen when a dose of hGH in enhancer - free formulation
was given 30 min after dosing of the test formulations; this resulted in
reduction in the AUC and Cmax by half.
Another group studied the enhancer effect of DDPC on the pharmacokinetics
and the biological activity of nasally administered hGH in GH - defi cient patients.
Three different doses — 0.05, 0.10, and 0.20 IU/kg with DDPC — were given by the
nasal route and 0.10 and 0.015 IU/kg were given by the subcutaneous and intravenous
routes. A short - lived serum GH peak was seen in the intravenous treatment,
showing a peak value of around 128 . g/L, whereas the subcutaneous route showed
a peak level of 13.98 . g/L and nasal doses showed peaks of about 3.26, 7.07, and
8.37 . g/L for the three treatment doses. The bioavailabilities of the nasal doses were
found to be 7.8, 8.9, and 3.8%, respectively, as compared with the Fabs of 49.5% for
the subcutaneous dose. It was also found that the serum insulinlike growth factor 1
(IGF - 1) increased only upon subcutaneous administration There was no change in
the serum IGF protein binding protein 3 levels in any of the nasal doses or in the
subcutaneous or intravenous doses [92] .
NASAL DELIVERY OF PEPTIDE AND HIGH-MOLECULAR-WEIGHT DRUGS 621
622 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
Leitner et al. [93] tried to overcome the limitations posed by the low bioavailability
of nasally delivered hGH due to the drug ’ s high molecular weight and
hydrophilicity. The strategy employed was to use dry polymer particles to enhance
absorption, taking advantage of the fact that these polymers would form a gellike
layer and hence be cleared slowly, giving a longer circulating drug. Due to the presence
of covalent immobilization of sulfhydryl groups on the backbone of the thiolated
polymers or thiomers, the permeation - enhancing properties of these thiomers
were improved. They have also been reported to have mucoadhesive and enzyme -
inhibitory properties, and the addition of glutathione (GSH) was found to increase
their permeation - enhancing properties further [93, 99 – 103] . A polycarbophil –
cysteine (PCP – Cys) microparticulate system was prepared using GSH and hGH,
and this was added to the formulation. The in vitro release profi les of the PCP – Cys
and unmodifi ed PCP containing hGH proved to be similar. Three different formulations
were tested for in vivo nasal absorption: PCP – Cys/GSH/hGH microparticles,
PCP/hGH microparticles, and mannitol/hGH powder against a subcutaneous hGH.
The Frel for mannitol/hGH powder was around 2.40%, the PCP – Cys/GSH/hGH
microparticles showed a Frel of 8.11%, and PCP/hGH showed 2.70%, which represents
a threefold increase in nasal uptake when thiomer/GSH is used in the formulation.
The microparticulate formulation of PCP – Cys/GSH/hGH showed a sixfold
higher plasma concentration when compared to the PCP – Cys/GSH/hGH gel
formulation.
5.6.7 NASAL DELIVERY OF NONPEPTIDE MOLECULES
5.6.7.1 Morphine
Morphine is a potent narcotic analgesic used preoperatively and as an anxiolytic
agent in pediatric patients; it is also used in the management of postoperative pain
as well in moderate to severe pain in cancer. Oral morphine in solution, immediate -
release, or controlled - release formulations shows a bioavailability of only about
20%. Morphine absorption in humans is poor, and only 10% bioavailability is
obtained when it is given as a solution rather than intravenously. Chitosan has been
reported to be a potent absorption enhancer and greatly improves the absorption
of small polar molecules and peptides. Intranasal administration was carried out in
a sheep model; various formulations were also tested in humans and comparisons
versus the intravenous route were made. The sheep model was used for nasal delivery
because it has been reported that testing in sheep is highly predictive of results
in humans [104, 105] . In sheep studies, when morphine HCl solution was given
nasally (control), the Cmax obtained was limited: 151 n M with a Frel of 10%, with a
Tmax of about 20 min, suggesting a slow rate of absorption. When 0.5% chitosan was
formulated with morphine as a solution, the Cmax increased to 657 n M , and Frel was
26.6%. The rate of absorption was also increased, as evidenced by a Tmax of 14 min.
With the formulation of chitosan into microspheres, the Cmax was 1010 n M, Tmax
about 8 min, and Frel about 54.6%. A further increase in nasal absorption was seen
when morphine was formulated as a powder consisting of starch microspheres and
L - . - lysophosphatidylcholine (LPC), with Cmax being 1875 n M, Tmax about 10 min, and
Frel about 75% (Figure 17 ). In the case of human phase I clinical trials, a dose of
10 mg morphine sulfate led to a mean C max of 336 n M following 30 min of intravenous
administration. A 0.5% chitosan and morphine HCl solution led to a C max of 98 n M
and a T max of about 16 min. The plasma half - life ( t 1/2 ) in the case of nasal administration
was found to be 2.98 h, as compared with 1.67 h via the intravenous route. The
mean bioavailability with the nasal solution of morphine plus chitosan was 56%.
Furthermore, the powder formulation comprising chitosan and morphine HCl
showed a C max of 92 n M, T max of 21 min, t 1/2 of 2.72 h, and F rel of about 56% (Figure
18 ). The nasal formulations were reported to be well tolerated.
5.6.7.2 Benzodiazepines
Diazepam has been the standard or preferred option for the treatment of all types
of seizures in both children and adults. However, it has disadvantages, including a
short duration of action, so that in some cases diazepam must be given rectally in
order to manage prolonged seizures. Moreover, its use in the community is restricted
because of the need for privacy, especially in the case of adult patients. Finally, the
intravenous route is also reported to be inconvenient, since nonprofessional caregivers
may not be comfortable enough to administer the drug in this way. Midazolam
has been reported to be clinically effective with both intravenous and oral administration
for the induction of sedation and reduction of anxiety. Owing to the drawbacks
mentioned above, intranasal formulations of benzodiazepines could be highly
benefi cial [106, 107] .
FIGURE 17 Morphine plasma concentration after nasal administration of morphine formulations
in sheep: Mor Sol, morphine solution; Mor Chi Sol, morphine solution containing
chitosan; Mor Chi PWD, morphine chitosan powder; Mor SMS LPC, starch microspheres
with lysophosphatidylcholine and morphine as a freeze - dried powder. ( Reproduced from
ref. 105 with permission of the American Society for Pharmacology and Experimental
Therapeutics. )
1500
1000
500
0
0 20 40 60 80 100 120
Time (min)
Morphine plasma concentration (nmol/L)
Mor Sol (F2)
Mor Chi Sol (F3)
Mor Chi PWD (F4)
Mor SMS LPC (F5)
NASAL DELIVERY OF NONPEPTIDE MOLECULES 623
624 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
Midazolam, Triazolam, and Flurazepam The feasibility of intranasal administration
of midazolam, fl urazepam, and triazolam has been studied and compared with
oral absorption in dogs. There was a 3.4 - fold increase in the C max after nasal administration,
from 5.5 – 8.7 ng/mL to 17.4 – 30.0 ng/mL. The mean t 1/2 showed comparable
values for both routes. The T max obtained after nasal administration of midazolam
was found to be 15 min, as compared with the 15 – 45 min observed for oral dosing,
while the C max after nasal administration was 6.5 – 20.3 ng/mL, as compared with
3.0 – 8.6 ng/mL observed for the oral route. Like midazolam and triazolam, fl urazepam
also showed a shorter half - life, 15 min, as compared with 15 – 45 min with oral
administration. The C max for oral administration was 0.14 – 0.59 ng/mL; after nasal
administration it was in the range of 2.6 – 11.1 ng/mL, a 16.4 - fold increase. Since the
gastrointestinal tract at bedtime is likely to be in the fed state, causing a twofold
decrease in the absorption of midazolam and triazolam, the nasal route may be a
better option for the treatment of amnesia, since these drugs cross the nasal mucosa
effectively without the use of an absorption enhancer, as shown in these studies
[108] .
In situ nasal absorption studies of midazolam were carried out in rats. The effects
of solution concentration, osmolality, and pH on nasal absorption were studied using
the in situ perfusion technique. The absorption of midazolam was reported to be
prevented at osmolalities in the range of 142 – 450 mOsm/kg; however, a hypoosmotic
3 - mOsm/kg solution resulted in signifi cant absorption, where the pH rose from 3.3
to 6.5. No lag time in absorption was observed when the solutions were buffered at
a pH of either 5.5 or 7.4; however, at pH 3.3, no absorption was seen, suggesting
FIGURE 18 Morphine plasma concentration in human volunteers after intravenous administration
of morphine and after nasal administration of morphine as chitosan solution and
powder formulations: Mor Chi Sol, morphine solution containing chitosan; Mor Chi PWD,
morphine – chitosan powder; IN, intranasal. ( Reproduced from ref. 105 with permission of the
American Society for Pharmacology and Experimental Therapeutics. )
IV Mor
IN Mor Sol
IN Mor PWD
Morphine plasma concentration (nmol/L)
1000
100
10
1
0 2 4 6 8 10 12
Time (h)
that pH was the main factor determining the absorption of midazolam (Figure 19 )
[109] .
The pharmacokinetics and pharmacodynamics of midazolam after nasal administration
were investigated in healthy volunteers in two different studies in comparison
with the intravenous route [110, 111] . Studies reported in 1997 demonstrated
that intranasal midazolam was rapidly absorbed, with maximal concentration
attained in the range of 10 – 48 min, with a mean of 25 min. These results were only
the maximum concentration achieved and the time taken to reach this maximum.
The maximum concentration reported after intranasal administration was in the
range of 91.0 to 224.3 ng/mL, with a mean of 147 ng/mL. Bioavailability reported in
this study [110] was about 50%, in line with the results obtained with oral administration,
as reported in an earlier study [112] . Knoester et al. [111] also carried out
similar studies using a concentrated intranasal spray in healthy volunteers. The
concentrated preparation was prepared by mixing midazolam HCl in a mixture of
water and propylene glycol, pH 4. A Spruyt Hillen (IJsselstein, Netherlands) intranasal
device was used to deliver the dose. Besides nasal irritation lasting 10 min and
teary eyes, no other discomfort was reported. Midazolam was rapidly absorbed on
nasal administration, showing a maximum concentration of 72 ng/mL within 14 min.
A mean bioavailability of 0.89 was obtained. It was reported that intraindividual
basal electroencephalogram (EEG) activity after intranasal administration was
comparable with that after intravenous administration (Figures 19 and 20 ) .
Diazepam As mentioned earlier, because of shortcomings of rectal administration,
the nasal delivery of diazepam has gained interest. The nasal bioavailability of
diazepam in sheep was estimated and further compared with results obtained earlier
in humans and rabbits [106] ; in this study, human and rabbit nasal bioavailability
for the fi rst 30 min was reported to be 37 and 54%, respectively [113] . Diazepam
solubilized in PEG 300 was used for nasal administration via a modifi ed nasal device,
a Pfeiffer unit dose (Princeton, NJ). The sheep received the nasal formulations in a
fi xed standing position such that the head was slightly tilted back. It was found that
the serum concentration after administration of a 7 - mg solution of diazepam was
FIGURE 19 Fit of composite model to concentration – time data for midazolam and 1 -
hydroxymidazolam in one volunteer. Solid lines indicate the time course of midazolam concentrations
( ) and 1 - hydroxymidazolam concentrations ( ) after intravenous administration.
Dotted lines indicate the time course of midazolam concentrations ( ) and 1 - hydroxymidazolam
concentrations ( ) after intranasal administration. ( Reproduced from ref. 111 with
permission of Blackwell Publishing. )
1000
100
10
1
0.1
0 60 120 180 240 300 360 420 480 540
Time (min)
Concentration (.g/L)
NASAL DELIVERY OF NONPEPTIDE MOLECULES 625
626 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
lower than that obtained with a 3 - mg solution, suggesting a low nasal bioavailability.
The bioavailability after the initial 30 min was found to be 15%, as compared to the
earlier mentioned bioavailability in human and rabbit, and a C max of 934 ng/mL with
a T max of 5 min. The difference in bioavailability between animal and human was
larger when periods shorter than 0 – 60 min were used in the calculations. When the
results among the three species were compared, it was found that the bioavailability
in sheep was higher than that in humans during the early or initial phases, after
which the reverse was observed. Lindhardt et al. [113] used a profi le that took into
account the observation period with respect to the rate of bioavailability and found
that similar profi les with respect to rate were observable in relation to all of the
three nasal formulations given in humans; moreover, an optimal correlation between
sheep and rabbit was observed. The authors suggested that use of the jugular vein
in sampling blood from sheep could have resulted in the low bioavailability.
5.6.7.3 Buprenorphine
Buprenorphine is a derivative of the morphine alkaloid thebaine and is a partial
opioid antagonist. It exerts an agonistic effect on the . - muscarinic receptors and an
antagonistic effect on the . type. Buprenorphine at lower doses produces suffi cient
agonist effect to enable opioid - addicted individuals to discontinue the use of opioids
without experiencing withdrawal symptoms. It has been reported that an intravenous
dose of 0.3 mg of buprenorphine is equivalent to 10 mg of morphine and that
oral delivery of buprenorphine results in a bioavailability of only about 15% due
to fi rst - pass metabolism. In addition to the intravenous formulation, there is a sublingual
formulation offering the advantage of avoiding the fi rst - pass metabolism
effect. A clinical trial of nasally administered buprenorphine was reported in 1989
[114] . Buprenorphine is highly lipophilic and hence easily absorbed across the nasal
epithelium. The buprenorphine formulation used in the clinical trial was prepared
in 5% dextrose solution and a Pfeiffer atomizing pump operated manually was used
to deliver it. The mean T max and C max for the intranasal dose were 30.6 min and
1.77 ng/mL, respectively. A relative nasal bioavailability of about 48.2% was attained.
FIGURE 20 Individual plasma concentration – time curves for midazolam (solid lines) and
1 - hydroxymidazolam (broken lines) after intranasal administration of 5 mg midazolam. The
bold curves represent the mean pharmacokinetic model fi t to the data. ( Reproduced from
ref. 111 with permission of Blackwell Publishing. )
1000
100
10
1
0.1
Concentration (.g/L)
0 60 120 180 240 300 360 420 480 540
Time (min)
Butorphanol, an analog of buprenorphine, showed a nasal bioavailability of 70%
and also a much lower Tmax after nasal absorption as compared with the sublingual
and buccal routes [115] . Lindhardt et al. [106] compared buprenorphine formulated
in 30% PEG - 300 in sheep with that of the 5% dextrose formulation mentioned
earlier. A unit - dose Pfeiffer device was again used to administer the formulation. It
was found that nasal bioavailability in sheep was about 70% when buprenorphine
was formulated in PEG - 300 and approximately 89% when it was formulated with
5% dextrose. The rate of absorption was reported to be very fast, with a Tmax of
10 min; the Cmax was found to be 37 and 48 ng/mL for PEG - 300 and dextrose, respectively.
In sheep, the pharmacokinetics of buprenorphine showed a two - compartment
model as compared to a three - compartment model in humans.
5.6.7.4 Hydralazine
Hydralazine is a vasodilator used in the treatment of malignant hypertension and
hypertensive emergencies and is generally used in conjunction with other antihypertensive
agents. Although the oral absorption is good, there is low oral bioavailability
due to fi rst - pass metabolism. Nasal absorption of hydralazine has been
studied in rats using both in vivo nasal absorption and in situ nasal perfusion
methods; the effect of surfactant and solution pH has also been reported. It was
found that the nasal absorption of hydralazine was increased in the presence of
surfactants such as sodium glycocholate and polyoxyethylene - 9 - lauryl ether. The
nasal absorption of hydralazine was reported to be a pH - dependent passive process,
with the absorption increasing as the pH was increased from 3.0 to 6.6. In nasal
absorption studies in rats, peak levels of hydralazine were reached in 30 min at pH
3.0. The in situ absorption of hydralazine as a function of perfusion pH was also
evaluated. The results of the in situ nasal perfusion studies demonstrated that
hydralazine is eliminated from the nasal cavity and the perfusate by fi rst - order
kinetics. Even in the ionized form, the drug was well absorbed, and it was suggested
that the aqueous channels in the nasal mucosa played an important role in the
transport of hydralazine [116, 117] .
5.6.7.5 Nitroglycerin
Nitroglycerin is delivered across the mucosal membranes in the management of
acute ischemic conditions. Nitroglycerin carries out this function by arterial vasodilatation
and venodilation, which leads to a decrease in both the preload and afterload
and also improved coronary blood fl ow. The intranasal action of nitroglycerin,
also called glyceryl trinitrate, appears to be similar [118] and brings about a reduction
in myocardial oxygen consumption. Like that of hydralazine, the oral bioavailability
of nitroglycerin is low; hence alternative routes of delivery and innovative
delivery systems have been preferred, such as sublingual patches, ointments, or
transdermal patches. The intravenous route ensures a rapid onset of action, but its
preparation and standardization procedures make it costly. Intranasal nitroglycerin
in various operative experiences has been found to have a rapid onset of action with
predictable and consistent therapeutic effects. A peak level of nitroglycerin is
reached 1 – 2 min after intranasal administration; it is barely detectable after 16 min.
These studies were carried out in fi ve patients who were undergoing elective coro-
NASAL DELIVERY OF NONPEPTIDE MOLECULES 627
628 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
nary artery bypass surgery. The plasma levels were reported to be similar with
intravenous administration and better than with sublingual administration [118 –
120] . The pressor response to endotracheal intubation in both normotensive and
hypertensive patients can be attenuated by intranasal nitroglycerin in operative
settings. It has been reported that when nitroglycerin was given 30 s before the
induction sequence in 40 hypertensive patients treated with . blockers, there was a
blunted pressor response to intubation. In comparison to the placebo control group,
the group that received nitroglycerin had a lower mean arterial pressure at 1, 3, and
5 min after induction. Thus intranasal nitroglycerin can be employed in the selective
control of hypertension [118, 121, 122] .
5.6.7.6 Propranolol and Other . - Adrenergic Blocking Agents
Propranolol is a nonselective . - adrenergic receptor blocking agent. It is clinically
used in the management of hypertension and the treatment of angina pectoris. When
given orally in humans, it has led to considerable variation in plasma drug levels.
This, as well as its subsequently low bioavailability, is believed to be due to its extensive
metabolism in the gut and in the liver. A study in the late 1970s showed that
nasal absorption of propranolol at a dose of 1 mg in rats produced blood levels
similar to those achieved with intravenous administration; however, the same dose
administered orally resulted in very low blood levels [47, 123] . The feasibility of
nasal absorption of propranolol in solutions and sustained - release formulations in
rats and dogs was studied. The procedure described by Hirai et al. [46] was used to
carry out the surgical operation in rats. In dogs, the formulations were given intranasally
using a micropipette and syringe. The mean blood levels of propranolol by
the nasal route were compared with those of the oral and intravenous routes in rats
and dogs. As in the case of the results obtained in humans, oral administration of
propranolol solutions resulted in low and variable drug levels in rats and dogs,
whereas the nasal administrations of propranolol solution showed plasma drug
levels that were similar to those achieved with intravenous administration. In the
case of sustained - release formulations, it was found that there was an initial low
level of drug; however, these levels were maintained for a longer time. The bioavailabilities
obtained from the AUC were found to be identical, although the maximum
blood levels in the case of sustained - release formulations were found to be much
lower than with the propranolol solutions. A propranolol formulation containing
2% methylcellulose gels in humans was studied by the same group. Identical
serum drug profi les were obtained after nasal administration as with intravenous
administration [124] .
The effect of intranasal propranolol on exercise parameters with the Bruce protocol
[118, 125] was studied in 16 patients with chronic, stable, effort - induced angina
pectoris. Propranolol was given as a single 5 - mg/puff nasal spray to the patients. A
mean plasma level of 20 ng/mL was obtained, and a signifi cant increase in total
exercise time was seen, from 460 to 530 s. This led to an increase in the time to 1 mm
ECG as well as to the onset of angina. Both maximum heart rate and systolic blood
pressure were lower than with placebo. This study demonstrated that propranolol
in the form of a nasal spray elicited immediate . blockade and was useful in treating
patients with angina pectoris, who showed improvement in exercise tolerance after
receiving the drug [118, 125] .
Although no adverse reactions have been reported with intranasal administration
of propranolol, complications may occur, as ocular administration has produced
some systemic side effects [118] . The infl uence of substrate lipophilicity on drug
uptake by the nasal route was reported in humans. Alprenolol and metoprolol, .
blockers with varying degrees of lipophilicity, were used. The fi ndings from these
studies demonstrate that the more hydrophilic drugs showed a lower bioavailability.
Alprenolol showed rapid uptake into the systemic circulation by the nasal route and
also a higher bioavailability [126, 127] .
5.6.7.7 Sex Hormones
The low oral bioavailability of hormone - replacement drugs due to intestinal and
fi rst - pass metabolism requires the use of higher doses of these drugs, which are
associated with many side effects. Parenteral administration of sex steroids as well
as use of the transdermal route has been viewed as an alternative. However, the
transdermal route has certain limitations, such as the visibility and palpability of the
patch as well as possible skin irritation. These drawbacks have limited the use of
this route. The intranasal route has therefore been considered as an alternative
[128] .
5.6.7.8 17 b - Estradiol ( E 2 )
The most common form of estrogen in clinical use is 17 . - estradiol. It is reported
to reduce bone turnover, prevent postmenopausal bone loss, and decrease the risk
of fracture in both early and late postmenopausal women. Lipophilic drugs such
as sex steroids pose the problem of going into solution, thus leading to low estrogen
levels. Hence a number of studies have attempted to solubilize 17 . - estradiol
using DM . - CD for its intranasal delivery [129, 130] . An E 2 spray has been developed
(S21400 or Aerodiol, Institut de Recherches Internationales Servier, France),
which is estradiol that has been solubilized in water with randomly methylated . -
CD. Estradiol delivered intranasally is rapidly absorbed by the nasal mucosa and
shows maximum plasma levels within 10 – 30 min. Plasma levels return to 10% of
the maximum plasma concentration within 2 h of administration and to untreated
postmenopausal levels within 12 h. Hence after intranasal administration, estradiol
has a pulsatile profi le as compared to the more sustained plasma profi le seen with
the oral and transdermal routes. However, whether sustained levels are required
for effi cacy has not been determined, although intranasally delivered estradiol does
increase serum estradiol to the same extent as is seen with oral administration
[128, 130 – 134] .
The short - and long - term effects of intranasal 17 . - estradiol on bone marrow
turnover and serum IGF - 1 were studied in a double - blind placebo - controlled clinical
trial and compared with oral 17 . - estradiol. Some 425 Caucasian postmenopausal
women with climacteric symptoms were studied. The nasal effi cacy of estradiol was
assessed using the Kupperman index (KI), which is a weighted evaluation of the
incidence and severity of 11 menopausal symptoms summarized in a menopausal
index as follows: hot fl ushes, the most heavily weighted ( . 4); night sweats ( . 2); and
sleep disturbances and nervousness (each . 2). The lower weighted symptoms
include depression, irritability, vertigo, fatigue, arthralgia, headache, tachycardia,
NASAL DELIVERY OF NONPEPTIDE MOLECULES 629
630 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
and vaginal dryness (each . 1). The highest possible score is 51 [128, 135 – 137] .
Various markers of bone resorption — such as urinary type I collagen telopeptides,
the formation of serum osteocalcin, serum type I collagen, N – terminal extension
propeptide (PINP), and serum bone marrow alkaline phosphatase (BAP) — were
determined at baseline and after 1, 3, and 15 months. Urinary - type collagen C telopeptides
were considerably reduced in all the treated groups within 1 month, and
this reduction continued even at 3 months. Neither serum osteocalcin nor PINP
showed any change at 1 month; however, they were reduced at 3 months with oral
dosage. There was an increase in the bone formation parameters at 1 month for the
higher doses of intranasal estradiol, but no reduction was seen at 3 months. No signifi
cant change from placebo - treated groups was observed at the end of 3 months
in the case of circulating IGF - I after intranasal estradiol, but a signifi cant decrease
was seen with oral estradiol. After a year of treatment with intranasal estradiol at
a dose of 300 . g/day, resorption and formation markers decreased to premenopausal
levels. This study concluded that normalization of bone turnover to premenopausal
levels can be achieved following 1 year of treatment with intranasal
17. - estradiol [131] .
5.6.7.9 Testosterone
Testosterone, the most potent natural male sex hormone, is generally given intramuscularly.
It is absorbed well orally but is extensively metabolized in the liver
and the gastrointestinal tract. Owing to fl uctuations in serum levels of testosterone
esters, other viable routes of delivery are being explored. Two transdermal patches
have become available commercially. However, due to the limitations associated
with this route, it is not preferred by patients, particularly because the site of
application is the scrotum [8, 138] . In order to improve systemic bioavailability,
the nasal absorption of testosterone has been evaluated versus intravenous and
intraduodenal administration in rats. When given nasally, the concentration of
testosterone in the circulation increased and peak levels were reached within
2 min; blood levels were similar to those seen with intravenous administration.
Intraduodenal administration produced low blood levels. A bioavailability of 99%
at 25 . g/dose was seen and 90% at 50 . g, but the intraduodenal route showed a
very low bioavailability of 1% [8] . Hussain et al. [139] showed in rats that testosterone
can be absorbed intranasally, and an elimination half - life of about 40 min
was obtained. However, since the problem of solubility is an obstacle to preparing
the formulation for nasal administration, the use of a prodrug was evaluated by
the same group. A water - soluble ester of testosterone, testosterone 17 . - N, N -
dimethylglycinate hydrochloride (TSDG), is completely absorbed when given
intranasally at much lower doses as compared with testosterone itself. After
absorption, conversion of the prodrug to testosterone begins almost immediately,
and the terminal elimination half - life of testosterone was found to be 55 min,
which is similar to that obtained after intravenous administration. Peak plasma
concentration was reached within 12 min for the lower dose (equivalent to
25 mg/kg of TS) and 20 min for the higher dose (50 mg/kg) by both routes. The
AUC also showed similarity, suggesting the complete absorption of the nasally
administered prodrug.
5.6.8 THE NOSE: OPTION FOR DELIVERY OF DRUGS TO CENTRAL
NERVOUS SYSTEM
The possibility of delivering agents to the central nervous system (CNS) via the nose
has long been known, an example being the sniffi ng of cocaine in order to produce
a sense of euphoria, which is attained within 3 – 5 min [53] . In short, nasal administration
is not only an exciting possibility in the fi eld of drug delivery but may also be
the means of solving delivery problems for the innumerable agents that cannot cross
the blood – brain barrier (BBB). Such agents are therefore being developed by
nanoparticle - based systems or by formulating them as prodrugs. The literature
offers numerous examples demonstrating ways of delivering such drugs to the brain.
In particular, use of a direct pathway, as in the case of cocaine, from the nasal cavity
to the CNS has been suggested. Illum [140] and Chow et al. [141] have shown in
animal models that, in the early time points after nasal administration, the brain
concentration of cocaine was higher than when the drug was given by the intravenous
route. The specifi c site through which nose - to - brain delivery is believed to take
place is the olfactory region. In the early 1900s it was shown that the olfactory region
was responsible for the uptake (or rather entry) of viruses, in particular the poliomyelitis
virus, into the brain [142 – 146] . Further work in support of this theory then
demonstrated the presence of the poliomyelitis virus in the cerebrospinal fl uid
(CSF) and also in the systemic lymphatics of the olfactory mucosa [147 – 150] . In the
period from 1970 to 1990, there were many reports of nose - to - brain delivery across
the olfactory epithelium for a number of different agents, including metals and
tracer materials such as colloidal gold, cadmium, potassium ferricyanide, and iron
ammonium citrate [151 – 154] .
There are certain aspects of drug delivery that must be clearly understood in
designing a nose - to - brain drug delivery system. For example, lipophilic drugs are
absorbed across the nasal epithelium almost immediately and effi ciently to enter
the systemic circulation. Therefore such drugs will show little sign of direct nose - to -
brain delivery. Drugs that are on the hydrophilic side or polar molecules will not be
readily absorbed across the nasal mucosa; these molecules generally undergo paracellular
transport as compared to transcellular transport in the case of lipophilic
drugs. Such molecules have a higher chance of being taken up by the olfactory
mucosa for delivery to the brain. In general, drugs travel from the nose to the brain
via (1) drug internalization into the olfactory epithelium ’ s primary neurons followed
by the intracellular axonal transport to the olfactory bulb or (2) drug absorption by
paracellular or transcellular pathways across the olfactory sustentacular epithelial
cells, following which the drug enters the CSF or CNS. In the human nervous system,
the olfactory region is the only site in direct contact with the surrounding environment.
It has been reported that the intracellular axonal pathway takes a longer time
to deliver drugs to the brain [140, 155 – 157] . Table 4 gives an account of some of the
drugs/molecules that have been delivered from the nose to the brain in human and
animal models. The direct delivery of drugs to the brain or CSF via the olfactory
epithelium is discussed in the following paragraph , briefl y describing the olfactory
mucosa.
The olfactory region is mainly involved in the detection of smell, and the makeup
and organization of the epithelial layer enhance the accessibility of air to the
OPTION FOR DELIVERY OF DRUGS TO CENTRAL NERVOUS SYSTEM 631
632 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
TABLE 4 Nose - to - Brain Delivery of Agents in Different Species
Drug/Molecule Path Followed Reference
Humans
Arginine - vasopressin — a 158
Adrenocorticotrophin — a 159
Cholecystokinin — a 160
Diazepam — a 161
Insulin — a 158 , 162
technetium - 99m -
diethylenetriaminepentacetic
acid
Direct from nose to brain 163
Apomorphine Nasal cavity > CSF 164
Melatonin/hydroxycobalamin Nasal cavity > CSF 165
Melanocortin, vasopressin, and
insulin
Nasal cavity > CSF 166
Rats
Zidovudine Nasal cavity > CSF, nasal cavity > systemic
circulation
167
Dextromethorphan HCl Direct from nose to brain 168
Cephalexin Nasal cavity > CSF, nasal cavity > systemic
circulation
169
Sulfonamides Nasal cavity > CSF, nasal cavity > systemic
circulation
170 , 171
Dextran Nasal cavity > olfactory mucosa > CNS and
also nasal cavity > systemic circulation
172
Dopamine Direct from nose to brain 173
Cocaine Direct from nose to brain 141
Dihydroergotamine Direct from nose to brain 174
Insulin Direct from nose to brain 175
Dopamine Nasal cavity > CSF 173
Methotrexate Nasal cavity > CSF 176
Nerve growth factor Direct from nose to brain via olfactory
pathway
177
WGA – HRP (wheat germ
agglutinin – horseradish
peroxidase)
Direct from nose to brain via olfactory nerve
and bulb
178
Zolmitriptan Direct from nose to brain 179
Leptin Direct from nose to brain 180
Morphine Direct nose to brain via olfactory pathway 181
Nimodipine Direct nose to brain via olfactory bulb and
nasal cavity > CSF
182
Meptazinol hydrochloride Nasal cavity > CSF 183
Estradiol Nasal cavity > CSF via olfactory neurons 184
a Facilitated transport to brain based on functional evidence in humans.
neuronal components comprising the odorant detectors. The olfactory region is
mainly located on the nasal septum and partly on the superior and middle turbinates.
It occupies only a small region in humans of about 10 cm 2 in the roof of the
nasal cavity, as compared to around 150 cm 2 in dogs. The olfactory epithelium is a
pseudostratifi ed epithelium comprising three types of cells: olfactory receptor cells,
supporting cells, and basal cells. The receptor cells are elongated bipolar neurons
located in the middle stratum of the epithelium, interspersed among the sustentacular
cells; the microvilli cover the supporting cells, which extend from the mucosal
surface to the basal membrane; while the basal cells are located in the basal surface
of the neuroepithelium. These basal cells go on to differentiate, becoming new
receptor cells [140, 185, 186] . The surface of the nasal cavity measures about 180 cm 2
in humans as compared to about 10 cm 2 in rats, and the olfactory region is reported
to constitute about 3% of the nasal cavity in humans and 50% in rats. Some other
differences include the fact that in adult humans the volume of CSF is about 160 mL
while it is only about 150 . L in rats; also, the CSF is replaced about three times daily
in humans, whereas in rats it is replaced hourly. Hence, though there is suffi cient
evidence regarding nose - to - brain delivery, especially in rats and in some cases in
humans, the impact of these factors on the interpretation of the results between the
two species could be signifi cant [155, 187] .
5.6.9 NASAL DELIVERY OF VACCINES
The discovery of vaccines for smallpox, cholera, and typhoid and the variety of vaccines
now available have led to a signifi cant reduction in the mortality and morbidity
due to many diseases, with smallpox being the fi rst to have been completely eradicated
and poliomyelitis targeted to be the next. At present, the World Health Organization
is working toward the complete elimination of poliomyelitis throughout
the world [188, 189] . However, since Jenner discovered the vaccine for smallpox
more than two centuries ago [190] , only some 50 vaccines have been approved for
use, and few additional vaccines have been discovered. Most of those in current use
are administered parenterally; they can induce only a systemic immune response,
not mucosal immunity. Obviously the latter is very important in the prevention and
treatment of infectious diseases, be they due to viral, bacterial, or parasitic pathogens
that attack via the mucosal surfaces [190] .
The criteria to be met in designing a vaccine formulation include the following:
The vaccine should have the capacity to produce lifelong immunity, be able to act
against the different strains and variants or the subtypes of the organisms, be effective
in all age groups, be able to act quickly and also to induce immunity in the fetus
when the mother is treated, be able to act effectively after a single treatment, and
ideally be administered noninvasively. Finally, such a vaccine must be relatively
inexpensive and remain active under a variety of conditions, especially not requiring
the cold chain [191] .
The following section addresses the need for needle - free vaccines with formulations
based on safer adjuvant and delivery systems.
5.6.9.1 Nasal Vaccines: Ideal Noninvasive Route
When we talk about targeting the pathogens entering through the mucosal surfaces,
the route that usually comes to mind is the oral route. However, this route has its
drawbacks for several reasons, such as the fact that the antigen used is degraded
along with the gastric contents; furthermore, there is also the diffi culty of reaching
the antigen - presenting cells [192] . In comparison to the oral route, nasal vaccination
NASAL DELIVERY OF VACCINES 633
634 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
has been shown to require a lower antigen dose, which is essential considering the
cost of the recombinant agents that may be used as antigens. Nasal administration
has been shown to induce immune responses in the respiratory and genital surfaces.
When compared with the other mucosal routes such as vaginal or rectal administration,
the nasal route is much more acceptable in terms of both accessibility and
overall convenience. Hence the nasal route is increasingly being seen as ideal for
the administration of vaccines.
The nasal route has traditionally been used as an effective route in the treatment
of respiratory infections, the rationale having been to target the infectious agents
at their port of entry. Since most infectious disease pathogens enter at various
mucosal sites, the nasal route has attracted increased attention as an alternative
route for the delivery of vaccines. A further advantage offered by the nasal route
in that it is capable of inducing both systemic and mucosal immunity as compared
to the parenteral route, which brings about only systemic immunity [190, 193, 194] .
Nasal immunization can result in distant as well as local mucosal immunity because
of the mucosal immune system ’ s common properties. This means that it is possible,
via nasal immunization, to attain adequate immunity at other mucosal sites such as
the respiratory, intestinal, and genital; hence vaccines administered nasally will have
an important role in the prevention of respiratory infections and, more importantly,
in the treatment of sexually transmitted diseases [190 – 196] .
The earliest vaccines were live attenuated, inactivated toxins, or inactivated
toxoids. But with advances in molecular biotechnology, it has become possible to
produce extremely pure vaccines. However, the main drawbacks of these vaccines
are their poor immunogenicity, so that the use of adjuvants is often required to
attain the necessary immunogenicity. A vaccine adjuvant is especially important in
subunit vaccines, which is how most of the vaccines available today are supplied.
Adjuvants can be best defi ned as particular agents that increase the immunity produced
when they are coformulated and delivered with the vaccine antigen. The
adjuvants are formulated in the vaccines so as to produce a longer lasting immunity.
The more time the adjuvant takes to be eliminated from the system, the longer it
is able to induce the required or intended lasting immune response.
Vaccine adjuvants may be classifi ed operationally as delivery or immunostimulating
adjuvants. Vaccine delivery adjuvants simply act as delivery agents, that is, they
present the vaccine antigens to the antigen - presenting cells (APCs), whereas the
immune - stimulating agents act by stimulating the APCs to elicit an appropriate
immune response. This results from the stimulation of the Toll - like receptors, present
on macrophages and dendritic cells, the activation of which shows increased antigen
presentation and cytokine release. The combination of both types of adjuvants may
lead to better formulations for nasal vaccines [196 – 199] .
5.6.9.2 Immunity after Intranasal Immunization
An understanding of the respiratory tract and the immune response following nasal
vaccination is necessary to understand how the antigen used in the vaccine interacts
with the surfaces of the human body and how the different adjuvants may interact,
modify, and aid in generating an immune response. The nose is a component of the
upper respiratory tract, which is composed of the mouth, nasopharynx, and larynx.
The nasal passages have an extensive surface area which is richly vascularized.
However, the nasal epithelium has little ability to break down drugs. The extensive
mucosal surface of the nose has a lining of pseudostratifi ed epithelium as well as
cilia and the goblet cells involved in the secretion of mucus. The lymphoid tissue
primarily involved in the mucosal immune responses is the mucosal - associated
lymphoid tissue (MALT). The different regions of the respiratory tract that play an
infl uencing role in the immune system are as follows:
• The epithelial surface, which comprises immunocompetent cells in the connective
tissue
• The lymphoid tissues linked to the respiratory tract, which are categorized into
three parts: larynx - associated lymphoid tissue (LALT), nose - associated lymphoid
tissue (NALT), and bronchus - associated lymphoid tissue
• The lymph nodes that drain the respiratory tract
The NALT, which is the organized lymphoid structure in the nasal passages and
occurs in abundance in the nasal mucosa, plays a signifi cant role in the mucosal
surface ’ s defense against invading pathogens. The NALT is equivalent to Waldeyer ’ s
ring, which is made up of the adenoids or tonsils situated in the roof of the nasopharynx,
bilateral lymphoid bands, the palatine tonsils, and the tonsil at the base of
the tongue (the lingual tonsil). The NALT is the main target site for vaccine antigens
in humans [193, 194, 197, 200, 201] . As the mucosal immune system develops, the
NALT is involved in a variety of important functions in relation to the host defense
mechanism, from being an impediment to drug absorption, serving as a guard
against attacking pathogens and antigens, facilitating the uptake of antigens, eliciting
the secretory antibody response, and inducing the immune response in the other
distant mucosal surfaces due to the function of the common mucosal immune
system. Owing to mucosal tolerance, it is also involved in preventing serious allergic
responses to inhaled antigens [194, 201, 202] .
On administration, the antigen interacts fi rst with the inductive tissue of the
MALT, thus initiating a primary response. The IgA serum cells are found in the
effector sites of the MALT, and local immunity results from the production of
the secretory IgA (s - IgA) response. As mentioned earlier, the NALT and the tonsils
form the main inductive sites in rodents and humans. These are composed of M cells
involved in the uptake of antigen and its presentation to the underlying lymphoid
tissues, the antigen being taken up by the M cells and the APCs, consisting of dendritic
cells, macrophages, and B cells; all these cells together with the T cells produce
the cellular and humoral immune responses [194] .
Vaccination by the nasal route produces a mucosal protection using mucus, the
epithelial surface, and both innate and acquired immune responses. The innate
defense mechanism plays a very important role in that it infl uences the type of
acquired immune mechanism, which mainly responds on the basis of “ immune
memory. ” The ability to attain these responses is the main principle of attaining
protection from infection.
5.6.9.3 Need for Adjuvants
As mentioned earlier, the subunit vaccines in particular require the use of adjuvants
in order to initiate an immune response leading to protection. The subunit vaccines
NASAL DELIVERY OF VACCINES 635
636 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
TABLE 5 Nasal Vaccination Delivery Systems Studied
Delivery
System/Adjuvant Antigen Employed Remarks Reference
Chitosan Diphtheria toxin Increased local as well as
systemic effects
208
Infl uenza virus Immune response
comparable to that
of intramuscular
administration
209
Filamentous hemagglutinin
and recombinant pertussis
toxin (single or bivalent
vaccine)
Chitosan stimulates
mucosal and systemic
effects
209 , 210
IL - 12 Tetanus toxoid IL - 12 - induced IgA
response
211
Infl uenza hemagglutinin and
neuraminidase
Induces protective
immunity
212
CpG motifs Hepatitis B surface antigen
(HBsAg)
Potent enhancement of
systemic and mucosal
immune responses
213
Formalin - inactivated
infl uenza virus
Enhances the serum IgG
and s - IgA responses
214
Liposomes Infl uenza subunit vaccine Induction of systemic IgG
and s - IgA responses
215
Inactivated measles virus Stimulation of mucosal
and systemic responses
216
Bovine serum albumin
(BSA)
— 217
Streptococcus mutans — 218
Infl uenza hemagglutinin and
neuraminidase (when
used in combination with
heat - labile toxin (HLT)
Good response in
presence of HLT
219
Infl uenza, hepatitis B,
tetanus toxoid
— 220
Yersinia pestis — 221
ISCOMs
(immune
stimulating
complex)
Infl uenza subunit Protective immunity to
challenge
222
Measles nucleoprotein Induces cytotoxic T - cell
response
223
Echinococcus surface
antigen
— 224
Respiratory syncytial virus
(RSV) envelope antigen
225
Poly(lactic - co -
glycolic acid
(PLGA)
microparticles
HBsAg Strong systemic and
mucosal immune
responses
226
Cationic
nanoparticles
(SMBV)
HBsAg and . - galactosidase Strong mucosal as well as
systemic antibody and
CTL responses
227
REFERENCES 637
available today, which are administered intramuscularly or subcutaneously, involve
alum as an adjuvant. The drive for initiating further research into vaccine adjuvants
has been stimulated by many factors, among the chief of which is that the aluminum -
based adjuvants currently available have failed in many candidate vaccines or have
not achieved the necessary immunity or induced a cytotoxic T - cell response. Nasally
formulated vaccines, mainly subunit vaccines, currently being purifi ed are less immunogenic
and also cannot elicit the necessary T - cell response. As a result, research is
now focusing on fi nding newer adjuvants for nasal DNA and subunit vaccines in
order to attain specifi c immune responses as well as the necessary antibody subtype
response. In addition to this, an adjuvant can help to reduce the dose of antigen
required and also the number of doses needed to achieve mucosal immunity [203 –
207] . Despite the extensive research going on in the fi eld of vaccine adjuvants, the
only FDA - approved adjuvant for human use is alum. There are several other adjuvants,
such as monophosphoryl lipid A (MPL), that have been approved in the
European market; another, Corixa, is used as an adjuvant in Fendrix, the hepatitis
B vaccine of GlaxoSmithKline Biologicals. The main hindrance to the approval of
many adjuvants that reach clinical trials is their potential to elicit toxic side effects
in clinical use. It has been reported that aluminum salts do induce some allergies in
humans. As more purifi ed and target - oriented or specifi c vaccines obtained by
recombinant technology are being launched, it becomes more diffi cult for vaccine
antigens alone to induce the necessary immune responses, as these recombinant
antigens or synthetic peptides cannot jump start an immune response.
A number of adjuvants are awaiting approval for human use. The main impediment
to the successful development of vaccine adjuvants is that their mechanism of
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systems and the various adjuvants that have been used in the development of nasal
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Delivery
System/Adjuvant Antigen Employed Remarks Reference
HBcAg HBsAg Stimulates strong Th1
response
228
Cholera toxin Group B streptococci High levels of IgA in
cervicovaginal
secretions
229
Haemophilus infl uenzae Effective nasal
vaccination
230
Infl uenza virus — 231
Synthetic peptide of RSV Complete protection 232
TABLE 5 Continued
638 NASAL DELIVERY OF PEPTIDE AND NONPEPTIDE DRUGS
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of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B
core antigen to stimulate a strong Th1 response against hepatitis B surface antigen ,
Immnol. Cell. Biol. , 82 , 539 – 546 .
229. Hordness , K. , Tynning , T. , Brown , T. A. , Haneberg , B. , and Jonsson , R. ( 1997 ), Nasal
immunization with group B streptococci can induce high levels of specifi c IgA antibodies
in cervicovaginal secretions of mice , Vaccine , 15 , 1244 – 1251 .
230. Kurono , Y. , Yamamoto , M. , Fujihashi , K. , Kodama , S. , Suzuki , M. , Mogi , G. , McGhee ,
J. R. , and Kiyono , H. ( 1991 ), Nasal immunization induces Haemophilis infl uenzae -
specifi c Th1 and Th2 responses with mucosal IgA and systemic IgG antibodies for protective
immunity , J. Infect. Dis. , 180 , 122 – 132 .
231. Hagiwara , Y. , Komase , K. , Chen , Z. , Matsuo , K. , Suzuki , Y. , Aizawa , C. , Kurata , T. , and
Tamura , S. ( 1999 ), Mutants of cholera toxin as an effective and safe adjuvant for nasal
infl uenza vaccine , Vaccine , 17 , 2918 – 2926 .
232. Bastein , N. , Trudel , M. , and Simard , C. ( 1999 ), Complete protection of mice from respiratory
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Vaccine , 17 , 832 – 836 .
651
5.7
NASAL POWDER DRUG DELIVERY
Jelena Filipovi - Gr i and Anita Hafner
University of Zagreb, Zagreb, Croatia
Contents
5.7.1 Introduction
5.7.2 Nasal Dry Powder Formulations
5.7.2.1 Benefi ts Associated with Nasal Powder Drug Delivery
5.7.2.2 Drug Powder or Drug/Polymer Powder Formulation for Nasal Drug
Delivery?
5.7.2.3 Powder Properties Affecting Nasal Deposition and Drug Delivery
5.7.3 Polymers in Nasal Powder Delivery System
5.7.4 Microspheres as Nasal Drug Delivery Devices
5.7.4.1 Preparation Methods
5.7.4.2 Microsphere Characterization
5.7.4.3 Chitosan - Formulated Spray - Dried Microspheres
5.7.5 Toxicological Considerations
References
5.7.1 INTRODUCTION
Intranasal drug administration has been practiced since ancient times. In Tibet
extracts of sandalwood and aloewood were inhaled to treat emesis. Egyptians
treated epistaxis and rhinitis using intranasal medication. North American Indians
relieved headaches inhaling crushed leaves of Ranunculus acris [1] . Due to the rich
vasculature and high permeability of nasal mucosa, the absorption rate and pharmacokinetics
of nasally administrated drug are comparable to that obtained by
intravenous drug delivery, while noninvasive nasal drug administration is more
convenient to patients. As nasally administrated drugs avoid fi rst - pass hepatic
metabolism, improved bioavailability can be expected. However, rapid mucociliary
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
c c c
652 NASAL POWDER DRUG DELIVERY
clearance reduces the residence time of nasal drug delivery system at the site of
absorption. Dry powders have been shown to delay mucociliary clearance, thus
prolonging the contact time between the drug delivery system and mucosa compared
to liquid formulations. Most of the dry powder investigations are based on
mucoadhesive swellable polymers as they can additionally improve drug absorption
and bioavailability. Dry powder delivery systems such as microspheres are of special
interest, offering the possibility of predictable and controlled drug release from the
polymeric device [2, 3] .
5.7.2 NASAL DRY POWDER FORMULATIONS
Liquid preparations are most frequently used nasal dosage forms at present.
However, such preparations are characterized with short residence time in the nasal
cavity, low drug concentration at the site of absorption, and problems linked to the
chemical stability of the drug and the stability of the preparation. In the case of
liquid formulations, drugs must be administered in small volumes. The maximum
volume of a single dose in one nostril is about 200 . L. The volume of therapeutic
dose should not exceed the capacity of the nasal cavity, as it would drain out of the
nose. Thus, only low - dose or highly soluble drugs can be administered nasally in the
form of a simple liquid formulation [4] . Dry powder formulations have been
recognized as effi cient nasal delivery systems offering numerous advantages over
liquid formulations, such as avoidance of preservatives, improved formulation
stability, and prolonged contact with the mucosa. For a powder formulation, the
maximum quantity is approximately 50 mg, depending upon the bulk density of the
material [5] .
5.7.2.1 Benefi ts Associated with Nasal Powder Drug Delivery
A powder form was found to be more effective than liquid formulations in a number
of investigations described in the literature [6 – 11] . Dry powders are characterized
by prolonged residence time and higher drug concentration at the site of deposition
as well as improved formulation stability with no requirement for preservatives [12] .
Prolonged residence time of the powder delivery systems at the absorption site
results in enhanced systemic bioavailability, compared to the liquid formulations. In
the case of powders, higher drug concentration at the site of absorption causes rapid
transmucosal diffusion and faster onset of action [13] . Most of the dry powder formulations
are based on mucoadhesive swellable polymers (e.g., starch, dextran,
chitosan) as they can additionally improve drug bioavailability, prolonging the residence
time in the nasal cavity or even promoting drug absorption. Powder formulation
with a water - insoluble and nonswellable drug carrier may also improve nasal
bioavailability of the polar drugs. Ishikawa et al. [14] found that a nasal powder
delivery system of elcatonin based on CaCO 3 signifi cantly increased the systemic
elcatonin bioavailability in rats and rabbits compared to the liquid formulation.
Enhanced bioavailability has been primarily ascribed to the retardation of the clearance
of the drug powder delivery system from the nasal cavity.
The use of dry powder formulations in nasal vaccine delivery has been extensively
reviewed elsewhere [15 – 18] . The association of vaccines to some of the par
ticulate systems has been proved to enhance the systemic and mucosal immune
responses against the antigens [19 – 21] . Dry powder formulations for nasal vaccine
delivery may also provide signifi cant advantages with respect to stability shortcomings
compared to conventional liquid intranasal and intramuscular formulations,
which require frozen storage or refrigeration [22] .
5.7.2.2 Drug Powder or Drug/Polymer Powder Formulation for
Nasal Drug Delivery?
The drug candidate for nasal administration should possess a number of attributes,
such as appropriate aqueous solubility and nasal absorption characteristics, minimal
nasal irritation, low dose, no offensive odor or aroma, and suitable stability characteristics
[23] . In the case of drug powder formulations it is possible to hide or alter
the unfavorable characteristics of a drug using suitable polymers as drug carriers.
Thus, improvement of the dissolution behavior of drugs of low aqueous solubility
after incorporation in polymeric powder devices such as microspheres has been
reported in the literature [24, 25] . The improvement of the drug dissolution rate
from the microspheres has been ascribed to several factors, such as high microsphere
surface – volume ratio, the hydrophilic nature of the polymer, and drug amorphization
due to drug – polymer interaction and/or the microsphere preparation method
[25, 26] .
Nasally administrated polymer – drug powders were also characterized by
improved drug absorption compared to pure drug powders [25, 27] . Teshima et al.
[27] monitored changes in the plasma glucagons and glucose concentrations after
nasal administration of the powder form of glucagon alone and glucagon mixed with
the carrier, microcrystalline cellulose (MCC). Glucagon and glucose plasma concentrations
remained unchanged after nasal administration of the powder form of glucagon
alone while it increased after glucagon – MCC administration in an MCC
content – dependent manner. Results of in vivo nasal administration of carbamazepine
- loaded chitosan microspheres revealed an increase in carbamazepine concentration
in serum compared to the pure carbamazepine powder [25] . Such an increase
in drug absorption has been ascribed to both improved dissolution of carbamazepine
and adhesion of the chitosan microspheres to the mucosal surface.
The infl uence of polymers on the drug stability in the powder formulation has
also been reported [8] . Green coloration of polyacrylic acid powder dosage forms
loaded with 60% apomorphine due to atmospheric drug oxidation upon storage has
been observed. Dosage forms with lower drug loadings (and higher polymer content)
showed no coloration, indicating the protective role of polymers against drug oxidation
in powder formulations.
Powders intended for nasal administration have to be optimized in terms of particle
size and morphology as these properties are related to potential irritation in
the nasal cavity [23] . Certain procedures (e.g., spray drying process) can modify the
particle size of the drug powder raw material, but in order to optimize the morphology
and fl owability properties of some pure drug powders, excipients need to be
used. Sacchetti et al. [28] reported that the use of mannitol as a fi ller and hydroxypropylmethyl
cellulose (HPMC) as a shaper of spray - dried caffeine microparticles
modifi ed the typical needle shape of spray - dried caffeine to a more convenient
roundish shape. Further addition of polyethylene glycol (PEG) resulted in increased
NASAL DRY POWDER FORMULATIONS 653
654 NASAL POWDER DRUG DELIVERY
cohesiveness between particles, producing agglomerates considered as benefi cial for
nasal deposition.
5.7.2.3 Powder Properties Affecting Nasal Deposition and Drug Delivery
Powder formulation effi ciency is strongly dependent on two technological aspects
of delivery: fi rst, the complete release of the dose through the powder delivery
device, that is, the quantitative aspect, and, second, the distribution of the powder
on the nasal mucosa, that is, the qualitative aspect. For example, rhinitis treatment
requires drug deposition over the entire mucosal area, while brain targeting requires
deposition strictly at the roof of the nose. The qualitative aspect is defi ned by the
aerodynamic properties of the powder cloud produced (puff) and is assessed by a
photographic technique providing information on the spray pattern and cloud
geometry. The parameters involved are shape, height, area, density, particle size, and
velocity. For nasal impacting the powder cloud should remain as compact as possible
to achieve an effi cient shot of powder to the nasal mucosa, whereas for sedimentation
a larger cloud would be preferred [12] .
The quantitative aspect of nasal powder delivery is largely dependent on the type
of delivery device. Devices for powder dosage forms, including insuffl ators, monodose
and multidose powder inhalers, and pressurized metered - dose inhalers, are
extensively reviewed elsewhere [29, 30] . Particles intended for nasal delivery should
have good fl ow properties to be reproducibly fi lled in the dose reservoir and easily
insuffl ated to obtain appropriate nasal deposition [28] . Since the particle size of the
applied powder formulation has a major impact on its nasal deposition, the characterization
of this parameter is very important. Only particles over 5 . m are deposited
in the nostrils while smaller particles can be inhaled into the lower parts of the
respiratory system [23] . For that reason, it is necessary to determine particle sizes
not only prior to fi lling the nasal delivery device but also after its actuation, since
the particle size of the formulation leaving the device depends on the device - disaggregating
properties [29] . Furthermore, it is known that powder deposition in the
anterior part of the nasal epithelium contributes to nasal absorption more than
powder deposition in the posterior part, since ciliated cells cover mainly the posterior
part of the nasal epithelium, providing a faster clearance of particles [31, 32] .
Thus, deposition studies must include both the amount of deposited particles and
the location of the particle deposition [29] . Distribution of the formulation in the
nasal cavity should be evaluated considering two different deposition types: the
initial deposition immediately after application and the secondary deposition, owing
to translocation by mucociliary clearance [29] . Cornaz et al. [33] investigated the
ability of microsphere delivery, studying the shape and area of the clouds of microspheres
emitted from a nasal device. The puff areas of loaded particles were smaller
compared to unloaded particles due to different particle porosities. Pringels et al.
[31] investigated the infl uence of the deposition pattern and spray characteristics of
nasal powder formulations on insulin bioavailability. It has been shown that posterior
deposition of the powder formulation in the nasal cavity lowered insulin bioavailability.
To study the spray characteristics, the shape and cross section of the
emitted powder cloud (puff) were analyzed. It has been concluded that the powder
bulk density of the formulation infl uenced the spray pattern. Comparing two
powders with different bulk densities and particle sizes, it has been revealed that
the powder with higher bulk density and smaller particle size was more compact
and was characterized by higher resistance to airfl ow, resulting in a slower spray
time and larger spray pattern [31] . No infl uence of the powder bulk density and
spray pattern on insulin bioavailability has been observed.
5.7.3 POLYMERS IN NASAL POWDER DELIVERY SYSTEM
It has been demonstrated that low absorption of drugs can be improved by using
absorption enhancers or prolonging contact between drug and absorptive sites in
the nasal cavity by delaying mucociliary clearance of the formulation. Some mucoadhesive
polymers can serve both functions. They are typically high - molecular -
weight polymers with fl exible chains which can interact with mucin through hydrogen
bonding, electrostatic, hydrophobic or van der Waals interactions [18, 34, 35] .
The mucoadhesive polymers are often hydrophilic and swellable, containing
numerous hydrogen bond – forming groups such as hydroxyl, carboxyl, or amine,
which favors adhesion. When used in a dry form they attract water from the mucosal
surface and swell, leading to polymer – mucus interaction, increased viscosity of
polymer – mucus mixture, and reduced mucociliary clearance [34] . Beside the type
of polymer functional groups, the mucoadhesive force of a polymer material is
dependent on the polymer molecular weight, concentration, fl exibility of the polymer
chain, spatial conformation, contact time, environmental pH, and physiological
factors such as mucin turnover and disease state [3] . There is a critical polymer
molecular weight for each polymer type below or above which there is reduced
adhesive power [34] . The mucoadhesive properties can also be affected by the
degree of cross - linking of the polymer since mucoadhesion requires an adequate
free chain length for interpenetration to occur. Hence, the more cross - linked the
polymer, the less strong the mucoadhesive interaction [36] . Hydration and swelling
present both polymer - and environment - related factors. Overhydration causes
extended swelling, resulting in slippery mucilage formation [30] . The polymer concentration
that is required for optimum mucoadhesion is different between gels and
solid mucoadhesives. In the liquid state, an optimum concentration exists for each
polymer for which best adhesion can occur, while with solid dosage forms, increased
polymer concentration leads to increased mucoadhesive power [35] . Studies have
shown that polymers with charge density can serve as good mucoadhesive agents,
although their mucoadhesive properties are affected by the pH of the surrounding
media. The presence of metal ions, which can interact with charged polymers, may
also affect the adhesion process [35, 36] . It has also been reported that polyanion
polymers are more effective bioadhesives than polycation polymers or nonionic
polymers [37] .
Research on nasal powder drug delivery has employed polymers such as starch,
dextrans, polyacrylic acid derivatives (e.g., carbopol, polycarbophil), cellulose derivatives
(microcrystalline cellulose, semicrystalline cellulose, hydroxypropylmethyl
cellulose, hydroxypropyl cellulose, carboxymethyl cellulose), chitosan, sodium alginate,
hyaluronans, and polyanhydrides such as poly(methyl vinyl ether - co - maleic
anhydride) (PVM/MA). Many of these polymers have already been used as excipients
in pharmaceutical formulations and are often referred to as fi rst - generation
bioadhesives [38 – 45] . In nasal dry powder a single bioadhesive polymer or a
POLYMERS IN NASAL POWDER DELIVERY SYSTEM 655
656 NASAL POWDER DRUG DELIVERY
combination of two or more polymers has been formulated as freeze - dried or spray -
dried particles or micropheres.
Crystalline cellulose, hydroxypropyl cellulose, and Carbopol 934 have been
studied in combination with lyophilized insulin as bioadhesive powder dosage forms
for nasal delivery. Each formulation tested resulted in an decrease in plasma glucose
level after nasal administration in dog and rabbit models. The most effective formulation,
crystalline cellulose blended with insulin, decreased the plasma glucose level
to 49% of the control value. In ternary systems the lyophilized Carbopol 934 and
insulin blend with crystalline cellulose powder has been the most effective, leading
to a hypoglycemia on the order of one - third of the effect obtained after intravenous
injection of the same dose of insulin. The plasma glucose levels obtained in the
volunteers after administration of the insulin – Carbopol – crystalline cellulose powder
formulation were quite variable [38] .
The various powder formulations were prepared by dry blending of octreotide
with microcrystalline cellulose, semicrystalline cellulose, hydroxyethyl starch, cross -
linked dextran, microcrystalline chitosan, pectin, and alginic acid [40] . Their potential
to enhance the nasal absorption of the somatostatin analogue peptide octreotide
was studied in vivo in the rat model. The powder mixtures were also characterized
in vitro regarding calcium binding, water uptake, and drug release. The bioavailabilities
obtained for all of the powder formulations were low, with the highest
values for alginic acid and cross - linked dextran powder formulations (4.1 and 5.56%,
respectively).
Callens and Remon [46] have shown improved nasal absorption of insulin in
rabbits by using a bioadhesive powder formulation containing drum - dried waxy
maize starch (mainly amylopectin) and Carbopol 974P. The bioavailability of 14%
has been obtained. They have shown [47] that the initial advantage of a longer residence
time of the powder formulation in the nasal cavity might turn into a disadvantage
after multiple administration and impact bioavailability. They investigated
the infl uence of eight daily administrations of two powder formulations to rabbits
on the bioavailability and therapeutic effect of the insulin [47] . The fi rst powder
formulation consisted of a co - spray - dried mixture of Amioca starch and Carbopol
974P and the second one has been a physical mixture of drum - dried waxy maize
starch and Carbopol 974P. By a single nasal administration to rabbits, absolute bioavailabilities
of 17.8 and 13.4% have been obtained, respectively. The lower insulin
bioavailabilities (4.4 and 3.6%, respectively) after multiple administrations were
observed with both formulations, mainly due to the high viscosity of the bioadhesive
powders in the nasal mucus, causing a physical barrier toward absorption and a
strongly decelerated mucociliary clearance. Long residence times of the powder
formulations were also reported by Ugwoke et al. [42, 48] , who noticed nasal residence
times of more than 24 h using powder formulations containing Carbopol 971P
and carboxymethyl cellulose.
Rhinocort is a commercially available mucoadhesive transnasal powder preparation
of beclomethasone dipropionate with hydroxypropyl cellulose (HPC) as a gel -
forming drug carrier developed by Suzuki and Makino [49] . The HPC has been
shown to promote the absorption of low - molecular - weight drugs, but it was not that
effective with a peptide drug salmon calcitonin. Microcrystalline cellulose has been
shown to be effective for the promotion of absorption of calcitonin in humans,
producing about 10% bioavailability with rapid absorption onset [49] . In the study
of the effect of an HPC and MCC combination on the development of nasal powder
preparations for peptide delivery, signifi cant absorption enhancement of leuprolide,
calcitonin, and fl uorescein isothiocyanate (FITC) – dextran in rabbits has been
obtained by the addition of 10 – 20% HPC to MCC. It has been suggested that MCC
works as an absorption enhancer by causing a locally high concentration of drugs
in the vicinity of the mucosa surface while HPC works to increase retention of drugs
on the nasal mucosa due to its gel - forming property. In a comparative study [50] of
a series of MCC nasal sprays and lyophilized powder formulations of ketorolac, the
spray formulations have been shown to be better absorbed than powder formulations.
The absolute bioavailability of ketorolac from a powder formulation has been
38%, and no signifi cant differences in absorption between different powder formulations
have been observed.
Lim et al. [51, 52] compared a number of mucoadhesive microspheres prepared
by solvent evaporation composed of hyaluronic acid (HA), chitosan glutamate
(CH), and a combination of the two with microcapsules of HA and gelatin prepared
by complex coacervation. Some other polymers — such as alginates [53, 54] , a natural
polymer of low toxicity, irritability, and immunogenicity; epichlorohydrine cross -
linked starch (Spherex) [55 – 61] and dextran (Sephadex) [62 – 65] ; poly(lactide - co -
glycolide) (PLGA) [66] ; and the biocompatible and biodegradable copolymer of
lactic and glycolic acids, which have also been approved by the Food and Drug
Administration (FDA) [67] — have mainly been used in microspheres for nasal dry
powder delivery and are referred to in more detail in the next section.
Recently, thiolated polymers or thiomers, a new generation of permeation -
enhancing agents, have been introduced in the pharmaceutical literature. Thiomers
are characterized by covalent immobilization of sulfhydryl groups on their polymeric
backbone, which are responsible for improved permeation - enhancing properties
combined with mucoadhesive and enzyme - inhibitory properties [68] . A further
improvement of the permeation - enhancing effect of thiomers has been achieved by
the addition of the permeation mediator glutathione [69] . The improvement of
human growth hormone (hGH) bioavailability (8.11%) by intranasal administration
of the microparticulate formulation composed of thiomer polycarbophil - cysteine
(PCP - Cys) and permeation mediator glutathione has been shown. Evaluation of the
effect of PCP - Cys on the ciliary beat frequency (CBF) of human nasal epithelial
cells in vitro has shown no ciliotoxic effect [70] .
Chitosan is a hydrophilic, biocompatible, and biodegradable polymer of low
toxicity, and it has been extensively investigated for pharmaceutical and medical
purposes. It has been included in the European Pharmacopoeia since 2002. Chitosan
is a polysaccharide composed of N - acetyl - d - glucosamine (approximately 20%) and
glucosamine (approximately 80%). It is derived by deacetylation of chitin, which
after cellulose is the most abundant polymer found in nature. It is a polycation at
acidic pH values where most of the amino groups are protonated and has an apparent
p K a of 5.5 [71 – 74] . In the context of drug delivery, chitosan has been used for
the preparation of microcapsules and microspheres with encapsulated small polar
molecules, proteins, enzymes, DNA, and cells, as a nasal delivery system for insulin
[75] , as a system for oral vaccination, and as a stabilizing constituent of liposomes.
Several studies have highlighted the potential use of chitosan as an absorption -
enhancing agent due to its mucoadhesive properties and ability to open the tight
junctions in the mucosal cell membrane [72] .
POLYMERS IN NASAL POWDER DELIVERY SYSTEM 657
658 NASAL POWDER DRUG DELIVERY
Its biodegradability and low toxicity in humans have aided the recent increased
interest in chitosan as an immunopotentiating agent. In vivo studies have demonstrated
that chitosan powder and solution formulations are able to enhance
the systemic and mucosal immune responses after nasal vaccine delivery [19, 22,
76] .
The nasal absorption of insulin after administration in chitosan powder was the
most effective formulation for nasal delivery of insulin in sheep compared to chitosan
nanoparticles and chitosan solution [11] . Similarly, chitosan powder formulations
have been shown to enable an effi cient nasal absorption of goserelin in a sheep
model where bioavailabilities of 20 – 40% were obtained depending on the nature
of the formulation [9] .
There has been a report on chitosan utility in improving the intranasal absorption
of high - molecular - weight ( > 10 - kDa) therapeutic protein. Chitosan glutamate
powder blend or granules with recombinant hGH have been evaluated for intranasal
administration in sheep. Relative to subcutaneous injection the nasal formulations
produced bioavailabilities of 14 and 15%, respectively [77] .
Various chitosan derivatives of enhanced solubility, mucoadhesive, and permeation
properties were developed. N - Trimethyl chitosan chloride (TMC) is a quaternized
derivative of chitosan with superior aqueous solubility over a broader pH
range and penetration - enhancing properties under physiological conditions [78] .
Carboxymethylated chitosan (CMChi) is a polyampholytic polymer able to form
viscoelastic gels in aqueous environments. CMChi appears to be less potent compared
with the quaternized derivative. Neither TMC nor CMChi have been found
to provoke damage of the cell membrane, and therefore, they should not alter the
viability of nasal epithelial cells [79] .
Thiolated chitosans, chitosan thioglycolic acid conjugates, chitosan – cysteine conjugates,
and chitosan - 4 - thio - butyl - amidine conjugates are new - generation polymers
that are pH sensitive. The pH range at which the gelation and mucoadhesion of
these polymers are optimal is within the physiological range (pH 5 – 6.5) of the nasal
mucosa, but these polymers have been primarily investigated for oral drug delivery
[80] .
5.7.4 MICROSPHERES AS NASAL DRUG DELIVERY DEVICES
Developing an appropriate drug delivery system for a given drug can completely
alter the drug ’ s unfavorable properties, such as improve its effectiveness or reduce
its side effects. Dry powder delivery systems such as microspheres are of special
interest. In the last two decades they have been extensively studied with respect to
nasal delivery and a considerable number of studies have been reported on that
subject [3, 23] .
In general, microspheres as specialized drug delivery systems represent spherical
polymeric devices that are small in size (from 1 to 1000 . m), are characterized by
high surface - to - volume ratio, and are able to provide targeted and predictable controlled
release of the drug [3] . In the scope of nasal delivery, except for controlled
drug release rate, microspheres are benefi cial due to their broad surface area, which
can provide extensive interaction with the mucin layer and protection of incorporated
drug from enzymatic degradation in the nasal cavity [10, 41] .
Microspheres prepared with bioadhesive polymers have some additional advantages;
they assure much more intimate and prolonged contact with the mucous layer
and improved drug absorption due to additional delay in mucociliary clearance.
Bioadhesive microspheres can signifi cantly improve patient compliance as all
the advantages described lead to reduction in the frequency of drug administration
[3, 74] .
Bioadhesive microspheres that have been extensively studied for nasal drug
delivery are water insoluble but they swell in contact with the mucosa. Swollen
microspheres form a gellike system that adheres onto the mucus, retaining drug at
the absorption site for prolonged periods [2, 81] . Swelling of the microspheres causes
mucosal dehydration and reversible shrinkage of the cells, resulting in the temporary
widening of the tight junctions and increased permeability of hydrophilic
compounds, or more precisely, paracellular absorption of the drug [62] . Oechslein
et al. [40] suggested that the opening of tight junctions could be related to the local
decrease in Ca 2+ concentration as well, since the absorption - promoting effect of
investigated particulate drug delivery systems correlated directly with their capability
to bind Ca 2+ . In order to additionally improve nasal drug absorption, bioadhesive
particulate systems have been combined with biological absorption enhancers
[55 – 57] .
A number of studies of the nasal mucociliary clearance rate of microspheres
confi rmed their potential to retain drug at the absorption site longer than liquid
formulations [58, 67, 81, 82, 83] . Methods to measure formulation clearance rates
from the nasal cavity can be divided into three groups, differing in the detected
substance [58] . The most exact method is gamma scintigraphy, which monitors the
deposition and clearance of radiolabeled drug delivery systems. The second method
involves mixing of a fl uorescent dye with the formulation and monitoring the cumulative
tracer amount in the pharynx. The third method is the saccharin test, in which
saccharin is mixed with the formulation and the clearance rate is determined by the
fi rst perception of sweet taste [84] . The fi rst study of the mucociliary clearance of
microspheres using gamma scintigraphy was reported by Illum et al. [81] . They
evaluated clearance rates of starch, dextran, and albumin microspheres: Three hours
after nasal administration about 50% of albumin and starch microspheres and 60%
of dextran microspheres were still detected at the site of deposition. Nasal clearance
study of melatonin starch microspheres and the melatonin solution applied revealed
that more than 80% of the starch microspheres remained in the nasal mucosa 2 h
after administration, compared to only 30% for the melatonin solution [58] . The
study of the clearance rate of alginate, PLGA, and Sephadex microspheres revealed
that alginate and PLGA were suitable for nasal delivery as they had the best mucoadhesive
properties [67] . It has also been shown that the limiting step of the mucociliary
clearance of nasally administrated microspheres was their clearance from the
initial deposition site. The same conclusion has been drawn for the Carbopol 971P
and carboxymethyl cellulose microspheres [82] . Soane et al. [83] evaluated the clearance
rate of chitosan microspheres and chitosan solutions, compared to the control
solution from the nasal cavity in sheep, by gamma scintigraphy. They found that
both chitosan systems had higher retention times compared to the control. Also,
chitosan microspheres were cleared at a slower rate than the chitosan solution, with
half times of clearance of 115 and 43 min, respectively. The nasal clearance rates
found in the sheep model were similar to the clearance rates found in their previous
MICROSPHERES AS NASAL DRUG DELIVERY DEVICES 659
660 NASAL POWDER DRUG DELIVERY
study carried out on human subjects [85] , indicating that the sheep could be a suitable
model for in vivo nasal clearance studies.
Starch Microspheres Bioadhesive starch microspheres in the context of the nasal
delivery system were fi rst introduced by Illum et al. [81] in a study that examined
human nasal mucociliary clearance. Since then starch microspheres have been shown
to promote the nasal absorption of a number of drugs. Animal studies using the sheep
model showed greatly improved absorption of gentamicin [59] and human growth
hormone [56] when administered in combination with starch microspheres in a
freeze - dried formulation. Similar fi ndings have been reported for insulin [55, 60] and
desmopressin [61] loaded starch microspheres compared to simple drug solutions
administered to animal models. Biodegradable starch microspheres have also been
investigated for nasal delivery of metoclopramide whereas enhanced bioavailability
was achieved compared to nasal spray [86] . The bioadhesive starch microspheres
were shown to act synergistically with the absorption enhancers improving the transport
of insulin across the nasal membrane [57] . Recently, the potential of starch
microspheres for the nasal delivery of melatonin was investigated [58] . An in vitro
release study revealed a sustained drug release profi le. Melatonin bioavailability
after nasal administration of starch microspheres was high, 84%. A good correlation
between the in vitro release profi le and in vivo absorption has been observed.
The use of degradable starch microspheres has proved to be well tolerated in
both experimental animals and humans. No alterations of nasal mucosa were
detected after eight weeks of nasal administration of starch microspheres to rabbits.
Additionally, a preliminary test on healthy volunteers also showed good acceptability
[62] . Another study of healthy volunteers revealed no changes in mucociliary
clearance or in the geometry of the nasal cavities after eight days of nasal administration
of dry starch microspheres [87] .
Dextran Microspheres Similar to starch microspheres, Illum et al. [81] introduced
dextran microspheres as a bioadhesive drug delivery system able to prolong the
residence time in the nasal cavity. However, Ryd e n and Edman [65] reported dextran
microspheres were not shown to improve nasal absorption of insulin in rats as
insulin was too strongly bound to the Diethylaminoethyl (DEAE) groups to be
released by a solution with an ionic strength corresponding to physiological conditions.
In a later study it has been shown that the localization of insulin infl uenced
the in vivo behavior of dextran microspheres [63] . The distribution of insulin at the
surface or inside the dextran microspheres after the lyophilization loading process
was determined by the cut - off limit of the microspheres. Microspheres with insulin
left at the surface showed higher insulin absorption – enhancing effect than the
microspheres with insulin inside the dextran matrix. Dextran microspheres have
also been evaluated in vivo as a delivery system for octreotide [40] and in vitro for
nicotine [33] . Ciliotoxicity studies performed in vitro on explants from rat trachea
showed that dextran microspheres had no effect on the ciliary beat frequency [64] .
The immediate recovery of the ciliary movement after dextran microspheres washing
off indicated that the cilia were not damaged by dextran microspheres.
Gelatin Microspheres Several studies characterizing gelatin microspheres as a
nasal drug delivery system have been reported. Gelatin microspheres were shown
to swell readily in contact with nasal mucosa and to have good bioadhesive properties
[65] . An in vitro release study using a Franz diffusion cell on levodopa - loaded
gelatin microspheres showed prolonged drug release as compared to drug alone
[88] . Negatively and positively charged gelatin microspheres intended for nasal and
intramuscular delivery of salmon calcitonin were prepared by Morimoto et al. [10] .
Both types of microspheres enhanced nasal absorption of salmon calcitonin compared
to the solution. Positively charged gelatin microspheres seamed to exhibit
greater enhancing effect on nasal absorption than negatively charged gelatin microspheres.
Recently gelatin and gelatin – poly(acrylic acid) microspheres were studied
with respect to oral and nasal delivery of oxprenolol [89] . Combining the gelatin
with poly(acrylic acid) resulted in microspheres with improved bioadhesive properties.
Also, nasal administration of gelatin – poly(acrylic acid) microspheres resulted
in improved bioavailability of the drug compared to nasal administration of the drug
solution.
Polyacrylate Microspheres Cross - linked polyacrylate microspheres as nasal
powder delivery systems have been investigated in several studies [39, 41, 90] . Microspheres
were produced by spray drying and emulsifi cation methods and their nasal
drug delivery potential has been evaluated only in vitro. Carbopol 934P microspheres
were shown to have the best bioadhesive properties compared to other
hydrophilic microspheres prepared with polyvinyl alchohol, chitosan, and hydroxypropylmethyl
cellulose [41] . Improved permeation - enhancing effect of polycarbophil
microparticles was obtained when microparticles were prepared with the
thiolated polycarbophil and the permeation mediator glutathione [68] .
Chitosan Microspheres As a specifi c chitosan - based delivery system, chitosan
microspheres have been extensively studied and number of reports has verifi ed their
potential regarding nasal drug delivery [9, 21, 25, 41, 53, 73, 91, 92] . Chitosan microspheres
have been prepared by the emulsifi cation solvent evaporation method [51,
52, 93] , emulsifi cation cross - linking process [91] , spray drying method [25, 53] , and
ionic gelation process [94] . They have been shown to signifi cantly reduce mucociliary
clearance from the nasal cavity of sheep and humans compared to solutions [83,
85] . The bioadhesive properties of chitosan microspheres were shown to be inversely
proportional to particle size: Among chitosan microspheres in the size class between
50 and 200 . m, smaller microspheres appeared to swell faster than large microspheres,
providing a more powerful mucoadhesive system [41] .
A modulated release rate of drug from the swellable chitosan microspheres has
been achieved with cross - linking agents such as glutaraldehyde [95] , citric acid [92] ,
ascorbic acid, or ascorbyl palmitate [91] that reacted with chitosan forming covalent
bonds with chitosan amino groups. However, to maintain the bioadhesive properties
of cross - linked chitosan microspheres, the amount of cross - linking agent should be
optimized [95] .
Chitosan molecular weight has also been reported to infl uence drug release. Jiang
et al. [94] studied Bordetella bronchiseptica dermonecrotoxin (BBD) release from
chitosan microspheres prepared by tripolyphosphate ionic gelation. It has been
shown that the BBD release rate increased with chitosan molecular weight decrease.
It has been explained by the weaker BBD interaction with chitosan of lower molecular
weight and lower content of free amine groups, responsible for their interaction.
MICROSPHERES AS NASAL DRUG DELIVERY DEVICES 661
662 NASAL POWDER DRUG DELIVERY
Chitosan microspheres were shown to enhance nasal bioavailability of several
peptide drugs such as insulin and goserelin. A simple chitosan – insulin powder formulation
provided about 20% of absolute insulin bioavailability in sheep [96] .
Improved bioavailability (of 44%, in rats) was obtained when insulin was loaded
into chitosan microspheres prepared with ascorbyl palmitate as cross - linking agent
[91] . Chitosan microspheres have also been shown to improve nasal goserelin
absorption providing about 40% bioavailability relative to goserelin intravenous
application [9] .
Krauland et al. [93] prepared the microparticles with thiolated chitosan (chitosan -
TBA; chitosan – 4 - thiobutylamidine conjugate) intended for nasal peptide delivery.
During the preparation process microparticles were stabilized by the formation of
inter - and intramolecular cross - linking via disulfi de bonds. Chitosan – TBA microparticles
were characterized by improved swelling ability and displayed 3.5 - fold higher
insulin bioavailability compared to unmodifi ed chitosan microparticles.
Besides the polymer derivatization, combining the polymers in microsphere
preparations can result in improved drug delivery and absorption characteristics.
Hyaluronic acid – chitosan microspheres appeared to improve the absorption of
incorporated gentamicin compared to the individual polymers, assembling the
mucoadhesive potential of both polymers and the penetration - enhancing effect of
chitosan [51, 52] .
Chitosan microparticulate systems have also been investigated for vaccine nasal
delivery and have proven to induce strong systemic and mucosal immune responses
[18, 21, 76] .
5.7.4.1 Preparation Methods
The design of bioadhesive microspheres includes selection of the most suitable
preparation method, considering the nature of the drugs and polymers used as well
as the route of administration. A number of methods for the preparation of microspheres
have been described in the literature [3, 97] . In the scope of nasal delivery,
the fi rst microspheres in use were starch and dextran microspheres, prepared by an
emulsion polymerization technique employing epichlorohydrine as a cross - linking
agent [55, 56, 59, 60, 62, 81] . Currently techniques based on solvent removal, such
as solvent evaporation [41, 51, 66, 93, 98] and solvent extraction [88, 99] , are most
frequently in use. There are tree processes involved in such microensapsulation
procedures: the preparation of emulsion, solvent removal, and separation of the
particles obtained. Selection of the type of (oil - and - water) emulsion system (O/W,
W/O, W/O/W, W/O/O, etc.) depends on the physicochemical properties of the drug
and polymer used. After the preparation of stable emulsion, solvent is removed from
the system at high or low temperature, at low pressure, or by addition of another
solvent that enables the extraction of polymer solvent to the continuous phase.
Hardened microspheres are then washed, centrifuged, and lyophilized.
Emulsion techniques are suitable for the preparation of microspheres intended
for nasal delivery since they allow controlling the size of the particles. Freiberg and
Zhu [97] reviewed solvent evaporation process parameters (e.g., polymer concentration,
viscosity, stirring rate, temperature and percentage of emulsifying agent) affecting
microsphere size. It can be assumed that the particle size is directly proportional
to polymer concentration and inversely proportional to stirring rate and percentage
of emulsifying agent [41, 88] , while there is a nonlinear correlation between particle
size and process temperature. Yang et al. [100] reported that larger microspheres
were produced at lower temperatures due to the higher viscosity of solution and at
higher temperatures due to the higher solvent fl ow pressure moving more material
from the microsphere center outward. In the same work encapsulation effi ciency
was also correlated with the temperature of solvent evaporation in the process of
microsphere preparation. It was found that the highest encapsulation effi ciencies
occurred at the lowest and highest temperatures tested.
Recently, the spray drying method has been extensively used for the preparation
of microspheres intended for nasal delivery [3, 25, 39, 44, 45, 53, 54, 101, 102] . Spray
drying is a single - step procedure transforming liquid into dry particulate form (e.g.,
microparticles, microspheres, microcapsules) applicable to drugs and polymers of
various solubility characteristics. It is a fast, simple, and reproducible technique that
is easy to scale up [3] . Spray drying can be described as follows: The liquid is fed to the
nozzle with a peristaltic pump, atomized by the force of the compressed air, and blown
together with hot air into the chamber, where the solvent in the droplets is evaporated.
The dry product is then collected in a collection bottle. Spray - dried microspheres
are reported to have relatively low production yields, rarely higher than 50%.
The loss of material during spay drying has been explained by the powder adhering
to the cyclone walls, small amounts of materials processed in each batch, and loss of
the smallest and lightest particles through the exhaust of the spray dryer apparatus,
which lacks a trap to recover the lighter and smaller particles [25, 53, 103, 104] .
Spray drying offers the possibility to control the particle size of the product.
Microparticles of desirable size can be obtained by optimizing the spray drying
process parameters, such as size of the nozzle, feeding pump rate, inlet temperature,
and compressed airfl ow rate. In accordance with this, He et al. [105] reported that
larger particles were formed at a larger size of nozzle and faster feeding pump rate,
while smaller particles were formed at a greater volume of air input [105] . The size
and other properties of spray - dried microspheres (e.g., morphology, density, shape,
porosity, and fl owability) can also be affected by the qualitative and quantitative
composition of the liquid feed [28] . Thus, feed concentration has been reported to
infl uence the particle size distribution, as spray drying of more concentrated liquid
feeds resulted in the formation of larger particles [104, 106, 107] .
5.7.4.2 Microsphere Characterization
Microspheres intended for nasal administration need to be well characterized in
terms of particle size distribution, since intranasal deposition of powder delivery
systems is mostly determined by their aerodynamic properties and particle sizes.
Commonly used methods for particle size determinations described in the literature
are sieving methods [108] , light microscopy [58] , photon correlation spectroscopy
[66] , and laser diffractometry [25, 41, 53, 93] . The morphology of the microparticles
(shape and surface) has been evaluated by optical, scanning, and transmission electron
microscopy [66, 95] .
Determination of the zeta potential is an important part of microsphere characterization,
as the zeta potential has a substantial infl uence on the adhesion of drug delivery
systems onto biological surfaces [109] . For example, Jaganathan and Vyas [110]
reported the reduction in the nasal clearance rate of PLGA microspheres modifi ed
with chitosan compared to unmodifi ed PLGA microspheres due to the change in zeta
potential from negative for PLGA microspheres to positive for surface - modifi ed
MICROSPHERES AS NASAL DRUG DELIVERY DEVICES 663
664 NASAL POWDER DRUG DELIVERY
PLGA microspheres. Methods to measure the zeta potential of microspheres are
laser doppler anemometry [105] and photon correlation spectroscopy [110] .
The physical state of the drug incorporated in a powder drug delivery system
(e.g., degree of crystallinity and possible interactions with the polymer) is assessed
by differential scanning calorimetry (DSC) or Fourier transform infrared (FTIR)
spectroscopy. These observations can clarify the results of other parameter investigations,
especially the results of in vitro drug release studies.
To predict microsphere performance in vivo, the swelling properties of nasal
powder delivery systems need to be evaluated. Methods described in the literature
are mostly based on the weight difference measurements between the dry and
swollen powder [40] . Swelling properties of nasal powders such as water - absorbing
capacity can be evaluated using a Franz diffusion cell [43, 107] . The swelling capacity
may also be expressed as the volume expansion of the microspheres that is determined
at equilibrium after placing the microspheres in water using a graduated
cylinder [108] . Gavini et al. [53] determined the swelling properties of microspheres
in vitro by laser diffractometry. That method allows us to evaluate the variation of
particle size versus time.
In vitro evaluation of mucoadhesive properties is essential in the development of
a nasal drug powder delivery system, since mucoadhesion is of great importance for
the in vivo performance of formulation. A large number of in vitro and in vivo
methods used to assess mucoadhesive properties of microspheres have been extensively
described in the literature [3, 111, 112] . Many in vitro methods are based on
the interaction of microspheres with mucin. Evaluation of that interaction can be
performed using scanning and transmission electron microscopy [95] or photon correlation
spectroscopy [45] . Scanning electron microscopy (SEM) provides the information
on morphological changes on the microsphere surface in contact with mucin,
while transmission electron microscopy confi rms SEM results and reveals the ultrastructural
features of the surface interactions between microspheres and mucin
chains [95] . He et al. [102] evaluated the mucoadhesive properties of chitosan microspheres
by measuring the amount of mucin adsorbed on the microspheres. Gavini et
al. [53] evaluated the mucoadhesive properties of metoclopramide - loaded microspheres
by determining the amount of microspheres that stuck to a fi lter paper saturated
with mucin after exposure to the air stream. Vidgren et al. [39] used a tensiometer
to measure the force required for the separation of two fi lter paper discs saturated
with mucin and with the examined microspheres placed between them.
In the work reported by Witschi and Mrsny [54] mucoadhesion of dry powder
microparticles was investigated using Callu - 3 cells as a surrogate for human nasal
epithelia: Microparticles were applied to the apical surface of cell sheets and at certain
time points were washed with phosphate - buffered saline (PBS) to remove poorly
adhering microparticles. Rango Rao and Buri [113] developed an in situ method to
evaluate the bioadhesive properties of polymers and microparticles, based on washing
off a mucous membrane covered with the formulation to be tested by simulated biological
fl ow. The mucoadhesion of gelatin microspheres [10] was measured by an in
situ nasal perfusion experiment. In the work reported by Lim et al. [51] the mucoadhesive
properties of microspheres were evaluated by determining the mucociliary
transport rate of the microparticles across an isolated frog palate. The boiadhesive
properties of microspheres can be evaluated by the everted sac technique using a
section of everted intestinal tissue or the CAHN dynamic contact angle analyzer [3] .
Santos et al. [112] correlated these two methods and concluded that each method
could be used alone as the relevant indicator of microsphere bioadhesion.
In vitro drug release experiments can be performed in order to characterize the
release behavior of microparticles in general. For that purpose microparticles can
be dispersed directly in the dissolution medium [51, 91] or a dynamic dialysis technique
can be employed [58] . However, to obtain results comparable with the in vivo
situation of nasal administration, it is necessary to provide experimental conditions
similar to those encountered in the nasal cavity as nasally administrated powders
are not being dispersed directly in the large quantity of liquid [8] . Such in vitro drug
release experiments can be performed by a modifi ed U.S. Pharmacopeia (USP)
XXII rotating basket [8] . The drug - loaded powder formulation is weighed on a
membrane fi lter placed between the fi lter holder and the cup and then immersed
in the released medium. Thus, the membrane fi lter separates the donor and acceptor
compartment but at the same time allows the powders to hydrate and to form a gel.
Drug is released to the release medium after diffusion through the swollen gel of
known surface area. Cornaz et al. [33] developed a special diffusion chamber that
simulated the hydration conditions of the nasal mucosa to study the in vitro release
of nicotine from dextran microspheres. A number of authors have used Franz diffusion
cells for in vitro release experiments since that model provides conditions
similar to those encountered in the nasal cavity and slow hydration of the microspheres
[41, 43 – 45, 107] .
5.7.4.3 Chitosan - Formulated Spray - Dried Microspheres
Chitosan, a biocompatible and biodegradable polycationic polymer with low toxicity,
is known for its swelling ability and permeation - enhancing properties and represents
a polymer of choice for the preparation of microspheres intended for nasal
administration [74] .
Spray drying has proved to be a suitable and simple technique for the preparation
of chitosan microspheres with preserved chitosan properties, offering numerous
advantages over other microencapsulation methods. It has been successfully used
to entrap both hydrophilic and lipophilic drugs into the chitosan matrix, since a
variety of colloidal systems (e.g., polymer solutions, emulsions, dispersions, suspensions)
can be subjected to spray drying. Chitosan - based spray - dried microspheres
have been prepared with chitosan alone (resulting in conventional microspheres)
or in combination with another polymer (resulting in composed microspheres).
Combining the polymers has been reported to result in microspheres with improved
properties regarding surface characteristics, entrapment effi ciency, or control over
the drug release rate. Recently, several drugs, such as carbamazepine [25] , propranolol
hydrochloride [45] , metoclopramide hydrochloride [53] , and loratadine [103,
107] , have been incorporated into chitosan - based nasal powder formulations produced
by the spray drying - method. They were characterized in terms of encapsulation
effi ciency, morphology, size distribution, zeta potential, physical state of the
drug, in vitro drug release behavior, and swelling and bioadhesive properties.
Chitosan – ethyl cellulose composed microspheres improved loratadine entrapment
compared to conventional chitosan microspheres, which infl uenced directly the
microsphere surface characteristics: Loratadine was less present at the surface of
the microspheres and consequently had less infl uence on their bioadhesive proper-
MICROSPHERES AS NASAL DRUG DELIVERY DEVICES 665
666 NASAL POWDER DRUG DELIVERY
ties. Thus, although showing moderate swelling ability, loratadine - loaded composed
microspheres were more bioadhesive than conventional chitosan microspheres.
Composed microspheres showed good loratadine - sustained release potential in
vitro, depending on the polymeric weight ratio and concentration of the spray - dried
system [103, 107] . Gavini et al. [53] produced metoclopramide - loaded alginate and/
or chitosan microspheres by the spray drying method. The results obtained revealed
that complexation of chitosan with alginate in the microsphere preparation provided
improved control of the drug release in vitro compared to chitosan alone.
Despite the chitosan complexation, composed microspheres showed good bioadhesive
properties. Ex vivo drug permeation tests carried out using sheep nasal mucosa
showed higher drug permeation from chitosan - based microspheres than from alginate
microspheres, confi rming the well - known chitosan permeation - enhancing
properties. Cerchiara et al. [45] developed spray - dried chitosan – poly(methyl vinyl
ether - co - maleic anhydride) microparticles for nasal delivery of propranolol hydrochloride.
Chitosan was combined with polyanhydride, able to enhance the formation
of hydrogen bonds between the polymers and mucosal components through carboxylic
acid groups generated after polyanhydride hydrolytical degradation. The
swelling and bioadhesive properties of chitosan – polyanhydride microparticles
increased in a pH - dependent manner. Both chitosan and chitosan – polyanhydride
microparticles provided sustained propranolol hydrochloride release.
Microparticulate spray - dried delivery systems have shown great potential for
nasal delivery of drugs characterized by poor water solubility. According to DSC
analysis, the spray drying method together with the carriers seemed to promote the
amorphization of loratadine [107] and carbamazepine [25] . Carbamazepine incorporated
into chitosan microspheres was characterized by increased dissolution rate
compared to carbamazepine raw material. It was explained not only by the promoted
drug amorphization but also by the chitosan well - known dissolution rate
enhancer properties and by the small size of microspheres (or high surface - to -
volume ratio). Results of in vivo nasal administration of carbamazepine - loaded
chitosan microspheres revealed a remarkable increase in carbamazepine concentration
in serum compared to the pure carbamazepine powder [25] . Such an increase
in drug absorption has been ascribed to both improved dissolution of carbamazepine
and adhesion of the chitosan microspheres to the mucosal surface.
The mucoadhesive function of chitosan has also been employed in vaccine dry
powder delivery. Alpar et al. [18] produced bovine serum albumin (BSA) – loaded
chitosan microspheres using the spray drying method. It has been shown that the
stability of encapsulated BSA was preserved in the microspheres prepared, indicating
that spray drying was appropriate even for the preparation of antigen - loaded
microspheres. BSA - loaded chitosan microspheres generated higher immune response
than the free BSA, thus proving to be a suitable system for nasal antigen delivery.
5.7.5 TOXICOLOGICAL CONSIDERATIONS
The possible toxicological effects of dry powder formulation on the nasal mucosa,
including local irritation, effect on mucociliary clearance, and epithelial damage and
recovery rate [114] , should be investigated in an early stage of its development.
There were some attempts to defi ne the categories of toxic effects as well of the
constituents of nasal formulations according to their toxic potential. Thus, Hvidberg
et al. [115] introduced a scale of irritation in the nose (0, no irritation; 1, slight irritation;
2, acceptable; 3, unwilling to accept the treatment again) that was later used to
evaluate the degree of nasal powder irritation after administration in human volunteers
[27] . Ugwoke et al. [82] classifi ed the degree of ciliary beat frequency change
caused by nasally applied liquid formulation as follows: no effect (less than 10%),
mild (10 – 20%), moderate (20 – 50%), and severe (more than 50%). Soon after, that
classifi cation was applied to a powder ciliotoxicity study [116] . Reversibility of ciliotoxic
effect after washing out the tested compound was classifi ed as well, resulting
in three categories: reversible, partially reversible, and irreversible effects [82] .
Merkus et al. [117] proposed the three categories of constituents of nasal formulations
based on the recovery of ciliary beat frequency after the tested compound was
washed out . The fi rst category is cilio friendly, with ciliary beat frequency recovery
of 75% or more; the second is cilio inhibiting, with recovery between 25 and 75%;
and the third category is ciliostatic, with recovery of 25% or less.
Methods to evaluate the possible toxicological effect of formulations on the nasal
mucosa described in the literature mainly refer to histopathological evaluation as a
standard method for cytotoxicity evaluation [64] and a study on the release of marker
enzymes [118] . In the work reported by Callens et al. [118] the possible toxicological
effects of multiple starch and carbopol powder nasal administration were evaluated
using rabbits by measuring the proteins and lactate dehyrogenase (LDH) release
from the nasal mucosa. Contrary to the invasive in situ perfusion method, performable
with anaesthetized animals [119] , this method has been shown to be noninvasive,
applicable to nonanaesthetized and nonsedated animals, and suitable for repeated
measurement of the marker protein release on the same animal during a long - term
administration study. A histopathological study has also been performed. In agreement
with attempting to replace the use of vertebrates in scientifi c experiments with
lower organisms such as invertebrates, plants, and microorganisms, Adriaens and
Remon [120] developed a new mucosal toxicity screening method using the slug
Arion lusitanicus as the model organism. The body wall of the slug resembles the nasal
mucosa since it consists of a single - layered epithelium containing both ciliated and
mucus - secreting cells. Callens et al. [118] successfully used that method for screening
the mucosal irritation potential of bioadhesive starch and carbopol powder formulations.
The possible toxicological effects of the powder formulations were evaluated
by measuring the proteins LDH and alkaline phosphatase (ALP) release from the
body wall of the slugs as well as the amount of mucus produced.
Among various nasal toxicity studies, ciliotoxicity studies are of special interest
due to the importance of maintaining optimal ciliary beating to protect the lower
respiratory system from infections. If the drug formulation inhibits the ciliary beating,
such inhibition needs to be completely reversible upon formulation removal [116] .
Methods to determine ciliary beat frequency and mucociliary transport in vitro
and in vivo have been extensively reviewed elsewhere [84, 121] . In general, in vivo
methods are more reliable for ciliotoxicity studies than in vitro methods and are
essential to confi rm the safety of nasal drug formulations. However, in vitro methods
are more suitable for the large number of screening studies required during formulation
development [122] .
In vitro methods to measure ciliary beat frequency can be performed on explants
of ciliated mucosa [64] or on different types of ciliated cells, such as ciliated chicken
embryo trachea cells [123] , nasal cell lines derived from carcinomas of epithelial
origin (RPMI 2650, BT, NAS 2BL), or human lung adenocarcinoma cell line Calu - 3
TOXICOLOGICAL CONSIDERATIONS 667
668 NASAL POWDER DRUG DELIVERY
[124] . In vitro ciliotoxicity of dextran microspheres was evaluated on explants from
rat trachea [64] .
Human nasal epithelial cell cultures can serve as a relevant screening tool for
prediction of nasal formulation toxicity in humans as long as the cells maintain differentiated
morphological and biochemical characteristics of the original tissue
[124] . Epithelial cells intended for initiation of primary cell culture should be taken
from the regions of the nasal cavity where formulations are supposed to be deposited
[125] . Considering toxicological investigations, human nasal epithelial cells
cultured in an air – liquid interface system are the most promising ones at the moment,
as in these culturing conditions cell differentiation closely resembles cell differentiation
in vivo [124] . Monolayer immersion feeding and air – liquid interface cultures
have already been used for ciliary beat frequency measurements. However, unstable
ciliary activity and their short life span in the culture have proved to be the main
shortcomings of these systems. Jorissen et al. [126] developed a suspension culture
system of human nasal epithelium with actively beating cilia for several months.
That system has been validated for ciliotoxicity investigations [122] and later applied
in other studies [116, 127] . At this time it is the most successful model of human
nasal epithelium culture useful for ciliary beat frequency determination [124] .
In vivo methods to determine the toxicological effects of drug formulations on
the nasal mucosa mainly refer to mucociliary clearance rate studies. Methods
described in the literature are mostly based on the gamma scintigraphic technique,
in which cleared formulations are labeled with the radiotracer [82] . In vivo scintigraphic
evaluation of the nasal clearance of drug delivery systems requires a nondiffusible
and stable radiotracer to prevent its absorption and decomposition. Another
method described in the literature is the saccharin test [87] , in which saccharin is
mixed with the formulation and the clearance rate is determined by the fi rst perception
of sweet taste [84] . Gamma scintigraphy [31] is more relevant for the ciliary
function monitoring than the saccharin test, since it investigates the whole mucosal
surface, while the saccharin test investigates only the fastest fl ow rate [82] .
A number of ciliotoxicity studies have pointed out a low correlation between the
results obtained using different in vitro and in vivo methods [121] . The effects of
nasal formulations on the ciliary beat frequency in vitro are usually more expressed
than in vivo, since in vivo, cilia are partially protected by the mucous layer and
investigated formulation is eventually cleared from the nasal cavity due to the
mucociliary clearance mechanism. Also, toxic effects of the formulations on the cilia
in vivo may be reversible due to the constant nasal mucosal cell turnover [121] .
In conclusion, in order to make predictions regarding the safety of the nasal formulation
on mucociliary clearance, both in vitro and in vivo studies have to be performed.
It is also essential to determine long - term use effects in animals and
in humans if the nasal formulation is intended for subchronic or chronic
administration.
Dry powder formulations for nasal delivery of peptides and proteins have been
investigated for the fi rst time by Nagai and others [38] . Since then, much research
work has been done on dry powders containing bioadhesive polymers for nasal drug
administration. The bioavailability and duration of action of drugs administered by
the nasal route are increased by the use of the principle of mucoadhesion and dry
powder formulations. Research work on dry powder formulation containing bioadhesive
polymers is summarized in Table 1 .
TABLE 1 Summary of Research Work on Nasal Dry Powder Formulations
Powder
Formulation Preparation Method Drug Polymer Studies Comments Reference
Microparticles Spray drying BSA Starch,
alginate,
chitosan, carbopol
In vitro Chitosan microparticles
provided most desirable
characteristics for protein
delivery
54
Microparticles
Budesonide Polymethacrylic
acid – polyethylene
glycol
[P(MAA – PEG)]
In rabbits Continuous drug release for
at least 8 h
High bioavailability
133
Microparticles Solvent evaporation
Gentamicin Hyaluronic
acid – chitosan
In rabbits Polymers improved
gentamicin absorption
synergistically
52
Microparticles Lyophilization hGH Polycarbophil –
cysteine
In rats Improved bioavailability
68
Microparticles W/O emulsifi cation
solvent
evaporation
Insulin Chitosan – TBA In rats Improved bioavailability
93
Microparticles Spray drying Propranolol
hydrochloride
CH - PVM/MA In vitro Sustained drug release 45
Microparticles,
nanoparticles
W/O/W solvent
evaporation
Model protein,
tetanus toxoid
PLA - PEG In rats Size - dependent mucosal
uptake
66
Microspheres Ionic gelation with
tripolyphosphate
Bordetella,
bronchiseptica,
dermonecrotoxin
Chitosan In mice Systemic and mucosal
immune responses induced
21
Microspheres Ionic gelation
process with
tripolyphosphate
Bordetella,
bronchiseptica,
dermonecrotoxin
Chitosan In vitro Chitosan molecular weight –
related drug release profi le
94
Microspheres Spray drying Carbamazepine Chitosan
hydrochloride,
chitosan
glutamate
In sheep Increased drug dissolution
rate and absorption
25
669
Powder
Formulation Preparation Method Drug Polymer Studies Comments Reference
Microspheres W/O/O emulsion
solvent
evaporation
. - Cobrotoxin PLGA/P(CPP:
CEFB)
In rats Increased strength and
duration of antinociceptive
effect
98
Microspheres Emulsion
polymerization
Desmopressin Starch In rats,
in
sheep
Increased bioavailability with
LPC
61
Microspheres W/O emulsifi cation
solvent
evaporation
FITC – dextran Carbopol 394P
Chitosan
HPMC
PVA
In vitro Initial release at a constant
rate
Chitosan microspheres
exhibited size - dependent
release effect
41
Microspheres
FITC – dextran Chitosan,
Carbopol
934P
In rabbits Improved bioavailability 131
Microspheres Cross - linking with
epichlorohydrin
Gabexate mesylate Starch cyclodextrin In vitro Fast release rate
108
Microspheres Solvent evaporation Gentamicin Chitosan,
hyaluronan,
gelatine
In vitro Prolonged release,
improved
mucoadhesive properties
51
Microspheres Emulsion
polymerization
Gentamicin Starch In sheep,
in
rats
Improved bioavailability with
microsphere/enhancer
(LPC) system
59
Microspheres Spray drying Gentamicin sulfate HPMC In vitro Modifi
ed drug release
44
Microspheres Solvent evaporation Heparin Poly(lactic acid)
In rats Sustained - release effect 134
Microspheres Emulsion
polymerization
hGH Starch In sheep Enhanced nasal absorption
with microspheres, rapid
and higher absorption with
microsphere/enhancer
(LPC) system
56
Microspheres Emulsion
polymerization
Insulin Starch,
dextran In rats Rapid absorption
62
Microspheres Emulsion
polymerization
Insulin Starch In sheep Improved bioavailability with
microsphere/enhancer
(LPC) system
55
TABLE 1 Continued 670
Powder
Formulation Preparation Method Drug Polymer Studies Comments Reference
Microspheres Emulsion
polymerization
Insulin Starch In rats Improved nasal absorption
60
Microspheres Emulsion
polymerization
Insulin Dextran In rats Promoted absorption 63,
65
Microspheres Emulsifi
cation – cross -
linking
Insulin Chitosan In rats Promising absolute
bioavailability of 44%
91
Microspheres Emulsifi cation –
solvent
evaporation
Insulin Hyaluronic acid
ester
In sheep Increased nasal absorption
128
Microspheres W/O emulsifi cation
solvent extraction
Levodopa Gelatin In vitro Initial fast release rate,
followed by a second
slower release rate
88
Microspheres Spray drying Loratadine Chitosan,
chitosan/
ethylcellulose
In vitro Moderate swelling behavior,
sustained drug release
107
Microspheres Emulsifi
cation – cross -
linking
Melatonin Starch In rabbits Increased residence time,
rapid absorption rate, high
absolute bioavailability
58
Microspheres Spray drying Metoclopramide Sodium alginate,
chitosan, sodium
alginate/chitosan
In vitro Controlled drug release,
promising properties as
nasal drug carriers
53
Microspheres Emulsifi
cation – cross -
linking
Metoprolol tartrate
Alginate In rabbits Improved therapeutic
effi cacy
132
Microspheres W/O emulsifi
cation Morphine Chitosan,
starch In sheep High bioavailability
73
Microspheres Emulsion
polymerization
Nicotine Dextran In vitro Rapid release,
good
dispersion ability
33
Microspheres W/O emulsifi cation
solvent extraction
Oxprenolol Gelatin – poly(acrylic
acid)
In rats Slow - release drug delivery
system with good adhesive
characteristics
89
Microspheres W/O emulsion
cross - linking
Pentazocine Chitosan In rabbits Matrix diffusion controlled
delivery, improved
bioavailability, in vitro/in
vivo correlation
129
Microspheres Emulsion solvent
evaporation
Salbutamol Chitosan In rabbits Prolonged and controlled
release
92
671
Powder
Formulation Preparation Method Drug Polymer Studies Comments Reference
Microspheres W/O emulsion
cross - linking
Salmon calcitonin Gelatin In rats Enhanced nasal absorption
10
Microsphers Emulsion
polymerization
Insulin Starch In sheep Microspheres and absorption
enhancers (LPC, GDC,
and STDHF) acted
synergistically to enhance
absorption
57
Powder Lyophilization Apomorphine Carbopol 971P,
polycarbophil
In rabbits Sustained release,
improved
bioavailability
8
Powder Lyophilization Apomorphine Carboxymethyl
cellulose
In rabbits Sustained plasma level 42
Powder Lyophilization Apomorphine Carbopol 971P,
carboxymethyl
cellulose
In rabbits Increased residence time
48
Powder Lyophilization Apomorphine HCl Carbopol 971P,
Carbopol 974P,
polycarbophil
In rabbits In vitro release but not in
vivo absorption has been
infl uenced by drug loading
130
Powder Spray drying Cyanocobalamin Microcrystalline
cellulose, dextran,
crospovidone
In rabbits Improved bioavailability 43
Powder Press - on force
method
Glucagon Microcrystalline
cellulose
In human
volunteers
Increased formulation
stability, decreased
irritability
27
Powder — Goserelin Chitosan In sheep Improved bioavailability
9
Powder Material mixing with
a pestle
Insulin Chitosan In rats,
in
sheep
Improved bioavailability
11
Powder Blending/
lyophilization
Insulin Microcrystalline
cellulose,
hydroxypropyl
cellulose,
Carbopo 934
In dogs Decreased plasma glucose
level
38
TABLE 1 Continued 672
Powder
Formulation Preparation Method Drug Polymer Studies Comments Reference
Powder Lyophilization Insulin Starch – Carbopol
974P,
maltodextrin –
Carbopol 974P
In rabbits The highest absolute
bioavailability obtained
was 14.4%
46
Powder Lyophilization Ketorolac Microcrystalline
cellulose
In rabbits Signifi cantly lower
bioavailability of drug
from powders compared
to spray formulation
50
Powder — Leuprolide,
calcitonin,
FITC – dextran
Hydroxypropyl
cellulose,
microcrystalline
cellulose
In rabbits Enhanced absorption 49
Powder Manual blending
using mortar and
pestle
Morphine Chitosan In human
volunteers
Rapid onset of pain relief,
formulations well tolerated
by patients
73
Powder Dry blending Octreotide Dextran,
microcrystalline
cellulose,
semicrystalline
cellulose,
hydoxyethyl
starch,
microcrystalline
chitosan, pectin,
alginic acid
In rats Correlation between carrier
calcium binding properties
and their potential as nasal
absorption enhancers for
peptides
40
Powder,
granules
Powder:
manual
mixing using
mortar and pestle
Recombinant hGH Chitosan In sheep Relative bioavailability of
hGH from powder and
granules have been 14 and
15%, respectively
77
Abbreviations:
PLGA:
poly(lactide - co - glycolide);
P(CPP:CEFB):
poly[1,3 - bis(p - carboxy - phenoxy) propane - co - p - (carboxyethylformamido) benzoic anhydride];
chitosan – TBA: chitosan – 4 - thiobutylamidine conjugate; LPC: lysophosphatidyl choline; hGH: human growth hormone; GDC: glycodeoxycholate sodium; STDHF:
sodium taurodihydroxyfusidate
673
674 NASAL POWDER DRUG DELIVERY
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c
c
680 NASAL POWDER DRUG DELIVERY
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683
5.8
AEROSOL DRUG DELIVERY
Michael Hindle
Virginia Commonwealth University, Richmond, Virginia
Contents
5.8.1 Introduction
5.8.2 Human Respiratory Tract and Aerosol Particle Deposition
5.8.2.1 Human Respiratory Tract
5.8.2.2 Mechanisms of Particle Deposition
5.8.2.3 Pharmacokinetics
5.8.3 Therapeutic Indications for Aerosol Delivery
5.8.3.1 Current Applications
5.8.3.2 Future Applications
5.8.4 Aerosol Drug Delivery Devices
5.8.4.1 Introduction
5.8.4.2 Characteristics of Ideal Delivery Device
5.8.5 Metered Dose Inhalers
5.8.5.1 Introduction
5.8.5.2 Metered Dose Inhaler and HFA Reformulation
5.8.5.3 Propellants
5.8.5.4 Excipients
5.8.5.5 Valves
5.8.5.6 Actuators
5.8.5.7 Canisters
5.8.5.8 Breath Actuation
5.8.5.9 Spacers
5.8.5.10 Dose Counters
5.8.6 Dry Powder Inhalers
5.8.6.1 Introduction
5.8.6.2 Size Reduction and Particle Formation Technologies
5.8.6.3 Drug – Lactose Formulations
5.8.6.4 Dry Powder Inhaler Design
5.8.6.5 Exubera
5.8.7 Nebulizers
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
684 AEROSOL DRUG DELIVERY
5.8.8 Emerging Technologies
5.8.8.1 Soft Mist Aerosols
5.8.8.2 Respimat
5.8.8.3 AERx
5.8.8.4 Mystic
5.8.8.5 Capillary Aerosol Generator
5.8.8.6 Staccato
5.8.9 Conclusions
References
5.8.1 INTRODUCTION
Aerosol drug delivery to the lungs has long been the route of choice for the treatment
of respiratory diseases, including asthma and chronic obstructive airway
disease. Metered dose inhalers (MDIs), dry powder inhalers (DPIs), and nebulizers
have been employed to successfully deliver a wide range of pharmaceuticals principally
to the lungs for local action. However, with their unique characteristics, the
lungs have now begun to be targeted as a means of noninvasive delivery of systemically
acting compounds, including genes, proteins, peptides, antibiotics, and other
small molecules [1, 2] . The primary function of the respiratory tract is gaseous
exchange, transferring oxygen from the inspired air to the blood and removing
carbon dioxide from the circulation. This pulmonary circulation offers rapid absorption
and systemic distribution of suitable drugs deposited in the airways. Due to its
anatomical structure, however, an important secondary role is the protection of the
body from inhalation of foreign particles (including aerosol drug particles). The
challenge of aerosol drug delivery is to overcome this highly effective barrier and
accurately and reproducibly deliver aerosol drug particles in suffi cient doses to their
targeted sites within the lungs for either local action or systemic absorption. Effective
aerosol drug delivery is tied to the aerosol inhaler that generates and delivers
the respirable aerosol. This chapter will primarily focus on aerosol drug delivery
devices, their development, and future prospects for pulmonary administration.
5.8.2 HUMAN RESPIRATORY TRACT AND
AEROSOL PARTICLE DEPOSITION
5.8.2.1 Human Respiratory Tract
The human respiratory tract can be divided into three main regions: fi rst, the upper
airways, including the nose, mouth and throat (oropharnyx), and the larynx [3] . The
conducting airways consist of the regions from the trachea to the respiratory bronchioles
and have airway diameters between 0.6 and 20 mm. The alveolar region
consists of respiratory bronchioles and alveolar sacs and has airway diameters
between 0.2 and 0.6 mm. The lungs are a branching system which commences asymmetrically,
dividing fi rst at the base of the trachea. The left and right bronchi branch
dichotomously into the conducting airways. There are approximately 23 generations
before the respiratory bronchioles give way to the alveoli, the site of gaseous
exchange [4] . This branching produces a progressive reduction in airway diameter
and also signifi cantly increases the total surface area of the lower airways [3] .
Another important characteristic with respect to drug delivery is the extensive vascular
circulation. The blood vessels supplying the conducting airways are part of the
systemic circulation. In contrast, the alveolar region is connected to the pulmonary
circulatory pathway; drugs absorbed into this circulation will avoid fi rst - pass hepatic
metabolism effects.
5.8.2.2 Mechanisms of Particle Deposition
Aerosol particles are deposited in the lungs by three main mechanisms, and the site
of deposition is dependent upon the predominating mechanism. Inertial impaction
occurs because a particle traveling in an air stream has its own momentum (the
product of its mass and velocity). As the direction of the airfl ow changes due to a
bend or obstacle, the particle will continue in its original direction for a certain distance
because of its inertia. Particles with a high momentum, due to high velocity
or large size, are often unable to change direction before they impact on the surface
in front of them [5] . Impaction of particles entering the mouth with a high velocity
occurs either at the back of the mouth or at the bend where the pharynx leads to
the trachea. Only a small fraction of particles greater than 15 . m will reach the
trachea following mouth breathing. The majority, due to their size, will impact in the
oropharyngeal region. Deposition by impaction will also occur as the trachea splits
into the left and right bronchus. As the velocity of the particles decreases, inertial
impaction becomes a less important mechanism of deposition in the smaller airways.
Following the removal of larger particles in the upper airways by inertial impaction,
gravitational sedimentation is the mechanism by which smaller particles (2 – 5 . m)
are deposited in the respiratory bronchioles and alveoli. These particles settle under
gravity and accelerate to a steady terminal velocity when the gravitational force is
balanced by the resistance of the air through which it is traveling [6] . It is a time -
dependent process which is aided by breath holding [7] . Brownian motion or diffusion
is a mechanism which signifi cantly affects only particles less than 0.5 . m in
diameter. These particles are subjected to bombardment by surrounding gas
molecules causing random movement of the particles. In this situation, the
diffusivity of a particle is inversely proportional to its diameter. For an extensive
mechanistic review of the area of particle deposition readers should consult Finlay
(2001) [8] .
Aerosol particle size and polydispersity are major determinants of the site and
mechanism of pulmonary deposition. Fundamental deposition studies using monodisperse
aerosols together with mathematical models have established the optimum
aerosol particle size for lung deposition [9 – 12] . Aerosols larger than 10 . m will
deposit in the oropharyngeal region and will not be inhaled. Particles less than 3 . m
will be capable of penetrating into the alveolar region. Aerosols in the size range
3 – 10 . m will be distributed in the central and conducting airways [13] . A polydisperse
aerosol containing a range of these particle sizes will allow deposition throughout
the lungs. In theory, lung site deposition targeting should be possible by
controlling the particle size of the inhalation aerosol [14] . However, a number of
HUMAN RESPIRATORY TRACT AND AEROSOL PARTICLE DEPOSITION 685
686 AEROSOL DRUG DELIVERY
other signifi cant variables can affect deposition within the respiratory tract and
these often confound any efforts at targeting [15] . The patient ’ s respiratory cycle,
both the rate and depth of breathing, will affect aerosol deposition, and this is also
the source of large intersubject variability in deposition [16] . Slow and deep inhalations
are required for deposition in the peripheral airways, and this is the technique
often recommended for inhalation with the MDI [17] . A different technique may
be required for DPIs, where the patient ’ s inspiratory effort is often the powder dispersion
and delivery force. Flow rates greater than 60 L/min are commonly employed
for powder inhalers [18, 19] . A fi nal respiratory maneuver can be employed to
promote deposition; breath holding up to 10 s is generally recommended to enhance
deposition by sedimentation [17] . Other parameters that will affect lung deposition
are the disease state within the lungs and its effect on airway caliber together with
the patient ’ s age and airway morphology [20 – 27] .
5.8.2.3 Pharmacokinetics
Once deposited on the surface of the airways, the particle is subject to absorption
and clearance processes depending upon its physical properties and the site of
deposition [28 – 30] . For example, a lipophilic small molecule deposited in the central
airways would have a different pharmacokinetic profi le than a 50 - kDa macromolecule
deposited in the alveolar region. The former may undergo mucociliary clearance
following deposition on a ciliated epithelial cell. Following dissolution, lipophilic
drugs may be transported across the epithelium by passive transcytosis, while hydrophilic
compounds are taken up by other pathways such as via tight junctions and
endocytosis. Having overcome the barrier of the epithelial layer, the drug is available
for distribution into the systemic circulation or to its site of action. Finally, the
drug may also be subject to metabolism within the airways. For the macromolecule
deposited in the peripheral airways, the absorption rate has been shown to be
dependent upon molecular size. Larger molecules are subject to active processes
such as caveolae or vesicular transport across the cell. Diffusion remains the predominant
mechanism for smaller lipophilic macromolecules. Insoluble molecules
can be phagocytosed by alveolar macrophages and removed via the lymphatic
system or the mucociliary escalator. The pharmacokinetics of inhaled drugs is complicated
by the fact that a signifi cant fraction of the delivered dose is deposited in
the oropharnyx or removed from the lungs via mucociliary clearance and in both
cases subsequently swallowed [31] . An often desired goal for a pulmonary formulation
is prolonged action within the lung. Rapid clearance or metabolism results in
short duration of action for most inhaled drugs. A number of approaches using formulation
excipient additives have been investigated to increase the residency or
prolong release of drug at its site of action within the lungs [32, 33] . Microspheres
containing nanoparticles have been formulated as dry powders for inhalation offering
sustained - release properties [34] . In addition, prodrugs which are activated
locally within the lungs have been used in an alternative approach [35 – 37] .
The pharmacokinetic process of absorption, distribution, metabolism, and excretion
within the lungs is an enormous subject area and readers are referred to specifi c
reviews for further details [38 – 43] . Of particular interest may be the subject of
absorption enhancer methodologies for lung delivery, which is beyond the scope of
this chapter [44] .
5.8.3 THERAPEUTIC INDICATIONS FOR AEROSOL DELIVERY
5.8.3.1 Current Applications
Aerosolized drug delivery is currently used to deliver a limited range of therapeutic
classes of compounds. These are mainly for asthma and chronic obstructive airway
disease. These classes of compounds include short - and long - acting . - adrenoceptor
agonist, corticosteroids, mast cell stabilizers, and muscarinic antagonists. Of recent
note is the popularity of combination products. These have obvious advantages from
a patient compliance perspective. In addition, certain combinations of drugs have
shown synergistic therapeutics benefi ts when compared to the drugs given by separate
inhalers [45] . Long - acting . - adrenoceptor agonists and corticosteroids formulated
as combination products are available as both MDIs and DPIs [46] . Also
recently introduced was a MDI formulation, the R enantiomer of albuterol, which
is believed to be mainly responsible for bronchodilation in the racemic mixture [47] .
Zanamivir is licensed in the United States as an inhaled antiviral agent for the treatment
of infl uenza [48] . Recombinant human deoxyribonuclease (rhDNAase) is
available as a nebulizer product for the treatment of cystic fi brosis, in which it acts
to liquefy viscous lung secretions [49] . And recently, insulin was approved as an
inhaled powder for glycemic control in type I and II diabetes (see Section 5.8.6.5 )
[50] .
5.8.3.2 Future Applications
Research and development are presently underway covering a vast array of novel
applications. Clark (2004) provides an extensive list of products and their current
state of development [51] . A signifi cant future advance will be the development
of inexpensive, noninvasive, stable, single - dose vaccine delivery via the lungs [52] .
Efforts in this area are being led by the World Health Organization in the Measles
Aerosol Project, and in a separate project, the Grand Challenges in Global Health
initiative has funded a program to further develop an inhalation aerosol measles
vaccine. Delivery of the measles vaccine via the lungs has been demonstrated to
be both safe and effective [53 – 58] . Now the challenge of each of these projects is
to produce stable inhalation vaccine formulations to be delivered via inexpensive
inhalers while maintaining both safety and effi cacy [59] . The use of inhaled
vaccinations in the event of a bioterrorism attack is also a potential application
[60, 61] .
The use of the inhalation route for the delivery of gene therapy is also an area
of signifi cant interest [60, 62] . Cationic liposomes and polymers together with adenoviral
vectors containing the reporting genes have been aerosolized using nebulizers
for the majority of clinical studies. However, there are a signifi cant number of challenges
that must be overcome before pulmonary gene delivery is deemed completely
successful, the most important being low gene transfer effi ciency at the
cellular level. This problem is not unique to inhalation therapy. Inhalation of a
recombinant adenovirus containing the cystic fi brosis transmembrane regulator
(Ad2/CFTR) demonstrated the feasibility of this approach for the treatment of
cystic fi brosis [63, 64] . However, the limited duration of transfection and low cellular
uptake effi ciency still remain a barrier to full utilization of this route [60, 65] . There
THERAPEUTIC INDICATIONS FOR AEROSOL DELIVERY 687
688 AEROSOL DRUG DELIVERY
are a number of reviews that provide updates as to recent developments in this area
[60, 66 – 70] .
Given the success of delivering insulin, other peptides and proteins are being
considered for pulmonary applications [71, 72] . Leuprolide is a nonapeptide which
has been investigated as both an MDI and DPI formulation for the treatment of
prostrate cancer [73 – 76] . Other hormones being investigated include calcitonin for
the treatment of Paget disease and osteoporosis, parathyroid hormone to treat
osteoporosis, growth hormone releasing factor for the treatment of pituitary dwarfism,
and vasoactive intestinal peptide (VIP) for the treatment of pulmonary diseases
[60, 77 – 81] .
Other potential inhalation applications include drugs for both local and systemic
delivery. Inhaled tobramycin is being investigated for the treatment of Pseudomonas
aeruginosa exacerbations in cystic fi brosis [82, 83] . Liposomal ciprofl oxacin is
being developed as a fi rst - line defense against biowarfare agents (e.g., anthrax)
[61] . Inhaled cyclosporine has been shown to improve survival rates and extend
periods of chronic rejection - free survival in lung transplant patients [84] . Apomorphine
has been proposed as an inhalation formulation for the treatment of erectile
dysfunction [85] . Aerosol delivery of chemotherapeutic drugs has been advocated
for the treatment of lung cancer [86] . Morphine and fentanyl have been investigated
for alternative routes of administering analgesics [87 – 90] . Heparin and low -
molecular - weight heparins have been aerosolized and advocated for the treatment
of emphysema and thrombosis [91 – 93] . Iloprost, a stable prostacyclin analog, has
been aerosolized by nebulization for use in the treatment of pulmonary hypertension
[94] . This list of potential new treatments approached via the inhalation
route is not exhaustive; among the other compounds under investigation are .1 -
antitrypsin, sumatriptan, ergotamine, nicotine as replacement therapy, pentamidine,
and ribavirin. Readers should be aware that a large number of these examples
are proof - of - concept studies that may not get beyond in vitro experiments and
animal studies.
5.8.4 AEROSOL DRUG DELIVERY DEVICES
5.8.4.1 Introduction
As can be seen from the previous section, aerosol drug delivery continues to be an
area of intensive research and development for the pharmaceutical industry. Not
only are new applications for the pulmonary route being investigated, but also new
delivery technologies are under development. The reformulation of MDIs with
hydrofl uoroalkane (HFA) propellants together with the potential of using the inhalation
route as a means of systemic administration has led to signifi cant technological
advances in delivery devices. In parallel to MDI research DPIs have been
developed from breath - actuated single - dose devices to both multiple - dose inhalers
and active - dispersion DPIs. There is an extensive literature detailing the fundamental
mechanisms of powder dispersion aimed at improving pulmonary deposition
from powder inhalers. In addition, novel particle production technologies have been
developed that provide alternatives to the traditional micronized powder for formulation
in both MDIs and DPIs. Nebulizer technology has evolved from previously
nonportable devices into high - effi ciency, hand - held nebulizers that offer alternatives
to the MDI and DPI for certain treatment regimes. Finally, novel soft mist inhalers
that generate aerosols by solution atomization have emerged on the inhaler landscape.
All this research has focused on improving aerosol deposition effi ciency and
reproducibility within the lungs, together with targeting the peripheral lungs for
systemic absorption. The efforts of the last decade culminated in two signifi cant
events. First, the regulatory approval of Proventil HFA and QVAR, the fi rst suspension
and solution HFA MDIs, respectively. Second, in 2006, the U.S. and European
regulatory authorities approved Exubera, an insulin DPI for the systemic treatment
of type I and II diabetes. Exubera offered a noninvasive alternative to subcutaneous
injections of insulin.
5.8.4.2 Characteristics of Ideal Delivery Device
With these developments, innovation continues toward development of the ideal
inhaler. A number of authors have compiled lists of desired characteristics for an
aerosol inhaler [95 – 97] . These can be grouped into patient - desired or industry -
driven properties. From the patient ’ s perspective the overriding requirement is a
device that is simple to operate. This is becoming increasing diffi cult to achieve as
evidenced by the intensive patient education initiative that is being planned for the
launch of the Exubera insulin inhaler. Poor compliance and adherence to prescribed
therapy may be related to patients ’ failure to use the inhaler correctly [98] . Inhalers
should be portable and contain a large number of doses. The device should also give
some indication to the patient when it is empty. The inhaler should be suitable for
use by all of the population, especially children and the elderly. Ganderton (1999)
cited that from a device perspective aerosol generation should be independent of
the patient ’ s inhalation and should continue for a substantial portion of the inspiratory
cycle. This would minimize the reliance on coordinating inhalation and actuation
of the device [99] . Breath - actuated devices have been developed to address this
issue. In order to achieve lung deposition targeting, the particle size distribution of
the aerosol should be capable of being altered depending upon the specifi c target
region. For example, the central airways may be targeted with a 3 – 5 . m aerosol for
the treatment of acute bronchoconstriction, while a smaller aerosol (1 – 3 . m) might
be used for deep lung deposition and subsequent systemic absorption [99] . In addition,
the dose should be delivered reproducibly with minimal oropharyngeal deposition,
perhaps as a low - velocity aerosol. There should be a minimal number of small
parts in the inhaler, and it should be robust and reliable when placed “ in use. ” The
manufacturer has the option of producing a disposable or refi llable unit; however,
the inhaler should protect the formulation from environment and not affect its
stability.
Dolovich et al. (2005) have provided an extensive evidence - based evaluation of
aerosol drug devices. They concluded that when selecting an inhalation delivery
system the following should be considered: device and drug combination availability,
clinical setting, patient age, the ability of the patient to use the device correctly,
device use with multiple medications, cost and reimbursement, drug administration
time, convenience in outpatient and inpatient settings, and patient and physician
preference [100] . Other reviews have compared the benefi ts and disadvantages of
inhalers from clinical and patients ’ perspectives [101, 102] .
AEROSOL DRUG DELIVERY DEVICES 689
690 AEROSOL DRUG DELIVERY
5.8.5 METERED DOSE INHALERS
5.8.5.1 Introduction
Since their development, MDIs have been widely used for pulmonary aerosol drug
delivery [103] . Despite their recognized limitations, they remain the device of choice
for many physicians around the globe. From a patient ’ s perspective, they are light,
portable, and robust and contain multiple doses of medication. They are also relatively
simple to operate (press and fi re); however, signifi cant numbers of patients
experience diffi culties correctly using the MDI due to coordination problems [104] .
To maximize lung drug deposition, actuation (pressing the MDI canister) by the
patient must be coordinated with a slow, deep inhalation. Studies have reported that
51% of patients fail to operate the MDI correctly [104] . This leads to low lung
deposition, high oropharyngeal deposition, and ultimately perhaps therapeutic
failure. From the pharmaceutical industry perspective, the components are relatively
inexpensive; however, the formulation and manufacturing have become increasingly
complex. There are numerous studies describing the multifaceted and interactive
effects of propellant [105 – 110] , excipient [111 – 115] , metering valve [110, 116] , and
actuator [116 – 119] on the aerosol particle size characteristics of the MDI [120, 121] .
To date, the success of the MDI has relied in part on the potency and relative safety
of the bronchodilators and corticosteroids commonly used for the treatment of
respiratory disorders rather than its delivery effi ciency. The relatively low and often
variable aerosol deposition effi ciency, only around 10 – 20% of the nominal dose
being delivered to the lungs, is the challenge that is beginning to be addressed as
the MDI looks to enter the next 50 years of aerosol drug delivery.
5.8.5.2 Metered Dose Inhaler and HFA Reformulation
The basic design and operation of the MDI has changed little over its lifetime.
Aerosols are generated from a formulation of drug (0.1 – 1% w/w) either suspended
or in solution in the liquefi ed propellant. The formulation is held under pressure in
a canister.
Figure 1 shows the basic components of the MDI, consisting of a canister sealed
with a metering valve which is inserted into a plastic actuator containing the mouthpiece.
Aerosol generation takes place when the canister is pressed against the actuation
sump by the patient. Actuation causes the outlet valve to open and the liquefi ed
propellant formulation is released through the actuator nozzle and subsequently
through the mouthpiece to the patient. Metered volumes between 20 and 100 . L
are dispensed, and as the pressurized propellant is released, it forms small liquid
droplets traveling at high velocity. These droplets evaporate to leave drug particles
for inhalation [117] . Purewal and Grant (1998) have assembled a defi nitive reference
source for issues relating to the design, manufacturing, and performance of
MDIs [122] .
The currently marketed MDIs may look similar to the devices that were fi rst
developed by Riker in 1950. However, due to the replacement of the ozone -
depleting chlorofl uorocarbon (CFC) propellants with HFA propellants, virtually all
of the components of the MDI have been altered. In 1987, the Montreal Protocol
was drawn up, leading to the eventual phase - out of CFC propellants. MDIs contain
ing CFC propellants were granted essential - use exemptions until viable alternatives
became available. Therefore, with this impending withdrawal, a consortium of pharmaceutical
companies (IPACT - I and IPACT - II) worked to identify and toxicologically
test alternative propellants for MDIs. HFA 134a and HFA 227 were identifi ed
as viable alternatives and the task of reformulation began. At fi rst look, it appeared
that the most expeditious route to replacing a CFC product would be to produce a
suspension HFA MDI with exactly the same in vitro characteristics as the CFC
MDI. This would prove to be a time - consuming route [123, 124] . While some manufacturers
focused on producing HFA products with identical characteristics to the
current CFC versions to accelerate the pathway through clinical testing to market.
Others undertook extensive research and development in the area of HFA formulation
options, and this has led to the possibility of utilizing the MDI for both local
and systemic administration. During this reformulation effort, the industry has
taken the opportunity to address some of the other shortcomings of the MDI [125] .
Among these issues were poor peripheral lung delivery, variable dose delivery, and
limitations as to the dose capable of being delivered to the lung (typically about
1 mg) [126] .
The replacement of CFC MDIs with inhalers formulated with the HFA propellants
is now well underway in Europe. Although progress in the United States has
been slower, with the introduction of suitable alternatives for albuterol inhalers, the
FDA has ordered that CFC albuterol MDIs be withdrawn from the market by the
end of 2008 [127] . Examples of reformulated products available in the United States
include Ventolin HFA, which is a suspension albuterol sulfate formulation using
HFA 134a alone. ProAir is an alternative albuterol sulfate product manufactured
by Ivax which contains ethanol and HFA 134a. Xopenex HFA has recently been
approved for marketing in the United States [128] . This product contains levalbuterol
tartrate (R - albuterol enantiomer) together with HFA 134a, dehydrated
alcohol, and oleic acid as a suspension formulation. Table 1 summarizes the HFA
FIGURE 1 Schematic of MDI.
METERED DOSE INHALERS 691
692 AEROSOL DRUG DELIVERY
products currently available in the United States and their excipients. The following
section will focus on the current options for formulation of drugs in HFA propellant
systems and the challenges that are encountered as products are reformulated as
HFA formulations.
5.8.5.3 Propellants
The CFC propellants primarily used in MDI formulations were CFC 11, 12, and 114.
Blends of these propellants were held liquefi ed under pressures of 50 – 80 psig within
the canister. Flocculated drug suspensions in CFC propellants were formulated
using a surfactant (e.g., oleic acid and lecithin). In a suspension formulation, the
aerosol particle size is dependent upon the initial micronized drug particle size
(typically between 2 and 5 . m) and the evaporation of the propellant droplets. It
has long been recognized that changes in CFC propellant vapor pressure result in
changes in droplet size and velocity of the aerosol. Newman et al. (1982) showed
that increasing the vapor pressure of the propellant blend in the MDI signifi cantly
increased whole - lung deposition and reduced oropharyngeal deposition [110] .
The fi rst challenge encountered during the reformulation with HFA propellants
was the altered physicochemical properties of HFA 134a and HFA 227 compared
to the CFC propellants [124] . Table 2 compares the physicochemical properties of
the CFC and HFA propellants [124] . The increased polarity of HFA 134a and HFA
227 is illustrated by the increased dipole moments and dielectric constant. From a
practical point of view, the altered solvency properties of the HFA propellant for
the drug, excipient, water, and, surprisingly, components of the MDI have been the
TABLE 1 Summary of HFA Metered Dose Inhaler Products Available in United States,
June 2006
Product Name Drug Approval Date Excipients Type
Proventil HFA
(3M)
Albuterol sulfate August 1996 HFA 134a, ethanol,
oleic acid
Suspension
Ventolin HFA
(GSK)
Albuterol sulfate April 2001 HFA 134a Suspension
Proair HFA
(IVAX)
Albuterol sulfate October 2004 HFA 134a, ethanol Suspension
QVAR (3M) Beclomethasone
dipropionate
September 2000 HFA 134a, ethanol,
oleic acid
Solution
Flovent HFA
(GSK)
Fluticasone
propionate
May 2004 HFA 134a, ethanol Suspension
Atrovent HFA
(BI)
Ipratropium
bromide
November 2004 HFA 134a, purifi ed
water, dehydrated
alcohol,
anhydrous citric
acid
Solution
Xopenex
(Sepracor)
Levalbuterol
tartrate
March 2005 HFA 134a,
dehydrated
alcohol, oleic acid
Suspension
METERED DOSE INHALERS 693
major issues during reformulation. Suspension formulations of micronized drug in
the liquefi ed CFC propellant blends with surfactants were replaced with either
suspension or solution HFA formulations, depending upon the solubility of the
individual drug in the HFA propellants. For suspension HFA formulations, however,
it was observed that the conventional surfactants used in the CFC products were
insoluble in the HFA propellants without the addition of a cosolvent (e.g., ethanol)
[129, 130] . An alternative approach to produce suspension formulations was to
develop a new class of surfactants suitable for use in the HFA systems [131] . It was
also observed that some of the commonly used inhalation drugs were slightly
soluble in the new propellants and therefore precluded their formulation as a suspension.
The potential to formulate as a solution offered a number of advantages
together with signifi cant problems. Perhaps most importantly, changing from a suspension
to a solution formulation altered the mechanism of aerosol particle formation.
In the case of solution formulations, drug is dissolved in the liquefi ed propellant
and a suitable cosolvent (if necessary) and particle formation takes place during
evaporation of the propellant. This leads to much smaller particles being formed
when propellant evaporation is complete. Stein and Myrdal (2006) recently described
the MDI aerosol generation for solution formulations as a two - step process [132] .
Droplet formation takes place as millions of atomized droplets are produced after
the formulation exits the metering valve through the actuator sump. Initial droplet
size is dependent upon the vapor pressure and surface tension of the formulation,
TABLE 2 Comparison of Physicochemical Characteristics of CFC and HFA Propellants
Property
CFC HFA
11 12 114 134a 227
Thermodynamic
RRM 137 121 171 102 170
Boiling point, ° C 24 . 30 4 . 26 . 16
Vapor pressure, 20 ° C, kPa 89 566 182 572 390
Enthalpy vap., 20 ° C, kJ/mol (J/g) 25.1
(183)
17.2
(142)
22.1
(130)
18.6
(182)
19.6
(115)
C p liquid, 20 ° C, J/mol · K (J/g · K) 120
(0.88)
118
(0.98)
168
(0.98)
143
(1.41)
210
(1.24)
Polarity
Dielectric constant 2.3 2.1 2.2 9.5 4.1
Dipole moment ( D ) 0.45 0.51 0.58 2.1 1.2
Induced polarization 2.8 2.3 3.2 6.1 6.1
Solubility parameter (Hild. units) 7.5 6.1 6.4 6.6 6.2
Kauri - butanol value 60 18 12 9 13
log P octanol/water 2.0 2.2 2.8 1.1 2.1
Water solubility (ppm) 130 a 120 a 110 a 2200 b 610 b
Liquid Phase
Density (g/cm 3 ) 1.49 1.33 1.47 1.23 1.42
Viscosity (mPa · s) 0.43 0.20 0.30 0.21 0.27
Surface tension (mN/m) 18 9 11 8 7
Source: From ref. 124 . a 30 ° b 25 ° .
694 AEROSOL DRUG DELIVERY
valve size, and actuator orifi ce diameter. The second step is an evaporative or
“ aerosol maturation ” phase, as the propellant and cosolvents (e.g., ethanol) rapidly
evaporate leaving inhalable drug particles. The fi nal size of these particles is dependent
upon the initial droplet size, the vapor pressure of the formulation mixture,
and the proportion of nonvolatiles in the formulation [132] .
Leach et al. (1998) compared the pulmonary deposition of a suspension CFC
formulation of beclomethasone diproprionate with a solution HFA formulation
[133] . The marketed CFC product had a mass median aerodynamic diameter
(MMAD) of 3.5 . m compared to 1.1 . m for the solution formulation, refl ecting the
altered aerosol formation mechanism. The gamma scintigraphy profi le for the solution
formulation showed the drug and label to be diffusely deposited throughout
the airways with approximately 55 – 60% deposited in the lungs. In contrast, the CFC
product was deposited mainly in the mouth and throat (90 – 94%), with only 4 – 6%
being deposited in the airways. In many ways, this study summarized the defi ciencies
of the suspension formulation CFC MDI and offered the alternative of improved
delivery effi ciencies with the solution HFA formulation. A signifi cant conclusion
from this and other studies supported the hypothesis that improved pulmonary
deposition and reduced oropharyngeal losses of aerosols would allow reduction in
the dose required by the patient to achieve the same therapeutic effect [108, 133,
134] .
The altered solubility profi le of the HFA propellant, while providing attractive
characteristics for solution formulations, also provide signifi cant challenges with
respect to their interactions with the basic MDI components. Leachables are compounds
that can be transferred from MDI component parts to the formulation
during the shelf life of the product. Berry et al. (2003) postulated that MDI orientation
could affect the amount of leachables that entered a formulation and affect the
particle size distribution of aerosol [135] . Extensive efforts are now required for
extractable and leachable testing of the component materials prior to formulation
of an MDI. Another by - product of replacing the CFC propellants was to tighten the
impurity specifi cations required for the new propellants. A proposed U.S. Pharmacopeia
(USP) monograph for HFA 134a has now been published detailing the
impurity profi le [136] .
Manufacturing processes for MDIs have also required adapting for the use with
the new propellant system [137] . There are two main manufacturing processes used
for MDIs: cold fi lling and pressure fi lling [138] . Cold fi lling requires cooling the
propellants to below . 50 ° F and fi lling at that temperature prior to crimping the
valve onto the canister. Pressure - fi lling techniques for MDIs are most commonly
employed. These can be accomplished in either a one - or two - step process. In the
single - step process, the formulation is placed in a pressurized mixing vessel. The
empty canister is purged with propellant to remove the air. The valve is then crimped
onto the canister and the formulation is metered through the valve. The absence of
a HFA propellant that was liquid at room temperature was a major difference
compared to the process employed for CFC manufacturing. In the two - step process,
the formulation (excluding the propellant) are mixed together to form a concentrate.
Previously, liquefi ed CFC 11 was used in this step of the process. However,
there is no suitable HFA propellant that is liquid at room temperature. Therefore,
cosolvents such as ethanol and glyercol are employed during this step to form the
product concentrate. The concentrate is metered into the empty canister. The valve
METERED DOSE INHALERS 695
is then crimped onto the canister and the propellant is fi lled through the valve.
Wilkinson (1998) provides an extensive history and review of the manufacturing
procedures for MDIs [138] .
5.8.5.4 Excipients
A number of excipients have been included in MDI formulations; however, the
nature of the excipients has changed with the introduction of the HFA propellant
aerosols. Oleic acid and sorbitan trioleate (SPAN 85) and lecithins were used in
CFC suspension MDIs as suspending agents and valve lubricants [112] . Typical
concentrations ranged from 0.1 to 2.0% w/w. Ethanol is now being used in HFA
formulations as a cosolvent for suspension and solution formulations. The addition
of ethanol to the formulation has a number of effects [121, 139] . Increasing the
ethanol concentration has been shown to increase the initial droplet size [121] . In
addition, ethanol can increase the hydrophilicity of the formulation and increase
moisture uptake. Glycerol and polyethyleneglycol have also been added as cosolvents
but also have the effect of increasing the residual droplet particle size due to
their lower volatility [111] . In general, a relationship can be observed between the
fraction of nonvolatile components (drug and nonvolatile excipients) in a solution
HFA formulation and the fi nal particle size of the aerosol. The MMAD was observed
to be linearly proportional to the cube root of the nonvolatile concentration [119,
121] . Oligolactic acids (OLAs) have been investigated for their use in a variety of
functions in HFA formulations. OLAs with repeating units of 6 – 15 units have been
proposed as suspending agents [131] . They are readily soluble in both HFA 134a
and 227. These molecules have also been shown to act as ion pair solubilizers for
certain drugs (e.g., albuterol). The addition of ethanol to these OLA formulations
synergizes the solubilizing effect [131] .
A word of caution is required when considering introduction of novel excipients
into any inhalation drug product formulation. Due to the unique toxicological challenges
associated with administration and clearance from the lung, the qualifi cation
of novel excipients for inhalation has proven to be an expensive and time -
consuming challenge. This has led to a limited number of compounds with an extensive
“ in - use ” profi le being commonly employed.
5.8.5.5 Valves
Metering valves are required to accurately meter and dispense the formulation upon
MDI actuation. In addition, they perform an important contact closure role preventing
moisture ingress and minimizing propellant evaporation. Figure 2 and Table 3
show the basic components of the metering valve. Currently, the most common valve
type is the retention valve, consisting of a plastic metering chamber and two rubber
gaskets. The remaining valve components are manufactured from plastic, metal, and
elastomeric materials.
Material component evaluation and selection are critical steps in the development
of a MDI formulation [140, 141] . The materials must be chemically resistant
and compatible with all components of the formulation. Gaskets must have appropriate
mechanical properties and work effectively as a seal, preventing leakage of
the formulation and moisture ingress. While the basic components themselves have
696 AEROSOL DRUG DELIVERY
remained unchanged during the introduction of the HFA propellants, the materials
used to manufacture the components have required signifi cant adaptation. Nitrile
was the most commonly employed elastomer in CFC MDIs; it has good mechanical
and elastic properties. However, it has been shown to swell when in contact with
HFA propellants and ethanol. Newer elastomers such as ethylene propylene diene
monomer (EPDM), chloroprene, and bromobutyl are now used in HFA MDIs [142] .
The ideal universal elastomer has yet to be developed and the newer materials must
be assessed on a case - by - case basis for formulation compatibility and the desired
moisture ingress characteristics. Among the many issues to be considered when
screening materials are formulation – material compatibility, extractable profi les, and
mechanical resistance. Manufacturers such as Valois, Solvay, and Bespak have extensive
knowledge of drug/excipient/material component compatibility and should be
used as the fi rst point of reference when considering a MDI formulation project.
Another concern to formulators is the ingress of moisture into HFA - formulated
MDIs [143] . HFA propellants have a higher moisture affi nity compared to the CFC
propellants, especially HFA 134a. In addition, the inclusion of ethanol in some formulations
increases its hydrophilicity. Moisture entering the canister can have
several effects; it may alter the physical or chemical stability of the formulation and
aerosolization performance of HFA MDIs. Due to its lower volatility compared to
FIGURE 2 Schematic of components of metering valve. (Courtesy of Valois Pharm.)
Upper stem
First gasket
Ring
Ferrule
Second gasket
Metering chamber
Neck gasket
Lower stem
Spring
Body
TABLE 3 Summary of Components and Materials Used in
Metered Dose Inhaler Valves
Component Material
Metering chamber Polyester
Core Polyester/acetal
Core extension Polyester/acetal
Body Polyester/nylon
Seats/gaskets EPDM/nitrile/butyl/chloroprene/
bromobutyl
Spring Stainless steel
Ferrule Aluminium
METERED DOSE INHALERS 697
the other components, water may affect aerosol generation and alter aerosol particle
size [144] . The increased water content may increase the solubility of suspended
polar drug particles or decrease the solubility of hydrophobic compounds [145] .
Corrosion in aluminum canisters may also increase over the shelf life of the product.
Williams and Hu (2000) reported that HFA 134a had a greater tendency to take up
moisture during storage than did HFA 227 [144] . The issue of moisture ingress
during storage has led to certain HFA MDIs being stored in moisture - protecting
pouches prior to initial use (e.g., Ventolin HFA). An alternative approach to minimize
the effects of moisture ingress has been taken by SkyePharma, which has
incorporated subtherapeutic doses of cromolyn sodium into its HFA MDI formulations.
Cromolyn sodium is used as a hygroscopic excipient to scavenge any moisture
that penetrates into the formulation. Cromolyn sodium has been used widely by
inhalation over the past 30 years and has an excellent safety profi le via the inhalation
route. Burel et al. (2004) reported that for a HFA 134a MDI formulation the
inclusion of a polyamide (nylon 66) molded ring around the valve body reduced
both the initial water content and the fi nal water content (6 months) when stored
under stress conditions [40 ° C and 75% relative humidity (RH)]. A combination of
a thermoplastic elastomer sealing gasket in the MDI valve and a polyamide ring
produced the lowest water ingress under these stress conditions [143] . For formulations
that might be susceptible to water - induced stability issues, HFA 227 may be
considered a more suitable propellant than HFA 134a. Given the possibility of
moisture ingress, there is also the issue of propellant leakage. Leak testing is among
the array of in - process quality assurance tests that are required. These include assay
of the suspension or solution, moisture level, consistency of fi lling of both the concentrate
and the propellant, valve crimp measurements, quality of sealing, in - line
leak testing under stress conditions, and performance of the valve.
Another signifi cant issue encountered during use of MDIs was related to loss of
prime and dose reproducibility [129, 146] . Loss of prime relates to the fact that in
conventional capillary retention metering valves the dose is fi lled into the valve
immediately following the last actuation. Capillary retention valves require priming
with one or two sprays prior to their fi rst use. In addition, if there is a signifi cant
interval between the actuations and the inhaler is stored upside down or on its side
or shaken, then the metering valve may actually partially empty, resulting in a low
and variable dose being delivered to the patient. A review of patient information
leafl ets indicated varying instructions on priming MDIs. This ranged from Atrovent
CFC and Combivent CFC requiring priming with 3 sprays “ after 24 hours of nonuse. ”
Ventolin HFA and Proventil HFA both required priming with 4 sprays after “ 2
weeks of nonuse. ” Flovent CFC required priming with 4 sprays after “ 4 weeks of
nonuse. ” Clearly, such instructions add to the complexity for patients using MDIs
and also contribute to drug waste issues. Loss of prime is also a signifi cant issue for
breath - actuated MDIs, where the opportunity to prime the inhaler is not readily
possible. A number of new valve designs have been developed to address this issue.
The fast - fi ll, fast - empty valves offer a solution to the priming and loss of prime
issues. In these valves [(e.g., 3M Shuttle valve (3M), 3M Face Seal valve (3M), ACT
(Valois), and Easifi ll valve (Bespak)], the metering valve is only isolated from the
formulation canister reservoir immediately prior to dose actuation. Therefore, the
metering chamber can be emptied and refi lled with a fresh dose from the reservoir
simply by shaking the canister prior to use.
698 AEROSOL DRUG DELIVERY
5.8.5.6 Actuators
Nonvolatile component concentration has previously been described as one of the
primary determinants of the initial droplet size for HFA solution formulations [119] .
Perhaps, equally important is the MDI actuator [147] . The actuator consists of the
sump block into which the metered dose is immediately delivered during MDI
actuation. As expansion and vaporization of the propellant take place, the aerosol
exits the sump via the actuator nozzle and then is inhaled through the actuator
mouthpiece. From a practical perspective, in general, reducing the size of the orifi ce
diameter for HFA solution formulations produced a relatively slower spray emitted
with less force compared to marketed CFC products [118] . The nozzle orifi ce diameter
has been considered to be the most important, although not the only, actuator
variable determining the particle size distribution of HFA solution formulations
[118, 147, 148] . Recently, Smyth et al. (2006) described three critical components of
the actuator that could affect the aerosol performance of a solution HFA formulation.
In addition to the orifi ce diameter, sump depth (and hence the expansion
chamber volume) together with orifi ce length was observed to have signifi cant
effects on the aerosol particle size distribution and should be considered for optimization
with an HFA formulation [147] . It has also been recognized that the electrostatic
charge of all components of the MDI and its formulation may affect the
aerosolization properties of the aerosol spray [149, 150] .
5.8.5.7 Canisters
Aluminum canisters are widely used in commercial MDI products mainly due to
their inert characteristics. Other materials, including stainless steel and glass, can be
employed depending upon the particular formulation characteristics. These canisters
were usually uncoated. Changes to the canister may be required when the formulation
interacts with the interior surface of the canister altering the chemical
stability of the formulation. The presence of ethanol in HFA formulations has also
increased the risk of metal corrosion. Drug migration or absorption to the metal
components of the canister and also the metal valve components has also been
reported [141] . The loss of drug to the walls of the canister will result in variability
in the delivered dose from the MDI during the shelf life of the inhaler. The use of
canister coating and anodized canisters has been advocated to mitigate this problem
[141] .
5.8.5.8 Breath Actuation
In order to overcome the problems associated with many patients ’ inability to coordinate
actuating the MDI and inhaling, breath - actuated MDIs were developed [107] .
These devices allow the MDI to be automatically actuated only when the patient
commences inhaling through the mouthpiece. Of critical importance here is ensuring
that the patient has suffi cient inspiratory fl ow rate to trigger actuation. While
these devices offer little improvement for patients with a good inhaler technique, it
has been shown that patients with poor coordination did have signifi cantly greater
lung drug deposition when inhaling using a breath - actuated MDI [151] . The 3M
Autohaler was the fi rst device marketed using this technology [151, 152] . In Europe,
METERED DOSE INHALERS 699
the Easibreathe and Autohaler breath - actuated MDIs are used to deliver . agonists
and corticosteroids for the treatment of obstructive airway. Recently, in the United
States, the MD Turbo has been launched, a device that allows patients to take their
regular MDI canister and actuator and insert it into the MD Turbo. The MD Turbo
acts as a generic breath actuator for a number of marketed MDIs and also incorporates
a dose counter.
5.8.5.9 Spacers
Spacer devices have been developed as another alternative to overcome the problems
associated with patients coordinating the beginning of their inspiratory effort
with actuation of the MDI [153] . This problem is extenuated by the fact that the
MDI emits a high - velocity, short - duration aerosol cloud. On actuation, the propellant
spray is delivered into the spacer that often incorporates a one - way inhalation
valve. The patient is now able to inhale the aerosol cloud. The large - volume spacers
have an additional effect in that they allow evaporation of large propellant droplets
prior to inhalation. These high - velocity droplets would previously have had a high
probability of impacting in the patient ’ s throat. Figure 3 shows the large number of
spacer chambers that are available [154] . Spacers are advocated for use by children
and elderly patients and people who experience diffi culty coordinating actuation of
the MDI. The use of spacers for the delivery of corticosteroids also minimizes oral
deposition of the inhaled dose and therefore reduces the incidence of steroid -
related side effects [155] . Both in vitro and clinical studies have shown the effectiveness
of spacers with CFC MDIs [156 – 158] . It has been shown that electrostatic
charge can have a signifi cant effect on the performance of a spacer chamber and
where possible the charge should be minimized to maximize drug delivery [159 –
161] . Finally, it should be noted that the use of any particular spacer – MDI combination
should be evaluated at least in vitro to confi rm the benefi cial effect, especially
when employed with solution - based HFA MDI formulations [162, 163] .
FIGURE 3 Example spacer chambers available for use with MDIs. (Reproduced from
ref. 154 with permission of Pharmacotherapy .)
OptiHaler® ACE®
MediSpacer® InspirEase®
OpriChamber®
Space ChamberTM
EasiVent®
EZ-Spacer®
Ellipse®
Gentle-Haler® AeroChamber®
BreatheRite®
6’’Tube
700 AEROSOL DRUG DELIVERY
5.8.5.10 Dose Counters
A guidance document from the Food and Drug Administration (FDA) recommends
the addition of a dose counter to the MDI. This would overcome a long - standing
problem with the MDI, the inability of a patient to accurately know the number of
doses remaining in the canister [164] . Dose counters have been incorporated successfully
into multiple - dose DPIs. In general, the counter should give a clear indication
of when approaching end of life and the actual end of life. It should be either
numeric or color coded. If numeric, it should count downward and should be 100%
reliable and avoid undercounting [165] .
5.8.6 DRY POWDER INHALERS
5.8.6.1 Introduction
Dry power inhalers have been in use for over 40 years. They were developed as an
environmentally friendly alternative to the MDI. The early DPIs were simple in
design, portable, but again, a relatively ineffi cient means of delivering drugs to the
lungs for local action [166] . The Spinhaler, the fi rst DPI, has been prescribed in
Europe since the late 1960s. In general, the acceptance and use of DPIs is much
greater in Europe than in the United States. However, with the reformulation efforts
for MDIs, there are an increasing number of DPIs becoming available in the United
States (Figure 4 ).
Research and development for dry powder inhalers have two main focuses: the
optimization of the powder formulation for use in these inhalers and investigations
of novel DPI device designs and technology. An enormous literature now exists in
each of these areas; for more extensive reviews readers should consult refs. 33, 167,
or 168 .
FIGURE 4 Example DPIs available in United States.
5.8.6.2 Size Reduction and Particle Formation Technologies
Dry powder inhaler formulations consist usually of either a drug - only formulation
or an ordered mixture of drug and excipient, most commonly lactose monohydrate.
In both cases, the fi rst challenge is the production of drug particles with suitable
size characteristics for inhalation (i.e., 1 – 5 . m). Traditionally, micronization or jet -
milling methods have been employed as the method of choice for conventional
small molecules. This method is identical to that employed for the production of
fi ne particles for suspension MDIs. Using this method it is possible to produce
primary particles between 1 – 5 . m. However, as a consequence of the particle size
reduction there are a number of undesirable effects with respect to the powder
properties. Micronized powders possess high intramolecular forces and are cohesive.
They readily form aggregates that are diffi cult to disperse to the primary particles.
Dispersion to its primary particle is essential for successful pulmonary deposition.
In addition, they often possess high inherent electrostatic charges which cause particle
adhesion to the components of the dry powder inhaler [169] . The high - energy
micronization process also causes disruption of the crystal lattice and results in the
formation of amorphous regions which may affect the long - term stability of the
formulation [170] . Finally, it is not possible to control the drug particle morphology.
Despite all of these problems, micronization remains the most common technique
employed for respirable particle formation. Modifi cations to conventional micronization
techniques have been investigated as alternative methods of particle size
reduction [171 – 173] .
A number of novel particle formation technologies now exist that are able to
produce respirable drug particles for formulation in both DPIs and MDIs. Depending
on the method of preparation, these particles offer unique and potentially
advantageous physical and aerodynamic properties compared to conventional crystallization
and micronization techniques. Some investigators have advocated that
major improvements in aerosol particle performance may be achieved by lowering
particle density and increasing particle size, as large, porous particles display less
tendency to agglomerate than (conventional) small and nonporous particles. Also,
large, porous particles inhaled into the lungs can potentially release therapeutic
substances for long periods of time by escaping phagocytic clearance from the lung
periphery, thus enabling therapeutic action for periods ranging from hours to many
days [174] .
Many of these techniques involve particle formation from solution formulations
that contain novel excipients. Spray drying is the most advanced of these technologies
and has been used to produce the powder formulation in the Exubera inhaler
[175] . Various modifi cations of this basic technique, including co – spray drying with
novel excipients, have been employed.
AIR particles are low - density lipid - based particles that are produced by spray
drying lipid – albumin – drug solutions. These particles are characterized by their
porous surface characteristics and large geometric diameter while having a low
aerodynamic diameter [176, 177] . This technology has been used to produce porous
particle powder formulations of L - dopa that have been investigated for the treatment
of Parkinson ’ s disease [178] .
Pulmospheres are produced using a proprietary spray drying technique, with
phosphatidylcholine as an excipient to produce hollow and porous particles with
DRY POWDER INHALERS 701
702 AEROSOL DRUG DELIVERY
low interparticulate forces. These particles have been formulated as suspended
particles in HFA MDIs. In comparison with conventional suspension MDIs, the
Pulmosphere MDI exhibited signifi cantly higher fi ne particle fractions. This technology
has been used to produce cromolyn sodium, albuterol sulfate and formoterol
fumarate microspheres [179] . Pulmospheres powder formulations containing tobramycin
and budesonide have also been tested clinically [83, 180] .
Technosphere technology has been developed as an alternative porous particle
for pulmonary delivery [181] . These porous microspheres are formed by precipitating
a drug - diketopiperazine derivative from an acidic solution. Para - thryroid
hormone (PTH) Technospheres have been investigated for the treatment of osteoporosis
following aerosol delivery [182] .
The use of supercritical fl uid processing technology has also been widely used
for its application in controlled microparticle formation. Conventional small molecules
and proteins for inhalation have been generated and formulated as powders
for inhalation. [183 – 186] .
The application of pulmonary delivery of nanoparticles ( < 1 um) for pharmaceuticals
remains to be developed [187 – 189] .
5.8.6.3 Drug – Lactose Formulations
The most common means of overcoming cohesion problems is by incorporation of
a carrier excipient. Lactose monohydrate is used most often; it is inert, cheap, widely
available, and a GRAS (generally regarded as safe) non - toxic excipient. A signifi -
cant area of research has been undertaken to optimize the critical parameters
involved in the formulation of drug – lactose blends. Micronized drug is typically
blended with lactose (50 – 100 . m) to produce an ordered mix. The blend ratio is
fi xed depending upon the dose of drug to be delivered and the mass of powder
blend in each dosage unit (typically between 5 and 25 mg). The aerosolization properties
of the blend are related to the adhesive forces between the drug and lactose
together with the cohesive forces between the drug particles. Reproducible dispersion
of the blend either by the dry powder inhaler (active DPI) or by the patients ’
inspiratory effort (passive DPI) is required. This allows the detached micronized
drug to be inhaled and deposited in the respiratory tract while the larger lactose
particles are deposited by inertial impaction in the oropharnyx.
Formulators have become increasingly aware of the criticality of the drug and
lactose powder surface characteristics and their relationship to the aerosolization
performance in a DPI [190, 191] . A number of investigators have shown in vitro the
importance of controlling the size of the lactose and the amount of “ fi nes ” (lactose
particles less than 5 . m in size) in the drug – lactose blend [192 – 194] . Inherent fi nes
are present in all lactose powders, and the fi nes are usually adhered to the surface
of the larger lactose particle. These fi nes are believed to occupy “ active ” or high -
energy sites on the lactose particle surface. Occupation of these sites by the lactose
fi nes prevents the micronized drug from adhering to these positions. This allows the
drug to adhere to less active sites and become detached easier from the lactose
surface during inhalation. Obviously any signifi cant change in the quantity of fi nes
present in the lactose may alter the distribution of the micronized drug on the
lactose particle and therefore the aerosolization characteristics of the powder blend
[193 – 195] . Batch - to - batch control of the fi nes content of inhalation lactose has been
recognized as critical to ensuring reproducible in vitro emitted and fi ne particle
doses. Jones and Price (2006) have recently surveyed the literature in this area and
provided a comprehensive review [196] . Modifi cation of the surface characteristics
of the lactose particle has been used as an alternative approach to control the adherence
of drug particles to the lactose surface [197 – 200] . Alternative sugar carriers
have also been investigated; these appear to possess many of the same performance -
limiting characteristics as lactose [201] . Finally, tertiary additives have also been
used to improve the aerosolization properties of DPI formulations [202] . The majority
of the studies described above relate to in vitro testing of DPI formulation performance,
and little is known about the clinical signifi cance of these studies.
Moisture ingress into a powder formulation is a particular concern as it may signifi
cantly decrease the aerosolization performance of the formulation [203, 204] .
Increased adhesion of particles is often seen following exposure to high - RH environments
[205, 206] . Moisture ingress has also been shown to affect drug stability
[170] . The pharmaceutical industry has used a number of approaches to protect
powder formulations from the ingress of moisture during storage and for their “ in -
use ” life. The Turbuhaler incorporates a desiccant in the base of the inhaler to keep
the power reservoir free from moisture [207] . Unit - dose blisters used in the Diskus
are sealed in a foil strip pack to protect each individual dose prior to inhalation
[208] . It is also essential that the patient not exhale into the DPI immediately prior
to inhaling the dose.
Electrostatic charge can also infl uence the performance of DPI formulations. A
number of studies have investigated the interactions of drug and lactose particle
charge with respect to aerosolization properties and drug retention by the plastic
components within the inhaler [209 – 212] .
5.8.6.4 Dry Powder Inhaler Design
Inhalation Flow Rate The main function of a DPI is to facilitate dispersion and
delivery of inhalable drug particles. An extensive patent and scientifi c literature
exists describing the ever - increasing number of DPI device designs [33] . Powder
dispersion in the early passive DPIs was provided in part by the inspiratory effort
of the patient. This removed the necessity to coordinate patient inhalation with
actuation and delivery of the dose (in contrast to MDIs). These passive DPIs were
“ breath actuated, ” with the patients ’ inspiratory effort dispersing, aerosolizing, and
delivering the powder during the inhalation cycle. The airfl ow rate through the
inhaler was determined by the inherent device resistance and the inspiratory force
exerted by the patient [18] . Devices such as the Spinhaler, Rotahaler, and Diskhaler
are low - resistance devices requiring relatively high inspiratory fl ow rates to disperse
the powder formulations by turbulent deaggregation. These simple devices have low
aerosolization effi ciencies with only 5 – 20% of the dose being delivered to the lungs
[166] The inhalation fl ow rate dependence of passive DPIs has been cited as a
potential problem in their use, especially given the large intersubject fl ow rate variability
within the patient population (especially for the young and older patients).
In vitro testing revealed that for certain DPIs there was large variability in both the
delivered dose and the aerodynamic particle size distribution as a function of the
inhalation fl ow rate [203, 213 – 216] . Similar clinical studies also revealed a fl ow rate
dependence for certain DPIs while others were observed to perform with a degree
DRY POWDER INHALERS 703
704 AEROSOL DRUG DELIVERY
of fl ow rate independence [214, 217 – 221] . When choosing a DPI, the effect of inhalation
fl ow rate should be assessed on a case - by - case basis for each individual DPI,
and readers should be aware of contradictory studies, especially when comparing in
vitro and clinical performance. The Turbuhaler is one such example, where some in
vitro studies show high variability; however, this is not refl ected in clinical studies
[215, 220, 222] .
From these and many other studies it can be concluded that a desirable characteristic
for any DPI is that its dose delivery performance is independent of inhalation
fl ow rate. A second generation of DPIs have been developed that incorporate
a combination of improved powder formulations, more effective turbulent dispersion
within the inhaler, and in some cases an active dispersion mechanism. The
Exubera inhaler releases a bolus of compressed air through the formulation and
actively generates an aerosol cloud from the powder (Figure 5 ). The cloud is held
within a reservoir chamber from which the patient then inhales the insulin dose
[175] . Active dispersion improves device aerosolization effi ciency, with greater than
50% of the dose being deposited in the lungs, while minimizing the reliance on the
patients ’ inspiratory effort.
Single - and Multiple - Dose DPI s Inhalation powder dose metering is one of the
problems encountered by DPI formulators. The powder dose can range from 250 . g
in the drug - only Pulmicort Turbuhaler formulation to 25 mg in the lactose - blended
FIGURE 5 Exubera Inhaler. (Reprinted from ref. 175 . Courtesy of Mary Ann Liebert,
Inc.)
Transparent
chamber
TransJector
(disperser)
Blister
pack
Pump
handle
Actuation
button
Spinhaler formulations. In each case, accurate and reproducible metering of the
powder is required for regulatory approval and therapeutic effi cacy. This proved to
be a technological challenge that was solved in a number of ways. Single - unit - dose
inhalers were the fi rst generation of DPIs, the unit dose being metered in the factory
and subsequently loaded into the inhaler by the patient immediately prior to each
dosing. Because metering takes place prior to batch release by the manufacturer,
this allows for quality control and release testing, ensuring that dosage units were
within acceptable criteria. Procedures such as capsule fi lling were common for early
devices such as the Spinhaler and Rotahaler. This approach is still used by some of
the newer devices being developed (e.g., Aerohaler and Cyclohaler) [185, 223] .
While popular with the pharmaceutical industry, the single - unit - dose device required
signifi cant patient handling to load and empty the inhaler for each inhalation (unlike
the MDI, which often contained up to 200 doses available for inhalation on demand).
Two approaches were taken toward the design of multiple - dose DPIs; the multiple -
unit - dose DPI (e.g., Diskhaler and Diskus) and the powder reservoir multidose DPI
(e.g., Turbuhaler) [207] . For the multiple - unit - dose DPI, manufacturers sought to
address the requirement for multiple doses while retaining the control of factory
premetering. Perhaps the most successful DPI in this respect is the Diskus, in which
the dose is premetered into a coiled foil covered strip containing individually sealed
blister reservoirs [208] . Each blister is opened immediately prior to inhalation and
up to 60 doses can be help in each foil strip. For the powder reservoir multidose
DPI, volumetric dose metering of the powder takes place within the DPI immediately
prior to inhalation in a manner analogous to MDIs. Among the devices
that use this approach are the Turbuhaler, Clickhaler, Pulvinal, and Easyhaler
[220, 224 – 226] . The Turbuhaler is used with a drug - only formulation (although
lactose blends have also been used) that employs a proprietary powder agglomeration
process to produce loosely bound aggregates that are easily dispersed by the
patient ’ s inhalation and by the turbulent fl ow path encountered in the DPI [227] .
Besides the Diskus and Turbuhaler, there are four other devices currently available
in the United States, the Asmanex Twisthaler, the Foradil Aerolizer, the Relenza
Diskhaler, and the Spiriva Handihaler. Other devices in development include
the Novolizer, a multidose, refi llable, breath - actuated DPI that delivers up to
200 metered doses of drug from a single cartridge [228, 229] . The Ultrahaler
offers yet another alternative DPI [230] . The Taifun inhaler, the JAGO inhaler, and
the Airmax are other multidose DPIs [231 – 234] .
5.8.6.5 Exubera
Systemic delivery of drugs via the lungs offers a noninvasive route of administration.
Perhaps the most important and widely investigated molecule considered for this
route has been insulin [235, 236] . Following over a decade of development, in
January 2006, Pfi zer and its partner Nektar received marketing approval for Exubera,
their insulin DPI. This offered diabetics a noninvasive route of insulin administration
rather than repeated subcutaneous injections [237] . Exubera has been indicated
for the treatment of adult patients with diabetes mellitus for the control of hyperglycemia
[238] . It has an onset of action similar to rapid - acting insulin analogs and
has a duration of glucose - lowering activity comparable to subcutaneously administered
regular human insulin [239] . Patton et al. (2004) provided an extensive review
DRY POWDER INHALERS 705
706 AEROSOL DRUG DELIVERY
of the clinical pharmacokinetics and pharmacodynamics of inhaled insulin [240] . In
patients with type I diabetes, Exubera should be used in regimens that include a
longer acting insulin [241] . In patients with type II diabetes, Exubera can be used
as monotherapy or in combination with oral agents or longer acting insulins [242 –
244] . Studies revealed that the same level of blood sugar control was achieved following
inhalation compared to subcutaneous injection, although different nominal
doses were required due to lung bioavailability issues [245] . The therapeutic effi cacy
and safety of inhaled insulin appears to have been proven, although there are a
signifi cant number of issues with its administration via this route [246] . It has been
noted that asthmatics absorb less insulin from the lungs than nonasthmatics. In
addition, smokers absorb more insulin than nonsmokers. Small and reversible
changes in pulmonary lung function have been observed in some studies with
inhaled insulin. Each of these issues has led to the development of specifi c prescribing
guidelines and an intensive physician/patient education program for the inhaled
insulin product. The Exubera insulin formulation is a spray - dried, amorphous insulin
powder containing 60% insulin in a buffered, sugar - based matrix [175] .
Other pharmaceutical companies are also continuing to develop their own inhalation
insulin products. Aradigm and NovoNordisk are using a liquid insulin formulation
in combination with the AERx IDMS inhaler [247 – 251] . Alkermes and Lily
are developing an insulin product derived from their research on geometrically
large, low - density particles that are formed by a spray drying process incorporating
a natural phospholipid. MannKind is using its Technosphere technology to produce
low - density porous insulin particles. This formulation is delivered using the MedTone
inhaler. Other companies working in this area include Kos Pharmaceuticals, Mircodose
Technologies, Coremed, and Biosante.
5.8.7 NEBULIZERS
Nebulization of liquid formulations has long been established as an effective, if not
effi cient, means of pulmonary drug delivery. The basic principle of nebulizer aerosol
generation has remained unchanged; however, a number of technological advances
have been made which have improved effi ciency and reduced variability. Aerosols
that were previously delivered in a continuous inhalation mode over 5 – 15 min are
now delivered only during the inspiratory cycle, thus reducing drug waste. In general,
nebulizers convert a liquid into a fi ne droplet mist, either by means of a compressed
gas (jet nebulizer) or by high - frequency sound (ultrasonic nebulizer) [252] . Ultrasonic
nebulizers use a piezoelectric source within the formulation reservoir to
induce waves at the surface of the nebulizer formulation. Interference of these
waves induces the formation of droplets which are then carried in a fl owing air
stream that is passed over the formulation. These devices are not suitable for the
nebulization of suspension formulations [253] . Rau (2002) also observed that ultrasonic
nebulizers can increase the solution reservoir temperature and may cause drug
degradation [254] . In the case of the jet nebulizer, an aerosol is produced by forcing
compressed air through a narrow orifi ce which is positioned at the end of a capillary
tube. The negative pressure created by the expanding jet causes formulation to be
drawn up to the capillary tube from the reservoir in which it is immersed. As the
liquid emerges from the tip of the capillary, it is drawn into the air stream and
broken up into droplets by the jet to produce an aerosol. Baffl e structures within
the nebulizer fi lter the large droplets from the aerosol by impaction and the deposited
drug solution is recycled back into the drug reservoir [255] . Only the small
aerosol droplets evade impaction on the baffl es and are delivered to the patient for
inhalation. Jet nebulizers can be categorized by function, for example, the DeVilbiss
646 is a conventional jet nebulizer with continuous drug output resulting in signifi -
cant waste during exhalation. The Pari LC Plus system incorporated a valve system
and operates as an active venturi jet nebulizer; although drug output is continuous,
there is an increased output during inhalation. The patient ’ s inspiratory effort
increases the nebulizer airfl ow, thus increasing drug output for these breath -
enhanced nebulizers. Finally, dosimetric jet nebulizers such as the Ventstream use a
one - way valve system to emit aerosol only during inspiration and are also breath -
enhanced nebulizers [256, 257] . It is this last type of nebulizer that offers the most
signifi cant advances in technology [258] .
Jet nebulizers are commonly used in nonambulatory settings such as hospitals or
the patient ’ s home. In vitro studies comparing the performance of commercial
nebulizers have concluded that there were large differences in drug delivery between
nebulizers of different classes and even between nebulizers of apparently the same
class [259 – 261] . The aerosolization performance of different nebulizers has been
found to be dependent upon a number of factors, including the drug being aerosolized,
the formulation fi ll volume, the compressed airfl ow rate, and breathing pattern
[254, 260, 262, 263] . These parameters ultimately control the aerosol droplet size and
rate of drug output [264] . However, probably the size of the conventional nebulizer,
the duration of the treatment cycle (5 – 15 min), and the cost of the nebulizers are
the main reasons that they are usually reserved for nonambulatory settings and
remain less popular than the MDI and DPI.
Solutions or suspensions are available as nebulizer formulations. Due to the relative
simplicity in formulating a liquid nebulizer formulation and because of the relatively
large range of doses available for delivery, the nebulization method is often
chosen as the aerosol method for proof - of - concept investigational studies.
Nebulizer technology continues to be developed to miniaturize and lower the
cost of the devices while maintaining the quality of the aerosols generated. The
Halolite incorporates adaptive aerosol delivery which monitors patients inspiratory
cycle and delivers drug to patients during the fi rst 50% of their inspiratory cycle
[265 – 267] . The Pari eFlow is a hand - held device that uses a vibrating membrane
nebulizer to generate a respirable aerosol [268] . Aerogen (now part of Nektar) has
a range of nebulizer - based technologies, including the Aeroneb and Aerodose
devices. Aerosols are generated as a liquid formulation passes through vibrating
apertures [269, 270] .
5.8.8 EMERGING TECHNOLOGIES
5.8.8.1 Soft Mist Aerosols
In recent years research has focused on a new method of pharmaceutical aerosol
generation that involves passing a solution formulation through a nozzle or series
of nozzles to generate a “ soft mist ” aerosol as a bolus dose [271] . Aerosol generation
EMERGING TECHNOLOGIES 707
708 AEROSOL DRUG DELIVERY
is achieved by mechanical, thermomechanical or electromechanical processes
depending upon the particular technology employed [272] . It is worth noting that
these devices are bolus dose delivery inhalers, rather than the new continuous -
generation nebulizers which generate aerosols by vibrating porous membranes at
ultrasonic frequencies. Such devices include the eFlow and Aerodose, which were
described earlier.
While the precise mechanism of soft mist aerosol generation may differ between
inhalers, a number of common characteristics can be observed. They are propellant
free and produce slow - moving aerosols over an extended duration with high in vitro
fi ne - particle fractions compared to MDIs and DPIs. The aerosols are often generated
from simple solution formulations containing pharmaceutically acceptable
excipients. Water and ethanol are the most commonly employed vehicles for soft
mist aerosols [273] . Perhaps the most simple and advantageous vehicle is water.
There is often a well - known and established stability profi le of many pharmaceuticals
in aqueous solutions, accelerating the route to the clinic in any development
program. Drug solubility can be manipulated by choice of water, ethanol, or mixtures
of the two to increase formulation options and doses. In multidose reservoir -
type devices, a preservative would be required to prevent microbial contamination.
This is in addition to the current federal regulations that all aqueous - based drug
products for oral inhalation must be manufactured to be sterile.
5.8.8.2 Respimat
The Respimat inhaler was recently launched in Germany as a combination product
of fenoterol and ipratropium hydrobromide (Berodual) and was licensed for the
treatment of chronic obstructive airway disease. A large body of literature now
exists documenting the aerosol characteristics and clinical performance of the
Respimat inhaler with a number of different drugs [274, 275] . Aerosolized formulations
include the steroids budesonide and fl unisolide in addition to the . agonist
fenoterol as well as the commercially available combination product of fenoterol
and ipratropium bromide [276 – 281] .
The Respimat device is a multidose reservoir system that is primed by twisting
the device base (Figure 6 ). This compresses a spring and transfers a metered volume
of formulation from the drug cartridge to the dosing chamber. The metered volume
is between 11 and 15 . l depending upon the drug formulation. When the device is
actuated (in coordination with the patient ’ s inspiration), the spring is released. This
forces a micropiston into the dosing chamber and pushes the solution through the
uniblock. The uniblock is the heart of the aerosol generation system and consists of
a fi lter structure with two fi ne outlet nozzle channels. The uniblock produces two
fi ne jets of liquid that converge at a precisely set angle and then collide. This collision
aerosolizes the liquid to form an aerosol [282] .
Aerosols generated from the Respimat inhaler have been characterized as having
a prolonged aerosol cloud duration compared to MDIs and have a slower cloud
velocity as measured using video camera imaging. Hochrainer et al. (2005) measured
the cloud duration of the Respimat aerosol to be 0.2 – 1.6 s compared to less than
0.2 s for HFA and CFC MDIs. Aerosol velocities have been reported as less than
1 m/s for the Respimat, compared to 6 – 8 m/s for CFC MDI inhalers [283] . While a
degree of patient coordination is required to actuate the Respimat and to inhale,
the longer duration of aerosol cloud generation makes this maneuver less critical
than with MDIs.
Aqueous and ethanolic formulations have been employed with the Respimat
and the in vitro aerosol performance determined. Zierenberg (1999) reported fi ne -
particle fractions of 66% for an aqueous fenoterol formulation and 81% for an
ethanolic fl unisolide formulation. The respective MMADs were 2.0 ± 0.4 . m for
the aqueous formulation and 1.0 ± 0.3 . m for the ethanolic formulation [284] .
5.8.8.3 AER x
The AERx system was developed for the systemic delivery of insulin. Unit - dose
aqueous solution formulations were produced in a blister strip design. The fi rst -
generation AERx device is a battery - operated device that guides the patient through
the inhalation technique required to successfully deliver a dose. It can also monitor
dose times and frequency together with the facility to download dosing data in
the clinic. A number of macromolecules, including insulin, and traditional small
molecules (e.g., morphine) have been investigated using the AERx technology
[89, 251] .
FIGURE 6 Respimat Inhaler. (Courtesy of Boehringer Ingelheim.)
Mouthpiece
Uniblock
Dose release button
Capillary tube
Upper housing
Transparent base
spring
Cartridge
EMERGING TECHNOLOGIES 709
710 AEROSOL DRUG DELIVERY
Aerosol generation using the AERx system is achieved by mechanically forcing
a dose of the liquid formulation though a nozzle array in its disposable unit - dose
blisters. The electronic version of the AERx inhaler guides the patient to inhale at
the required fl ow rate. A cam - operated piston mechanism is actuated to compress
the blister and extrude the dose as an aerosol through the nozzle array into warmed
fl owing air. The nozzle array consists of a number of laser - drilled holes. Nozzle
design characteristics can be altered depending upon the formulation characteristics
and the desired droplet particle size. The single - use nature of the blister avoids
potential problems such as microbial contamination from a dosing solution reservoir
and nozzle - clogging issues.
A number of prototype versions of the AERx system have been investigated. In
general, the in vitro aerosol characteristics revealed that about 50 – 60% of the
loaded dose was emitted from the device, of which over 90% was respirable. MMADs
ranged from 1 to 3 . m depending upon the formulation and nozzle array [285] . In
a scintigraphic study, lung deposition following inhalation from the AERx was
53.3% (expressed as a percentage of the radioactivity in the AERx blister) compared
to 21.7% for an MDI [285] .
A number of clinical studies delivering insulin to diabetic patients using the
AERx system are currently ongoing. Hermansen et al. (2004) concluded that in type
II diabetics, preprandial inhaled insulin via the AERx was as effective as preprandial
subcutaneous insulin in achieving glycemic control [286] . Clinical studies with morphine
revealed comparable analgesic effi cacy for a matched dose of inhaled and
intravenous morphine in a postsurgical pain model [251] . In addition, the AERx
inhaler has been employed for the topical delivery of rhDNase to cystic fi brosis
patients. A mean relative increase in forced expiratory volume in 1 s (FEV1) of 7.8%
was observed after 15 days treatment compared to control [287] .
5.8.8.4 Mystic
The Mystic inhaler offers a soft mist aerosol generated from solution or suspension
formulations. Unlike the previously described soft mist inhalers which use purely
mechanical forces to generate the aerosol, the Mystic inhaler applies an electric fi eld
to the formulation within the spray nozzle [288] . An electric charge builds on the
fl uid surface and, as the droplets exit the nozzle, the repelling force of the surface
charge overcomes the surface tension of the droplets to form a soft mist droplet
aerosol. This process is known as electrohydrodynamic aerosolization or electrospray.
The particle size characteristics of the aerosol can be controlled by adjusting
the physical and chemical characteristics of the formulation together with the formulation
fl ow rate and electrical fi eld properties. The inhaler consists of a number
of components, a drug containment system, metering system, aerosol nozzle, power
supply, and microprocessor, all enclosed in a housing. To date, Ventaira reports that
the inhaler has been successfully employed to generate aerosols from small -
molecule formulations (albuterol, triamcinolone, cromolyn, budesonide, and terbutaline)
and macromolecules, including insulin [288] .
5.8.8.5 Capillary Aerosol Generator
In the capillary aerosol generator (CAG) system, the aerosol is formed by pumping
the drug formulation through a small, electrically heated capillary. Upon exiting the
capillary, the formulation is rapidly cooled by ambient air to produce an aerosol.
The generated aerosol characteristics are dependent upon the formulation employed.
Using propylene glycol as a condensing vehicle, drug containing condensation aerosols
are generated [289] . When using water, ethanol, or combinations of both as
noncondensing excipients, a stream of solid particles is delivered as a soft mist
aerosol. In vitro studies using budesonide, cromolyn sodium, buprenophine, albuterol,
and insulin have been performed to demonstrate various applications of the
CAG technology. These studies are characterized by high emitted doses and high
fi ne - particle fractions. Using noncondensing excipients, it is possible to produce
aerosols with vastly different size characteristics, depending upon the required
application.
5.8.8.6 Staccato
This technology utilizes a rapid heating technique to vaporize a thin fi lm of drug.
Following vaporization, the drug particles condense in the inhalation fl ow stream
to form a respirable aerosol and are inhaled. Single - and multiple - dose breath -
actuated inhalers are currently in development. As with any method involving
heating of a formulation, drug degradation must be minimal. Rabinowitz et al.
(2006) described the absorption of prochlorperazine from human lungs as similar
to the pharmacokinetic profi les observed following intravenous administration [290,
291] .
5.8.9 CONCLUSIONS
Pharmaceutical aerosol drug delivery has been established for over 50 years. Pulmonary
administration remains the route of choice for local treatment of respiratory
diseases. Over the past decade there have been changes in both the diseases treated
by this route and the devices used for aerosol generation. Future advances will see
pulmonary delivery of gene therapy and vaccines, together with improved drug
targeting within the respiratory tract using novel inhalers.
ACKNOWLEDGMENTS
The author would like to thank Suparna Das Choudhuri and Deepika Arora
for their assistance and discussions during the preparation of this chapter. In
addition, he is grateful to Guillaume Brouet (Valois Pharm), Michael Spallek
(Boehringer Ingelheim), and Joanne Peart (RDD) for their help in obtaining
fi gures and tables used in this chapter. Finally, the review of soft mist inhalers
has previously been published in the Drug Delivery Company Report (Autumn/
Winter 2004), and the author acknowledges PharmaVentures and the Drug
Delivery Company Report, which allowed reproduction of an abridged form of
this paper.
The author received a research grant from Chrysalis Technologies, a division of
Philip Morris USA, for the development of the CAG technology.
ACKNOWLEDGMENTS 711
712 AEROSOL DRUG DELIVERY
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729
5.9
OCULAR DRUG DELIVERY
Ilva D. Rupenthal and Raid G. Alany
The University of Auckland, Auckland, New Zealand
Contents
5.9.1 Introduction
5.9.2 Challenges in Ocular Drug Delivery
5.9.2.1 Anatomical and Physiological Considerations
5.9.2.2 Pharmacokinetic Considerations
5.9.2.3 Formulation Considerations
5.9.3 Formulation Approaches to Improve Ocular Bioavailability
5.9.3.1 Conventional Dosage Forms
5.9.3.2 Polymeric Delivery Systems
5.9.3.3 Colloidal Delivery Systems
5.9.3.4 Other Delivery Approaches
5.9.4 Conclusion
References
5.9.1 INTRODUCTION
Due to the accessibility of the eye surface, topical administration of ophthalmic
medications is the most common method for treating conditions affecting the exterior
eye surface. However, the unique anatomy and physiology of the eye renders
it diffi cult to achieve an effective drug concentration at the target site. Therefore,
effi cient delivery of a drug past the protective ocular barriers accompanied with
minimization of its systemic side effects remains a major challenge.
Conventional eye drops currently account for more than 90% of the marketed
ophthalmic formulations [1] . However, after instillation of an eye drop, typically less
than 5% of the applied drug penetrates the cornea and reaches the intraocular
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
730 OCULAR DRUG DELIVERY
tissues. This is due to the rapid and extensive precorneal loss caused by drainage
and high tear fl uid turnover.
As a consequence, the typical corneal contact time is limited to 1 – 2 min and the
ocular bioavailability is usually less than 10% [2] . Furthermore, there is an initial
peak dose of the drug, which is usually higher than that needed for a therapeutic
effect, followed by a sharp drop - off in concentration to subtherapeutic levels.
Various ocular delivery systems, such a ointments, suspensions, micro - and nanocarriers,
and liposomes, have been investigated during the past two decades pursuing
two main strategies: to increase the corneal permeability and to prolong the contact
time on the ocular surface [3] .
On the other hand, the most effi cient method for drug delivery to the posterior
chamber of the eye so far has been intravitreal injection. This chapter focuses on
the topical application of drugs to the surface of the eye and discusses the most
recent formulation approaches in this area.
5.9.2 CHALLENGES IN OCULAR DRUG DELIVERY
5.9.2.1 Anatomical and Physiological Considerations
In order to research and develop an effective ophthalmic delivery system, a good
understanding of the anatomy and physiology of the eye (the globe) is necessary.
Figure 1 shows a cross section through the human eye. This chapter will mainly focus
on the precorneal area and the transport barriers present in the eye.
FIGURE 1 Cross section of the human eye and cornea.
Precorneal Area
Precorneal Tear Film Corneal transparency and good visual function require a
uniform eye surface. This is achieved by the tear fi lm, which covers and lubricates
the cornea and the external globe. It is about 7 – 8 . m thick and is the fi rst structure
encountered by topically applied drugs. The trilaminar structure of the tear fi lm is
shown in Figure 2 .
Attached to the glycocalix of the corneal/conjunctival surface is a mucous layer,
which consists mainly of glycoproteins. This layer is produced by the conjunctival
goblet cells and the lacrimal gland. It plays an important role in the stability of the
tear fi lm as well as in the wetting of the corneal and conjunctival epithelium. The
middle aqueous layer constitutes about 98% of the tear fi lm and is mostly secreted
by the main and accessory lacrimal glands [4] . It is composed of water, electrolytes,
and various proteins such as lipocalin, lysozyme, and lactoferrin [5 – 8] . The outermost
lipid layer is derived from the Meibomian and sebaceous Zeiss glands and
prevents the evaporation of the tear fl uid. It consists of sterol esters, triacylglycerols,
and phospholipids and is spread over the aqueous layer during blinking.
Nasolacrimal Drainage System Figure 3 illustrates the nasolacrimal drainage
system. The lacrimal gland, which is situated in the superior temporal angle of the
orbit, is responsible for most of the tear fl uid secretion. Secreted fl uid is spread over
the surface of the cornea during blinking and ends up in the puncta when the upper
eye lid approaches the lower lid. The blinking process creates a suction mechanism
which results in tears fl owing through the lacrimal canaliculi into the lacrimal sac.
Fluid from the lacrimal sac then drains into the 12 - mm - long nasolacrimal duct,
which empties into the inferior nasal passage. This passage is a highly vascular area
FIGURE 2 Structure of the precorneal tear fi lm.
CHALLENGES IN OCULAR DRUG DELIVERY 731
732 OCULAR DRUG DELIVERY
and is responsible for most of the systemic drug absorption and subsequent systemic
side effects.
The cul - de - sac normally holds 7 – 9 . L of tear fl uid, with the normal tear fl ow rate
being 1.2 – 1.5 . L/min [4] . The loss from the precorneal area by drainage, tear fl uid
turnover, and noncorneal absorption plays an important role in determining the
ocular bioavailability of a drug. As the drainage rate is much faster than the ocular
absorption rate, most of the topically applied drug is eliminated from the precorneal
area within the fi rst minute [4] .
Tear production can be divided into basal, refl ex, and emotional tearing [9] .
Refl ex tearing can be induced by many pharmaceutical/formulation factors, including
the drug itself as well as pH and tonicity of the ocular dosage form.
Transport Barriers in the Eye Topical administration is the most common route
for ocular drug delivery. Consequently, the cornea, conjunctiva, and sclera form the
most essential barriers for drug penetration into the intraocular tissues.
Cornea The cornea is an important mechanical barrier protecting the intraocular
tissues. It is considered to be the main pathway for ocular penetration of topically
applied drugs. However, due to its unique structure, with the hydrophilic stroma
sandwiched between the highly lipophilic epithelium and the less lipophilic endothelium,
the penetration of compounds through the cornea depends on their n -
octanol – water partition coeffi cient. Only drugs with a partition coeffi cient between
10 and 100 that show both lipid - and water - soluble properties can readily pass
through the cornea.
The cornea is composed of fi ve layers (see Figure 1 ): epithelium, Bowman ’ s
membrane stroma, Descemet ’ s membrane, and endothelium.
The epithelium is the outermost layer and consists of fi ve to six cell layers. These
can be subdivided into one to two outermost layers of fl attened superfi cial cells with
microvilli on their anterior surface enhancing the cohesion and stability of the tear
FIGURE 3 Schematic of the nasolacrimal drainage system.
fi lm [10] , two to three layers of polygonal wing cells, and a single layer of basal
columnar cells, allowing for minimal paracellular transport. The superfi cial cells
adhere to one another via desmosomes and the cells are encircled by tight junctions
[11] . Due to the nature of the tight junctions, the epithelium represents the rate -
limiting barrier for hydrophilic compounds.
Bowman ’ s membrane is composed of a layer of collagen fi bers which form a relatively
tight and impermeable barrier against microorganisms and therefore protect
the stroma.
The stroma makes up approximately 90% of the corneal thickness and is mainly
composed of hydrated collagen fi brils. It is highly hydrophilic, porous, and can be
considered as an open structure, as it allows free passage of hydrophilic substances
with a molecular weight below 500,000 Da but acts as a diffusion barrier to all lipophilic
drugs [4, 12] .
Descemet ’ s membrane is the basement membrane of the endothelial cells. It is
comprised of collagen fi bers arranged in a hexagonal pattern and embedded in a
matrix.
The endothelium is a single layer of fl attened polygonal cells with microvilli
which increase the surface area for removal of waste and absorption of nutrients
[12] . It plays an important role in the maintenance of corneal hydration and transparency
via active ion and fl uid transport mechanisms.
Since the cornea exhibits hydrophilic as well as lipophilic characteristics, it represents
an effective barrier for diffusion of both hydrophilic and lipophilic substances
[3, 13] .
Conjunctiva The conjunctiva is a thin and vascular mucous membrane consisting
of two to three layers of epithelial cells overlying a loose, highly vascular connective
tissue. The tight junctions present on the apical surface of the epithelium act as the
main barrier for drug penetration (molecules > 20,000 Da) across the tissue, although
not as tight as the corneal epithelium, which is impermeable to molecules larger
than 5000 Da [14, 15] . The conjunctiva covers the anterior surface of the globe
(bulbar conjunctiva), with the exception of the cornea, and is folded at the fornix
(fornix conjunctiva) to form the palpebral conjunctiva, which lines the inner surface
of the eyelids. The bulbar conjunctiva represents the fi rst barrier for permeation of
topically applied drugs via the noncorneal route [16] .
Sclera The sclera is the outermost fi rm coat of the eye that serves as a protective
barrier for the sensitive inner parts. It is composed of the same type of collagen
fi bers as the corneal stroma. However, the fi bers are arranged in an irregular network
rather than a lattice pattern, which makes the tissue appear opaque compared to
the transparent cornea. The white sclera constitutes the posterior fi ve - sixths of the
globe, whereas the transparent cornea comprises the anterior one - sixth [17] .
Iris – Ciliary Body The ciliary body comprises the ciliary muscle, which mainly
enables accommodation, and the ciliary processes, which produce the aqueous
humor.
The iris separates the anterior and posterior chambers and consists of the pigmented
epithelial cell layer, the iridial sphincter and dilator muscles, and the stroma.
The amount of melanin present in the stroma determines the color of the iris: few
CHALLENGES IN OCULAR DRUG DELIVERY 733
734 OCULAR DRUG DELIVERY
melanocytes exhibit blue, grey, or green, while many melanocytes are responsible
for the brown appearance of the iris. There is often a considerable quantitative difference
in drug response between light and heavily pigmented eyes [18] . The binding
of drugs with melanin can decrease the aqueous humor concentration of free drug
and is therefore likely to reduce the pharmacological response [19] .
Lens The lens is the transparent biconvex structure situated behind the iris and
in front of the vitreous. It plays an important role in the visual function of the eye
and also enables accommodation together with the ciliary muscle. The lens is made
up of slightly more than 30% protein (water - soluble crystallins) and therefore has
the highest protein content of all tissues in the body [20] . The lens receives its nutrients
from the aqueous humor and its transparency depends on the geometry of the
lens fi bres.
Blood – Ocular Barriers The blood – ocular barriers can be divided into the blood –
aqueous barrier and the blood – retinal barrier.
The blood – aqueous barrier is located in the anterior part of the eye and is formed
by the endothelial cells of the blood vessels in the iris and the nonpigmented cell
layer of the ciliary epithelium [21] . It regulates the solute exchange between the
blood and the intraocular fl uid, preventing unspecifi c passage of solutes that could
infl uence the transparency of the ocular tissues. The outward movement into the
systemic blood circulation is less restricted, allowing especially small and lipophilic
drug molecules to enter the uveal blood circulation [22] . These molecules are consequently
removed more rapidly from the anterior chamber than larger, hydrophilic
molecules, which are eliminated by the aqueous humor turnover only [23] .
The blood – retinal barrier can be found in the posterior part of the eye. It prevents
toxic molecules, plasma components, and water from entering the retina. It also
forms a barrier for passage of systemically administered drugs into the vitreous,
typically resulting in only 1 – 2% of the drug ’ s plasma concentration in the intraocular
tissues [24] .
5.9.2.2 Pharmacokinetic Considerations
After topical application of an ophthalmic solution, the solution is instantly mixed
with the tear fl uid and then spread over the eye surface. However, various precorneal
factors such as the drainage of the instilled solution, induced lacrimation,
normal tear turnover, noncorneal absorption, drug metabolism, and enzymatic degradation
limit the ocular absorption by shortening the contact time of the applied
drug with the corneal surface [25] . As a result, typically less than 10% of the instilled
dose is delivered into the intraocular tissues, whereas the rest is absorbed into the
systemic circulation, leading to various side effects [3, 25, 26] . A summary of the
drug deposition model in the eye after topical application as described by Lee and
Robinson [27] is given below.
Upon instillation, the topically applied drug solution is instantly diluted by the
resident tears, resulting in a signifi cant decrease in the concentration gradient
(driving force) and hence in the reduction of the transcorneal fl ux. Drainage of
lacrimal fl uid towards the nasolacrimal sac during blinking leads to a rapid elimination
of the ocular solution via the canaliculi.
Any drug remaining on the ocular surface for a suffi cient period of time can be
absorbed into the anterior chamber via either the corneal or the conjunctival – scleral
route.
Corneal absorption is considered to be the major penetration pathway for topically
applied drugs. There are two mechanisms for absorption across the corneal
epithelium, namely transcellular and paracellular diffusion [28] . Lipophilic drugs
prefer the transcellular route while hydrophilic drugs penetrate primarily via the
paracellular route. Transcorneal transport includes simple diffusion, facilitated diffusion,
active transport, and endocytosis. Transport along the paracellular route is
passive and is only limited by the pore size and the charge of the intracellular spaces.
Only relatively small molecules can permeate through the pores. Negatively charged
molecules permeate at a slower rate than positively charged and neutral ones [29] .
In addition, a positive charge may also decrease the permeation due to the possible
ionic interaction with the negatively charged carboxylic acid residues of the tight
junction proteins [30] .
The noncorneal route of absorption via the conjunctiva and the sclera is usually
nonproductive, as most of the drug reaches the systemic circulation before gaining
access to the intraocular tissues. As the surface area of the conjunctiva is much larger
than that of the cornea and with the highly vascularized conjunctiva being more
permeable, especially to larger hydrophilic molecules, drug loss through this route
of absorption may be signifi cant. Both transconjunctival and transnasal absorption
via the nasolacrimal duct are generally undesirable, not only because of the loss of
drug into the systemic circulation, but also because of the possible side effects [1] .
5.9.2.3 Formulation Considerations
Irritation of the eye following the use of an ocular delivery system can be induced
by a number of factors, including the instilled volume, the pH, and the osmolality
of the formulation, as well as by the drug itself [31] . All these factors may induce
refl ex tearing and as a result increase lacrimal drainage. This is likely to reduce the
ocular bioavailability of the drug and thus needs to be considered during the formulation
process. In addition, general safety considerations such as sterility, ocular
toxicity and irritation, and the amount of preservative used, need to be taken into
account when formulating an ocular dosage form.
Physicochemical Drug Properties On the one hand, factors such as the chemical
nature or the concentration of a drug can cause irritation of the ocular tissues, inducing
refl ex tearing and therefore reducing the retention time of the formulation in
front of the eye.
However, more important physicochemical properties in terms of ocular bioavailability
are the ones that affect the corneal permeability of the active compound.
These include the lipophilicity of the drug as refl ected by its n - octanol – water partition
coeffi cient [32] , the molecular size and shape [33] , the charge [34] , and the
acid – base properties as determined by its p Ka [35] .
According to Kaur and Smitha [36] , the optimum lipophilicity for corneal absorption
is found in drugs with an n - octanol – water partition coeffi cient between 10 and
100. For drugs with smaller partition coeffi cients (highly hydrophilic drugs), the
lipophilic epithelium forms the rate - limiting barrier, whereas the hydrophilic stroma
CHALLENGES IN OCULAR DRUG DELIVERY 735
736 OCULAR DRUG DELIVERY
represents the primary barrier for transcorneal diffusion of highly lipophilic drugs
[1, 3, 25] . In general, more lipophilic drugs penetrate via the transcellular pathway
while the more hydrophilic drugs enter the cornea via the paracellular route.
In the case of ionized compounds (weak acids and bases), drug permeation
depends on the chemical equilibrium between the ionized and the un - ionized form,
both in the delivery system itself and in the lacrimal fl uid. In general, un - ionized
molecules penetrate lipid membranes more readily than ionized ones.
Besides lipophilicity and degree of ionization, the charge of the drug molecule
may have an effect on its penetration. Cationic drugs permeate the cornea more
easily than anionic compounds, which are repelled by the negative charge of the
mucin layer on the ocular surface as well as the negatively charged pores present
in the corneal epithelium [1, 3, 25, 29] . However, a positive charge may also decrease
permeation in some cases, due to possible ionic interactions between the positively
charged molecules and the negatively charged carboxylic acid groups of the tight
junction proteins [21, 30] .
Finally, the molecular size of the drug has an effect on the corneal absorption.
The cornea is impermeable to molecules larger than 5000 Da, whereas the conjunctival
tissues allow compounds of up to 20,000 Da to penetrate [14, 15] .
Buffer Capacity and pH The normal pH of the tear fl uid is 7.4. Ocular formulations
should ideally be formulated between pH 7.0 and 7.7 to avoid irritation of the
eye [31] . However, in most cases the pH necessary for maximal solubility or stability
of the drug is well outside this range. The tear fl uid has only a limited buffering
capacity, which is mainly due to the dissolved carbon dioxide and bicarbonate. It is
therefore recommended to formulate using buffers with a low buffering capacity to
allow the tears to regain their normal pH more rapidly [31] .
Instillation Volume The cul - de - sac normally holds 7 – 9 . L [37] , but can momentarily
accommodate up to 30 . L without overfl owing. Most commercial eye droppers
however, deliver a volume of approximately 50 . L. The excess volume is rapidly
removed, either by spillage from the conjunctival sac or through the puncta to the
nasolacrimal drainage system, until the tears return to their normal volume [38] .
Chrai et al. [39, 40] determined the infl uence of drop size on the rate of drainage of
a solution instilled into the conjunctival sac of rabbits. The authors reported that
the drainage process followed fi rst - order kinetics and found that the rate of solution
drainage from the conjunctival sac (as refl ected by the elimination rate constant)
was directly proportional to the instilled volume [40] . Similar observations were
reported with other dosage forms such as suspensions [41] and liposomes [42] .
Therefore, keeping the applied dose constant while decreasing the instilled volume
substantially increases ocular bioavailability and decreases systemic absorption
[3, 26, 43] .
Osmotic Pressure The osmolality of the lacrimal fl uid is mainly dependent on the
number of ions dissolved in the aqueous layer of the tear fi lm and normally ranges
between 310 and 350 mOsm/kg [44] . When an ophthalmic solution is instilled into
the eye, it mixes with the tear fl uid, resulting in an osmotic pressure that is dependent
on the osmolality of the tears as well as that of the formulation and the amount
of the formulation instilled. In general, hypotonic solutions are better tolerated by
the ocular tissues than hypertonic ones, which lead to increased lacrimation. If the
tonicity of the formulation is lower than 260 mOsm/kg or higher than 480 mOsm/kg,
the formulation becomes irritant [45] , induces refl ex tearing and blinking, and is
therefore likely to reduce the bioavailability of topically instilled drugs.
5.9.3 FORMULATION APPROACHES TO
IMPROVE OCULAR BIOAVAILABILITY
One of the major problems encountered with topical administration is the rapid
precorneal loss caused by nasolacrimal drainage and high tear fl uid turnover,
which leads to drug concentrations of typically less than 10% of the applied drug.
Approaches to improve the ocular bioavailability have been attempted in two
directions: to increase the corneal permeability by using penetrations enhancers or
prodrugs and to prolong the contact time with the ocular surface by using viscosity -
enhancing or in situ gelling polymers. Table 1 summarizes conventional and novel
ocular drug delivery approaches. Marketed ophthalmic delivery systems based on
recent formulation approaches are listed in Table 2 . An optimal ocular delivery
systems would be administered in the form of an eye drop, causing neither blurred
vision nor ocular irritancy, and would only need to be instilled once a day [1] .
5.9.3.1 Conventional Dosage Forms
Conventional dosage forms such as solutions, suspensions, and ointments account
for almost 90% of the currently accessible ophthalmic formulations on the market
[1, 169] . They offer some advantages such as their ease of administration by the
patient, ease of preparation, and the low production costs. However, there are also
signifi cant disadvantages associated with the use of conventional solutions in particular,
including the very short contact time with the ocular surface and the fast
nasolacrimal drainage, both leading to a poor bioavailability of the drug. Various
ophthalmic delivery systems have been investigated to increase the corneal permeability
and prolong the contact time with the ocular surface. However, conventional
eye drops prepared and administered as aqueous solutions remain the most commonly
used dosage form in ocular disease management.
Solutions The reasons behind choosing solutions over other dosage forms are
their favorable cost advantage, the simplicity of formulation development and production,
and the high acceptance by patients [170] . However, there are also a few
drawbacks, such as rapid and extensive precorneal loss, high absorption via the
conjunctiva and the nasolacrimal duct leading to systemic side effects, as well as the
increased installation frequency resulting in low patient compliance.
Some of these problems have been encountered by addition of viscosity -
enhancing agents such as cellulose derivates, which are believed to increase the
viscosity of the preparation and consequently reduce the drainage rate. The use of
viscosity enhancers will be discussed later in this section.
Suspensions Suspensions of the micronized drug ( < 10 . m) in a suitable aqueous
vehicle are formulated, where the active compound is water insoluble. This is the
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 737
738 OCULAR DRUG DELIVERY
TABLE 1 Summary of Conventional and Novel Ocular Drug Delivery Approaches
Drug
Formulation
Approach Polymers/Bases References
Pilocarpine Viscosity enhancer Methylcellulose [ 40 ]
Fluorescein Viscosity enhancer +
collagen shields
Collagen shields in
methylcellulose vehicle
[ 46 ]
Ciprofl oxacin Viscosity enhancers
+ penetration
enhancer
Carbopol 934P/HPMC +
dodecylmaltoside
[ 47 ]
Pilocarpine Viscosity enhancers,
mucoadhesives,
in situ gelling
systems
Gellan gum, xanthan gum, HEC,
HPMC, PVA
[ 48 ]
Progesterone Mucoadhesive Cross - linked acrylic acid [ 49 ]
Levobetaxolol Mucoadhesive Polyacrylic acid [ 50 ]
Tropicamide Mucoadhesive CMC, HPCL, HPCM, PVP, PVA [ 51 ]
CMC - Na, HA - Na, PAA [ 52 ]
Pilocarpine,
tropicamide
Mucoadhesive Hyaluronic acid, polyacrylic acid [ 53 ]
Tertrahydrozoline Mucoadhesive Hyaluronic acid, polyacrylic acid,
chitosan, gelatin
[ 54 ]
Tobramycin Mucoadhesive Chitosan [ 55 ]
Tobramycin,
ofl oxacin
Mucoadhesive Chitosan [ 56 ]
Ofl oxacin Mucoadhesive N - Trimethyl and N - carboxy -
methyl chitosan
[ 57, 58 ]
Pilocarpine Mucoadhesive Carbopol 934P [ 59 ]
Carbomer 974P and 1342 [ 60 ]
PVA, PVP, dextran, HPMC, HEC,
MC, PAA, Na - hyaluronate, Na -
alginate, gellan gum, chitosan
[ 61 ]
Timolol Mucoadhesive Xyloglucan [ 62 ]
Xanthan gum [ 63 ]
Carrageenan, gellan gum [ 64 ]
Carrageenan, locust bean gum,
guar gum, xanthan gum,
scleroglucan, xanthan gum,
sodium alginate, . - cyclodextrin
[ 65 ]
Hyaluronic acid [ 66 ]
HPMC, PVA, hyaluronic acid [ 67 ]
Carbomer, hyaluronic acid [ 68 ]
PAA, PVP [ 69 ]
In situ gelling system Gellan gum [ 70 – 78 ]
Indomethacine In situ gelling system Gellan gum [ 79 ]
Pefl oxacin
mesylate
In situ gelling system Gellan gum [ 80 ]
Gatifl oxacin In situ gelling system Alginate/HPMC [ 81 ]
Ofl oxacin In situ gelling system Carbopol 940/HPMC [ 82 ]
Pilocarpine In situ gelling system Alginate [ 83 ]
Carteolol In situ gelling system Alginate [ 84 ]
Ciprofl oxacin In situ gelling system Poloxamer/hyaluronic acid [ 85 ]
Drug
Formulation
Approach Polymers/Bases References
Pilocarpine In situ gelling system Pluronic F127, MC, HPMC [ 86 ]
Pluronic F127/carbopol [ 87 ]
Pluronic F127, xyloglycan [ 88 ]
Timolol In situ gelling system Pluronic F127, MC, HPMC, CMC [ 89 ]
Doxorubicin In situ gelling system Chitosan/glycerophosphate [ 90 ]
Pluronic F127 and F68, sodium
hyaluronate
[ 91 ]
Pilocarpine,
hydrocortisone
Nanoparticles Gelatin [ 92 ]
Hydrocortisone Nanoparticles Albumin [ 93 ]
Pilocarpine Nanoparticles Albumin [ 94, 95 ]
Poly(methyl)methacrylate – acrylic
acid copolymer (Piloplex)
[ 96 – 98 ]
Cellulose acetate hydrogen
phthalate (CAP)
[ 99 – 101 ]
Betaxolol Nanoparticles Poly - . - caprolactone (PECL), poly
(isobutyl)cyanoacrylate,polylac
tic -co - glycolic acid (PLGA)
[ 102 ]
Carteolol Nanoparticles Poly - . - caprolactone (PECL) [ 103 ]
Pilocarpine Nanoparticles Poly(butyl)cyanoacrylate
(PBCA)
[ 104 – 107 ]
Poly(hexyl)cyanoacrylate
(PHCA)
[ 104, 106,
107 ]
Ciclosporine Nanoparticles Chitosan [ 108 ]
Indomethacine Nanoparticles Chitosan - and poly - L - lysine -
coated poly - . - caprolactone
nanocapsules
[ 109 ]
Rhodamine Nanoparticles Chitosan - and PEG - coated poly -
. - caprolactone nanocapsules
[ 110 ]
Aciclovir Nanoparticles PEG - coated PLA nanospheres [ 111 ]
Epinephrine Nanoparticles Poly - N - isopropylacrylamide [ 112 ]
Ibuprofen Nanoparticles Eudragit RS100 [ 113, 114 ]
Flurbiprofen Nanoparticles Eudragit RS100 and RL100 [ 115 ]
Diclofenac Nanoparticles Eudragit RLPM and RSPM [ 116 ]
Tobramycin Nanoparticles Various lipids [solid lipid
nanoparticles (SLN)]
[ 117 ]
Gentamicin Microspheres Eudragit RS100 and RL100 [ 118 ]
Piroxicam Microspheres Pectin [ 119 ]
Albumin [ 120 ]
Pilocarpine Liposomes +
mucoadhesive
coating
Carbopol - coated liposomes [ 121, 122 ]
Radioactive -
labeled BSA
Liposomes +
mucoadhesive
coating
Chitosan - coated liposomes [ 123 ]
Oligonucleotides
(pdT16)
Liposomes + in situ
gelling system
Liposomes in poloxamer 407 gel [ 124 – 126 ]
Tropicamide Liposomes + in situ
gelling system
Liposomes in polycarbophil gel [ 127 ]
TABLE 1 Continued
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 739
740 OCULAR DRUG DELIVERY
Drug
Formulation
Approach Polymers/Bases References
Timolol Niosomes Chitosan - and carbopol - coated
niosomes
[ 128 ]
Indomethacine Submicrometer
emulsions
Phospholipids,
lauroamphodiacetate
[ 129 ]
Pilocarpine Submicrometer
emulsions
Mono - dodecylphosphoric acid [ 130 ]
Microemulsions Lecithin - based microemulsions [ 131 ]
Retinol Microemulsions Tween 60 and 80, soy bean
lecithin, n - butanol, triacetin,
PG
[ 132 ]
Crodamol EO, Crill 1 and
Crillet 4
[ 133 ]
Epinephrine Prodrug Dipivalyl epinephrine [ 134, 135 ]
Pilocarpine Prodrug O, O. (Xylylene)bispilocarpic
acid esters
[ 136 ]
Ganciclovir Prodrug Ganciclovir acyl ester [ 137 ]
Atenolol,
betaxolol,
Timolol
Penetration
enhancers
Polyoxyethylene alkyl ethers
(Brij), bile salts
[ 138 ]
Cromoclycin Penetration
enhancers
EDTA [ 33 ]
Carbonic
anhydrase
inhibitors
Cyclodextrins . - Cyclodextrin,
hydroxypropyl -. - cyclodextrin
[ 139 ]
Ciprofl oxacin Cyclodextrins Hydroxypropyl - . - cyclodextrin [ 140 ]
Pilocarpine Cyclodextrins Hydroxypropyl - . - cyclodextrin [ 141 ]
Ocular fi lms Hydroxypropyl cellulose [ 142 ]
Poly(2-hydroxypropyl-methacrylate) [ 143 ]
Pefl oxacin
mesylate
Ocular fi lms HPC, HPMC, PVP, PVA [ 144 ]
Ocular inserts PVP, Eudragit RS and RL [ 145 ]
Mitomycin C Ocular inserts Collagen implant [ 146 ]
Pilocarpine Ocular inserts Collagen shield [ 147 ]
PAA, PVP, HPMC [ 148 ]
PVA, glyceryl behenate, xanthan
gum, carrageenan, HPMC, HA;
coated with Eudragit RS and
RL
[ 149 ]
Timolol Ocular inserts HPC, coated with Eudragit RS
and RL
[ 150 ]
Tilisolol Ocular inserts Poly(hydroxypropyl-methacrylate) [ 151 ]
Poly(2 - hydroxypropyl -
methacrylate), polypropylene
tape
[ 152 ]
Ciprofl oxacin Ocular inserts Sodium alginate, Eudragit,
polyvinyl acetate
[ 153 ]
Pradofl oxacin Ocular inserts Hydrogel coating on thin metallic
wire (OpthaCoil)
[ 154 ]
TABLE 1 Continued
Drug
Formulation
Approach Polymers/Bases References
Fluorescein Ocular inserts HPMC lyophilisate on carrier
strip
[ 155 ]
HPMC lyophilisate on poly(tetra
fl uoroethylene) carrier strip
[ 156 ]
Carbopol 974P, maize starch [ 157, 158 ]
Gentamicin Ocular inserts CAP, carbomer, HPMC, HPC, EC [ 159, 160 ]
Oxytetracycline Ocular inserts Silicone, PAA, PMA [ 161 ]
Oxytetracycline,
prednisolone,
gentamicin
Ocular inserts PEO 400, PEO 900 [ 162 ]
Ofl oxacin Ocular inserts PEO 200, 400, 900, and 2000 [ 163 ]
PEO 400, Eudragit L100 [ 164 ]
PEO 900, PEO 2000, chitosan -
thiolated PAA
[ 165 ]
Fluorescein,
diclofenac
Ocular inserts [ 166 ]
Gentamicin Iontophoresis Hydroxyethyl methacrylate [ 167 ]
Pilocarpine,
tropicamide
Dendrimers Various poly(amidoamine)
(PAMAM) dendrimers
[ 168 ]
TABLE 1 Continued
case for most of the steroids. It is assumed that the drug particles remain in the
conjunctival sac, thus promoting a sustained release effect [171] . There have been
many studies trying to prove this assumption, but none of them has revealed a pronounced
prolonged release profi le [35, 172, 173] .
According to Davies [174] , topical ophthalmic suspensions have a number of
limitations compared to solutions. They need to be adequately shaken before use
to ensure correct dosing, a process which can result in poor patient compliance. In
addition, they need to be sterilized, which may cause physical instability of the formulation.
Furthermore, the amount of drug required to achieve only a moderate
increase in bioavailability is very high, rendering suspensions expensive in terms of
their production costs [175] .
The drug particle size plays the most important role in the formulation process
of suspensions. Particles greater than 10 . m cause patient discomfort. As they are
perceived as foreign substances, they cause refl ex tearing in order to eliminate the
particles from the ocular surface [176] . A study by Schoenwald and Stewart [177]
showed the infl uence of the particle size of dexamethasone on its bioavailability.
The in vivo dissolution rate decreased with increasing particle size to the point when
particles were removed from the conjunctival sac before the dissolution was
complete.
As a result, achieving a near - solution state with small particles that are easy to
resuspend and show minimal sedimentation remains the goal when formulation of
a suspension is unavoidable [176] .
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 741
742 OCULAR DRUG DELIVERY
TABLE 2 Marketed Ophthalmic Delivery Systems Based on Recent Formulation
Approaches
Formulation
Approach Polymer/Base Product Company
Suspensions/
microparticulates
Carbomer ion exchange
resin
Betoptic S Alcon
Ointments Wool fat, paraffi n Polyvisc Alcon
Liquid paraffi n, white soft
paraffi n
LacriLube Allergan
Viscosity enhancers/
mucoadhesives
Polyethylene glycol (PEG),
propylene glycol (PG),
HP-guar
Systane Alcon
Dextran, HPMC Bion Tears Alcon
Carboxymethylcellulose
sodium (CMC-Na)
Refresh Celluvisc Allergan
Refresh Liquigel Allergan
Polyvinyl alcohol (PVA) Liquifi lm Tears Allergan
HPMC Lacrigel Sunways
Carbomer Viscotears Novartis
Carbomer, polyvinyl
alcohol (PVA)
Nyogel Novartis
Hyaluronic acid (HA) Hy - Drop Bausch & Lomb
Fidia Oftal
Sodium hyaluronate Vismed TRB
Chemedica
Carbomer Pilopine HS Alcon
Polyacrylic acid (PAA) Fucithalmic Leo Pharma
In situ gelling
systems
Gellan gum Timoptic XE Merck
Polycarbophil DuraSite InSite Vision
Polyacrylic acid, poloxamer Smart Hydrogel Advanced
Medical
Solutions
Prodrugs Dipivefrin hydrochloride
(epinephrin prodrug)
Propine Allergan
Ocular inserts Alginic acid Ocusert Alza
Hydroxylpropyl cellulose Lacrisert Merck
Silicone elastomer Ocufi t SR Escalon Medical
Collagen shield MediLens Chiron
ProShield Alcon
Ointments Ointments generally consist of a dissolved or dispersed drug in an
appropriate vehicle base. They are the most commonly used semisolid preparations
as they are well tolerated and fairly safe and increase the ocular bioavailability of
the drug. The instilled ointment breaks up into small oily droplets that remain in
the cul - de - sac as a drug depot. The drug eventually gets to the ointment – tear interface
due to the shearing action of the eyelids [178] .
Sieg and Robinson [35] compared the bioavailability of fl uorometholone in a
solution, a suspension, and an ointment. They found that the peak concentration
(cmax ) of the drug in the aqueous humor of rabbits was comparable with all three
formulations, whereas the time to peak concentration ( tmax ) occurred much later
with the ointment, leading to a signifi cantly greater total bioavailability of the
drug.
Overall, ophthalmic ointments offer the following advantages: reduced dilution
of the medication via the tear fi lm, resistance to nasolacrimal drainage, and an
increased precorneal contact time [179, 180] . However, oily viscous preparations for
ophthalmic use (such as ointments) can cause blurred vision and matting of the
eyelids and may also be associated with discomfort by the patient as well as occasional
ocular mucosal irritation. Ointments are therefore generally used in combination
with eye drops, which can be administered during the day, while the ointment
is applied at night, when clear vision is not required.
5.9.3.2 Polymeric Delivery Systems
Polymeric systems used for ocular drug delivery can be divided into three groups:
viscosity - enhancing polymers, which simply increase the formulation viscosity,
resulting in decreased lacrimal drainage and enhanced bioavailability; mucoadhesive
polymers, which interact with the ocular mucin, therefore increasing the contact
time with the ocular tissues; and in situ gelling polymers, which undergo sol - to - gel
phase transition upon exposure to the physiological conditions present in the eye.
However, there are no defi ned boundaries between the different groups and most
polymers exhibit more than one of these properties.
Viscosity -Enhancing Polymers In order to reduce the lacrimal clearance (drainage)
of ophthalmic solutions, various polymers have been added to increase the
viscosity of conventional eye drops, prolong precorneal contact time, and subsequently
improve ocular bioavailability of the drug [40, 51, 181 – 184] . Among the
range of hydrophilic polymers investigated in the area of ocular drug delivery are
polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), cellulose derivates such
as methylcellulose (MC), and polyacrylic acids (carbopols).
Chrai and Robinson [40] evaluated the use of an MC solution of pilocarpine in
albino rabbits and found a decrease in the drainage rate with increasing viscosity.
Patton and Robinson [185] investigated the relationship between the viscosity and
the contact time or drainage rate and demonstrated an optimum viscosity of 12 –
15 cps for an MC solution in rabbits. The infl uence of different polymers on the
activity of pilocarpine in rabbits and human was reported by Saettone et al. [182] .
Trueblood et al. [183] used lacrimal microscintigraphy to evaluate the corneal
contact time for saline, PVA, and hydroxpropyl methylcellulose (HPMC) and
observed the longest contact time for the formulation with HPMC as a viscosity -
enhancing agent.
The ocular shear rate ranges from 0.03 s . 1 during interblinking periods to 4250 –
28,500 s . 1 during blinking [186] . It has a great infl uence on the rheological properties
of viscous ocular dosage forms and consequently the bioavailability of the incorporated
drug [187] . Newtonian systems do not show any real improvement of bioavailability
below a certain viscosity and blinking becomes painful, followed by refl ex
tearing, if the viscosity is too high [188] . While the viscosity of Newtonian systems
is independent from the shear rate, non - Newtonian pseudoplastic or so - called
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 743
744 OCULAR DRUG DELIVERY
shear - thinning systems exhibit a decrease in viscosity with increasing shear rates.
This pseudoplastic behavior is favorable for ocular drug delivery systems as it offers
less resistance to blinking and therefore shows greater acceptance by patients than
Newtonian systems of the same viscosity [189] .
Mucoadhesive Polymers Bioadhesion refers to the attachment of a drug molecule
or a delivery system to a specifi c biological tissue by means of interfacial forces. If
the surface of the tissue is covered by a mucin fi lm, as is the case for the external
globe, it is more commonly referred to as mucoadhesion.
In order to be an effective mucoadhesive excipient, polymers must show one or
more of the following features [190] : strong hydrogen binding group, strong anionic
charge, high molecular weight, suffi cient chain fl exibility, surface energy properties
favoring spreading onto the mucus, and near - zero contact angle to allow maximum
contact with the mucin coat.
The most commonly used bioadhesives are macromolecular hydrocolloids with
numerous hydrophilic functional groups capable of forming hydrogen bonds (such
as carboxyl, hydroxyl, amide, and sulfate groups) [191] . Hui and Robinson [49] were
the fi rst to demonstrate the usefulness of bioadhesive polymers in improving the
ocular bioavailability of progesterone. Saettone et al. [53] evaluated a series of
bioadhesive dosage forms for ocular delivery of pilocarpine and tropicamide and
found hyaluronic acid to be the most promising mucoadhesive polymer. Lehr et al.
[192] suggested that cationic polymers, which are able to interact with the negative
sialic acid residues of the mucin, would probably show better mucoadhesive properties
than anionic or neutral polymers. They investigated the polycationic polymer
chitosan and demonstrated that the mucoadhesive performance of chitosan was
signifi cantly higher in neutral or slightly alkaline pH as it is present in the tear
fl uid.
However, according to Park and Robinson [193] , polyanions are better than
polycations in terms of binding and potential toxicity. In general, both anionic and
cationic charged polymers demonstrate better mucoadhesive properties than nonionic
polymer, such as cellulose derivates or PVA [194, 195] .
The mechanism of mucoadhesion involves a series of different steps. First, the
mucoadhesive formulation needs to establish an intimate contact with the corneal
surface. Prerequisites are either good wetting or swelling of the mucoadhesive
polymer as well as suffi cient spreading across the cornea. The second stage involves
the penetration of the mucoadhesive polymer chains into the crevices of the tissue
surface and also the entanglement with the mucous chains [196] . On a molecular
level, mucoadhesion is a results of van - der - Waals forces, electrostatic attractions,
hydrogen bonding, and hydrophobic interactions [36] .
Mucoadhesive polymers increase the contact time of a formulation with the tear
fi lm and simulate the continuous delivery of tears due to a high water - restraining
capacity. As such, they allow a decrease in the instillation frequency compared to
common eye drops and are therefore useful as artifi cial tear products [1, 197, 198] .
In Situ Gelling Systems In situ gelling systems are viscous polymer - based liquids
that exhibit sol - to - gel phase transition on the ocular surface due to change in a
specifi c physicochemical parameter (ionic strength, temperature, pH, or solvent
exchange). They are highly advantageous over preformed gels as they can easily be
instilled in liquid form but are capable of prolonging the residence time of the formulation
on the surface of the eye due to gelling [199] .
The principal advantage of in situ gelling systems is the easy, accurate, and reproducible
administration of a dose compared to the application of preformed gels
[198] .
The concept of forming gels in situ (e.g., in the cul - de - sac of the eye) was fi rst
suggested in the early 1980s, and ever since then various triggers of in situ gelling
have been further investigated.
Polymers that may undergo sol - to - gel transition triggered by a change in pH are
cellulose acetate phthalate (CAP) and cross - linked polyacrylic acid derivates such
as carbopols, methacrylates and polycarbophils. CAP latex is a free - running solution
at pH 4.4 which undergoes sol - to - gel transition when the pH is raised to that of the
tear fl uid. This is due to neutralization of the acid groups contained in the polymer
chains, which leads to a massive swelling of the particles. The use of pH - sensitive
latex nanoparticles has been described by Gurny et al. [100, 200] . Carbopols have
apparent p Ka values in the range of 4 – 5 resulting in rapid gelation due to rise in pH
after ocular administration.
Gellan gum is an anionic polysaccharide which undergoes phase transition under
the infl uence of an increased ionic strength. In fact, the gel strength increases proportionally
with the amount of mono - or divalent cations present in the tear fl uid.
As a consequence, the usual refl ex tearing, which leads to a dilution of common
viscous solutions, further enhances the viscosity of gellan gum formulations due to
the increased amount of tear fl uid and thus higher cation concentration [201] .
Several studies have been performed comparing Gelrite formulations (low acetyl
gellan gum) to conventional ophthalmic solutions of the same active compound.
Shedden et al. [76] compared the plasma concentrations of timolol following multiple
applications of Timoptic - XE and a timolol maleate solution. They found that a
once - daily application of the in situ gelling formulation was suffi cient to reduce the
intraocular pressure to levels comparable to a twice - daily application of the solution,
leading to better patient compliance as well as a reduction in systemic side
effects.
Poloxamers or pluronics are block copolymers consisting of poly(oxyethylene)
and poly(oxypropylene) units. They rapidly undergo thermal gelation when the
temperature is raised to that of the ocular surface (32 ° C), while they remain liquid
at refrigerator temperature. Poloxamers exhibit surface active properties, but even
if used in high concentrations (usually between 20 and 30%), Pluronic F127 was
found no more damaging to the cornea than a physiological saline solution [202] .
In order to reduce the total polymer concentration and achieve better gelling properties,
several poloxamer combinations have been tested. Wei et al. [91] used a
mixture of Pluronic F127 and F68 resulting in a more suitable phase transition
temperature with a free - fl owing liquid under 25 ° C.
Combining thermal - with pH - dependent gelation, Kumar et al. [86] developed
a combination of methylcellulose 15% and carbopol 0.3%. This composition exhibited
a sol - to - gel transition between 25 and 37 ° C with a pH increase from 4 to 7.4
[203] . A possible mechanism for the thermal effect could be the decrease in the
degree of the methylcellulose hydration, while the polyacrylic acid can transform
into a gel upon an increase in pH due to the buffering properties of the tear
fl uid [1] .
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 745
746 OCULAR DRUG DELIVERY
5.9.3.3 Colloidal Delivery Systems
Colloidal carriers have been investigated as drug delivery systems for the past 30
years in order to achieve specifi c drug targeting, facilitate the bioavailability of drugs
through biological membranes, and protect the drug against enzymatic degradation.
Their use in topical administration and especially in ocular delivery however has
only been studied for the last two decades [1, 3] .
Colloidal carriers are small particulate systems ranging in size from 100 to 400 nm.
As they are usually suspended in an aqueous solution, they can easily be administered
as eye drops, thus avoiding the potential discomfort resulting from bigger
particles present in ocular suspensions or from viscous or sticky preparations [38] .
Most efforts in ophthalmic drug delivery have been made with the aim of increasing
the corneal penetration of the drug. Calvo et al. [204] have shown that colloidal
particles are preferably taken up by the corneal epithelium via endocytosis. It has
also been stated by Lallemand and co - workers [205] , that the cornea acts as a drug
reservoir, slowly releasing the active compound present in the colloidal delivery
system to the surrounding ocular tissues.
Nanoparticles Nanoparticles have been among the most widely studied particulate
delivery systems over the past three decades. They are defi ned as submicrometer
- sized polymeric colloidal particles ranging from 10 to 1000 nm in which the drug
can be dissolved, entrapped, encapsulated, or adsorbed [206] . Depending on the
preparation process, nanospheres or nanocapsules can be obtained. Nanospheres
have a matrixlike structure where the drug can either be fi rmly adsorbed at the
surface of the particle or be dispersed/dissolved in the matrix. Nanocapsules, on the
other hand, consist of a polymer shell and a core, where the drug can either be dissolved
in the inner core or be adsorbed onto the surface [207] .
The fi rst nanoparticulate delivery system studied was Piloplex, consisting of pilocarpine
ionically bound to poly(methyl)methacrylate – acrylic acid copolymer
nanoparticles [44] . Klein et al. [1, 98] found that a twice - daily application of Piloplex
in glaucoma patients was just as effective as three to six instillations of conventional
pilocarpine eye drops. However, the formulation was never accepted for
commercialization due to various formulation - related problems, including the nonbiodegradability,
local toxicity, and diffi culty of preparing a sterile formulation
[208] .
Another early attempt to formulate a nanoparticulate system for the delivery of
pilocarpine was made by Gurny [99] . This formulation was based on pilocarpine
dispersed in a hydrogen CAP pseudolatex formulation. Gurny and co - workers [101]
compared the formed nanoparticles to a 0.125% solution of hyaluronic acid some
years after their fi rst investigation and found that the viscous hyaluronic acid system
showed a signifi cantly longer retention time in front of the eye than the pseudolatex
formulation.
The most commonly used biodegradable polymers in the preparation of nanoparticulate
systems for ocular drug delivery are poly - alkylcyanoacrylates, poly - . -
caprolactone, and polylactic - co - glycolic acid copolymers. Marchal - Heussler et al.
[102, 103] compared the three particulate delivery systems using antiglaucoma drugs
including betaxolol and cartechol. Results showed that poly - . - caprolactone (nanospheres
and nanocapsules) exhibited the highest pharmacological activity when
loaded with betaxolol. It seemed that the higher ocular activity was related to the
hydrophobic nature of the carrier and that the mechanism of action seemed to be
directly linked to the agglomeration of the particles in the conjunctival sac [1] . In
general, nanocapsules displayed a much better effect than nanospheres probably
due to the fact that the active compound was in its un - ionized form in the oily core
and could diffuse faster into the cornea. Diffusion of the drug from the oily core of
the nanocapsule to the corneal epithelium seems to be more effective than diffusion
from the internal, more hydrophilic matrix of the nanospheres [1, 209] .
In order to achieve a sustained drug release and a prolonged therapeutic activity,
nanoparticles must be retained in the cul - de - sac and the entrapped drug must be
released from the particles at a certain rate. If the release is too fast, there is no
sustained release effect. If it is too slow, the concentration of the drug in the tears
might be too low to achieve penetration into the ocular tissues [208] . The major
limiting issues for the development of nanoparticles include the control of particle
size and drug release rate as well as the formulation stability.
So far, there is only one microparticulate ocular delivery system on the market.
Betoptic S is obtained by binding of betaxolol to ion exchange resin particles. Betoptic
S 0.25% was found to be bioequivalent to the Betoptic 0.5% solution in lowering
the intraocular pressure [208] .
Liposomes Liposomes were fi rst reported by Bangham in the 1960s and have been
investigated as drug delivery systems for various routes ever since then. They offer
some promising features for ophthalmic drug delivery as they can be administered
as eye drops but will localize and maintain the pharmacological activity of the drug
at its site of action [1] . Due to the nature of the lipids used, conventional liposomes
are completely biodegradable, biocompatible, and relatively nontoxic [1] .
A liposome or so - called vesicle consists of one or more concentric spheres of
lipid bilayers separated by water compartments with a diameter ranging from 80 nm
to 100 . m. Owing to their amphiphilic nature, liposomes can accommodate both
lipohilic (in the lipid bilayer) and hydrophilic (encapsulated in the central aqueous
compartment) drugs [207] . According to their size, liposomes are classifi ed as either
small unilamellar vesicles (SUVs) (10 – 100 nm) or large unilamellar vesicles (LUVs)
(100 – 300 nm). If more than one bilayer is present, they are referred to as multilamellar
vesicles (MLVs). Depending on their lipid composition, they can have a positive,
negative, or neutral surface charge.
Liposomes are potentially valuable as ocular drug delivery systems due to their
simplicity of preparation and versatility in physical characteristics. However, their
use is limited by instability (due to hydrolysis of the phospholipids), limited drug -
loading capacity, technical diffi culties in obtaining sterile preparations, and blurred
vision due to their size and opacity [42] .
In addition, liposomes are subject to the same rapid precorneal clearance as
conventional ocular solutions, especially the ones with a negative or no surface
charge [127] . Positively charged liposomes, on the other hand, were reported to
exhibit a prolonged precorneal retention due to electrostatic interactions with the
negative sialic acid residues of the mucin layer [2, 127, 208, 210 – 213] .
There have been several attempts to use liposomes in combination with other
newer formulation approaches, such as incorporating them into mucoadhesive gels
or coating them with mucoadhesive polymers [210] . Mucoadhesive polymers inves-
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 747
748 OCULAR DRUG DELIVERY
tigated in this regard were poly(acrylic acid) (PAA), hyaluronic acid (HA), chitosan,
and poloxamer [36, 43, 121 – 122, 214] .
Durrani and co - workers [122] reported on the infl uence of a carbopol coating on
the corneal retention of pilocarpine - loaded liposomes, and demonstrated a biphasic
response with an initial low intensity followed by a sustained reaction.
Bochot et al. [124, 125] developed a novel delivery system for oligonucleotides
by incorporating them into liposomes and then dispersing them into a thermosensitive
gel composed of poloxamer 407. They compared the in vitro release of the
model oligonucleotides pdT16 from simple poloxamer gels (20 and 27% poloxamer)
with the ones where pdT16 was encapsulated into liposomes and then dispersed
within the gels. They found that the release of the oligonucleotides from the gels
was controlled by the poloxamer dissolution, whereas the dispersion of liposomes
within 27% poloxamer gel was shown to slow down the diffusion of pdT16 out of
the gel.
Niosomes In order to circumvent some of the limitations encountered with
liposomes, such as their chemical instability, the cost and purity of the natural
phospholipids, and oxidative degradation of the phospholipids, niosomes have been
developed. Niosomes are nonionic surfactant vesicles which exhibit the same bilayered
structures as liposomes. Their advantages over liposomes include improved
chemical stability and low production costs. Moreover, niosomes are biocompatible,
biodegradable, and nonimmunogenic [215] . They were also shown to increase the
ocular bioavailability of hydrophilic drugs signifi cantly more than liposomes. This is
due to the fact that the surfactants in the niosomes act as penetrations enhancers
and remove the mucous layer from the ocular surface [209] .
A modifi ed version of niosomes are the so - called discomes, which vary from the
conventional niosomes in size and shape. The larger size of the vesicles (12 – 60 . m)
prevents their drainage into the nasolacrimal drainage system. Furthermore, their
disclike shape provides them with a better fi t in the cul - de - sac of the eye [26] .
Vyas et al. [216] demonstrated that discomes entrapped higher amounts of timolol
maleate than niosomes and that both niosomes and discomes signifi cantly increased
the bioavailability of timolol maleate when compared to a conventional timolol
maleate solution.
Microemulsions Microemulsions (MEs) are colloidal dispersions composed of an
oil phase, an aqueous phase, and one or more surfactants. They are optically isotropic
and thermodynamically stable and appear as transparent liquids as the droplet
size of the dispersed phase is less than 150 nm. One of their main advantages is their
ability to increase the solubilization of lipophilic and hydrophilic drugs accompanied
by a decrease in systemic absorption [217] . Moreover, MEs are transparent
systems thus enable monitoring of phase separation and/or precipitation. In addition,
MEs possess low surface tension and therefore exhibit good wetting and
spreading properties.
While the presence of surfactants is advantageous due to an increase in cellular
membrane permeability, which facilitates drug absorption and bioavailability [218] ,
caution needs to be taken in relation to the amount of surfactant incorporated, as
high concentrations can lead to ocular toxicity. In general, nonionic surfactants are
preferred over ionic ones, which are generally too toxic to be used in ophthalmic
formulations. Surfactants most frequently utilized for the preparation of MEs are
poloxamers, polysorbates, and polyethylene glycol derivatives [219] .
Similar to all other colloidal delivery systems discussed above it was hypothesized
by numerous research teams that a positive charge (provided by cationic surfactants
[220] ) would increase the ocular residence time of the formulation due to electrostatic
interactions with the negatively charged mucin residues. However, toxicological
studies contradicted this assumption regarding the ocular effects, and so far there
has been no publication demonstrating a distinct benefi cial effect of charged surfactants
incorporated into MEs.
Microemulsions can be classifi ed into three different types depending on their
microstructure: oil - in - water (o/w ME), water - in - oil (w/o ME), and bicontinuous ME.
They have been investigated by physical chemists since the 1940s but have only
gained attention as potential ocular drug delivery carriers within the last two
decades.
Gasco and co - workers [221] investigated the potential application of o/w lecithin
MEs for ocular administration of timolol, in which the drug was present as an ion
pair with octanoate. The ocular bioavailability of the timolol ion pair incorporated
into the ME was compared to that of an ion pair solution as well as a simple timolol
solution. Areas under the curve for the ME and the ion pair solution respectively
were 3.5 and 4.2 times higher than that of the simple timolol solution. A prolonged
absorption was achieved using the ME with detectable amounts of the drug still
present 120 min after instillation.
Various lecithin - based MEs were also characterized by Hasse and Keipert [131] .
The formulations were tested in terms of their physicochemical parameters (pH,
refractive index, osmolality, viscosity, and surface tension) and physiological compatibility
(HET - CAM and Draize test). In addition, in vitro and in vivo evaluations
were performed. The tested MEs showed favorable physicochemical parameters
and no ocular irritation as well as a prolonged pilocarpine release in vitro and in
vivo.
Muchtar and co - workers [129] prepared MEs with poloxamer 188 and
soybean lecithin to deliver indometacin to the ocular tissues. They found a threefold
increased indomethacin concentration in the cornea and aqueous humor 1 h
post - instillation.
Beilin et al. [222] demonstrated a prolonged ocular retention of a submicrometer
emulsion (SME) in the conjunctival sac using a fl uorescent marker (0.01% calcein)
as well as the miotic response of New Zealand Albino rabbits to pilocarpine. They
found that the fl uorescence intensity of calcein in SME was signifi cantly higher than
that of a calcein solution at all time points. Moreover, the pilocarpine SME exhibited
a longer duration of miosis than the simple pilocarpine solution. It should be mentioned
that SMEs are true emulsions, being different from MEs. They do not form
spontaneously and are kinetically rather than thermodynamically stable. They generally
require high - shear homogenization to form and are more susceptible to phase
inversion. Furthermore, they are neither transparent nor translucent but rather
turbid due their larger droplet size compared to MEs. While the two terms are used
interchangeably in the the scientifi c literature, they actually refer to two distinct
categories of dispersed systems and should be differentiated from each other.
The w/o MEs composed of water, Crodamol EO, Crill 1, and Crillet 4 were investigated
as potential ocular delivery systems by Alany et al. [133] . It was hypothesized
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 749
750 OCULAR DRUG DELIVERY
that w/o MEs undergo phase transition into lamellar liquid crystals (LCs) upon
aqueous dilution by the tears, prolonging the precorneal retention time due to an
increase in the formulation ’ s viscosity. HET - CAM studies revealed no ocular irritancy
by the excipients used. Ocular drainage was evaluated via . - scintigraphy and
demonstrated a signifi cantly higher precorneal retention of the tested microemulsions
compared to an aqueous solution.
The use of MEs in ocular delivery is very attractive due to all the advantages
offered by these formulations. They are thermodynamically stable and transparent,
possess low viscosity, and thus are easy to instill, formulate, and sterilize (via fi ltration).
Moreover, they offer the possibility of reservoir and/or localizer effects. All
these factors, in addition to the ones previously mentioned, render MEs promising
ocular delivery systems.
5.9.3.4 Other Delivery Approaches
Many other ocular delivery approaches have been investigated over the past decades,
including the use of prodrugs, penetration enhancers, cyclodextrins, as well as different
types of ocular inserts. In addition, iontophoresis, which is an active drug
delivery approach utilizing electrical current of only 1 – 2 mA to transport ionized
drugs across the cornea, offers an effective, noninvasive method for ocular delivery.
Another more recent approach is the use of dendrimers in ocular therapy. Dendrimers
are synthetic spherical molecules named after their characteristic treelike
branching around a central core with a size ranging from 2 to 10 nm in diameter
[223] . So far, PAMAM (polyamidoamine) has been the most commonly studied
dendrimer system for ocular use [224, 225] .
Prodrugs Prodrugs are pharmacologically inactive derivates of drug molecules
that require a chemical or enzymatic transformation into their active parent drug
[226] . To be effective, an ocular prodrug should show an appropriate lipophilicity to
facilitate corneal absorption, posses suffi cient aqueous solubility and stability to be
formulated as an eye drop, and demonstrate the ability to be converted to the active
parent drug at a rate that meets therapeutic needs [227] .
When considering ophthalmic drug molecules as prodrug candidates, the following
factors need to be considered: the pathway and mechanism of ocular drug
penetration, the functional groups of the drug candidate amenable to prodrug
derivatization, and the enzymes present in the ocular tissues, which are necessary
for prodrug activation [28] .
The majority of ophthalmic prodrugs developed so far are chemically classifi ed
as ester. They are derived from the esterifi cation of the hydroxyl or carboxylic acid
groups present in the parent molecule. Of all enzymes participating in the activation
of prodrugs, esterases, which are present in all anterior segment tissues except the
tear fi lm, have received the most attention [228, 229] .
Prodrugs were introduced into the area of ocular drug delivery about 25 years
ago [230] , and steroids were probably the fi rst ones to be utilized as prodrugs.
However, the concept of prodrugs was not fully exploited until the introduction of
dipivefrin (epinephrine prodrug) in the late 1970s. Kaback and co - workers [134]
found that a 0.1% dipivalyl epinephrine solution lowered the intraocular pressure
as effective as a 2% epinephrine solution, while signifi cantly lowering the systemic
side effects. Wei et al. [135] compared the ocular penetration, distribution, and
metabolism of epinephrine and dipivalyl epinephrine and found the partition coef-
fi cient of the later to be 100 – 600 times higher than that of epinephrine, therefore
leading to a 10 - times faster absorption into the rabbit eye.
Dipivefrin was the only commercially available ophthalmic prodrug at that time.
However, numerous prodrug derivates have been designed to improve the effi cacy
of ophthalmic drugs ever since.
Jarvinen and co - workers [136] synthesized unique O, O. - (xylylene)bispilocarpic
acid esters containing two pilocarpic acid monoesters linked with one moiety. The
found that prodrug showed a two - to seven - fold higher corneal permeability than
pilocarpine itself despite the high molecular weight.
Tirucherai et al. [137] formulated an acyl ester prodrug of ganciclovir. The
increased permeability was associated with a linear increased susceptibility of the
ganciclovir esters to the esterases present in the cornea.
So far, aims that have been achieved by using prodrugs include the modifi cation
of the drug ’ s duration of action, reduction of the systemic absorption, and reduction
of ocular and systemic side effects. Although prodrugs are commonly used to treat
diseases of the anterior segment, there have also been attempts to treat conditions
associated with the posterior segment of the eye.
Penetration Enhancers The transport process across the corneal tissue is the rate -
limiting step in ocular drug absorption. Increasing the permeability of the corneal
epithelium by penetration enhancers is likely to enhance the drug transport across
the corneal tissues and therefore improve ocular bioavailability of the drug.
Penetration enhancers act by increasing the permeability of the corneal cell
membrane and/or loosening the tight junctions between the epithelial cells, which
primarily restrict the entry of molecules via the paracellular pathway. Classes of
penetration enhancers include surfactants, bile salts, calcium chelators, preservatives,
fatty acids, and some glycosides such a saponin.
Surfactants are perceived to enhance drug absorption by disturbing the integrity
of the plasma membranes. When present at low concentrations, surfactants are
incorporated into the lipid bilayer, leading to polar defects in the membrane, which
change the membrane ’ s physical properties. When the lipid bilayer is saturated,
micelles start to form, enclosing phospholipids from the membranes, hence leading
to membrane solubilization [36] . Saettone et al. [138] found an increased corneal
permeability for atenolol, timolol, and betaxolol by including 0.05% Brij 35, Brij 78,
and Brij 98 into their formulations.
Bile salts are amphiphilic molecules that are surface active and self - associate to
form micelles in aqueous solution. They increase corneal permeability by changing
the rheological properties of the bilayer [231] . A number of bile salts such as deoxycholate,
taurodeoxycholate, and glycocholate have been tested so far, and it was
suggested, that a difference in their physicochemical properties (solubilizing activity,
lipophilicity, Ca 2+ sequestration capacity) is probably related to their performance
as permeability - enhancing agents [36] .
Another class of penetration enhancers includes calcium chelators such as ethylenediaminetetraacetic
acid (EDTA). These molecules induce Ca 2+ depletion in the
cells. This leads to a global change within the cell and as a result loosens the tight
junctions between superfi cial epithelial cells, thus facilitating paracellular transport
FORMULATION APPROACHES TO IMPROVE OCULAR BIOAVAILABILITY 751
752 OCULAR DRUG DELIVERY
[138, 232] . Grass et al. [33] were among the fi rst to emphasize the enhancing effects
of chelating agents for ocular drug delivery. They found that 0.5% EDTA doubled
the corneal absorption of topically applied glycerol and cromoclycin sodium.
Large numbers of penetration enhancers have been investigated to date. However,
the unique structure of the corneal/conjunctival tissues requires caution. When
selecting penetration enhancers for ocular delivery, their capacity to affect the
integrity of the epithelial surfaces needs to be considered. Studies have shown that
penetration enhancers themselves can penetrate ocular tissues, which could lead to
potential toxicity. EDTA concentrations in the iris – ciliary body, for example, were
found to be high enough to alter the permeability of the blood vessels in the uveal
tract, therefore indirectly accelerating the drug ’ s removal from the aqueous humor
[233] . Similarly, benzalkonium chloride (BAC), a cationic surfactant which shows
the highest penetration - enhancing effect among the currently used preservatives,
was found to accumulate in the cornea for several days.
Cyclodextrins Cyclodextrins were introduced into the area of ocular drug delivery
in the early 1990s. They are a group of homologous cyclic oligosaccharides with a
hydrophilic outer surface and a lipophilic cavity in the center. Their initial aim
was to increase the solubility of lipophilic drugs by forming inclusion complexes.
Cyclodextrin complexation generally results in improved wettability, dissolution,
solubility, and stability in solution as well as reduced side effects [234] .
It is assumed that cyclodextrins are too large and hydrophilic to penetrate biological
membranes. However, they act as penetration enhancers by assuring a high
drug concentration at the corneal surface, from where the drug then partitions into
the ocular tissues [235] .
Even though cyclodextrins drug complexes seem to decrease ocular toxicity of
irritant drugs, cyclodextrins themselves may exhibit ocular toxicity and should therefore
be screened by performing corneal sensitivity studies. Among all cyclodextrin
derivates investigated, hydroxy - propyl - . - cyclodextrin showed the most favorable
properties in terms of toxicity [1] .
Nijhawan and Agarwal [140] investigated inclusion complexes of ciprofl oxacin
hydrochloride and hydroxy - propyl - . - cyclodextrin and found that the complexes
exhibited better stability, biological activity, and ocular tolerance than the uncomplexed
drug in solution.
Aktas et al. [141] showed an increased permeation of pilocarpine nitrate complexed
with hydroxy - propyl - . - cyclodextrin using isolated rabbit cornea. They found
a signifi cant reduction in the pupil diameter compared to a simple aqueous solution
of the same active compound.
Cyclodextrins improve chemical stability, increase the drug ’ s bioavailability, and
decrease local irritation. However, the improvement of ocular bioavailability seems
to be limited by the very slow dissociation of the complexes in the precorneal tear
fl uid.
Several studies have shown that combinations of cyclodextrins drug complexes
and viscosity enhancers can signifi cantly improved ocular absorption [141, 236 – 237]
and should therefore be further investigated.
Ocular Inserts Solid ocular dosage forms such as fi lms, erodible and nonerodible
inserts, rods, and shields have been developed to overcome the typical pulse - entry -
type drug release associated with conventional ocular dosage forms. This pulse entry
is characterized by a transient overdose, a relatively short period of appropriate
dosing, followed by a prolonged period of underdosing. Ocular inserts were developed
in order to overcome these disadvantages by providing a more controlled,
sustained, and continuous drug delivery by maintaining an effective drug concentration
in the target tissues and yet minimizing the number of applications [238] .
Ocular inserts probably represent one of the oldest ophthalmic formulation
approaches. In 1948 the British Pharmacopoeia described an atropine - in - gelatin
wafer and ever since then numerous systems have been developed applying various
polymers and different release principals. However, the diffi culty of insertion by the
patient, foreign - body sensation, and inadvertent loss of inserts from the eye make
these systems less popular, especially among the elderly. Furthermore, the high cost
involved in manufacture prevented the insert market from taking off [197] .
Two products, Alza Ocusert and Merck Lacrisert, have been marketed, although
Ocusert is no longer available. Ocusert is a membrane - controlled reservoir system
for the treatment of glaucoma. It contains pilocarpine and alginic acid in the core
reservoir, sandwiched between two transparent, lipophilic ethylenevinyl acetate
(EVA) rate - controlling membranes, which allow the drug to diffuse from the reservoir
at a precisely determined rate for a period of seven days. This system is nonbiodegradable
and must therefore be removed after use. Lacrisert, on the other
hand, is a soluble minirod of hydroxypropylmethyl cellulose without any active
ingredient. The system is placed in the conjunctival sac, where it softens within an
hour and completely dissolves within 14 – 18 h. Lacrisert stabilizes and thickens the
precorneal tear fi lm and prolongs the tear fi lm break - up time, which is usually
accelerated in patients with dry - eye syndrome (keratoconjunctivitis sicca) [239] .
A number of ocular inserts using different techniques, namely soluble, erodible,
nonerodible, and hydrogel inserts with polymers such as cellulose derivates, acrylates,
and poly(ethylene oxide), have been investigated over the last few decades.
An example of a degradable matrix system is the pilocarpine - containing inserts
formulated by Saettone et al. [148] . Pilocarpine nitrate and polyacrylic acid were
incorporated into a matrix containing polyvinyl alcohol and two types of hydroxypropyl
methylcellulose. It was shown that all inserts signifi cantly increased the pharmacological
effect (miotic response) compared to a solution of pilocarpine nitrate.
Sasaki et al. [151] prepared nondegradable disc - type ophthalmic inserts of
. - blockers using different polymers. They found that inserts made from
poly(hydroxypropyl methacrylate) were able to control the release of tilisolol
hydrochloride.
Numerous studies have also been performed on soluble collagen shields. Collagen
shields are fabricated from porcine scleral tissue, which has a similar collagen
composition to that of the human cornea. Drug loading is typically achieved by
soaking the collagen shield in the drug solution prior to application. Designed to
slowly dissolve within 12, 24, or 72 h, collagen shields have attracted much interest
as potential sustained ocular drug delivery systems over the last years [240] .
5.9.4 CONCLUSION
Although conventional eye drops still represent about 90% of all marketed ophthalmic
dosage forms, there have been signifi cant efforts towards the development
of new drug delivery systems.
CONCLUSION 753
754 OCULAR DRUG DELIVERY
Only a few of these new ophthalmic drug delivery systems have been commercialized
over the past decades, but research in the different areas of ocular drug
delivery has provided important impetus and dynamism, with the promise of some
new and exciting developments in the fi eld.
An ideal ophthalmic delivery system should be able to achieve an effective drug
concentration at the target site for an extended period of time while minimizing
systemic side effects. In addition, the system should be comfortable and easy to use,
as the patient ’ s acceptance will continue to play an important role in the design of
future ocular formulations.
All delivery technologies mentioned in this chapter hold unlimited potential for
clinical ophthalmology. However, each of them still bears its own drawbacks. To
circumvent these, newer trends are directed toward combinations of the different
drug delivery approaches. Examples for this include the incorporation of particulates
into in situ gelling systems or coating of nanoparticles with mucoadhesive
polymers.
These combinations will open new directions for the improvement of ocular
bioavailability, but they will also increase the complexity of the formulations, thus
increasing the diffi culties in understanding the mechanism of action of the drug
delivery systems.
Many interesting delivery approaches have been investigated during the past
decades in order to optimize ocular bioavailability, but much remains to be learned
before the perfect ocular drug delivery system can be developed.
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tolerability of pilocarpine prodrug in rabbits , J. Pharm. Pharmacol. , 48 ( 3 ), 263 – 269 .
238. Sultana , Y. , Jain , R. , Aqil , M. , and Ali , A. ( 2006 ), Review of ocular drug delivery , Curr.
Drug Deliv. , 3 ( 2 ), 207 – 217 .
239. Ranade , V. V. , and Hollinger , M. A. ( 2003 ), Intranasal and ocular drug delivery. in Drug
Delivery Systems , 2nd ed., CRC Press , Boca Raton, FL , pp. 249 – 287 .
240. Lee , V. H. ( 1990 ), New directions in the optimization of ocular drug delivery , J. Ocul.
Pharmacol. , 6 ( 2 ), 157 – 164 .
769
5.10
MICROEMULSIONS AS DRUG
DELIVERY SYSTEMS
Raid G. Alany and Jingyuan Wen
The University of Auckland Auckland, New Zealand
Contents
5.10.1 Historical Background, Terminology, and Defi nition
5.10.2 Structure and Formation of Microemulsion Systems
5.10.3 Role of Cosurfactants/Cosolvents in Formation and Stabilization of Microemulsion
Systems
5.10.4 Pharmaceutical Formulation of Microemulsions
5.10.4.1 Selection of Microemulsion Ingredients
5.10.4.2 Phase Behavior Studies
5.10.5 Techniques Used to Characterize Microemulsions and Related Systems
5.10.5.1 Polarized Light Microscopy
5.10.5.2 Transmission Electron Microscopy
5.10.5.3 Electrical Conductivity Measurements
5.10.5.4 Viscosity Measurements
5.10.5.5 Other Characterization Techniques
5.10.6 Microemulsions as Drug Delivery Systems
5.10.6.1 Oral Drug Delivery
5.10.6.2 Transdermal Drug Delivery
5.10.6.3 Parenteral Drug Delivery
5.10.6.4 Ocular Drug Delivery
5.10.7 Concluding Remarks
References
5.10.1 HISTORICAL BACKGROUND, TERMINOLOGY,
AND DEFINITION
Hoar and Schulman coined the term microemulsion (ME) in 1943 to defi ne a transparent
system obtained by titrating a turbid oil - in - water (o/w) emulsion with a
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
770 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
medium - chain alcohol, namely hexanol. Since then the term has been used to
describe systems comprising a nonpolar component, an aqueous component, a surfactant,
and a cosurfactant. While a cosurfactant is usually present, a ME can be
formulated without a cosurfactant, that is, using a single surfactant. It is important
to point out that the term ME was (and occasionally is) used in the literature to
describe various liquid crystalline systems (lamellar, hexagonal, and cubic), surfactant
systems (micelles and reverse micelles), and even coarse emulsions that are
micronized using external energy (submicrometer emulsions). To avoid such confusion,
Danielsson and Lindmann [1] proposed the following defi nition: “ Microemulsion
is defi ned as a system of water, oil and amphiphile which is optically isotropic
and a thermodynamically stable liquid solution. ” By this defi nition the following
systems were excluded:
• Aqueous solutions of surfactants (micellar and nonmicellar) without additives
or with water soluble nonelectrolytes as additives
• Liquid crystalline phases (mesophases)
• Coarse emulsions, including micronized coarse emulsions
• Systems that are surfactant free
The term ME is often incorrectly used in the literature to describe oil and water
dispersions of small droplet size produced by prolonged ultrasound mixing, high -
shear homogenization, and microfl uidisation, that is, submicrometer emulsions. The
major differences between a microemulsion and a coarse emulsion are shown in
Table 1 .
5.10.2 STRUCTURE AND FORMATION OF
MICROEMULSION SYSTEMS
A ME system can be one of three types depending on the composition: oil in water
(o/w ME), in which water is the continuous medium; water in oil (w/o ME), in which
oil is the continuous medium, and water - and - oil bicontinuous ME, in which almost
equal amounts of water and oil exist [3] . While the three types are quite different
in terms of microstructure, they all have an interfacial amphiphile monolayer separating
the oil and water domains.
The formation of a ME system can be explained using a simplifi ed thermodynamic
approach and with reference to the equation
TABLE 1 Comparison of Emulsions and Microemulsions
Property Emulsion Microemulsion
Disperse - phase droplet size 0.2 – 10 . m Less than 0.2 . m
Visual appearance Turbid to milky Transparent to translucent
Stability Thermodynamically unstable Thermodynamically stable
Formation Requires energy input Spontaneous
Source : From ref. 2.
. . . G A T S = . . (1)
where . G - free energy of ME formation
. - interfacial tension at oil – water interface
. A - change in interfacial area (associated with reducing droplet size)
S - system entropy
T - absolute temperature
The process of ME formation is associated with a reduction in droplet size, which
results in an increase in the value of . A due to an overall increase in surface area
that is associated with droplet size reduction. This is compensated by a very low
interfacial tension that is normally achieved by using relatively high amphiphile
concentrations. Furthermore, the process of ME formation is accompanied by a
favorable entropy contribution (increased value of . S ) that is due to the mixing of
the two immiscible phases, surfactant molecules partitioning in favor of the interface
rather than the bulk and monomer – micelle surfactant exchange. The net outcome
is a negative value for . G which translates into a spontaneous ME formation [4] .
The simplest representation of the ME microstructure is with reference to the
droplet model in which an interfacial fi lm consisting of amphiphile (surfactant/
cosurfactant) molecules surrounds the dispersed droplets (Figures 1 a and b ). The
orientation of the amphiphile at the interface differs depending on the type of the
ME. Whether an o/w or w/o ME forms is dependent to a great extent on the volume
fraction of oil and water as well as the nature of the interfacial fi lm as refl ected
by the geometry of the amphiphile molecules forming the fi lm. It follows that the
FIGURE 1 Diagrammatic representation of different types of ME systems: ( a ) w/o ME;
( b ) o/w ME; ( c ) water - and - oil bicontinuous ME. Droplet diameter for ( a ) and ( b ) is typically
less than 140 nm.
(a) (b)
(c)
STRUCTURE AND FORMATION OF MICROEMULSION SYSTEMS 771
772 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
presence of o/w ME droplets is more likely to happen in systems where the oil
volume fraction is low, whereas w/o ME droplets form when the water volume fraction
is low and oil is present in abundance. Interestingly, in systems containing
comparable amounts of water and oil, a bicontinuous ME may exist (Figure 1 c ). In
such systems both oil and water exist as microdomains that are separated by an
amphiphile - stabilized interface with a zero net curvature.
Mitchell and Ninham [5] extended the theory of self - assembly of surfactant molecules
forming micelles and bilayers [6] to ME systems. Accordingly, if the volume
of the surfactant is v , its head group surface area a , and its length l , it follows that
when the critical packing parameter (CPP = v/al ) has values between 0 and 1, o/w
MEs are likely to be formed. On the other hand, when CPP is greater than 1, w/o
MEs are favored. When using surfactants with critical packing parameters close to
unity (CPP . 1) and at approximately equal volumes of water and oil, the mean
curvature of the interfacial fi lm approaches zero and droplets may merge into a
bicontinuous structure (Figure 1 c ). It should be noted, however, that this approach
is based solely on geometric considerations and does not account for penetration
of oil and cosurfactant molecules into the interface and the hydration of surfactant
head groups.
The ratio of the hydrophilic and the hydrophobic groups of the surfactant
molecules, that is, their hydrophile – lipophile balance (HLB), is also important in
determining interfacial fi lm curvature and consequently the structure of the ME.
The HLB system has been used for the selection of surfactants to formulate MEs
and accordingly the HLB of the candidate surfactant blend should match the
required HLB of the oily component for a particular system; furthermore a
match in the lipophilic part of the surfactant used with the oily component is favorable
[7] .
Shinoda and Kuineda [8] highlighted the effect of temperature on the phase
behavior of systems formulated with two surfactants and introduced the concept of
the phase inversion temperature (PIT) or the so - called HLB temperature. They
described the recommended formulation conditions to produce MEs with surfactant
concentration of about 5 – 10% w/w being (a) the optimum HLB or PIT of a surfactant;
(b) the optimum mixing ratio of surfactants, that is, the HLB or PIT of the
mixture; and (c) the optimum temperature for a given nonionic surfactant. They
concluded that (a) the closer the HLBs of the two surfactants, the larger the cosolubilization
of the two immiscible phases; (b) the larger the size of the solubilizer, the
more effi cient the solubilisation process; and (c) mixtures of ionic and nonionic
surfactants are more resistant to temperature changes than nonionic surfactants
alone.
5.10.3 ROLE OF COSURFACTANTS/COSOLVENTS IN FORMATION
AND STABILIZATION OF MICROEMULSIONS
Cosurfactants are molecules with weak amphiphilic properties that are mixed with
the surfactant(s) to enhance their ability to reduce the interfacial tension of a system
and promote the formation of a ME [3] . Cosolvents have also been described as
weak amphiphilic molecules that tend to distribute between the aqueous phase, the
oily phase, and the interfacial layer and act by making the aqueous phase less hydrophilic,
the oily phase less hydrophobic, and the interfacial fi lm more fl exible and less
condensed [9, 10] .
Most single - chain surfactants do not suffi ciently lower the oil – water interfacial
tension to form MEs, nor are they of the right molecular structure (i.e., HLB) to
act as cosolvents. To overcome such a barrier, cosurfactant/cosolvent molecules are
added to further lower the interfacial tension between oil and water, fl uidize the
hydrocarbon region of the interfacial fi lm, and infl uence the curvature of the fi lm.
Typically small molecules (C3 – C8) with a polar head (hydroxyl or amine) group
that can diffuse between the bulk oil and water phase and the interfacial fi lm are
suitable candidates [11] .
All the abovementioned mechanisms are expected to facilitate the formation and
stabilization of ME systems.
5.10.4 PHARMACEUTICAL FORMULATION OF MICROEMULSIONS
5.10.4.1 Selection of Microemulsion Ingredients
Pharmaceutically acceptable ME systems are formulated using at least GRAS -
(generally regarded as safe) and preferably pharmaceutical - grade ingredients, that
is, ones already in use in pharmaceutical formulation and devoid of serious side
effects and toxicity in humans [12] . Nonionic and zwitterionic surfactants are among
the most commonly used ingredients to formulate pharmaceutical MEs while vegetable
oils, medium - and long - chain triglycerides, and esters of fatty acids are the
most commonly used oils [2] . Among the range of nonionic surfactants used are
sucrose esters [13] , polyoxyethylene alkyl ethers [14] , polyglycerol fatty acid esters
[15] , polyoxyethylene hydrogenated castor oil [16] , and sorbitan esters [17] . Furthermore,
systems formulated with zwitterionic phospholipids, particularly lecithin, have
been widely investigated because of their biocompatible nature [9, 18 – 22] .
The effect of the oily component on the phase behavior of o/w ME - forming
systems formulated with nonionic surfactants was reported [23] . The authors showed
that it is possible to formulate cosurfactant - free o/w ME systems suitable for use as
drug delivery vehicles using either polyoxyethylene surfactants or amine - N - oxide
surfactants. The major advantage of these ME systems is their ability to be diluted
without destroying their integrity; however both classes of surfactants were shown
to be sensitive to electrolytes.
The choice of cosurfactants to formulate pharmaceutically acceptable MEs is
challenging as most of the cosurfactants investigated in fundamental ME research
cannot be used for the development of pharmaceutically acceptable systems due to
biocompatibility considerations. Among the pharmaceutically acceptable cosurfactants
are ethanol [24] , medium - chain mono - and diglycerides [25 – 28] , 1,2 - alkanediols
[29, 30] and sucrose – ethanol combinations [31] , alkyl monoglucosides, and
geraniol [32] . Kahlweit et al. [29, 30] reported on the usefulness of certain 1,2 -
alkanediol cosurfactants as nontoxic substitutes to the physiologically incompatible
short - and medium - chain alcohols. They suggested the possible use of these components
for the formulation of nontoxic ME systems.
PHARMACEUTICAL FORMULATION OF MICROEMULSIONS 773
774 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
5.10.4.2 Phase Behavior Studies
Before a ME can be used as a drug delivery vehicle, the phase behavior of the
particular combination of the candidate ingredients should be established. This is
necessary due to the diverse range of colloidal and coarse dispersions that could
be obtained when oil, water, and an amphiphile blend are mixed. Coarse emulsions,
vesicles, lyotropic liquid crystals, and micellar systems are some examples.
A variety of multiphase systems may coexist and the demarcations of the regional
boundaries become important. One of the most suitable methods to study the
phase behavior of such systems is to construct a ternary phase diagram using a
Gibbs triangle (Figure 2 ). A ternary phase diagram can be constructed by two
methods [33] :
• Titrating a mixture of two components with the third component
• Preparing a large number of samples of different composition
If all mixtures reach equilibrium rapidly, both methods give identical results. For
mixtures that do not reach equilibrium quickly, the second method is recommended,
as with the titration method the change in the ratio of components during titration
may occur too fast, not allowing suffi cient time to visually recognize phase changes
[33] .
As the formulation may contain more than three components, the complete
phase behavior cannot be fully represented using a triangular diagram. However,
FIGURE 2 Ternary phase diagram used to elucidate ME formation regions. Each of the
three corners represents 100% of the individual components. Apex S = 100% w/w surfactant
(0% oil and water), apex W = 100% w/w water (0% oil and surfactant), and apex O = 100% w/
w oil (0% water and surfactant). The three lines joining the corner points represent two -
component systems. The area within the triangle represents all possible combinations of the
three components.
W 0
70
10
20
50
60
40
30
80
90
100 0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100
S
O
Increasing
S
Increasing
W
Increasing O
the phase behavior of a four - component mixture at fi xed pressure and temperature
can be represented using a tetrahedron. Full characterization of such systems is a
tedious task requiring a large number of experiments [34] . One acceptable approach
for representing such systems is by fi xing the mass ratio of two components
(such as the two amphiphiles) and as such considered a single component. Such an
approach, although regarded by many as an oversimplifi cation of the systems, is yet
acceptable for the purpose of phase behavior studies. Such systems are described
as “ pseudoternary ” as they comprise more than three components (four or possibly
fi ve) yet are represented using a Gibbs triangle, which is a used to describe the phase
behavior of a three - component system.
A novel approach to reduce the experimental effort associated with constructing
pseudoternary phase diagrams is by using expert systems to predict the phase
behavior of multicomponent ME - forming systems. Artifi cial neural networks have
been investigated and were shown to be promising in phase behavior studies
[17, 35, 36] as well as in the process of ingredient selection [37] .
5.10.5 TECHNIQUES USED TO CHARACTERIZE MICROEMULSIONS
AND RELATED SYSTEMS
The physicochemical and analytical techniques used to characterize MEs and related
systems could be categorized into those used to:
• Elucidate the microstructure and monitor phase behavior changes
• Determine the droplet size of the disperse phase
The choice of a particular technique is limited by factors such as availability,
feasibility, and the nature of the information sought. Pharmaceutical scientists are
more focused on the usefulness of a particular ME system for a drug delivery application
and the infl uence of the microstructure on that, rather than on the fundamental
understanding of aspects such as microstructure and phase behavior.
Polarized light microscopy is a readily available technique that could be used at the
early formulation development stage to differentiate between isotropic and anisotropic
systems. Transmission electron microscopy (TEM) is another available
technique that has been shown to provide microstructural as well as size - related
information on droplet and bicontinuous ME systems.
The main disadvantages of TEM applications (such as freeze fracture and cryo -
TEM) are the lengthy and sophisticated experimental procedures associated with
sample preparation and the possibility of creating artefacts during sample preparation.
Other readily available and more user friendly techniques are electrical conductivity
and viscosity measurements. Electrical conductivity measurements are
widely used for their simplicity, feasibility, and sensitivity to structural changes in
systems with increasing water content, particularly systems undergoing percolation
transitions. Viscosity measurements, on the other hand, require more sophisticated
instrumentation yet provide useful information on changes in the fl ow properties
associated with structural changes of the systems. A brief overview of the key characterization
techniques will follow.
CHARACTERIZE MICROEMULSIONS AND RELATED SYSTEMS 775
776 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
5.10.5.1 Polarized Light Microscopy
When a mixture of oil, water, and surfactant(s) is examined under a polarized light
microscope, the textures observed depend on the nature of the surfactant aggregate
formed and the relative ratio of the comprising constituents. If the resulting aggregates
are anisotropic, they tend to show strong birefringence and characteristic
textures could be viewed when examined using a polarized light microscope. On the
other hand, if the resulting aggregates are isotropic (as with ME systems or coarse
emulsions), then polarized light microscopy would be of less value in disclosing
structural information. Thermotropic and lyotropic liquid crystalline (LC) systems
such as lamellar, hexagonal, and reverse hexagonal mesophases are anisotropic and
exhibit characteristic textures when viewed under a polarizing light microscope. The
only type of LC systems that is isotropic and would not display birefringence when
viewed under a cross - polarizer is the cubic mesophase.
Polarized light microscopy is a simple technique to learn and use, readily available,
and of great value to differentiate between various anisotropic LC systems. It
is also of value to formulation scientists investigating amphiphile – oil – water mixtures
with emphasis on colloidal systems in general and MEs in particular. This is
mainly due to the fact that many LC systems may appear transparent to the naked
eye and can be easily misinterpreted as isotropic ME systems. Thus it becomes
essential when investigating systems of amphiphile – oil – water to confi rm fi ndings
based on visual appearance with polarized light microscopic examination.
5.10.5.2 Transmission Electron Microscopy
Transmission electron microscopy was one of the earliest techniques used to investigate
MEs [38, 39] . Freeze fracture along with replication is a sophisticated sample
preparation method for TEM that requires careful attention to a variety of details
to avoid formation of sample artefacts. The technique involves rapidly freezing the
sample by immersing it in a cryogen (slush nitrogen, propane, ethane, and freons).
For systems with volatile ingredients the freezing must be achieved rapidly to avoid
phase separation or crystallization. Thus, high cooling rate and adequate environmental
control of the samples before freezing are critical to prevent loss of volatile
components. The frozen sample is then transferred to a vacuum chamber and split
under vacuum by a fracturing device. The fractured surface is then shadowed with
metal (usually platinum) deposited from one side. The shadowed surface is then
coated with a layer of carbon that is directly deposited from above the specimen.
The carbon layer is transparent when examined and forms a supportive backing for
the shadowing metal deposited on the fractured surface. The specimen is then
removed from the vacuum and treated with solvents of different polarities, leaving
the metal carbon fi lm as a replica of the fracture surface.
Although freeze - fracture TEM provides direct visualization of ME structures, it
is not currently in wide use probably due to the experimental diffi culties associated
with the technique. The points to consider when preparing conventional TEM replicas
are the physical and chemical sample properties, freezing, cleaving, etching,
replication, cleaning, and mounting steps of the procedure.
Jahn and Strey [40] investigated systems with varying water - to - oil ratios at constant
amphiphile concentration. The TEM images support the notion of a bicontinu
ous network for systems containing comparable amounts of the aqueous and oil
components. The author also showed images of w/o droplet ME systems which
showed a reduction in number densities of the dispersed phase droplets upon dilution
with the organic phase.
Freeze - fracture TEM combined with nuclear magnetic resonance and quasi -
elastic light scattering was used to study the microstructure of surfactant – water
systems and dynamics of o/w and bicontinuous ME systems [41] . The authors
reported a rather abrupt transition from a discontinuous droplet (o/w) to bicontinuous
(oil - and - water) microstructure occurring at low surfactant concentration, close
to a three - phase region in the constructed phase diagram of pentaethylene glycol
dodecyl ether, water, and octane [41] .
Direct imaging of the ME microstructure using cryo - TEM involves directly investigating
a thin proportion of the specimen in the frozen hydrated stage by using a
cryo stage in the transmission electron microscope. Cryo - TEM was used in combination
with nuclear magnetic resonance (NMR), small - angle X - ray diffraction, and
small - angle neutron scattering to investigate four - component nonionic systems
composed of 1 - dodecane, octa - ethylene glycol mono dodecyl ether, n - pentanol, and
water [42] . These authors reported on the existence of at least two different colloidal
microstructures, swollen spherical micelles with a diameter of around 8 nm and
lamellar structures. Both o/w and w/o MEs were also visualized using cryo - TEM
[43] .
In the pharmaceutical fi eld, very little has been done to elucidate the microstructure
of ME systems using electron microscopy. Bolzinger et al. [44] reported on
bicontinuous sucrose ester - based ME systems for transdermal drug delivery. The
microstructure of the investigated ME systems was viewed by freeze - fracture TEM.
The authors showed images of a bicontinuous structure and reported that incorporating
the anti - infl ammatory drug nifl umic acid into the system did not alter the ME
microstructure. Alany et al. (2001) reported on the microstructure of ME systems
formulated using a blend of two nonionic surfactants, ethyl oleate and water with
and without 1 - butanol. They described two distinct microstructures, namely droplet
w/o and bicontinuous MEs [35] . Their TEM observations were complemented by
electrical conductivity and viscosity measurements.
5.10.5.3 Electrical Conductivity Measurements
Electrical conductivity measurements can provide valuable information concerning
the structure and phase behavior of ME systems [45] . Schulman et al. [38] measured
the conductivity of ME systems, but only in a qualitative way, as they did not monitor
changes in conductivity that are associated with phase changes.
Shah and Hamlin [46] studied the changes in electrical resistance associated with
change in the water - to - oil ratio in an ME system during inversion into various LC
systems and coarse dispersions. In such systems the electrical conductivity may
exhibit both maxima and/or minima, refl ecting changes in ion mobility caused
by variation in viscosity. The most important feature of systems undergoing ME - to -
LC transition is the gradual change in electrical conductivity with changing
composition.
On the other hand, a large electrical conductivity transition has been observed
in several w/o ME systems [47] . A well - known feature of w/o ME systems is the
CHARACTERIZE MICROEMULSIONS AND RELATED SYSTEMS 777
778 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
steep rise in electrical conductivity as the water concentration increases [48] . This
sudden change in electrical conductivity has been attributed to the percolation of
spherical droplets (water droplets surrounded by an amphiphile shell, that is, water -
swollen reverse micelles) in the oil phase [49] . The conductivity remains low up to
a certain water volume fraction due to the nonconducting nature of the continuous
phase of the w/o system. However, as the volume fraction of water reaches and
exceeds the percolation threshold ( . p ), some of these conductive droplets begin to
contact each other and form clusters which are suffi ciently close to each other. This
causes an effi cient transfer of charge carriers between the dispersed droplets by
charge hopping or transient merging of connected droplets resulting in an exponential
increase of conductance from an almost zero value to much higher levels. It is
reasonable to imagine a continuous pathway of water extending through the ME
system, which by some authors is recognized as a sign of emergence of a bicontinuous
structure [50] . Different methods were used to estimate the percolation threshold
( . p ) from the conductivity data by various investigators [35, 48, 51 – 54] .
5.10.5.4 Viscosity Measurements
The viscosity of ME systems is also sensitive to structural changes and Newtonian
fl ow is usually observed. The low viscosity of ME refl ects the fl uid character of the
overall structure, which is a favorable feature for most ME applications. The pioneering
work of Attwood et al. [55] on ME systems formulated using liquid paraffi n,
water, Span 60, and Tween 80 made reference to the equation
. . ..
rel = 1 . ( ) a (2)
where . rel - relative viscosity
a - viscosity constant with theoretical value of 2.5 for solid spheres
. - volume fraction of disperse phase
. - hydrodynamic interaction coeffi cient
In the same study Attwood et al. [55] investigated the effect of increasing the surfactant
concentration on the overall viscosity of an o/w ME system and obtained
values for the viscosity constant a of 3.19 – 4.17. The authors concluded that allowance
for the hydration of the polyoxyethylene chain of the used surfactant reduced
the value of the viscosity constant a toward the theoretical value of 2.5 for a solid
sphere. They also concluded that changing the ratio of the nonionic surfactants did
not signifi cantly affect the viscosity of the system.
Baker et al. [56] studied the viscosity of w/o ME systems containing water, xylene,
sodium alkylbenzenesulfonate, and hexanol using Equation (1) . They reported
values of the viscosity constant a of 3.3 – 6.0, which is above the theoretical value of
2.5 for a sphere with an increase in the surfactant concentration. This fi nding was
attributed to the increase in the ratio of surfactant layer thickness to droplet core
radius as the surfactant concentration was increased. However, deviation in values
of the viscosity constant a from the solid - sphere theoretical value of 2.5 could also
be attributed to changes in the droplet shape or symmetry [57] .
Viscosity studies have also been carried out to investigate the effect of the surfactant
and cosurfactant concentrations as well as the surfactant – cosurfactant mass
ratio on the hydration of the disperse - phase droplets for o/w ME systems [58] . A
study was conducted on systems composed of isopropyl myristate, water, polysorbate
80, and sorbitol. The results showed an increase in the viscosity constant and
a decrease in the hydrodynamic interaction coeffi cient with decreasing surfactant –
cosurfactant mass ratio. The increase in the viscosity constant resulted from greater
hydrodynamic volume of droplets as well as the associated increase in the bound
solvent layer of the droplet core radius from 7 to 22%.
Kaler et al. [50] reported on the viscosity changes in association with a percolative
phenomenon for systems containing the commercial surfactant TRS 10 – 80, octane,
tertiary amyl alcohol, and various brines. Their viscosity results were interpreted as
evidence for a smooth transition from an oil - continuous to a bicontinuous one in
which both oil and water span the sample. A second transition was observed and was
attributed to a transition from a bicontinuous to a water - continuous system.
Borkovec et al. [59] also reported on a two - stage percolation process for the ME
AOT (Aerosol OT, bis(2 - ethylhexyl)sodium sulfosuccinate) system AOT – decane –
water. The structural inversions were investigated using viscosity, conductivity, and
electro - optical effect measurements. The viscosity results showed a characteristic
profi le with two maxima, which was interpreted as evidence for two symmetrical
percolation processes: an oil percolation on the water - rich side of the phase diagram
and a water percolation process on the oil - rich side.
Alany et al. [11, 35] reported on the phase behavior of two pharmaceutical ME
systems showing interesting viscosity changes. The viscosity of both systems increased
with increasing volume fraction of the dispersed phase to 0.15 and fl ow was Newtonian.
However, formation of LC in one of the two systems, namely the cosurfactant
- free system, resulted in a dramatic increase in viscosity that was dependent on
the volume fraction of the internal phase and a change to pseudoplastic fl ow. In
contrast, the viscosity of the bicontinuous ME was independent of water volume
fraction. The authors used two different mathematical models to explain the viscosity
results and related those to the different colloidal microstructures described.
5.10.5.5 Other Characterization Techniques
Among the other techniques used to characterize ME systems with emphasis on
droplet size determination are optical techniques such as static and dynamic light
scattering and nonoptical techniques such as small - angle X - ray scattering and small -
angle neutron scattering [22, 45, 60 – 67] , pulsed fi eld gradient NMR [21, 42, 61, 68 –
71] , and dielectric measurements [72 – 74] . One limitation of dynamic light scattering
or so - called photon correlation spectroscopy is the need to either dilute or heat the
ME system to overcome droplet – droplet interactions as well as microstructure
changes that are associated with increased viscosity. Such techniques are valuable
to obtain useful information regarding such interactions, but the downside would
be the inevitable phase behavior changes that would render these techniques of
some limited value in determining the original droplet size [75] .
5.10.6 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
ME systems have been attracting increasing interest as vehicles for drug delivery
via the various routes. Particular emphasis has been put on the oral, transdermal,
ocular, and parenteral routes. Moreover, these systems have been investigated for
MICROEMULSIONS AS DRUG DELIVERY SYSTEMS 779
780 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
other applications that are relevant to the pharmaceutical, chemical, and biological
sciences. The most signifi cant development from a pharmaceutical perspective to
this date would be undoubtedly the launch of the fi rst oral cyclosporin A ME formulation,
namely Sandimmnue Neoral. Other breakthroughs are likely to follow.
Some of the key advantages related to ME systems include their thermodynamic
stability, transparency, ease of preparation, low viscosity, and ultralow interfacial
tension, to mention a few.
Furthermore, the presence of nanodomains of different polarity along with an
interfacial surfactant/cosurfactnat fi lm within the same systems allow for hydrophilic,
lipophilic, and amphiphilic drugs to be accommodated together if needed.
For o/w ME systems, their main advantage would be to improve the oral bioavailability
of class two (II) drugs. These are drugs that display a dissolution -
dependent bioabsorption [76] . The rationale would be to dissolve the poorly
water soluble drug in the oil phase, thereby rendering it available in a molecular
form ready for dissolution. However, it is important to remember that most drugs
are insoluble in hydrocarbon and to some extent in vegetable oils, rather they
would require more polar oils such as mixtures of mono - and diglycerides along
the lines of Miglyol 840. Using such oils results in systems where the poorly
soluble drug is dispersed as molecules in nanometer - sized oil droplets and as
such are readily available for dissolution. Moreover, it is worth noting that the
use of o/w MEs for delivery routes where extensive aqueous dilution is likely is
less problematic than using w/o ME systems. This is because the external phase
of an o/w ME will be diluted by water (being the main constituent of biological
fl uids) and will therefore retain its microstructure. Conversely, the potential use
of w/o ME systems in the area of drug delivery where there is extensive aqueous
dilution is rather complicated by the fact that such systems will be diluted by the
aqueous biological fl uids. This will result in droplet growth and subsequent phase
changes with potential dose dumping. Despite the aforementioned disadvantages
w/o MEs offer an option for the formulation of class III drugs. These drugs show
permeability - limited bioabsorption in vivo [76] . Peptides/proteins, antisense oligonucleotides,
deoxyribozymes, and small interfering ribonucleic acids (siRNAs)
are good examples on class II drugs. Such molecules display little or no activity
when delivered orally and are highly susceptible to the harsh gastrointestinal
(GI) tract conditions and to their degrading enzymes in vivo. Water - in - oil MEs
offer an exciting opportunity for optimizing the delivery of such molecules as
they can be successfully incorporated in the internal (aqueous) phase and therefore
are denied access to the harsh external conditions. Moreover the internal
water droplets are likely to act as a nanoreservoir to control the mass transfer
process of the loaded drug, that is, offer a mechanism for controlled release. In
addition, the presence of surfactants/cosurfactants as constituents of the formulation
can serve to increase membrane permeability, thereby improving drug
absorption and possibly bioavailability. Bicontinuous MEs are the least investigated
as drug delivery vehicles. They are highly fl uid with low viscosity and
possess ultralow interfacial tension. These properties render them potential candidates
for topical and ocular drug delivery where their wetting and spreading
properties come as an advantage.
The following section highlights some of the main drug delivery areas where ME
systems have been researched as potential drug carriers.
5.10.6.1 Oral Drug Delivery
The most common method for drug delivery is through the oral route as it offers
convenience and high patient compliance. However, recent advances in combinatorial
chemistry is resulting in new molecules with very low water solubility. This leads
to poor dissolution in the GI tract and subsequent erratic and unpredictable bioabsorption
post – oral administration. Self - emulsifying drug delivery systems
(SEDDSs) and self - micro - emulsifying drug delivery systems (SMEDDSs) offer an
interesting option to optimize the delivery of such problematic drug molecules.
SMEDDSs can be defi ned as isotropic, anhydrous systems comprising oil and surfactant
that form o/w MEs upon mild agitation in the presence of water [77, 78] .
The usefulness of SEDDSs in the area of oral drug delivery has been previously
reported by Charman and co - workers, who showed improved pharmacodynamic
properties of the investigational lipophilic drug WIN 54954 [79] . Systems comprising
the medium - chain triglycerides Captex 355 and 800 in combination with Capmul
MCM (medium chain mono - and di - glyceride mixture) and polyoxyethylene 20
sorbitan mono - oleate (Tween 80) were formulated by Constantinides et al. [26, 28,
80] . The authors reported on the improved bioavailability of calcein, RGD peptide,
and a water - soluble marker when incorporated using a ME preconcentrate and w/o
ME in comparison with an aqueous solution serving as a control. The same group
also reported on the use of ME systems to increase oral absorption of poorly water
soluble drugs [25] . The authors reported on the ability of the investigated systems
to increase drug aqueous solubility and improve dissolution and oral bioavailability.
Surprisingly, the formation of lamellar LC systems upon aqueous dilution of SEDDSs
was reported to be a characteristic feature of the most effective SEDDSs [81] . Tenjarla
summarized the factors that are likely to have an effect on the in vivo absorption
of drugs from ME systems as being phase volume ratios, in vivo droplet size,
partition coeffi cient of the drug between the two immiscible phases, the presence
of a drug in an emulsifi ed form or dispersed in oil, site or path of absorption, metabolism
of the oil in the formulation, excipients that may act as absorption promoters,
gastric emptying, and drug solubility in the ME excipients [2] . It was also reported
that peptide uptake from MEs in the GI tract is dependent on particle size, type of
ME lipid phase, digestability of lipid used, presence of bile slats, lipase, type of surfactants
in ME, pH, and shedding of enterocytes [82] .
The most remarkable story of success with ME research and development has
to do with the oral delivery of cyclosporin A, marketed nowadays under the commercial
name Sandimmune Neoral. Cyclosporin A is a cyclic peptide used posttransplantation
surgery as an immune - suppressing agent. Unlike most peptides,
cyclosporin A is hydrophobic and possesses very limited water solubility. The conventional
cyclosporin oral formulation (Sandimmune) is in the form of a drug solution
in olive oil along with ethanol and polyethoxylated oleic acid glycerides. Once
given orally, the oily solution forms a coarse emulsion and as such behaves as a
SEDDS with a bioabsorption process that is slow and incomplete. The net outcome
is fl uctuating drug plasma levels, poor and variable bioavailability, and pronounced
inter - and intrapatient variability [83] . In an attempt to overcome some of these
problems, Tarr and Yalkowsky [84] demonstrated that particle size reduction using
high - shear homogenization can enhance absorption in rats. This improvement in
oral absorption was attributed to the increased dosage form surface area that is
MICROEMULSIONS AS DRUG DELIVERY SYSTEMS 781
782 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
associated with droplet size reduction. The Neoral formulation is composed of a
concentrated blend of two surfactants based on medium - chain partial glycerides
along with an equivalent chain length triglyceride serving as oil, a cosolvent, and
the drug and is described by the manufacturer as “ microemulsion preconcentrate. ”
Exposure of this “ preconcentrate ” to water results in the formation of a w/o ME
that upon further dilution undergoes a phase change into an o/w ME. The improved
in vivo performance of Neoral over Sandimmune has been demonstrated on multiple
occasions [83, 85, 86] . Furthermore, the available pharmacokinetic data have
been reviewed and it was concluded that Neoral offers better predictable and more
extensive drug absorption than Sandimmune. Other poorly water soluble molecules
that have been recently formulated in the form of SMEDDSs with the aim of
improving bioavailability include simvastatin [87] and paclitaxel [88] .
Water - in - oil microemulsions, on the other hand, offer an exciting opportunity to
enhance the oral bioavailability of water - soluble peptide drugs. Because of their low
oral bioavailability, peptide drugs are mostly available as parenteral formulations.
However, parenteral peptides have an extremely short biological half - life and would
therefore require multiple daily injections. This is likely to be problematic in chronic
conditions (insulin for diabetes management is a good example) where patient
compliance is likely to be an issue. Hydrophilic peptide drugs of this nature can be
successfully accommodated into the internal aqueous phase of w/o ME systems
where they are provided with protection from enzymatic degradation post – oral
administration [89] . Furthermore, the presence of surfactant and some cosurfactants
(such as medium - chain glycerides) can act to increase GI membrane permeability
through interacting with the cell membrane bilayer and as such improve oral bioavaialibility
[25, 26, 80, 89 – 92] .
One major concern regarding the safety profi le of ME systems intended for oral
administration is the comparatively high amphiphile content. Both o/w and w/o
ME systems are amphiphile - rich systems compared to conventional emulsions and
would contain in the most conservative case up to 15 – 20% w/w surfactant – cosurfactant.
This is further complicated by the limited models available to evaluate chronic
toxicology in comparison to conventional oral dosage forms such as tablets [91] .
5.10.6.2 Transdermal Drug Delivery
Transdermal drug delivery to the systemic circulation is one of the oldest routes
that have been exploited using ME systems. This route offers distinct advantages
compared to traditional routes by avoidance of fi rst - pass metabolism, potential of
controlled release, ease of administration, and possibility of immediate withdrawal
of treatment when necessary [93] . Transdermal drug delivery aims at maximizing
drug fl ux into the systemic circulation through the skin, whereas dermal drug delivery
aims at targeting either the epidermis or the dermis of the skin. The key challenge
in both cases is to provide suffi cient increase in drug fl ux with minimal or no
signifi cant irreversible alteration to the skin barrier function [93] .
Several studies have reported on the enhanced bioavailability of cutaneous drugs
using o/w and w/o MEs compared to conventional emulsions, gels or solutions,
mesophases, micellar and inverse micellar systems, and vesicles [93] . Moreover, a
diverse range of drug molecules such as ketoprofen, apomorphine, estradiol, lidocaine
[94 – 97] , indomethacin and diclofenac [98] , prostaglandin E 1 [99] , aceclofenac
[100] , vinpocetine [101] , azelaic acid [102] , methotrexate [103] , piroxicam [104] ,
triptolide [105] , fl uconazol [106] , and ascorbyl palmitate [107] were incorporated
into different ME systems.
Kantaria et al. [108, 109] reported on gelatin ME - based organogels (MBGs) as
potential iontophoretic systems for the transdermal delivery of drugs. The microstructure
of the proposed MBG was elucidated using small - angle neutron scattering
where w/o ME droplets were entrapped in an extensive network of gelatin – water
percolative channels. Theses MBGs were found to be electrically conducting and
were shown to successfully deliver a model drug (sodium salicylate). Theses systems
were formulated using pharmaceutically acceptable ingredients, including Tween 80
as a surfactant and isopropyl myristate as the oily component [108] .
Transdermal delivery of proteins and/or DNA vaccines for needle - free immunization
has been attracting increasing interest. Cui et al. [110] reported on ethanol -
in - fl uorocarbone (E/F) MEs for topical immunisation. The authors showed that
plasmid DNA incorporated into E/F MEs was found to be stable. Furthermore, after
topical application to the skin, signifi cant enhancements in luciferase expression,
antibody production, and T - helper type 1 based immune response compared to an
aqueous or ethanolic solutions of DNA were observed [110] .
One major concern with the topical application of ME systems is their biocompatibility
and toxicity potential, mostly due to their high surfactant – cosurfactant content.
Fundamental ME research utilizes ionic surfactants and medium - chain alcohols.
While these ingredients are interesting from a physicochemical perspective, they pose
serious biocompatibility and toxicity concerns [75] . Nonionic and zwitter ion – based
surfactants (such as certain phospholipids) offer a more pharmaceutically acceptable
alternative. Several research groups have been focusing on formulating ME systems
using a single surfactant such as lecithin [98, 111, 112] or n - alkyl POE (polyoxyethylene
ethers) [14, 112, 113] . This approach tends to compromise the phase behavior
of ME - forming systems. This is usually seen when the ME region in the constructed
ternary phase diagrams tends to become smaller in size. This translates to less choice
in terms of ME composition and possibly stability. Alternative cosurfactants with
improved biocompatibility and lower skin irritation potential have been recently
introduced. Plurol isostearique has been shown to be compatible with a range of
surfactants and oils and was capable of providing sizable ME regions.
In conclusion, topically applied MEs have been shown to signifi cantly increase
the cutaneous uptake of both lipophilic and hydrophilic drugs. The favorable properties
of ME systems include the large concentration gradient (between vehicle and
skin) due to the high drug solubilization power of ME systems without increasing
drug affi nity to the vehicle compared to conventional topical delivery systems
[93] . Moreover, the penetration - enhancing properties of the individual surfactant/
cosurfactant ingredient, ease of preparation and “ infi nite ” physical stability, and
good wetting and spreading properties make ME promising for future topical
applications.
5.10.6.3 Parenteral Drug Delivery
Flubiprofen o/w ME systems were prepared and evaluated as vehicles for parenteral
drug delivery [114] . These systems were formulated using POE 20 sorbitan monolaurate
(Tween 20) as the surfactant and ethyl oleate as the oil phase. Flubiprofen
MICROEMULSIONS AS DRUG DELIVERY SYSTEMS 783
784 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
solubility in the o/w ME systems was eight times higher than that in an isotonic
buffer; however, there was no signifi cant differences in the pharmacokinetic parameters
in rats between the ME formulation and the buffer [114] . Bicontinuous MEs
designed for intravenous (i.v.) administration have been prepared and characterized
[71] . The bicontinuous ME system underwent a phase change into an o/w emulsion
upon aqueous dilution. In vitro investigations revealed small droplets with mean
size radii of 60 – 200 nm. While solubilization studies were conducted using two drugs,
namely felodipine and an antioxidant experimental drug (H 290/58), in vivo evaluations
were conducted using the drug - free formulation. Doses of up to 0.5 mL/kg
given i.v. to rats did not show any undesirable effects and had no signifi cant effects
on acid – base balance, blood gases, plasma electrolytes, arterial blood pressure, or
heart rate [71] .
Lecithin - based o/w MEs for parenteral use were formulated using polysorbate
80, IPM (Isopropyl myristate) , lecithin, and water at different lecithin – polysorbate
80 weight ratios [115] . The formulated systems were shown to be highly stable and
of minimal toxicity when evaluated in vitro. Phospholipid - based ME formulations
of all - trans retinoic acid (ATRA) for parenteral administration were prepared and
tested in vitro [116] . ATRA is effective against acute promyelocytic leukemia with
highly variable oral bioavailability. Parenteral ME of ATRA was prepared using
pharmaceutically acceptable ingredients, namely phospholipids and soybean oil. The
inhibitory effect of ATRA on two human cancer cell lines (HL - 60 and MCF - 7) was
not affected by incorporation into a ME formulation.
ME systems intended for parenteral application have to be formulated using
nontoxic and biocompatible ingredients. The o/w ME systems would be suitable to
improve the solubility of poorly water soluble drug molecules whereas w/o ME
systems would be best suited for optimizing the delivery of hydrophilic drug molecules
that are susceptible to the harsh GI conditions. Moreover, w/o systems can
serve to prolong the release and mask any potential tissue irritation and site toxicity
that are caused by intramuscular (i.m.) administration of hydrophilic drug
molecules.
5.10.6.4 Ocular Drug Delivery
Aqueous solutions account for around 90% of the available ophthalmic formulations,
mainly due to their simplicity and convenience [117] . However, extensive loss
caused by rapid precorneal drainage and high tear turnover are among the main
drawbacks associated with topical ocular drug delivery. Only 1 – 5% of the topically
applied drug reaches the intraocular tissue with the remainder of the instilled dose
undergoing nonproductive absorption via the conjunctiva or drainage via the nasolacrimal
duct. This results in drug loss into the systemic circulation and provides
undesirable systemic side effects [118] . Many strategies have been implemented to
overcome such delivery challenges. These include the use of thermosetting in situ
gelling polymer - based systems [119] , nanoparticles, liposomes, and niosomes [120 –
123] . However, MEs offer a promising alternative as they comprise aqueous and
oily components and can therefore accommodate both hydrophilic and lipophilic
drugs. Moreover, they are transparent and thermodynamically stable and possess
ultralow interfacial tension and therefore offer excellent wetting and spreading
properties. Further advantages result form possible improvement of solubility and
REFERENCES 785
stability of incorporated drugs with potential increase in bioavailability; hence these
systems could be a suitable alternative to conventional ocular formulations. So far,
only few investigators [124 – 127] have considered the use of MEs for ocular drug
delivery. Their work was solely focused on o/w MEs as ocular delivery carriers.
Recently Alany et al. [128] reported on w/o MEs formulated using a blend of two
nonionic surfactants (Crillet 4 and Crill 1), an oily component (Crodamol EO), and
water. These systems were shown to be capable of undergoing a phase change to
lamellar liquid crystals upon aqueous dilution. The ocular irritation potential of the
individual components and fi nal formulations was assessed using a modifi ed hen ’ s
egg chorioallantoic membrane test (HET - CAM), and the preocular retention was
investigated in the rabbit eye using gamma scintigraphy. The authors demonstrated
that the retention of ME systems was signifi cantly greater than an aqueous solution.
The rapid clearance of the w/o ME formulated with 10% water compared to the
LC system indicated that phase change is less likely to take place in the rabbit eye
[128] . It was also concluded that w/o MEs may be of value as vehicles for the ocular
drug delivery of irritant hydrophilic drugs as they appear to have a protective effect
when evaluated using a modifi ed HET - CAM test [128] . The potential of bicontinuous
ME systems as vehicles for ocular drug delivery is yet to be investigated. A
recent review reported on the potential of MEs as ocular drug delivery systems;
however, submicrometer emulsions and systems requiring energy input to prepare
were also covered and classifi ed as MEs [129] .
5.10.7 CONCLUDING REMARKS
Microemulsions represent an exciting opportunity for pharmaceutical formulators
and drug delivery scientists. They are easy to prepare and thermodynamically stable.
Moreover, they can accommodate drugs of different physicochemical properties and
protect those that are labile. They have the potential to increase the solubility of
poorly water soluble drugs, enhance the bioavailability of problematic drugs, reduce
patient variability, and offer an option for controlled drug release. A critical look at
the current literature shows that exciting and promising research is taking place. It
is only a matter of time before new ME - based products will fi nd their way to the
market following the successful introduction of Sandimmune Neoral.
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792 MICROEMULSIONS AS DRUG DELIVERY SYSTEMS
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793
5.11
TRANSDERMAL DRUG DELIVERY
C. Scott Asbill and Gary W. Bumgarner
Samford Unversity, Birmingham, Alabama
Contents
5.11.1 Introduction
5.11.2 Physiology and Characteristics of Human Skin
5.11.2.1 Transepidermal Water Loss and Occlusion
5.11.2.2 Skin Lipids
5.11.3 Diffusion
5.11.4 Drug Candidates for Transdermal Drug Delivery
5.11.5 In Vitro Testing of Transdermal Devices and Drug Candidates
5.11.6 Transdermal Patch Design
5.11.6.1 Membrane - Moderated Patches
5.11.6.2 Adhesive Matrix
5.11.7 Commercially Available Patches
5.11.7.1 Transderm Scop
5.11.7.2 Catapres TTS
5.11.7.3 Androderm
5.11.7.4 Estradiol Transdermal Systems
5.11.7.5 CombiPatch
5.11.7.6 Duragesic
5.11.7.7 Ortho Evra
5.11.7.8 Oxytrol
5.11.7.9 Emsam
5.11.7.10 Daytrana
5.11.8 Chemical and Physical Approaches to Transdermal Delivery
5.11.8.1 Chemical Penetration Enhancers
5.11.8.2 Physical Enhancement Methods
5.11.9 The Future of Transdermal Drug Delivery
References
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
794 TRANSDERMAL DRUG DELIVERY
5.11.1 INTRODUCTION
During the last decade there has been an explosion of drug candidates in clinical
trials and a high number of dosage forms approved by the Food and Drug Administration
(FDA) that utilize the transdermal route of drug delivery (Table 1 ). The
interest in transdermals can be attributed to the many advantages offered by this
route of delivery [1] . These advantages include maintaining steady - state drug concentrations
(Figure 1 ), bypassing fi rst - pass metabolism, mimicking an intravenous
(IV) infusion, less frequent dosing, and increased patient compliance (Table 2 ).
The disadvantages of transdermal patches are patient allergies to adhesives found
in transdermal patches, patch excipients that sometimes produce local irritation,
potential for abuse or misuse, temperature that can affect the delivery of drugs from
certain patches, and the inability of most drugs to be delivered transdermally
(Table 3 ) [2] .
In the 1960s a proposal was made that suggested the transdermal exposure of
nitroglycerin to munitions workers resulted in tolerance of the workers to nitroglycerin.
Long - term exposure of nitroglycerin in high doses can lead to tolerance.
Workers who were abruptly removed from exposure to the nitroglycerin often
experienced cardiac arrest and cases of sudden death were sometimes reported [3] .
Soon after, a correlation was made between the physical and chemical properties
of nitroglycerin and its signifi cant transdermal penetration [4] .
TABLE 1 Selected FDA - Approved Transdermal Patches
Trade Name Generic Name Indication Company
Androderm Testosterone Testosterone defi ciency Watson Pharma
Catapres TTS Clonidine Hypertension Boehringer
Ingelheim
Climara Estradiol Hormone replacement Berlex
CombiPatch Estradiol/norethindrone
acetate
Hormone replacment Novartis
Daytrana Methylphenidate Attention - defi cit
hyperactivity disorder
Shire
Pharmaceuticals
Duragesic Fentanyl Pain managment Janssen
Emsam Selegiline Depression Somerset
Pharmaceuticals
Estraderm Estradiol Hormone replacement Novartis
Nicoderm CQ Nicotine Smoking cessation GlaxoSmithKline
Nicotrol Nicotine Smoking cessation Pfi zer
Ortho Evra Ethinyl
estradiol/norelgestromin
Contraception Ortho - Mcneil
Oxytrol Oxybutynin Urinary incontinence Watson Pharma
Transderm
Nitro
Nitroglycerin Angina Novartis
Transdrerm
Scop
Scopolamine Motion sickness Novartis
Vivelle Estradiol Hormone replacement Noven
Vivelle Dot Estradiol Hormone replacement Noven
Another historical perspective on transdermal drug delivery involved tobacco
farmers receiving transdermal doses of nicotine from handling tobacco leaves. A
paper published by Gehlback et al. in 1974 introduced the concept of transdermal
nicotine [5] . It was common for farm workers who had direct contact with tobacco
plants during rainy and humid conditions to exhibit symptoms often associated with
nicotine poisoning. This condition is called green tobacco sickness and has affected
many farm workers, particularly in states such as Kentucky and North Carolina,
where the farming of tobacco is signifi cant. Moisture on tobacco leaves from rain
or dew may contain signifi cant amounts of nicotine due to its high water solubility.
Farm workers directly handling tobacco leaves often retained the moisture -
FIGURE 1 Steady - state drug levels achieved by transdermal delivery.
Steady-state levels of transdermal dose
Time (h)
0 10 20 30 40
Drug
plasma
concentration
Minimum toxic
concentration
Minimum therapeutic
concentration
TABLE 2 Advantages of Transdermal Drug Delivery
Excellent for drugs with short half - lives
Analogous to IV infusion
Route bypasses fi rst - pass metabolism
Reduced side effects
Decreased dosing
Zero - order kinetics
Self - administration
Increased patient compliance
TABLE 3 Disadvantages of Transdermal Drug Delivery
Allergies to adhesives
Product of local irritation
Potential for abuse or misuse
Temperature affects delivery
Drug needs to be potent and have desired physical and
chemical properties
INTRODUCTION 795
796 TRANSDERMAL DRUG DELIVERY
containing nicotine on their clothes or skin. Nicotine, because of its physical and
chemical properties, is low molecular weight and has excellent transdermal penetration,
and signifi cant blood levels can lead to many adverse reactions, such as nausea
and vomiting [6] .
5.11.2 PHYSIOLOGY AND CHARACTERISTICS OF HUMAN SKIN
The skin is the largest organ in the human body and has many physiological functions.
The skin serves to regulate overall body homeostasis, protect the body from
external pathogens and chemicals, as well as control water loss from the body. The
skin has three main layers. The epidermis, which is the outermost layer, is the thinnest
layer of the skin and provides the most signifi cant barrier function [7] . Beneath
the epidermis, the dermis provides mechanical support to the skin and the third
layer, immediately under the dermis, is a layer of subcutaneous fat called the
hypodermis.
The epidermis consists of fi ve principal layers and is an area of both intense biochemical
activity and differentiation. These layers are the stratum corneum, stratum
lucidum, stratum granulosum, stratum spinosum, and stratum basale. The stratum
corneum (horny layer) is the uppermost layer of the epidermis and the skin. The
stratum corneum is composed of dead keratinocytes, which are called corneocytes,
and has an abundance of keratin and lipid structures [8] . The stratum corneum is
considered the rate - limiting barrier for the diffusion of chemical compounds across
the skin. The stratum lucidum (clear layer) is composed of two to three layers of
dead fl attened keratinocytes which appear translucent under a microscope and are
present only in thick glabrous skin.
Stratum granulosum (granular layer) is an epidermal layer than consists of three
to fi ve layers of keratinocytes which have started to fl atten out, and their nuclei and
organelles have begun to disintegrate. Also, lipid and keratin granules have started
to form inside the keratinocytes. Eventually the granules will give rise to the brick -
and - mortar structure of the stratum corneum.
The stratum spinosum (prickly layer) is composed of several layers of keratinocytes
which are starting to exhibit histological and biochemical changes that mark
the beginning of the differentiation process. The shape of the keratinocytes has
become irregular and enzymes responsible for lipid synthesis are present.
The stratum basale is the deepest layer of the epidermis and is composed mainly
of keratinocytes with melanocytes making up approximately 10% of the cell population.
The stratum basale is one cell layer thick and is a layer of rapid cell division
where keratinocytes are rapidly dividing and giving rise to the uppermost layers of
the epidermis.
The dermis is the largest layer of the skin. It is a region of strong and fl exible
connective tissue. The dermis consists of two primary layers, the papillary layer and
the reticular layer. The papillary layer is the smallest layer of the dermis and is
composed mainly of collagen and elastin fi bers. The reticular layer is the largest
layer of the dermis and is composed of mainly dense connective tissue. The layer of
subcutaneous fat found directly beneath the dermis provides insulation and additional
mechanical support to the skin.
5.11.2.1 Transepidermal Water Loss and Occlusion
Transepidermal water loss (TEWL) is a natural occurrence that takes place in the
skin layers. TEWL is the result of movement of water from the deep skin layers
across the epidermis into the outside atmosphere. It is a tightly regulated process
that is controlled by the stratum corneum [9] . Occlusive topical bases and devices,
such as transdermal patches, block TEWL and cause increased hydration of the skin.
Hydration of the skin increases the permeation rates of compounds transdermally.
The occluding effect of transdermal patches is an important mechanism that
promotes increased diffusion of the compound across the skin into the systemic
circulation [10] .
5.11.2.2 Skin Lipids
As keratinocytes differentiate and move toward the stratum corneum, they synthesize
an abundance of lipids, much of which are packaged into small organelles called
lamellar granules [11] . When reaching the stratum corneum, the corneocytes eject
these lipid granules forming a principal component of the brick - and - mortar structure
of the stratum corneum. Lipids synthesized in the skin layers are thought to
arrive from carbon sources derived from acetate obtained from the systemic circulation.
The role of epidermal lipids in the brick - and - mortar structure of the stratum
corneum and the barrier function of the skin is well established in the literature
[12] . Many studies have suggested that when organic solvents such as dimethyl
sulfoxide (DMSO) are used to dissolve epidermal lipids, an increase in skin permeability
is found [13] .
The major lipids found in the stratum corneum are ceramides, fatty acids, and
cholesterol. Free fatty acids make up 10 – 15% of the lipid mass of the stratum
corneum and predominantly consist of straight - chain saturated species ranging from
14 to 28 carbons in length. Cholesterol, a major lipid found in the stratum corneum,
represents approximately 25% of the total stratum corneum lipid while cholesterol
sulfate accounts for another 5%.
Ceramides are considered to be the largest group of lipids found in the stratum
corneum, representing 50% of the total lipid weight. Six distinct ceramide fractions
have been isolated and characterized [14] . Ceramide 1 is derived from the linoleate
- rich acylglucosylceramide. Ceramide 2 consists of straight - chain saturated fatty
acids amide linked to sphingosine and dihydrosphingosine bases. Ceramide 3 consists
of saturated fatty acids amide linked to phytosphingosine, which has an additional
hydroxyl group on carbon 4. Ceramides 4 and 5 both consist of . - hydroxyacids
amide linked to sphingosines and dihydrosphingosines. Ceramide 6 contains . -
hydroxyacids amide linked to phytosphingosines.
It has been suggested that these ceramides form a gel - phase membrane domain
within the skin. Straight fatty acid chains as well as the small polar head groups on
the ceramides are thought to produce a tightly packed domain which is less fl uid
and thereby less permeable than other liquid crystalline domains which are also
present. Recent evidence using differential scanning calorimetery (DSC) and infrared
absorption spectroscopy analyses verifi es the presence of gel phases within the
stratum corneum.
PHYSIOLOGY AND CHARACTERISTICS OF HUMAN SKIN 797
798 TRANSDERMAL DRUG DELIVERY
5.11.3 DIFFUSION
The diffusion of an active ingredient from a transdermal patch through the skin
layers and into the systemic circulation can be attributed to Fick ’ s law. Fick ’ s law is
best defi ned as a linear relationship between the fl ux of a chemical and the chemical
’ s concentration gradient. The concentration gradient is the engine which triggers
drug diffusion in all directions [15] . The chemicals move from a region of higher
concentration to a region of lower concentration. Transdermal patches are typically
loaded with large amounts of active ingredients in order to maximize diffusion.
Once the drug is absorbed systemically the concentration gradient is maintained
with “ sink conditions ” existing between the dosage form and the systemic
circulation.
5.11.4 DRUG CANDIDATES FOR TRANSDERMAL DELIVERY
Several characteristics are important in determining which drugs are candidates for
transdermal drug delivery. These are half - life, molecular weight, lipophilicity, and
potency. Some pharmaceutical active ingredients have short biological half - lives
whereas other have very long half - lives. For drugs such as estradiol with half - lives
of less than 2 h, for the drug to be delivered orally, a frequent dosing schedule must
be followed due to the rapid clearance of the drug from the systemic circulation.
This high dosing frequency may be inconvenient and may lead to poor patient
compliance. However, drugs with short half - lives are excellent candidates for transdermal
drug delivery because the rate of drug delivery is constant and mimics the
kinetics offered by an IV infusion. The steady - state drug delivery allows for less
frequent dosing, which may result in better patient compliance.
Molecular weight is another property that must be considered when selecting
potential candidates for transdermal drug delivery. Molecular weight is a major
determinant in whether a molecule may pass through the restrictive barrier of the
stratum corneum. Drugs that have low molecular weights have a better chance of
penetrating the stratum corneum compared to high - molecular - weight compounds
such as proteins and oligonucleotides, which are too large to passively diffuse across
the stratum corneum. It has been proposed in the literature that compounds with a
molecular weight of less than 400 daltons are potential candidates for transdermal
drug delivery (Table 4 ) [16] . Lipophilicity of the drug is also an important factor
concerning the chemical ’ s ability to undergo transdermal delivery. Pharmaceutical
active ingredients must have suffi cient solubility in both the lipid portion and hydrophilic
regions of the skin in order to have signifi cant permeation. Drugs with a log P
between . 1.0 and 4.0 can potentially traverse the brick - and - mortar structure of the
stratum corneum.
TABLE 4 Physico Chemical Factors That Affect
Permeation of Compounds
Small molecular weight
Melting point < 200 ° C
Suitable partition coeffi cient > 0.5 . 10 . 3 cm/h
Another important parameter when selecting transdermal candidates is the
drug ’ s potency. Only drugs that provide therapeutic effects at low steady - state
plasma levels are viable candidates. The input rate needed for a transdermal patch
fl ux can be determined from multiplying the clearance of the drug with the desired
plasma concentration of the drug at steady state. This is a useful predictor when
selecting the feasibility of using an active ingredient in a transdermal patch.
5.11.5 IN VITRO TESTING OF TRANSDERMAL DEVICES AND
DRUG CANDIDATES
The ability to test the transdermal penetration of compounds in vitro has been
investigated extensively over the last decade. Hundreds of studies have shown that
the permeation rate of compounds can be accurately measured using several available
in vitro diffusion cells and various types of skin models [17] .
A typical in vitro permeation cell is composed of both a donor compartment and
a receptor compartment (Figure 2 ). The model skin or membrane for permeation
testing is placed between the donor and receptor compartments. The donor compartment
is where the drug solution, ointment, or transdermal patch is applied. The
compound then permeates across the membrane into the receptor compartment.
The receptor compartment typically contains a buffered solution maintained at
37 ° C and is continuously stirred [18] . Often antibiotics and preservatives are added
to the receptor compartment to restrict growth of microorganisms and to maintain
the integrity of the skin model that is being utilized for the permeation study [19] .
Samples are withdrawn from the receptor compartment at predetermined intervals
for analysis. Following the withdrawal of samples from the receptor compartment,
an equal amount of diffusion buffer is added to the volume.
5.11.6 TRANSDERMAL PATCH DESIGN
5.11.6.1 Membrane - Moderated Patches
Membrane - moderated patches have been utilized in many FDA - approved transdermal
patches such as Duragesic, Transderm Scop, and Catapress TTS. This type
of patch utilizes a rate - controlling membrane to precisely control the release of
FIGURE 2 Typical in vitro permeation cell.
Sampling port
Donor compartment
Receptor compartment
IN VITRO TRANSDERMAL PATCH DESIGN 799
800 TRANSDERMAL DRUG DELIVERY
active ingredient from the transdermal patch and provides release profi les of active
ingredients that exhibit zero - order kinetics [20] . The layers of a membrane -
moderated patch are as follows: The uppermost layer is an occlusive backing that
is impermeable; next is the reservoir layer, which consists of active ingredient dissolved
in an appropriate solvent such as mineral oil or ethyl alcohol. The next layer
is a rate - controlling membrane that is typically composed of ethylene vinyl acetate
copolymer. Underlying the rate - controlling membrane is a layer of adhesive and
then fi nally the release liner (Figure 3 ). Commonly burst doses of the active ingredient
are placed in the adhesive layer to saturate the skin layers and reduce the lag
time to steady - state blood levels [21] (Figure 1 ).
5.11.6.2 Adhesive Matrix
Adhesive matrix patches are increasingly common, and most of the recently
approved transdermal drug delivery devices utilize this type of technology [22 – 24] .
Advantages of this type of system are easier to manufacture than membrane -
moderated patches, smaller in size, and resistant to manufacturing defects which
could lead to dose dumping. The layers of an adhesive matrix patch consists of the
backing followed by a layer containing the active ingredient dissolved and mixed
with adhesive and then the release liner [25] (Figure 3 ).
5.11.7 COMMERCIALLY AVAILABLE PATCHES
5.11.7.1 Transderm Scop
In 1978 Tranderm Scop was the fi rst transdermal patch to receive FDA approval.
Scopolamine, the active ingredient, is a belladonna alkaloid that is frequently
used to treat motion sickness and nausea resulting from anesthetics and analgesics.
Transderm Scop is a membrane - moderated patch that has a three - day life span. It
has a circular shape with an area (2.5 cm 2 ) approximately the size of a quarter
[26] .
FIGURE 3 Diagram of membrane - moderated and adhesive matrix transdermal patches.
Membrane moderated patch
Backing
Reservoir
Rate-controlling
membrane
Adhesive layer
Adhesive matrix patch
Adhesive layer
Backing
Drug adhesive
5.11.7.2 Catapres TTS
Catapres TTS is the fi rst and only transdermal patch approved by the FDA for the
treatment of hypertension. Catapres TTS contains the active ingredient clondidine
and was approved in 1985 [27] . Catapres TTS is a seven - day patch and is a membrane
- moderated transdermal patch. The patch comes in three sizes, 0.1, 0.2, and
0.3 mg/day. In addition there is also a burst dose of clonidine in the adhesive layer.
The presence of the burst doses provides an immediate - release dose of clonidine
which promotes rapid systemic levels of the drug.
5.11.7.3 Androderm
Androderm is an FDA - approved membrane - moderated transdermal patch that
delivers testosterone. Androderm is manufactured in two sizes and both are 24 - h
patches. The round Androderm patch contains 12.2 mg of testosterone and delivers
a 2.5 - mg dose over a 24 - h period. The larger oval patch contains 24.3 mg of testosterone
and delivers 5 mg over a 24 - h period. The reservoir of Androderm contains
testosterone gelled with alcohol and glycerin.
5.11.7.4 Estradiol Transdermal Systems
There are several FDA - approved estradiol patches currently on the market. These
patches are three - to four - day patches used to treat symptoms associated with
menopause. Estraderm is the only membrane - moderated estradiol system that is on
the market. Estraderm is available in two sizes: 0.05 and 0.1 - mg/day patches. Vivelle,
Vivelle Dots, Alora, and Climara are examples of commercially available estradiol
patches that utilize adhesive matrix patch design.
5.11.7.5 CombiPatch
CombiPatch is a three - or four - day patch that delivers both estradiol and norethindrone
acetate. CombiPatch is available in two sizes: a 9 - cm 2 patch that delivers
0.05 mg of estradiol per day and 0.14 mg of norethindrone acetate per day and a
16 - cm 2 patch that delivers 0.05 mg of estradiol per day and 0.25 mg of norethindrone
acetate per day. Estradiol is a lipophilic compound with a molecular weight of 272.
The molecular weight of norethindrone acetate is 340. The design of the patch is
considered an adhesive - matrix - type patch that consists of three layers. The backing
is comprised of polyolefi n and the adhesive layer contains a silicone adhesive, acrylate
adhesive, estradiol, norethindrone acetate, oleic acid, and oleyl alcohol.
5.11.7.6 Duragesic
Duragesic is a transdermal patch that delivers the potent opioid analgesic fentanyl.
The life span of the patch is three days and it is manufactured in fi ve sizes: 12, 25,
50, 75, and 100 . g/h. Duragesic is a membraned - moderated patch and consists of
four patch layers: a backing layer of polyester fi lm, a drug reservoir that contains
fentanyl, and U.S. Pharmacopeia (USP) alcohol gelled with hydroxyethyl cellulose,
COMMERCIALLY AVAILABLE PATCHES 801
802 TRANSDERMAL DRUG DELIVERY
a rate - controlling membrane made of ethylene - vinyl acetate copolymer, and a layer
of silicone adhesive with a burst dose of fentanyl. Fentanyl has a molecular weight
of 336.5 and is a lipophilic drug with signifi cant transdermal permeation.
5.11.7.7 Ortho Evra
Ortho Evra was approved by the FDA in 2002 as the world ’ s fi rst transdermal contraceptive
patch. Ortho Evra contains both ethinyl estradiol and norelgestromin as
active ingredient. Ortho Evra is a three - layered patch that is a matrix - type design.
The molecular weight of ethinyl estradiol is 296.41 and the molecular weight of
norelgestromin is 327.47. The adhesives used in this system are polyisobutylene and
polybutene. Ortho Evra is a seven - day patch which is cycled three weeks on and
one week patch free.
5.11.7.8 Oxytrol
Oxytrol is a three - or four - day patch used for the treatment of urinary incontinence
that was recently approved by the FDA. It delivers the active ingredient oxybutynin.
Oxybutynin is an antispasmotic and anticholinergic agent with a molecular weight
of 357. Oxybutynin is considered to be a lipophilic drug. Oxytrol is a matrix - type
patch with a surface area of 39 cm 2 and contains 36 mg of oxybutynin. Oxytrol has
an in vivo delivery rate of approximately 3.9 mg/day.
5.11.7.9 Emsam
Emsam is a once - a - day patch that delivers an active ingredient selegiline that is used
to treat depression. Selegiline is an irreversible monoamine oxidase inhibitor and
has a molecular weight of 187.30. Emsam is a matrix - type patch that has three layers
and is available in three size. The 6 - mg/day patch has a surface area of 20 mg/20 cm 2 ,
the 9 - mg/day patch has a surface area of 30 mg/30 cm 2 , and the 12 - mg/day patch has
a surface area of 40 mg/40 cm 2 .
5.11.7.10 Daytrana
Daytrana is a newly approved methylphenidate transdermal system. It is a 9 - h
adhesive matrix patch and comes in four sizes: a 12.5 - cm 2 patch that has a delivery
rate of 1.1 mg/h, an 18.75 - cm 2 patch that has a delivery rate of 1.6 mg/h, a 25 - cm 2
patch that delivers 2.2 mg/h, and a 37.5 - cm 2 patch that delivers 3.3 mg/h.
5.11.8 CHEMICAL AND PHYSICAL APPROACHES TO
TRANSERMAL DELIVERY
Due to the brick - and - mortar structure of the stratum corneum, the skin is a diffi cult
layer to permeate across for most active pharmaceutical ingredients. Because of this
diffusional barrier, new strategies have been developed to allow compounds to
better penetrate the stratum corneum [28] . These strategies can be defi ned as either
chemical or physical approaches to disrupting the barrier function of the skin.
5.11.8.1 Chemical Penetration Enhancers
Many approaches that have been investigated over the last several decades enhance
the permeation of compounds across the skin using novel chemical compounds [29] .
These permeation enhancers are compounds which partition into the stratum
corneum and promote the passage of topically applied compounds across the skin
layers by using three possible mechanisms of action (Table 5 ). The mechanisms by
which these compounds enhance permeability of the skin have been previously
described by Williams and Barry and are often referred to as the lipid – protein partitioning
theory [30] . One proposed mechanism of action of permeation enhancers
and the most common method by which chemical enhancers increase permeation
is by fl uidizing the intercellular lipid structures that are found within the stratum
corneum. By interacting with and disorganizing these lipid structures, channels can
be formed which allow the compound to better diffuse across the rate - limiting
barrier of the stratum corneum.
Another method is based on the ability of some permeation enhancers to interact
with intracellular proteins such as keratins inside corneocytes. The disassembly of
these proteins structures within the corneocytes allows some compounds to transcellularly
penetrate the stratum corneum. Also, some enhancers act as vehicles and
cosolvents, increasing and promoting the partitioning of compounds into the stratum
corneum (Table 6 ).
5.11.8.2 Physical Enhancement Methods
Microneedles Microneedles are a new type of transdermal device that is receiving
tremendous attention by pharmaceutical companies because of the potential for the
transdermal delivery of high - molecular - weight compounds. This type of transdermal
device contains microscopic needles or projections that can be loaded with active
ingredient [31] . The projections when placed on the skin penetrate beyond the
stratum corneum into the living epidermis. This allows the compound to bypass
passage directly through the stratum corneum, which is the rate - limiting barrier for
TABLE 5 Desired Properties of Chemical Permeation
Enhancers
Provide reversible effects in skin layers
Low systemic bioavailability
High stability and compatibility with formulations
Possess no pharmacological activity
Should be nontoxic and nonirritating
TABLE 6 Investigated Chemical Permeation Enhancers
Azone Oleic acid
Ethano Oxazolidnesl
Dimethyl sulfoxide Propylene glycol
Fatty acids Sodium lauryl sulfate
Lecithin Terpenes
CHEMICAL AND PHYSICAL APPROACHES TO TRANSERMAL DELIVERY 803
804 TRANSDERMAL DRUG DELIVERY
passive diffusion. The drug can be loaded into the microneedles and then released
into the lower skin levels or the microneedle patch may contain a reservoir and the
drug will penetrate through channels in the stratum corneum that have been created
by the microneedles. This allows compounds that normally would not passively
diffuse across the stratum corneum, such as therapeutic proteins, to be delivered
transdermally. Currently various formulations of microneedle patches are being
investigated in clinical trials in the United States [32 – 34] .
Iontophoresis and Sonophoresis In the early 1900s it was discovered that some
chemical compounds could be delivered into the systemic circulation across the skin
using an electric current. This phenomenon was later described as iontophoresis.
Iontophoresis occurs when an electric potential difference is created across the skin
layers by an electric current and this gradient drives the penetration of both charged
and uncharged drugs across the skin [35] .
One of the earliest FDA - approved products that utilized iontophoresis was the
iontophoretic delivery of pilocaripne as a method for diagnosing cystic fi brosis [36] .
The sweat of individuals with cystic fi brosis contains large amounts of both sodium
and chloride ions. The pilocarpine that is delivered into the skin promotes increased
sweating, which can be easily collected and analyzed.
Another product that uses iontophoresis has been recently approved by the FDA.
This device is called Ionsys and is an iontophoretic system that delivers fentanyl
hydrochloride transdermally [37, 38] . This is a patient - controlled device that provides
on - demand delivery of fentanyl for up to 24 h or 80 doses. This device contains
10.8 mg of fentanyl hydrochloride and is designed to deliver a 40 - . g dose of fentanyl
over a 10 - min period upon activation of the dose button by the patient.
Another popular physical enhancement method that has routes in physical
therapy and sports rehabilitation clinics is sonophoresis. Sonophoresis involves the
use of ultrasound as a source of disrupting intercellular lipid structures in the
stratum corneum [39, 40] . The sound waves produced by the device induce cavitation
of the lipids found within the stratum corneum, which then opens channels and
allows the chemical compound to easily penetrate the skin. This is a safe and reversible
process that has received much attention in the literature and by pharmaceutical
companies.
5.11.9 FUTURE OF TRANSDERMAL DRUG DELIVERY DEVICES
Through practical application of recombinant deoxyribonucleic acid (DNA) technology
there has been a recent explosion of biotech drugs that are in clinical trials,
and the FDA has approved many. Unfortunately, due to the high molecular weights
and other physical and chemical characteristics of these macromolecules, many
delivery obstacles exist that prevent these compounds from being delivered transdermally.
Many physical and chemical approaches are being investigated that
enhance the delivery of the biotech agents across the skin.
TransPharma - Medical, an Israeli - based pharmaceutical company, is investigating
the transdermal delivery of human parathyroid hormone fragment for the treatment
of osteoporosis in addition to the delivery of human growth hormone [41] . This
technology utilizes a 1 - cm 2 patch that creates small channels or holes in the stratum
corneum much like the microneedle technology that was previously discussed. The
delivery of insulin has been studied for over a decade using transdermal systems.
Unfortunately, due to the high molecular weight and dosing considerations with
insulin, it has been diffi cult. However, insulin delivery transdermally has recent successes
clinically using sonophoresis, permeation enhancers, iontophoresis, and
microneedles [42, 43] .
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REFERENCES 807
809
5.12
VAGINAL DRUG DELIVERY
Jose das Neves , Maria Helena Amaral , and
Maria Fernanda Bahia
University of Porto, Porto, Portugal
Contents
5.12.1 Introduction
5.12.2 The Human Vagina
5.12.2.1 Anatomy
5.12.2.2 Histology
5.12.2.3 Physiology
5.12.2.4 Childhood, Pregnancy, and Menopause
5.12.3 General Features of Vaginal Drug Delivery
5.12.3.1 Advantages and Disadvantages of Vaginal Drug Delivery
5.12.3.2 Permeability and Drug Absorption
5.12.3.3 First - Uterine - Pass Effect
5.12.4 Vaginal Drug Delivery Systems
5.12.4.1 Overview
5.12.4.2 Excipients
5.12.4.3 Solid Systems
5.12.4.4 Semisolid Systems
5.12.4.5 Liquid Systems
5.12.4.6 Vaginal Rings
5.12.4.7 Vaginal Films
5.12.4.8 Medicated Vaginal Tampons
5.12.4.9 Vaginal Foams
5.12.4.10 Vaginal Sponges
5.12.4.11 Other Strategies and Vaginal Drug Delivery Systems
5.12.4.12 Packaging and Vaginal Applicators
5.12.5 Pharmaceutical Evaluation of Vaginal Drug Delivery Systems
5.12.5.1 Legal and Offi cial Compendia Requirements
5.12.5.2 Drug Release and Permeability
5.12.5.3 pH and Acid - Buffering Capacity
5.12.5.4 Rheological Studies
5.12.5.5 Textural Studies
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
810 VAGINAL DRUG DELIVERY
5.12.5.6 Mucoadhesion
5.12.5.7 Vaginal Distribution and Retention
5.12.5.8 Safety and Toxicology
5.12.5.9 Other Characteristics
5.12.6 Clinical Usage and Potential of Vaginal Drug Delivery
5.12.6.1 Microbicides
5.12.6.2 Antimicrobials
5.12.6.3 Hormonal Contraceptives and Hormonal Replacement Therapy
5.12.6.4 Spermicides
5.12.6.5 Labor Inducers and Abortifacients
5.12.6.6 Proteins and Peptides
5.12.6.7 Vaccines
5.12.6.8 Other Uses
5.12.7 Acceptability and Preferences of Women Related to Vaginal Drug Delivery
5.12.8 Veterinary Vaginal Drug Delivery
5.12.9 Conclusions and Future Trends
References
5.12.1 INTRODUCTION
The vagina has been used for a long time as a route for drug administration, being
as old as medicine and pharmacy themselves. Throughout the history of human
civilization, vaginal administration of drugs has been practiced and recorded until
the modern era. Some of the fi rst records were found in Egypt, where the Kahun
Papyrus, the oldest of the surviving medical papyri (ca. 1850 b . c .), included references
to vaginal “ preparations ” containing substances such as mud, frankincense,
oil, malachite, ass urine, myrrh, crocodile dung, honey, and sour milk, normally used
in female genitalia – related conditions and contraception [1] . Latter papyri such as
the Ramesseum Papyrus (ca. 1700 b . c .), the Ebers Papyrus (ca. 1550 b . c .), and the
Greater Berlin Papyrus (ca. 1300 b . c .) also contained drug formulations to be
administered in the vagina. Vaginal administration of drugs continued to be carried
out by other civilizations from ancient Greece and Rome to the Middle Ages,
in the Arabic and Oriental cultures, passing through the Renaissance, until our
days [1, 2] .
Although traditionally used for local action, some drugs can permeate the vaginal
mucosa and reach the bloodstream in suffi cient concentrations to have systemic
effects. Current understanding of the vaginal anatomy, physiology, and pathophysiology
is very considerable, contrasting with the still limited knowledge of the possibilities
of vaginal drug delivery. Nonetheless, interest and contribution of
pharmaceutical scientists toward vaginal drug delivery development have increased
in the last years in response to the specifi c needs of this route of drug administration.
Indeed, vaginal drug delivery has seen recent advances that make it very
promising, in particular therapeutic fi elds such as the prevention of human immunodefi
ciency virus (HIV) and other sexually transmitted infections, contraception,
hormone replacement therapy in menopausal women, and labor induction.
Therefore, this chapter discusses the main features of vaginal anatomy, physiology,
and histology related to drug delivery as well as vaginal drug delivery systems
and their evaluation. Also, past and current usage of the vagina as a route of drug
administration, ongoing investigation, and promising strategies in this fi eld are
addressed.
5.12.2 THE HUMAN VAGINA
The knowledge of the human vagina ’ s anatomy, histology, and physiology is fundamental
in the design of drug delivery systems, helping researchers optimize vaginal
products. Pharmaceutical scientists must comprehend the particularities of this
organ in order to overcome its natural limitations and enhance its advantages over
other drug delivery routes.
5.12.2.1 Anatomy
The organs of the female reproductive tract are classically divided into the internal
and external genitalia [3] . The internal genital organs include the vagina, cervix,
uterus, oviducts, ovaries, and surrounding supporting structures, as seen in Figure 1 .
The external genital organs, also known as the vulva or pudendum, are composed
by the structures that surround the vaginal entrance, which are visible in the perineal
area. These include the mons pubis, labia majora, labia minora, hymen, clitoris,
vestibule, urethra, vestibular glands (Skene glands and Bartholin glands), and vestibular
bulbs.
THE HUMAN VAGINA 811
FIGURE 1 Female reproductive system and related structures: vagina (1), cervix (2), uterus
(3), ovary (4), fallopian tube (5), urinary bladder (6), urethra (7), anus (8), rectum (9), colon
(10), vestibule (11), and pubic symphysis (12). (Courtesy of Lu i s Paup e rio.)
812 VAGINAL DRUG DELIVERY
The vagina is a tubular, fi bromuscular organ approximately 9 cm long, extending
from the cervix to the vestibule, being positioned between the urinary bladder and
the rectum (Figure 1 ). Because of the protrusion of the cervix into the upper part
of the vagina, two deep recessions (anterior and posterior) are formed, called fornices,
the posterior fornix being considerably larger than the anterior one. Also, two
lateral fornices can be considered [3, 4] . In the adult woman, the anterior wall of
the vagina is approximately 7.5 cm long, while the posterior wall extends by approximately
9 cm. The width of the vagina varies throughout its length, the transverse
diameter being higher at the level of the fornices, decreasing progressively toward
the vaginal entrance. The vagina is commonly misunderstood as a straight tube,
descending from the cervix to the introitus. The axes of the upper and lower vagina
are different, forming a slight S - shaped curve: The axis of the lower vagina in relation
to a standing woman is vertical and posterior, while the upper part (from the
pelvic diaphragm to the cervix) becomes more horizontal, with the fi nal portion of
the vagina curving toward the hollow of the sacrum [3, 5] . The angle between these
two portions is approximately 130 ° C. This fact is important in drug delivery, taking
into consideration that pharmaceutical formulations must be retained in the vagina,
resisting to gravity forces. The walls of the vagina are covered by a mucosal tissue
that forms a series of transverse folds denominated rugae (more prominent in the
lower third of the vagina), thus increasing the available surface for absorption and
allowing considerable distension of this organ during penile penetration and childbirth
[4, 6] . Indeed, the surface area of the vaginal mucosa is an important parameter
concerning its coverage by vaginal products or drug absorption. Results by Pendergrass
et al. varied through a wide range of values (65.73 – 107.07 mm 2 ), with a mean
surface area of 87.46 mm 2 , although these results can be an understatement because
of limitations in the determination method [7] . In a more recent study, Barnhart
et al. found higher surface values which ranged from 103.9 to 165.0 mm 2 . These variable
results suggest that one volume of vaginal product may not be appropriate for
all women [5] . The walls of the vagina are normally in apposition and fl attened in
the anteroposterior diameter. Thus the vagina has the appearance of the letter H in
cross section, although a W shape may also be observed [8] .
The vagina has a high blood supply, which is primarily provided by the vaginal
branches of the internal iliac artery. To a minor extension, blood is also supplied
through branches of the uterine, middle rectal, and internal pudendal arteries. Blood
drainage from the vagina returns through vaginal veins (the perineum venous
plexus), running parallel to the course of the arteries, which fl ow into the pudendal
vein, internal iliac vein, and then the vena cava, thus bypassing the portal circulation.
Blood supply increases with sexual excitement, particularly to the clitoris and vestibular
bulbs, causing the erection of these structures along with the expansion and
elongation of the vagina, allowing the accommodation of the male penis [4] . The
lymphatic drainage is characterized by its wide distribution and frequent crossovers
between both sides of the pelvis. Generally, the upper third of the vagina drains to
the external iliac nodes, the middle third to the common and internal iliac nodes,
and the lower third to the common iliac, superfi cial inguinal, and perirectal nodes
[3] .
Sensory innervation of the vagina is provided by the pudendal plexus and is
particularly developed near the vaginal orifi ce. The nervous sympathetic innervation
of the vagina is provided by the hypogastric plexus, while the nervous parasympa
thetic innervation derives from the second and third sacral nerves. There is a lack
of free nerve endings in the upper two - thirds of the vagina [3, 4] . Thus, objects placed
in this area, such as vaginal drug delivery systems, are likely to be unperceived by
women.
5.12.2.2 Histology
The vaginal tissue is composed of four distinct layers: stratifi ed squamous epithelium,
lamina propria, muscular layer, and tunica adventitia, the fi rst two usually
referred to as vaginal mucosa (Figure 2 ). The epithelial layer is about 150 – 200 . m
thick, corresponding approximately to 30 – 45 layers of cells, being nonkeratinized.
The epithelium presents different types of cells, making it possible to identify fi ve
different layers, namely the basal, parabasal, intermediate, transitional, and superfi -
cial layers. Epithelial cells are closely joined by numerous desmosomes and occasionally
tight junctions, the last being particularly abundant in the basal layer.
Deposits of a lipid material are found near the surface of the epithelium, acting as
a permeability barrier to large water - soluble molecules. The lamina propria, or
tunica, is composed of fi brous connective tissue richly supplied by small blood and
lymphatic vessels. Although being a mucosal surface, the vaginal mucosa does not
FIGURE 2 Detail of human vaginal mucosa of fertile adult (H & E, . 40). Image shows
stratifi ed squamous epithelium (E), which is nonkeratinized, and lamina propria (LP), richly
supplied by small blood vessels and deprived of glands (Courtesy of Manuel Dias.)
THE HUMAN VAGINA 813
814 VAGINAL DRUG DELIVERY
have mucus - secreting glands (Figure 2 ). The muscular layer has a large number of
interlacing fi bers, making it possible to distinguish an inner circular layer and an
outer longitudinal layer. These muscle fi bers allow substantial elongation during
childbirth, increasing approximately fi ve - fold. The tunica adventitia consists of connective
tissue containing a large plexus of blood vessels, lymphatic vessels, and
nerves [9] .
The characteristics of the vaginal mucosa depend on sexual hormones undergoing
changes throughout the menstrual cycle. During the proliferative phase, estrogens
increase vaginal blood fl ow and the integrity of the mucosa, inducing the
proliferation of the vaginal epithelium, peaking at approximately midcycle. Nonetheless,
this change in epithelial thickness is small and probably clinically insignifi -
cant [10, 11] . In the secretory phase progesterone opposes those actions, although
without signifi cantly infl uencing the epithelium thickness [12] .
5.12.2.3 Physiology
Functions of the vagina include receiving the erect penis and semen during coitus
and ejaculation and serving as a passageway for fetus and menses to the outside of
the body [4] .
The vaginal milieu is typically acidic in healthy women during their fertile years,
playing an important role in preventing the proliferation of pathogenic microorganisms.
Vaginal pH is around 3.5 – 4.5, changing during the menstrual cycle being lower
in the middle cycle and higher around menses [13] . Also, vaginal pH may be altered
by several conditions, including the presence of semen (pH 7.2 – 8.0) or bacterial
infections. In this last case vaginal pH usually increases to values of 5.0 – 6.5 [14] .
These and other variations of vaginal pH may alter the effi cacy of administered
drugs through quite a few mechanisms, including variable release from the delivery
system, alteration in drug absorption and/or metabolism, and modulation of drug
activity in the cervicovaginal milieu [15] . Lactobacillus species are present in the
vagina of healthy women, being responsible for its acidity. These bacteria use glycogen,
which is synthesized by epithelial cells under estrogen ’ s infl uence in order to
produce lactic acid, thus reducing vaginal pH [16] . Besides their contribution to
vaginal pH, lactobacilli also have the ability to regulate the proliferation of microorganisms,
namely pathogenic species, by other mechanisms: adherence to the
mucus, forming a barrier which prevents colonization by pathogens or competition
for the receptors of the epithelial cells, and the production of antimicrobial compounds
such as hydrogen peroxide, lactic acid, bacteriocin - like substances, and possibly
biosurfactants [17] . Although lactobacilli are the main organisms responsible
for vaginal acidity, recent research suggests that other acid - producing microorganisms,
such as Atopobium sp., Megasphaera sp., and Leptotrichia sp., may also contribute
to the acidity of the vaginal tract [18] . The composition of vaginal fl ora is
complex, being infl uenced by hormonal changes, although lactobacilli levels of colonization
seem to remain relatively constant during the menstrual cycle [19] . Also,
other factors such as glycogen content, pH, sexual intercourse, medication, and
immunity status are known to infl uence the vaginal ecosystem [20] .
Although deprived of secreting glandules, the vaginal mucosa is covered with a
watery acidic fl uid. This fl uid includes contributions from vaginal transudation,
Bartholin and Skene glands, exfoliated epithelial cells, residual urine, and fl uids from
the upper reproductive tract such as cervical mucus and endometrial and tube fl uids.
Vaginal fl uid consists of 90 – 95% water, inorganic and organic salts, urea, carbohydrates,
glycerol, mucins, fatty acids, albumins, immunoglobulins, enzymes, leukocytes,
and epithelial debris. Although quite variable, normal daily production of vaginal
fl uid is estimated at around 6 mL, increasing at midcycle and decreasing around the
menstruation period [21] . In fact, the menstrual cycle plays an important role in
vaginal fl uid characteristics, particularly on pH values, rheological properties, color
(from milky white to transparent), and antimicrobial activity. Estrogens induce the
production of vaginal fl uid, leading to the lubrication of the mucosa. Decline of
serum estrogen levels, as during prepubertal or postmenopausal periods, results in
a reduction of vaginal moisture [22, 23] . This variability may infl uence drug release,
dissolution, absorption, and removal and thus its activity. Also, vaginal fl uid is selectively
antimicrobial, lactic acid and to a lesser extent antimicrobial peptides and
proteins (calprotectin, lysozyme, histones, and others) being partially responsible by
the resistance of the normal vagina to colonization by exogenous microorganisms
[24] .
Cervical mucus is an important component of the vaginal fl uid, although it presents
substantial differences when compared with whole vaginal fl uid, mainly in pH
value (approximately 7.0). It is produced in the cervix, leaking down into the vagina,
particularly during the three - to fi ve - day interval that precedes ovulation. Its properties
are also infl uenced by the menstrual cycle, particularly pH (range of 5.4 – 8.0)
and antimicrobial activity [25] . Also, estrogens stimulate the secretion of abundant
and fl uid cervical mucus, while progesterone induces the formation of thick cervical
mucus. These changes in viscosity infl uence the capacity of sperm and other substances,
such as drugs, to pass the cervix and migrate to the uterus [22, 23] .
Interaction between vaginal fl uids and drug delivery systems is an important
aspect that has to be managed during drug design, as it may infl uence the fl ow,
retention, drug delivery kinetics, and bioactivity of vaginal formulations [21] . Since
fl uids present in the vaginal environment are diffi cult to obtain, simulated fl uids
have been developed in order to emulate their physical and chemical properties.
Recently, a vaginal fl uid simulant was proposed by Owen and Katz [21] , whether
Burruano et al. [26] developed a synthetic cervical mucus formulation. These fl uids
have been successfully used to evaluate vaginal formulations in vitro, being able to
mimic with considerable accuracy the physiological fl uids. Also, the standardization
of a single composition provides the possibility of comparing results obtained by
different investigators. Table 1 presents the main features of these two simulants.
Enzymes present in the vagina may infl uence drug delivery, as they can degrade
and infl uence the permeability of the administered drugs. Although the enzymatic
activity in the vagina is not as high as in other drug delivery sites, several enzymes
can be found in the vaginal fl uid and in different vaginal epithelium cells, namely
succinic and lactic dehydrogenase, acid and alkaline phosphatases, . - glucuronidase,
phosphoamidase, lactate dehydrogenase, aminopeptidase, and esterases [27, 28] .
This enzymatic activity can limit drug bioavailability and decrease the stability of
prolonged delivery formulations.
Understanding the immune mechanisms responsible for the defense of the female
genital tract is of extreme importance concerning the development of vaccines that
are effective against pathogens. The female genital tract has several mechanisms of
defense against infectious agents, which appear complementary, additive, and even
THE HUMAN VAGINA 815
816 VAGINAL DRUG DELIVERY
synergistic. The immune response can be classifi ed in three levels: nonimmune,
preimmune, and acquired or specifi c (Table 2 ) [29] . Nonimmune response is very
effective in limiting the infectious inoculum, while preimmune mechanisms hold up
the infection long enough so that the immune response can be activated. These
defense strategies are largely infl uenced by pathogenic agents presented in the
vagina and the hormonal milieu [29, 30] .
The genital tract is part of the common mucosal immune system, which comprises
all mucosal tissues of the body, sharing similarities and common immunization
mechanisms, being able to disseminate acquired immunity between them. Nonetheless,
it has become clear that the genital tract presents several unique features that
differentiate it from other mucosal sites [31] . One of the most important is the ability
to only induce local immune responses. In fact, most of the antibodies are produced
locally at the mucosa, while those derived from the circulation represent only a small
fraction. Also, contrasting with other mucosal tissues, vaginal secretions contain
more immunoglobulin (Ig) G than secretory IgA [30, 32] . Hence, immunity of the
genital tract is conferred by local production of IgA and, to a less extent, by transudation
of serum IgG, although cellular immune response should also be considered
[33] . Local immune cell population in the vaginal mucosa includes Langerhans
cells, macrophages, T cells, and neutrophils. Systemic immune response is also impor-
TABLE 1 Main Features of Vaginal Fluid Simulant a and Synthetic Cervical Mucus b
Vaginal Fluid Simulant Synthetic Cervical Mucus
Composition NaCl (3.51 g), KOH (1.40 g), Ca(OH) 2
(0.222 g), bovine serum albumin
(0.018 g), lactic acid (2.00 g), acetic
acid (1.00 g), glycerol (0.16 g), urea
(0.4 g), glucose (5.0 g), HCl (to
adjust pH), and water (e.q. 1 L)
Guar gum (1.00%), dried porcine
gastric mucine (type III)
(0.50%), imidurea, (0.30%),
methylparaben (0.15%),
propylparaben (0.02%), dibasic
potassium phosphate (0.26%),
monobasic potassium
phosphate (1.57%), and water
(96.20%)
pH 4.2 7.4
a From ref. 21 .
b From ref. 26 .
TABLE 2 Immune Response of Female Genital Tract
Nonimmune Passive
Synthesis of protective mucus
pH
Epithelial barrier
Active
Infl ammatory reaction
Secretion of humoral soluble factors
Preimmune Humoral response, cellular response
Immune Humoral response, cellular response
Source : From ref. 29 .
tant in the reinforcement of the acquired mucosal immunity or to take over when
this one has faded, although they are independent from one another [29, 30] .
Sexual hormones, particularly estrogens, also infl uence the regulation of local
immunity, as IgA levels in genital secretions, antigen presentation by vaginal cells,
and lymphocyte proliferation vary throughout the menstrual cycle [34, 35] . Estrogens
can decrease the concentrations of immunoglobulins, while progestogens can
increase their levels [36] .
Alterations in vaginal physiology before, during, and after sexual intercourse are
important factors in the performance of those formulations intended to be used in
this period. During sexual arousal, genital vasocongestion occurs, leading the clitoris
and the labia minora to become enlarged with blood and the vagina to increase in
length and diameter as a result of relaxation of the smooth muscular wall. The
vaginal canal is lubricated by secretions from the uterus, Bartholin and Skene glands,
and a fl uid that transudates from the subepithelial vascular tissues, being passively
transported through the intercellular spaces. Engorgement of the vaginal wall raises
the pressure inside the capillaries, thus increasing the transudation of plasma through
the vaginal epithelium [6] . The resulting vaginal fl uid is increased in quantity, less
acidic, and more diluted, allowing penile penetration and thus preventing the male
and female genitalia from becoming irritated [3, 4] .
5.12.2.4 Childhood, Pregnancy, and Menopause
The vagina undergoes several lifetime changes that may infl uence vaginal formulation
performance. These changes are particularly important when developing vaginal
products that are intended to be used in specifi c situations, such as labor inducers
in pregnant women or hormonal supplements in postmenopausal women.
The vagina of the newborn exhibits the infl uence of residual maternal estrogens,
presenting a stratifi ed squamous epithelium rich in glycogen and becoming colonized
with lactic acid – producing microorganisms shortly after birth. By the fourth
postnatal week these estrogenic effects disappear, and the vaginal epithelium loses
its stratifi cation and glycogen content, becoming much thinner and exhibiting alkaline
or neutral pH because of acid - producing microorganism depletion. These characteristics
remain throughout childhood, until puberty. By this time, the vagina
experiences changes due to adrenal and gonadal maturation. This organ increases
in size, vaginal fornices develop, cervicovaginal secretions start being produced, and
vaginal milieu becomes acidic. Also, vaginal epithelium thickens and intracellular
glycogen production increases [37] .
When the fertile woman becomes pregnant, the connective tissue of the vulva,
vagina, and perineum relaxes, and the muscle fi bers of the vaginal wall increases in
size. These alterations prepare the vagina for childbirth. During delivery, perineal
and vaginal musculature relaxes and the vaginal rugae fl atten, allowing full expansion
of the vaginal tract, accommodating the passage of the newborn. Normal morphology
and dimensions of the vagina are recovered after 6 – 12 weeks [37] . The
vaginal blood supply is substantially increased during pregnancy, which can enhance
systemic absorption of drugs. High levels of estrogens during pregnancy lead to
thickening of vaginal epithelium and stimulation of glycogen production. This
increased glycogen content promotes lactobacilli growth, consequently decreasing
vaginal pH by the enhancement of lactic acid synthesis.
THE HUMAN VAGINA 817
818 VAGINAL DRUG DELIVERY
Menopausal women experience a decline in estrogens, which leads to several
changes in the genital organs, namely in the vagina. Such changes include atrophy
of the labia majora and shortening and loss of elasticity of the vaginal barrel. The
number of epithelial cell layers decreases (from 8 – 10 in premenopausal to 3 – 4 in
postmenopausal), leading to alterations in the barrier capacity and potential increase
of mucosal damage and pain and burning sensation during sexual intercourse.
Vaginal fl uids decrease approximately 50% because of the Bartholin glands atrophy
and a decrease in the number and maturity of vaginal cells. A decrease in the colonization
by lactobacilli species is observed in menopausal women as a result of the
reduction of vaginal glycogen levels, leading to a low production of lactic acid and
consequently to increased vaginal pH. In postmenopausal women without estrogen
treatment the vaginal pH is estimated to be 5.5 – 6.8 or even higher [37, 38] .
5.12.3 GENERAL FEATURES OF VAGINAL DRUG DELIVERY
Vaginal drug delivery is mostly used in gender - specifi c conditions, although it can
be a viable alternative for drugs usually administered by other routes. Also, traditionally
problematic drugs from a delivery point of view (e.g., peptides) may fi nd in
the vaginal route an interesting and promising way for nonparental administration.
Limitations and potentialities of vaginal drug administration are intimately connected
to this route ’ s idiosyncrasy. Hence, acquaintance of particular features of
vaginal drug delivery is required.
5.12.3.1 Advantages and Disadvantages of Vaginal Drug Delivery
The administration of drugs through the vagina, and eventually their absorption, is
a function for which this organ is not physiologically conceived. Nonetheless, the
vaginal route of administration presents some advantages. Substances absorbed
through the vaginal mucosa bypass the liver before entering systemic circulation,
avoiding hepatic fi rst - pass metabolism. Thus, drugs that undergo extensive hepatic
fi rst - pass metabolism can benefi t from vaginal administration, usually requiring less
amount of drug to achieve the same biological effects. Steroids used in hormone
replacement therapy or contraception are a good example of molecules that are
largely metabolized in the liver, with approximately 95% of orally administered
estrogens undergoing hepatic metabolism. Also, these molecules are able to damage
the liver when administered by the oral route, an event that can be minimized with
vaginal administration. Gastrointestinal side effects are common for many oral
administered drugs; the vaginal route may be an alternative to their administration,
with the benefi t of increased patient compliance. Additionally, vaginal enzymatic
activity is lower when compared with gastrointestinal activity, lacking even some
important enzymes enrolled in drug metabolism, such as trypsin and chymotrypsin.
The vaginal route offers women the possibility of easy self - insertion and removal
of drug delivery systems as well as avoidance of the pain, tissue damage, and eventual
infection often associated with parenteral routes. Ocular and buccal administration
sites frequently become irritated after prolonged contact with drug delivery
systems; conversely, the vagina presents less sensitivity, allowing the presence of
drug formulations for long periods of time. Although absorption of substances is
not a function of the vagina, features such as its relatively large surface area and
rich blood supply contribute to this organ ’ s high permeability to several drugs,
allowing higher bioavailability of some active substances when compared to other
routes [39 – 41] . On the other hand, the vaginal drug delivery route has some limitations.
Gender specifi city is the most important, as it restricts its use to females only.
Others, such as misperceptions and cultural issues about genital manipulation and
insertion of objects in the vagina, personal hygiene, infl uence with sexual intercourse,
and variability in drug absorption related with menstrual cycle, menopause,
and pregnancy, can also limit vaginal drug delivery route usage [41, 42] .
5.12.3.2 Permeability and Drug Absorption
Although some substances are not desired to be absorbed, such as those targeted
for local action, permeation of drugs through the vaginal wall into the bloodstream
must occur if one seeks to obtain a systemic effect. Although vaginal administration
of drugs has been performed for a long time, the capability of systemic drug absorption
through this organ was not clarifi ed until the early twentieth century by investigators
such as Macht or Robinson [43, 44] . Both conducted independent experiments
with substances such as potassium iodide, morphine, atropine, sodium salicylate,
quinine, sucrose, and phenol red, which evidenced the permeability of this mucosal
tissue, thus opening the possibility of systemic drug delivery through the vagina.
Nonetheless, the currently acknowledged variability of vaginal drug absorption with
the hormonal status of women, which can limit the potential for systemic drug
delivery, was documented in the 1940s by authors such as Rock et al. These researchers
reported that although the vaginal administration of drugs, namely penicillin,
could provide therapeutic blood levels, the extension of their absorption was highly
variable [45] .
Recently, several in vitro experiments substantiated the potential of the human
vaginal mucosa as a good administration route relating to the degree of permeation
when compared with other mucosal surfaces. In fact, the vagina can be more permeable
to some commonly used model substances, such as water, 17 . - estradiol (Figure
3 ), arecoline, arecaidine, and vasopressin, than colonic or small intestinal mucosa,
or at least as permeable as when compared to human buccal mucosa [46, 47] .
In general, systemic drug absorption requires three steps: drug release from the
delivery system, drug dissolution in the vaginal fl uid, and permeation of the vaginal
mucosa. Knowledge of the permeability characteristics of the vaginal mucosa is an
important step when developing a pharmaceutical product, with both the physicochemical
properties of chemical substances (e.g., chemical nature, degree of ionization,
molecular weight and size, conformation, and oil/water partition coeffi cient)
and the biophysicochemical nature of the tissue infl uencing absorption. The epithelial
layer of the vaginal mucosa presents itself as the main permeability barrier for
drug absorption. As the epithelium is hormonally dependent, its permeability also
changes, usually decreasing with higher estrogen levels because of the induced
membrane thickening. However, contradictory fi ndings of enhanced vaginal absorption
in postmenopausal women treated with estrogen have been reported [48] .
These results can be explained by the increment of vaginal mucosa blood fl ow that
is also induced by estrogen. The transport mechanism of most vaginal absorbed
substances is simple diffusion. Lipophilic substances are absorbed through the
GENERAL FEATURES OF VAGINAL DRUG DELIVERY 819
820 VAGINAL DRUG DELIVERY
intracellular (or transcellular) pathway, whereas hydrophilic substances are absorbed
through the intercellular (or paracellular) pathway or across aqueous pores present
in the vaginal mucosa [49] . Also, receptor - mediated transport mechanisms can be
involved in the absorption of some substances.
The magnitude of the fl ux rate across the vaginal mucosa is mainly related to the
molecular size and hydrophobicity of the permeating substances. In general, compounds
with molecular weight over 300 Da have decreased fl ux rates, while hydrophobic
properties usually increase permeation [50 – 52] . The infl uence of the penetrant
hydrophobicity/hidrophilicity in the rate and extent of absorption through the
vaginal mucosa was demonstrated by Corbo et al. [53, 54] . Experiments performed
in rabbits showed that hydrophilicity infl uenced mucosal permeability of drugs such
as progesterone, with increasing hidrophilicity leading to decrease in rate and extent
of vaginal absorption. Nonetheless, one should keep in mind that a minimum degree
of aqueous solubility is always required in order to ensure that the drug dissolves
in the vaginal fl uid. Also, pH, viscosity, and volume of this fl uid change throughout
the menstrual cycle, potentially infl uencing the extension of drug dissolution: Different
pH values infl uence the drug degree of ionization and thus its solubility;
increased viscosity of the vaginal fl uid may enhance drug retention, although it may
also present a barrier to drug absorption; and higher volumes of fl uid benefi t drug
dissolution but also increase its clearance from the vagina.
However, permeability - enhancing strategies may be required, as many substances
cannot permeate the vaginal mucosa in signifi cant pharmacological levels. An interesting
and helpful option is the use of several permeation enhancers which can
increase drug absorption by interacting with epithelial tight junctions, providing a
new intercellular penetration pathway [55] . Some of these substances, such as citric
acid, benzalkonium chloride, laureth - 9, lysophosphatidylglycerol, sodium taurodihydrofusidate,
lysophosphatidylcholine, palmitoylcarnitine chloride, lysophosphatidylglycerol,
and sodium glycodihydrofusidate, have been successfully tested [56 – 60] .
FIGURE 3 Overall mean fl ux values of 17 . - estradiol across human vaginal, colonic, and
small intestinal mucosa, as determined by fl ow - through diffusion cells. Flux values ( J ) were
calculated as J = Q /( At ), where Q is quantity of 17 . - estradiol crossing mucosa (in dpm), A
is mucosa area exposed (in cm 2 ), and t is time of exposure (in min). SEM: standard error of
the mean. (Reprinted with permission from P. van der Bijl and A. D. van Eyk, International
Journal of Pharmaceutics , 261, 147 – 152, 2003. Copyright 2003 by Elsevier.)
Vagina Colon Intestine 500
400
300
200
100
0
0
5 10 15 20 25
Time (h) Bar represents SEM
Although this strategy may enhance permeability, it presents some disadvantages,
particularly the possibility of mucosal damage. These unwanted effects are variable
and not always observed; however, they may often be severe [61] . Also, some drugs
achieve poor systemic levels after being delivered by the vaginal route, not because
of poor permeation through the mucosa, but due to their fast inactivation by local
enzymes, particularly when therapeutic peptides and proteins are considered.
In these cases, enzymatic inhibitors may be a helpful solution. Vaginal peptidase
inhibitors, such as ethylene diamine tetraacetic acid (EDTA), thimerosal, amastatin,
bestatin, leuptin, and pepstatin A, were shown to be useful, promoting peptide
absorption in rats, as they prevent drug degradation [60, 62] . At this point it is also
important to notice that some substances can reduce the permeability of the vaginal
mucosa [63] , this possibility always being important when designing a drug delivery
system. In addition, other approaches, such as the use of mucoadhesive polymers,
in situ gelling formulations, or solubility enhancers, have been shown to be useful
in improving vaginal permeability of several drugs [40] .
5.12.3.3 First - Uterine - Pass Effect
The fi rst - uterine - pass effect can be defi ned as a preferential transfer of vaginally
administered drugs to the uterus. This effect is due to a countercurrent mode of
exchange, with an upward vagina - to - uterus transport of substances absorbed in
vaginal and lymphatic vessels and diffusing to nearby arteries [64] . Evidences of
higher than expected uterine concentration after vaginal administration of drugs,
namely progesterone, terbutaline, or danazol, led to the postulation and verifi cation
of this hypothesis [65, 66] . This effect can be of the utmost importance when the
uterus is the desired locale for a drug to exert its effects, opening new therapeutic
options for uterus - related conditions.
Further investigations showed that the placement of a formulation in different
areas of the vagina dramatically infl uences the observation of the fi rst - uterine pass -
effect. Experimental fi ndings suggest that this preferential transfer to the uterus is
only observed when absorption occurs in the outer one - third of the vagina [67] .
Thus, drugs intended to exert their effects in the uterus should not be inserted
deeply in the vagina, as it is often recommended, instead they should be placed near
the vaginal entrance.
5.12.4 VAGINAL DRUG DELIVERY SYSTEMS
5.12.4.1 Overview
A wide range of drug delivery systems have been used, although many of them are
not specifi cally designed for intravaginal administration. Traditionally used vaginal
drug delivery systems include solutions, ointments, creams, vaginal suppositories,
and tablets. Recently, others, such as vaginal rings or vaginal fi lms, have been developed.
Also, several strategies and improvements have been tested in order to overcome
natural limitations of drug delivery through this route, particularly low
retention, limited absorption, and cyclic variations.
Most of the currently available vaginal formulations, particularly those that have
been marketed for a longer period of time, have serious limitations such as poor
VAGINAL DRUG DELIVERY SYSTEMS 821
822 VAGINAL DRUG DELIVERY
spreadability, messiness, and small capacity of retention in the vagina. Nonetheless,
recent advances allowed circumventing some of the major diffi culties that hold back
the use of this route of drug delivery as a serious alternative to the most traditional
ones, with the consequent increase of commercially available drug delivery systems
[39] . Drug release of most traditional formulations is rapid, needing frequent administrations
to sustain therapeutic drug concentrations. Thus, in recent years, sustained
release has been a new approach to deliver several active substances through the
vaginal route. Also, vaginal drug delivery systems should ensure either an adequate
penetration of the drug within the mucosa, in order to enhance the local effects and
reduce systemic absorption, or an ideal permeation of the active substances into the
bloodstream in order to assure an effective systemic response.
Before formulators choose a delivery system for a selected drug, several issues
should be taken into consideration: physicochemical properties of the active substance,
intended effect of the active substance, required drug release profi le, excipients
to be used and their compatibility with the active substance and vaginal mucosa,
women ’ s preferences, and economical implications.
5.12.4.2 Excipients
When vaginal drug delivery is considered, formulators must select a number of suitable
excipients in order to design a drug delivery system able to ensure the therapeutic
success of the active substance(s). In fact, it is known that excipients used in
vaginal formulations can infl uence the pharmacological performance of active substances,
being able to improve or diminish their activity [68, 69] . The decision of
which excipients to use depends to a great extent on the fi nal dosage form and
desired characteristics of the drug delivery system. Some excipients can infl uence
drug delivery system performance by changing some properties, such as viscosity,
mucoadhesion, and distribution [70] . Although these variations do not interfere
directly with the pharmacological effect of the active substances, their availability
and thus the formulation clinical outcome can be compromised. Thus, excipient
selection must be performed with utmost caution, taking into consideration the
quality, safety, and functionality aspects of these materials. Indeed, Garg et al.
recently compiled a list of excipients that are currently approved or have already
been investigated for vaginal administration [71] .
Although by defi nition excipients are deprived of pharmacological effects, some
have showed that this is not always true. For instance, chitosan, an excipient that
has attracted a lot of interest in the formulation of vaginal drug delivery systems,
exhibits antimycotic effects, particularly against the common vaginal pathogen
Candida albicans [72] . Also, other polymers commonly used in tablets and capsules,
such as cellulose acetate phthalate, have been investigated in the formulation of
vaginal microbicides, due to their antiviral effects against HIV [73] .
Some commonly used excipients can interact with vaginal and cervical fl uids,
altering their properties. These interactions should be taken into consideration when
designing a drug delivery system, as they can infl uence in vivo performance. For
example, small amounts of nonionic (e.g., polyethylene glycol) and cationic (e.g.,
polyvinylpyridine) polymers are able to modify the gel structure of the cervical
mucus, altering its barrier properties, while like - charged molecules (e.g., polyacrylic
acid) interact little with this biological fl uid. This approach has been taken into
account, particularly as a new prevention strategy for pathogens that infect via the
mucosa, as a new treatment option for diseases that affect the mucous layer itself
or even as a strategy for systemic drug delivery routes [74, 75] .
5.12.4.3 Solid Systems
Solid systems commonly administered by the vaginal route include tablets, capsules,
and vaginal suppositories.
Tablets are frequently used as vaginal drug delivery systems, being inexpensive
and easy to manufacture. They are also easily administered in the vagina, allowing
a “ clean ” insertion that contrasts with the typical messiness of semisolid drug delivery
systems. Although very similar to oral tablets, these systems have some particularities,
such as being round or oval shaped and devoid of sharp edges, in order to
avoid damage of mucosal tissue. These drug delivery systems are usually designed
to rapidly release their active substances after being placed in the vagina. In fact,
disintegration or dissolution problems, mainly due to the scarce amount of vaginal
fl uid, are important issues to be managed by formulators. This rapid release and
solubility enhancement of the active substances can be important because of the
rapid vaginal wash - off and low in situ retention. Increased and faster release of drug
content has been achieved using effervescent tablets [76] or including specifi c excipients
that can enhance its disintegration in vaginal fl uids [77] . For instance, Karasulu
et al. proposed an effervescent tablet made of a mixture of mucoadhesive microcapsules
loaded with ketoconazole and effervescent granules [76] . This combination
showed ability to improve retention with rapid onset of action. Additionally, other
strategies have been used, such as inclusion complexes of poorly soluble drugs with
cyclodextrins, in order to improve drug solubility, allowing a rapid onset of the
pharmacological effect.
Although fast release of the active substances is a frequent goal, controlled -
release tablets can be used in order to enhance their effi cacy, because of their prolonged
release, and prevent the irritation of the vaginal mucosa that may be caused
by some drugs [78] . Nonoxynol - 9, a commonly used microbicide and spermicide,
known for its irritability when administered in the vagina, is a good example of a
drug that can benefi t from controlled release. Formulation of double - layer tablets
(fast - release outer layer and slow - release core) obtained from coprecipitates of
nonoxynol - 9 with polyvinylpyrrolidone, can provide extended drug release, allowing
a more prolonged spermicidal effect while reducing its irritating effect [79] . Also,
controlled release prevents peaks in serum concentration of absorbable drugs, limiting
possible systemic effects [80] .
Vaginal tablets containing lactobacilli have been used in order to restore the
normal vaginal fl ora. Formulation of these delivery systems requires specifi c proceedings
in order to provide viability of lactobacilli and stability of the fi nal product.
Freeze drying of bacterial suspensions has been tested to obtain lyophilized powders
for tablet production [81] . These powders were shown to be processable and tablet
production was easy and reproducible. Also, the use of double - layer tablets (fast -
release layer and slow - release layer) seems to be an interesting approach to lactobacilli
administration.
It is common to use tablets designed for the oral route in order to deliver drugs
through the vagina. Nonetheless, issues such as delivery system retention and
VAGINAL DRUG DELIVERY SYSTEMS 823
824 VAGINAL DRUG DELIVERY
distribution and drug release can infl uence the fi nal performance of the formulation,
being preferable to use specifi cally vaginal designed drug delivery systems, or at
least study the pharmacokinetics of oral tablets after vaginal administration [82] .
Capsules, particularly soft capsules, have been used as vaginal drug delivery
systems, but with modest popularity. These systems are relatively stable, particularly
when compared with semisolid formulations or vaginal suppositories, being an
adequate way to deliver liquid drugs within a solid dosage form.
Vaginal suppositories, also referred as ovules or pessaries, are ovoid - shaped, solid
(but generally malleable) dosage forms specifi cally designed for vaginal administration.
These systems usually weigh 2 – 3 g, although formulations with up to 16 g have
been used in the past [83] . Vaginal suppositories have a long history of use as vaginal
drug delivery systems, mainly in the management of local conditions. Major advantages
are their reduced price and ease of manufacture. However, they present some
inconveniences, such as messiness, low retention in the vagina, and poor stability,
the last feature due to their temperature and moisture sensibility.
Vaginal suppositories are very close to rectal suppositories in terms of excipient
nature and manufacturing process. Thus, they are usually prepared by fusion of the
excipient(s) (referred as “ base ” ) and incorporation of the active substance(s), this
mixture being subsequently poured into molds and allowed to solidify. Other
methods, such as by compression, can also be used. Several substances have been
utilized as bases for the formulation of vaginal suppositories: gelatin and glycerin,
cocoa butter, semisynthetic glycerides, and polyethylene glycol, among others [83] .
Composition of vaginal suppositories is importantly related to their melting or dissolution,
thus infl uencing drug release profi le. Generally, it can be stated that drug
release rate increases as the melting temperature of a suppository decreases or as
its dissolution time in vaginal fl uids increases. Also, affi nity of the drug for the base
infl uences its release from vaginal suppositories: Greater release of drug is expected
when there is less affi nity between the active substance(s) and the base [84] . The
melting temperature and melting process of vaginal suppositories can be characterized
by several techniques, such as differential scanning calorimetry and viscosity
and dilatometry methods, among others [85] . Specifi c pharmaceutical characterization
of vaginal suppositories includes the determination of disintegration time and
breaking hardness. Also, other standard quality control tests include appearance
description, surface texture evaluation, pH determination, uniformity of content,
and microbial limit testing [86, 87] .
Recently, sustained - release vaginal suppositories have been developed in order
to attain drug delivery systems with improved performance. Sustained release can
reduce the number of administrations, thus improving patient compliance. A base
composition consisting of a polymeric gum (carboxymethylcellulose and xanthan
gum), a dispersing agent (colloidal silicone dioxide), and polyethylene glycol,
referred as long acting, sustained release of spermicide (LASRS), has recently been
studied by Zaneveld et al. in order to deliver contraceptives and microbicides [88] .
Results showed that a LASRS base is able to spread quickly and evenly over the
mucosa, being retained in place for prolonged periods of time and allowing long -
lasting effi cacy for several active drugs. Preliminary human trials have confi rmed
these results [89] . In another study, Mandal developed hydrophilic vaginal suppositories
comprising mixtures of miconazole cross - linked with poly(vinyl alcohol) by
freeze thawing and different polyethylene glycols that were able to sustain release
this antifungal drug for up to 108 h [90] .
5.12.4.4 Semisolid Systems
Semisolid systems present several advantages over other drug delivery systems:
They are easy to use and generally inexpensive and have good acceptability. Among
their disadvantages, leakage has been one of the most disturbing, mainly because
many conventional formulations are not mucoadhesive. The simplest way of dealing
with this problem has been the recommendation for night administration, as the
supine position diminishes leakage. Also, messiness and discomfort upon application
and diffi culties in dispensing an accurate dose are important limitations.
Once widely used, ointments have been largely substituted by creams and gels.
Nonetheless, some of these drug delivery systems may still be encountered, particularly
as hydrophilic bases.
Creams have been used for quite some time as vaginal drug delivery systems,
particularly for the administration of sexual hormones and antimicrobials. The main
advantage of creams over other semisolid systems is their ability to easily dissolve
both hydrophobic and hydrophilic drugs. As most conventional creams do not
possess bioadhesive properties, incorporation of bioadhesive polymers is an effective
approach to improve their retention in the vagina. Recently, a new approach
to vaginal drug delivery was developed using Site Release (SR) technology (KV
Pharmaceutical, St. Louis, MO). This technology is based on bioadhesive controlled -
release water - in - oil emulsions, being formulated as a vaginal cream (SR cream). The
outer oily phase repels moisture (thereby resisting dilution) and retains the dispersed
water phase containing the drug (allowing controlled release) [91] . The SR
cream allows minimizing leakage and enhancing clinical outcome, requiring less
total drug exposure per course of therapy. Site Release technology is currently available
in the United States in two commercial products: one containing butoconazole
nitrate 2% (Gynazole - 1, Ther - Rx Co., St. Louis, MO) and the other containing
clindamycin phosphate 2% (Clindesse, Ther - Rx Co.). Clinical fi ndings demonstrated
that a single application of Gynazole - 1 makes it possible to achieve more rapid relief
of vaginal candidiasis symptoms than standard oral therapy with fl uconazole [92] .
Similarly, Clindesse was show to be able to achieve prolonged local effective concentrations
while presenting lower systemic bioavailability and thus less systemic
adverse effects when compared with conventional formulations in the treatment of
bacterial vaginitis [93] . Also, other drugs have been studied in order to further evaluate
the potential of this versatile technology [94] .
Since the pioneer work by Wichterle and Lim in the 1960s [95] , gels have evolved
greatly from simple formulations to advanced drug delivery systems. These systems
were soon demonstrated to be good candidates to deliver drugs in the vagina,
particularly because of their high bioavailability (mainly because of mucoadhesive
properties), biocompatibility, spreadability, ease of usage, and economical savings
[96] . Gels are extremely versatile, being used to deliver most of the currently used
drugs through the vaginal route.
Recent advances in gel and polymer technology boosted research, opening new
possibilities for vaginal drug delivery [97] . Indeed, the development of new and
VAGINAL DRUG DELIVERY SYSTEMS 825
826 VAGINAL DRUG DELIVERY
improved gelling agents, particularly concerning to their mucoadhesive properties,
has been of great importance. For example, polycarbophil (Noveon AA - 1, Noveon,
Cleveland, OH), a mucoadhesive polyacrilic acid polymer, has been widely used as
a gelling agent in vaginal gel formulations. This polymer is acidic in nature, which
can be useful in reducing the elevated pH associated with bacterial vaginosis [98] .
Additionally, acidic polycarbophil gels may be used in the treatment of dry vagina
and menopause - related stress incontinence [99] .
Also, gel microemulsions have been recently reported as safe and devoid of
mucosal toxicity drug delivery systems, presenting intrinsic spermicide activity and
the possibility of improving vaginal bioavailability of poorly soluble antimicrobial
agents [100] .
Although most currently available vaginal gels rapidly release their active
substance(s), they can also be formulated to achieve modifi ed drug release profi les
[101] . It is not clear how controlled release is achieved, but the analysis of most
formulations that claim to possess this feature suggests a combination of dissolution
and diffusion control [102] . Gels are also known to be promising drug delivery
systems in protein and peptide administration through the vagina, proving to be
adequate to accommodate and stabilize sensible molecules such as leuprolide
[103] .
5.12.4.5 Liquid Systems
Vaginal douching with liquids containing antimicrobial drugs such as povidone -
iodine has been a common practice among women, with the intention of improving
personal hygiene and treat vaginitis [104] . These liquids are almost immediately
removed from the vagina after administration, thus being inadequate for controlled
release. Although vaginal washing is frequently performed by women all over the
world, its practice is discouraged, as it is associated with increased risk of acquiring
HIV, particularly when soap or other substances rather than water are used [105,
106] . Also, bacterial vaginitis and other adverse reproductive health effects are possible
when vaginal douching is a frequent practice [107, 108] .
In addition, several solutions are utilized by gynecologists in their offi ce practice.
For example, glacial acetic acid solutions (3 – 5%) are used to identify cervical dysplasia
during colposcopy, and Lugol solution is employed to perform Schiller ’ s test
(diagnosis of cervix cancer).
5.12.4.6 Vaginal Rings
Vaginal rings are doughnut - shaped drug delivery systems designed to provide controlled
release of drugs. Developed systems are made of fl exible, inert, and nonirritating
polymeric materials, presenting different dimensions, usually 54 – 58 mm in
diameter and 4 – 9.5 mm in cross - sectional diameter [109, 110] . Vaginal rings present
several advantages particularly important for hormonal contraceptives delivery:
(1) They do not require daily attention, allowing higher compliance than with daily
dosage forms; (2) fl exibility of current rings allow them to be easily inserted and
removed by the woman herself, not requiring medical assistance as in the case of
subcutaneous or intrauterine devices; (3) the continuous and prolonged delivery
(three weeks to one year) of hormones avoids the high peak concentrations and
fl uctuations seen with daily oral administration; (4) rings are not associated with
adverse local effects, including cytological and normal fl ora changes; and (5) contraceptive
rings may be removed from the vagina during sexual intercourse and up
to 2 h, without compromising their pharmacological effect [110 – 112] . Although
vaginal rings have been essentially investigated and used for the delivery of sexual
hormones with contraceptive purposes or as hormone replacement therapy, these
drug delivery systems can be also useful for the administration of other drugs
such as bromocriptine mesylate, danazol, oxybutynin, antigens, and microbicides
[113 – 117] .
In the 1960s, fi rst reports that implants made of polysiloxane, containing sexual
steroids, could release their content at constant rates in saline solutions provided
early information that led to the development of the fi rst vaginal rings [118] . Vaginal
rings were initially developed in the 1970s as contraceptives. The fi rst system was
composed of a silicone rubber ring containing medroxyprogesterone acetate as the
active substance [119] . Nonetheless, the fi rst vaginal rings have just recently reached
the market, due to several unpredictable obstacles such as formulation diffi culties,
safety issues, and poor ovulation suppression [120, 121] . Table 3 presents some
vaginal rings currently available in the market.
Vaginal rings may present several designs, as seen in Figure 4 . The fi rst
vaginal rings were made of a homogeneous matrix containing the mixture of
poly(dimethylsiloxane) (matrix - forming polymer) and the active drug, usually
referred as a matrix design. Unfortunately, these rings showed an initial burst effect
due to rapid release of the drug contained in the system ’ s surface followed by persistent
linear decrease of the drug release rate. This later phenomenon is related to
the gradually thickening of a drug - depleted boundary between the inner drug -
loaded region and the release surface, which is created by continuous drug release
from the outer layers. Thus, their use in clinical practice was compromised, namely
TABLE 3 Selected Vaginal Rings Currently Available
Commercial
Name
Active
Substance(s)
Clinical
Indications Availability Company
Nuvaring Etonogestrel
and ethinyl
estradiol
Contraception United States,
Europe,
Brazil, Chile
Organon
Progering Progesterone Contraception
in lactating
women
Chile Laboratorios
Silesia
Femring Estradiol
acetate
Relief of vaginal
and urogenital
symptoms in
menopausal
women
United States,
Netherlands
Warner Chilcott
Laboratories
Estring Estradiol Relief of vaginal
and urogenital
symptoms in
menopausal
women
United States,
Canada,
Europe,
South Africa
Pfi zer
VAGINAL DRUG DELIVERY SYSTEMS 827
828 VAGINAL DRUG DELIVERY
as contraceptive devices. Later, and in order to improve control of the drug release,
a layer of poly(dimethylsiloxane) without active substance was applied over the core
containing the drug, acting as a drug release – limiting sheath (core or reservoir
design). This strategy allowed achieving a near zero - order drug release profi le. The
diffusion rate of reservoir design rings is dependent on the drug concentration in
the core, its partition coeffi cient between the core and membrane, the thickness and
surface area of the membrane, and the diffusion coeffi cient of the drug in the membrane.
In order to achieve a constant release rate, the drug should be much more
permeable through the core than through the membrane [122] . Rings with several
independent reservoirs containing different drugs have been obtained, thereby
allowing the administration of two or more active substances from the same device.
Also, another design has been developed in order to overcome drug release drawbacks,
comprising a core of poly(dimethylsiloxane), an intermediate layer of
poly(dimethylsiloxane) containing the active substance, and an outer drug release –
limiting membrane of poly(dimethylsiloxane) (sandwich or shell design). As the
drug is closer to the releasing surface, this strategy is particularly suited for substances
presenting poor polymer diffusion characteristics [123] . As with reservoir
design rings, a near zero - order drug release profi le is obtained.
Besides poly(dimethylsiloxane), other elastomeric polymers have been employed
in the manufacturing of vaginal rings, such as poly(dimethylsiloxane/vinylmethylsiloxane),
styrene – butadiene – styrene block copolymer, and poly(ethylene - co - vinyl
acetate) [123 – 125] . In fact, poly(ethylene - co - vinyl acetate) (commonly referred as
EVA) appeared in the mid 1990s as an alternative to poly(dimethylsiloxane), when
the manufacturer of this last material stopped supplying it for human use, demonstrating
it to be very suitable for the production of controlled - release systems.
At the laboratory scale, silicone vaginal rings are usually obtained by injection
molding, where poly(dimethylsiloxane) is mixed with a polymerization catalyst and
the drug, being subsequently injected in ring - shaped molds. The mixture is allowed
to cure for a period of time at a preestablished temperature, which can range from
FIGURE 4 Cross sections of three vaginal rings presenting different designs: matrix design
(left), core or reservoir design (center), and sandwich or shell design (right). Light gray represents
drug – polymer mixture and dark gray represents polymer only.
room temperature to 150 ° C and over (higher temperatures allow increasing the
speed of the ring curing). In fact, curing time and temperature should be optimized
as they infl uence the fi nal performance of the ring, particularly drug release (Figure
5 ). This step leads to the formation of a three - dimensional network by means of a
cross - linking reaction between polymer chains [126] . Afterward, other layers can be
added, a step that is usually performed by injection molding or a dipping process.
Although vaginal rings produced at the laboratory scale are useful during preclinical
and clinical experimentation, the pharmaceutical industry needs other manufacture
solutions to allow large - scale and fi nancially viable production of these drug delivery
systems. This process scale - up requires proof of bioequivalence between rings
obtained by both processes [109] .
The most common process of obtaining vaginal rings in the pharmaceutical
industry is hot - melt extrusion (or hot - melt spinning), where the polymer, either
alone or mixed with drugs or other additives, is melted (usually between 105 and
120 ° C) and forced by single - or twin - extrusion screws to pass through a die. After
leaving the die, the obtained coaxial fi ber is cooled and cut, the obtained fragments
being shaped as rings by gluing both ends with an adequate pharmaceutical adhesive.
Figure 6 presents a simplifi ed scheme of the manufacturing process of a reservoir
design ring similar to one used to produce Nuvaring (NV Organon, Oss, The
Netherlands), an EVA vaginal ring containing etonogestrel and ethiny lestradiol.
The core [polymer and active substance(s)] and the surrounding membrane polymer
mixtures are extruded separately through two single - screw extruders (coextrusion)
FIGURE 5 Effect of curing conditions on in vitro release profi le of estradiol ( . g) from
core design rings containing estradiol and progesterone: T: polymer - only outer sheath
[poly(dimethylsiloxane/vinylmethylsiloxane)]; E2 + P: core sheath, comprising mixture of
polymer [poly(dimethylsiloxane/vinylmethylsiloxane)], estradiol, and progesterone.
(Reprinted with permission from S. I. Saleh et al., Journal of Pharmaceutical Sciences , 92,
258 – 265, 2003. Copyright 2003 by Wiley - Liss.)
T
E2 + P
Days
Cured at 105°C, 30 min
Cured at 140°C, 15 min
Cured at 140°C, 30 min
Amount released, .g/day
800
700
600
500
400
300
200
100
0
0 5 10 15 20 25 30 35
VAGINAL DRUG DELIVERY SYSTEMS 829
830 VAGINAL DRUG DELIVERY
that are connected to a spinning pump. In these cylindrical pipelines the polymers
are melted and extruded through a die at an accurate fl ow rate. Then, the core and
membrane polymers are combined in a spinneret, forming a coaxial fi ber. The ratio
between fl ow rates of both spinning pumps determines the thickness of the membrane.
After leaving the spinneret, this fi ber is cooled, fi rst by air exposure and then
by immersion in a water bath. At this stage, the fi ber diameter is adjusted to the
desired value by elongation with take - up rolls. In fact, after leaving the die, the
obtained fi ber expands its diameter as a result of the viscoelastic behavior of
the polymers used [122, 127] .
The drug release profi le is conditioned by the polymeric structure of the systems,
which is infl uenced by several parameters such as polymer composition, melt spinning
process variables (namely feeding of polymer mixture and spinning velocity,
extrusion temperature, spinline stress, cooling rate, and drawing back elongating
force), and storage conditions [122, 128] . Also, drug release characteristics are largely
infl uenced by the active substances ’ molecular weight and diffusion coeffi cient and
solubility in the polymer. For example, extremely hydrophobic drugs and molecules
with molecular weight above 450 Da are poorly released from silicone. Modulation
of the drug solubility by adding various excipients (e.g., propylene glycol, polyethylene
glycol, gelatin, and fl uid silicone) can be used to change the release profi le [129,
130] . The physical state of the drug is also an important parameter related to drug
release. Taking the example of steroids, these compounds can be either in a solid
crystalline state or in a molecularly dissolved state. In the fi rst case, the concentration
of the drug is fi xed by its saturation solubility, making it possible to control the
release rate by the thickness and the permeability of an outer membrane. When two
drugs are present, this concept cannot be used: Both drugs need to be completely
dissolved, their release rates being controlled by their concentrations [123] .
FIGURE 6 Schematic of manufacturing process of reservoir design ring by hot - melt
extrusion.
5.12.4.7 Vaginal Films
Vaginal fi lms are polymeric drug delivery systems shaped as thin sheets, usually
ranging from 220 to 240 . m in thickness. These systems are often square (approximately
5 cm . 5 cm), colorless, and soft, presenting a homogenous surface. Vaginal
fi lms have some advantages, such as portability, ease of application, long time of
retention in the vagina, and good drug stability [131] . Once placed in the vagina,
the fl uid present in the mucosa hydrates the polymer, covering the mucosa with the
active substance. This coating may also be helped during sexual intercourse due to
the spreading motion of the male penis.
Vaginal fi lms are produced with polymers such as polyacrylates, polyethylene
glycol, polyvinyl alcohol, and cellulose derivatives. Proper combination of these
polymer is essential to achieve adequate mucoadhesion and optimal drug release
profi les. Vaginal fi lms can be produced by casting [132] , in which polymer solutions
containing the active substance(s) are poured into adequate molds and dried until
a thin, solid, and fl exible polymeric sheet is formed. Afterward, the sheet is cut in
small pieces (individual fi lms) and peeled off.
Vaginal fi lms have been mostly used as spermicides, although they present inferior
contraceptive success than hormonal methods, condoms, and intrauterine
devices. A contraceptive fi lm containing 28% nonoxynol - 9 in a polyvinyl alcohol
base (VCF, Apothecus Pharmaceutical) is currently available in the United States.
Alternative contraceptive fi lms containing different drugs and fi lm - forming polymers
have also been investigated, in order to obtain more effective and acceptable
formulations [133] .
5.12.4.8 Medicated Vaginal Tampons
Vaginal tampons have been studied for their feasibility as vaginal drug delivery
systems. First experiments used commercially available tampons that were impregnated
with active substances by a simple dipping process [134] . Currently, a medicated
vaginal tampon, approved as a medical device by the Food and Drug
Administration (FDA), is available (Ela Tampon, Rostam, Israel). This bifunctional
tampon contains a polymeric delivery system (strips) that absorbs menstrual fl uid
while gradually releasing lactic acid and citric acid. These two drugs act by preserving
the acid vaginal milieu, preventing the proliferation of potential pathogenic
bacteria [135] .
5.12.4.9 Vaginal Foams
Vaginal foams have been tested to deliver drugs in the vagina, mainly microbicides
or spermicides [136, 137] . The effi cacy of these systems was shows to be limited when
compared to other available options, leading to a decline of their use. Nonetheless,
foams are easy to use, providing a good coverage of the vaginal mucosa with
minimal leaking. Also, most foam bases are nonirritating, unlike some other conventional
formulations which are reported to cause burning and itching. Thus,
improving their formulation can be an interesting approach to obtaining new vaginal
drug delivery systems. In fact, foams containing antimicrobials, local anesthetics, and
hormones have the potential to gradually substitute several currently available
dosage forms, namely creams and ovules.
VAGINAL DRUG DELIVERY SYSTEMS 831
832 VAGINAL DRUG DELIVERY
5.12.4.10 Vaginal Sponges
Vaginal sponges were once widely used as vaginal contraceptives, the oldest reference
to their use in the Talmud (c. 500 b . c. ), where a sponge soaked in vinegar was
recommended in order to prevent pregnancy [138] . Although these devices have
been disapproved by some researchers, since their use encouraged colonization and
proliferation of bacteria in the vagina, predisposing women to vaginitis and other
genital disorders [139] , sponges containing spermicides are still used as contraceptives.
These contraceptive devices have the ability to deliver the active drugs while
absorbing semen and blocking the cervical canal. Also, vaginal sponges are inexpensive
devices; less messy than creams, gels, and foams; and easier to insert and remove
than diaphragms [140] . Nonetheless, these systems have demonstrated less effi cacy
than other contraceptive methods, such as the diaphragm [141] .
A vaginal sponge (Today, Whitehall Robins) made of soft polyurethane foam
(diameter of 5.5 cm and 2.5 cm thick) and saturated with 1 g of nonoxynol - 9 was
authorized as a spermicide in the United States in 1983 [140] . Advantages of this
formulation include the possibility of being used up to 24 h without requiring additional
application of spermicide. Although in 1995 the manufacturer discontinued
its production because of increased costs related to FDA guideline compliance,
Allendale Pharmaceuticals purchased the rights to the sponge, reintroducing the
product in U.S. and Canadian markets [142] . Similar sponges are available outside
the United States containing benzalkonium chloride alone (Pharmatex, Innotech
International) or a combination of nonoxynol - 9 and sodium cholate (Protectaid,
Pirri Pharma).
5.12.4.11 Other Strategies and Vaginal Drug Delivery Systems
Bioadhesion may be defi ned as the state in which two materials, at least one of which
is biological in nature, are held together for extended periods of time by interfacial
forces. Mucoadhesion is a particular case of bioadhesion where one of these materials
is a mucous membrane or the mucus [143] . Mucoadhesive drug delivery systems
can circumvent the poor retention that most traditional vaginal formulations present,
improving residence time, and even their specifi c location in the mucosa. Prolonged
time of contact and intimate interaction with the vaginal mucosa are able to increase
drug absorption and bioavailability and thus its therapeutic effect. Currently used
mucoadhesive materials are polymeric in nature, many of them previously used for
other specifi c purposes (e.g., as gelling agents in vaginal gels). Several polymers,
alone or in combination, have been used or investigated for both systemic and local
vaginal drug delivery in order to obtain pharmaceutical systems with mucoadhesive
properties (see Table 4 ) [144] . Virtually almost all drug delivery systems may benefi t
from these mucoadhesives, particularly gels, creams, tablets, vaginal suppositories,
and fi lms. To date, polyacrylates are the most used and explored mucoadhesive
polymers, although others such as chitosan, carrageenan, or sodium alginate have
proved to be advantageous. Also, some widely used mucoadhesives in vaginal formulations,
such as polycarbophil (Noveon AA - 1, Noveon, Cleveland, OH) present
the advantage of being useful as controlled - and sustained - release matrices [145] .
Recently, thiolated polymers, usually referred as thiomers, have been studied in
vaginal mucoadhesive systems, exhibiting higher mucoadhesion than nonmodifi ed
polymers. Although thiomers can improve the mucoadhesion of the original polymers,
they do not interfere with other advantageous characteristics such as the
ability of controlled release [146, 147] .
Environmentally sensitive drug delivery systems are characterized by their ability
to respond to changes in pH, temperature, ionic strength, solvent composition, magnetic
fi elds, light, and electric current, among others, most commonly used for the
fi rst two [148] . Thermoreversible systems are fl uids that can be introduced into the
body in a minimally invasive manner prior to solidifying or gelling within the local
of administration because of temperature increase from room temperature to body
temperature [149] . This innovative approach is particularly interesting in vaginal
drug delivery, as a liquid system is easily introduced in the vaginal cavity, while the
in situ formed gel facilitates retention. Indeed, some thermosensitive formulations
have already been suggested to obtain vaginal drug delivery systems, poloxamer
gels being the most extensively studied. A clotrimazole - containing vaginal thermosensitive
gel (poloxamer 188 and polycarbophil) was demonstrated to be useful for
the effective and convenient treatment of vaginal candidiasis in female rats, with
the advantages of facilitating administration and reducing dosing interval when
compared with conventional therapy [150] .
The incorporation of drug - loaded liposomes in adequate formulations can
improve their stability, allowing their applicability in vaginal drug delivery [151] .
Additionally, entrapment of drugs in liposomes may improve their solubility and
TABLE 4 Examples of Mucoadhesive Polymers Used or Investigated in Formulation of
Vaginal Drug Delivery Systems
Polymer Classes Examples
Carrageenan Iota - carrageenan
Cellulose derivatives Sodium carboxymethylcellulose (NaCMC)
Methylcellulose (MC)
Hydroxypropyl methylcellulose (HPMC)
Hydroxypropyl cellulose (HPC)
Hydroxyethyl cellulose (HEC)
Chitosans
Gelatin
Gums Pectin
Tragacanth
Dextran
Xanthan
Hyaluronic acid and derivatives Sodium hyaluronate
Polyacrylates Carbopol 974P
Polycarbophil (Noveon ® AA-1)
Polyethylene glycols (PEGs) PEG 600
Povidone
Alginates Sodium alginate
Starch
Sulfated polysaccharides Cellulose sulfate
Thiomers Carbopol ® 974P – cysteine
Chitosan – thioglycolic acid (TGA)
Chitosan – 4 - thio - butylamidin - conjugates (TBA)
Note : As reviewed in ref. 144 .
VAGINAL DRUG DELIVERY SYSTEMS 833
834 VAGINAL DRUG DELIVERY
availability at the site of administration, reducing the administered dose and systemic
effects. In order to administer liposomes, adequate dosage forms must be
selected. Polyacrylate gels have emerged as good candidates, showing the potential
to accommodate liposomes for vaginal drug delivery [151, 152] . Incorporation of
liposomes in these gel bases can be achieved by gently mixing both components,
which is an advantage over other dosage forms. Also, these novel drug delivery formulations
enable sustained drug release, combining both the liposome limiting
release effect and the bioadhesive properties of these gels.
When liposomes are considered for vaginal administration, their stability should
be assessed under in situ conditions presented by both pre - and postmenopausal
women. Drug formulation can help improve their stability. Several gels made of
Carbopol 974P NF were shown to be suitable in improving the stability of liposomes
containing clotrimazole, metronidazole, or acyclovir in a vaginal environment simulating
media when compared to liposomal dispersions [153, 154] . Also, compatibility
between liposomes and vaginal mucosa (animal or human) should be checked [155] .
Nonetheless, liposomes still have some stability - related problems that limit their
shelf life. Formulation of liposomes as proliposomes can prevent these stability
issues. Proliposomes are dry, free - fl owing products which on addition of water disperse
to form liposomal suspensions [156] . As proliposomes are dry powders, they
can be formulated as solid dosage forms, which can be more convenient to administer
in the vagina. Recent work by Ning et al. demonstrated that clotrimazole -
containing proliposomes can be suitable for vaginal administration, allowing a
rapid conversion to sustained - release liposomes upon contact with physiological
fl uids [157] .
Niosomes (nonionic surfactant vesicles) have been experimented as controlled -
and prolonged - release drug delivery systems to administer insulin through the
vagina. These particles might be good carriers for protein delivery, showing the
potential to enhance the effects of these drugs when compared to the vaginal administration
of free insulin. Results obtained in rats showed that niosomes containing
insulin can achieve comparable bioavailability with subcutaneous administration of
this protein [158] . Also, experiments performed with vaginal gels containing both
niosomes and liposomes have been shown to be a promising strategy for the prolonged
release and safe vaginal administration of clotrimazole [159] .
Microparticles and nanoparticles present some advantageous features, namely
mucoadhesive properties. They have demonstrated some potential in vaginal drug
delivery, particularly in the formulation of delivery systems for vaccines or peptides
and proteins [160, 161] . Nonetheless, these particles have to be incorporated in
adequate carrier systems in order to be delivered. This task has been shown to be
complex, it being hard to achieve controlled - release and steady - release profi les.
Cyclodextrins are commonly used in pharmaceutics, their applicability being no
exception in vaginal drug delivery. Drug/cyclodextrin complexes allowed enhancement
of solubility and achievement of prolonged - release properties of drugs such
as clotrimazole and itraconazole when included in suitable vaginal drug delivery
systems such as gels, creams, or tablets [162 – 164] .
Cervical barrier devices such as diaphragms can be modifi ed to include a reservoir
that releases active substances such as spermicides or microbicides. Although
these devices have limited applicability, they can be particularly helpful in prevent
ing pregnancy, as they combine a physical barrier device with a chemical spermicide
that faces the vagina, after correct placement in the cervix [165] . Lee et al. presented
a diaphragm made from silicone containing 35% nonoxynol - 9 that was able to
achieve controlled drug release depending on the device design and size [166] . The
diaphragm was prepared by using compression molding in a single - cavity aluminum
mold.
Patches can provide delivery of very toxic drugs in very limited areas of the
vaginal mucosa without leaking to the circumventing tissue. McCarron et al.
described a bioadhesive patch that delivered 5 - aminovulinic acid to intraepithelial
neoplasia lesions. The proposed delivery system is based on a poly(methyl -
vinylether/maleic anhydride) matrix that contains the active drug, providing local
retention of up to 4 h and enhancing effi cacy without damaging healthy epithelium
[167] . Patchs with bilayer design may also be an option for the treatment of these
neoplasic lesions, where a bioadhesive drug - loaded matrix bonded to a drug -
impermeable backing layer is able to prevent drug spillage to healthy tissues, promoting
unidirectional and deep drug penetration. Woolfson et al. presented a
5 - fl uorouracil vaginal patch with a bilayer design, comprising a fl exible polyvinyl
chloride (PVC) emulsion as a backing layer and a drug - loaded bioadhesive fi lm
made of 2% Carbopol 981 and 1% glycerin as a plasticizer [168] .
Propess (Ferring Pharmaceuticals) is a commercially available controlled - release
vaginal insert presented as a thin macrogol 8000 matrix (rectangular in shape with
radiused corners) containing 10 mg of dinoprostone being used for labor inducement.
This drug - loaded matrix is included within a knitted polyester retrieval system
that ends in a long tail to help retrieval at the end of the dosing interval. This insert
is capable of releasing the active substance at a rate of 0.8 mg/h over 12 h, after being
exposed to vaginal moisture [169] . Clinical studies indicate that Propess is as equally
effective and safe in achieving cervical ripening as other commercially available
vaginal drug delivery systems containing dinoprostone [170] .
Multiple emulsions have been studied as drug vehicles for vaginal administration
that are able to include several active substances in the different phases. An antimicrobial
water – oil – water (W/O/W) multiple emulsion containing lactic acid in the
internal aqueous phase, octadecylamine in the oily phase, and benzalkonium chloride
in the external aqueous phase was tested, proving to stabilize the included
active substances while providing adequate viscosity properties to vaginal administration
[171] . Nonetheless, the structure of these formulations is destroyed at high
shear rates (e.g., shear rates observed during coitus), losing or diminishing their
intended activity and thus limiting their applicability.
Recently, an interesting approach for protein delivery using genetically engineered
normal vaginal fl ora as delivery systems has been proposed. This strategy is
based on the natural affi nity that these microorganisms have to adhere tightly to
the epithelial surface, providing a direct delivery of the drugs to the mucosa and
thus minimizing enzymatic and bacterial degradation. Lactic acid bacteria, transformed
with plasmids that contained a gene encoding for the therapeutic protein
to be administered, have been used as delivery systems. Obtained results showed
enhancement of protein delivery when compared with a conventional solution containing
the same molecule. This strategy looks particularly appealing for the development
of vaccines that induce mucosal immunization [172, 173] .
VAGINAL DRUG DELIVERY SYSTEMS 835
836 VAGINAL DRUG DELIVERY
5.12.4.12 Packaging and Vaginal Applicators
Vaginal packaging and applicators are an integral part of vaginal products. Packaging
is designed to accommodate and protect formulations, while applicators should
allow their convenient administration in the vagina. Several materials have been
used to manufacture these devices, such as plastics (e.g., polypropylene and polyethylene)
and nonlatex rubber. Besides compatibility, stability, and suitability issues,
these materials should be selected regarding the fi nal cost of packaging and
applicators.
Applicators are intended to be introduced in the vagina, adequately deliver the
product, and then be removed. Their design relates to safety (e.g., relationship with
product purity and stability, avoidance of local trauma associated with insertion or
use), effi cacy (e.g., consistent delivery of the required amount of product in the
intended location), and acceptability (comfort, ease of use, convenience, aesthetic
appeal) [174] . In general, they can be divided as single - use or reusable applicators.
Single - use applicators are usually prefi lled, while reusable applicators are fi lled by
women prior to vaginal insertion. Several applicator designs have been used, such
as barrel - and - plunger and squeeze tube, but they all should be easy to insert, comfortable,
and deprived of cutting edges. Also, some specifi c formulations, such as
those intended for vaginal douching, require other types of applicators. Typically,
squeeze plastic bottles with variable volumes (approximately 100 – 200 mL) are
used.
5.12.5 PHARMACEUTICAL EVALUATION OF VAGINAL DRUG
DELIVERY SYSTEMS
Evaluation of pharmaceutical systems is consensually recognized as an important
component of their development and quality control. Although evaluation of general
features (e.g., drug content) is also required for vaginal formulations, this section
focuses only upon some of the most important parameters that are intimately
related to drug delivery systems specifi cally designed to be administered by this
route.
5.12.5.1 Legal and Offi cial Compendia Requirements
There is a lack of well - defi ned guidelines and regulations for vaginal products in
most countries as well as offi cial compendia information and requirements on
quality control and other important aspects of vaginal drug delivery systems [39] .
In fact, the latest editions of the U.S. Pharmacopeia, European Pharmacopoeia, and
Japanese Pharmacopeia include limited or even no information related to the quality
control and evaluation of vaginal drug delivery systems. Thus, most of the currently
used evaluation procedures require standardization, making it diffi cult to compare
results obtained by different research groups.
5.12.5.2 Drug Release and Permeability
When formulating vaginal drug delivery systems, it is important to consider the
release of the active substances, as different formulations can greatly affect the
release rate and ultimately their pharmacological effects. The choice of the dissolution
method should be done on a case - by - case basis, while dissolution profi les can
then be fi tted to commonly used mathematical models, as reviewed by Costa and
Sousa Lobo [175] . Conventional procedures and apparatus have been adapted
taking into consideration vaginal physiological specifi cations such as pH, fl uid
volume, and temperature, among others. The experimental method may not inevitably
imitate the vaginal environment, but it should test the main key performance
indicators of the formulation. Although offi cial methods are not available, some
have been recommended for specifi c vaginal drug dosage forms [176] . For semisolid
vaginal formulations, the Franz diffusion cell is considered the most promising
apparatus for drug release investigation. In the case of hydrophilic vaginal suppositories,
a basket apparatus, a paddle apparatus, or fl ow - through cells can generally
be considered as suitable; for hydrophobic vaginal suppositories, modifi ed fl ow -
through cells would be preferable. Dissolution methods that use a basket instead of
a paddle can be advantageous for vaginal solid formulations that tend to fl oat,
particularly those that may include a modifi cation that prevents the formulations to
form a cake inside the basket, limiting their dissolution [177, 178] . Drug release from
vaginal rings is usually determined by placing these systems in conical fl asks containing
an adequate dissolution medium. Flasks are then placed in a water bath controlled
at 37 ° C and shaken during the time of the assay, with samples being taken
and release medium being replaced typically every 24 h [113, 129] . Correlation of
these in vitro release tests with in vivo results proved to be satisfactory for the
majority of the evaluated vaginal rings [125] .
Absorption of drugs through the vagina is an important parameter to be evaluated,
particularly when a systemic effect is required. Also, assessment of the absorption
potential of drugs intended to locally exert their effects needs to be evaluated,
as this event can lead to unwanted systemic effects. The evaluation of both formulated
and unformulated drugs can be performed by in vitro or in vivo methods.
In vitro permeability studies have been performed in fl ow - through diffusion cells
using either animal or human vaginal mucosa [179, 180] . After isolation and adequate
treatment of vaginal mucosa specimens, small tissue disks are mounted in the
apparatus in order to perform the permeation experiments. At the end of these
procedures, routine histological examination of the used tissues can be performed
in order to identify any changes in the normal structure of the vaginal mucosa.
Several alterations can suggest potential mechanisms of permeation of tested drugs
[181, 182] . Animal mucosa used in these experiments can be obtained from several
species, namely rabbits and pigs. Also, porcine vaginal mucosa was demonstrated to
be a good in vitro permeability model of human vaginal mucosa, particularly when
hydrophobic substances are tested [50] . This feature can be explained because both
mucosal tissues are very similar in many aspects, namely their lipid composition and
histological structure. On the other hand, high molecular weight or charged molecules,
such as oxytocin, may show different permeability profi les when tested with
either porcine or human vaginal mucosa. Thus, researchers must be careful when
interpreting and extrapolating results to human tissues because unexpected differences
often occur. When human vaginal mucosa is used, samples are usually obtained
from excess tissue removed from postmenopausal women after vaginal hysterectomy
[181, 182] . Use of postmenopausal vaginal mucosa is advantageous as it is less
altered (particularly in thickness) by hormonal stimulation, leading to more uniform
PHARMACEUTICAL EVALUATION OF VAGINAL DRUG DELIVERY SYSTEMS 837
838 VAGINAL DRUG DELIVERY
results. Nonetheless, these features do not refl ect the normal histological architecture
of fertile women, being able to signifi cantly alter the permeability profi les for
many drugs. Along with vaginal mucosal tissue, other model membranes, such as
vaginal and cervical cell monolayer membranes, have been suggested in recent years
in order to predict in vivo absorption [172] .
It is also noteworthy that many of the in vitro results of vaginal permeability
studies cited in the literature have limitations related to the experimental conditions
that were used, particularly pH values at which they have been performed. Differences
in permeability values are particularly expected when ionization characteristics
change between experimental pH and vaginal pH [183] . However, in vitro
results should only be considered as evidence that the vaginal mucosa is able to be
permeated.
In vivo studies performed in animals are an important step before considering
human experimentation. Animal species commonly used in vaginal permeability
studies include rabbits, rats, and mice [53, 158, 184] . Although potentially more
accurate in predicting human vaginal absorption of drugs, animal experimentation
have some limitations. A major problem is the variability of the vaginal epithelium
properties throughout the estrous cycle, thus infl uencing drug absorption [184, 185] .
In order to minimize this variability and standardize the thickness of the epithelium,
animals are usually ovariectomized. Also, vaginal enzymatic activity is an important
parameter in choosing animal models. It is recommended that the enzymatic profi le
of such animal should be comparable to that of the woman. Taking this into consideration,
rats and rabbits seem to be good models for vaginal permeability studies,
particularly when protein and peptide drugs are considered [27] .
5.12.5.3 p H and Acid - Buffering Capacity
As already referred, pH is an important parameter concerning the health and
normal physiology of the vagina, being important that vaginal formulations do not
interfere with its normal value. Also, the pH of the vagina can be elevated due to
changes in its normal physiology (e.g., bacterial vaginitis) or the presence of semen.
Vaginal formulations presenting good acid - buffering capacity have the potential to
reestablish normal pH or to prevent it from rising.
The acid - buffering capacity of a vaginal formulation can be measured by simple
titration with an inorganic alkali, such as sodium hydroxide. The physiologically
relevant acid - buffering capacity can be defi ned as the amount of alkali required to
elevate the pH from its initial value to the maximum desirable value when considering
the healthy vagina [186] . Also, mixtures of vaginal formulations with semen may
be useful in determining their buffering capacity, this proceeding being particularly
helpful when testing products used during sexual intercourse.
Variations in vaginal pH of the vagina can infl uence drug stability, particularly
when extreme values are observed. Thus, the adjustment of the formulation pH can
also be important in order to assure maximum stability or pharmacological activity
of the active substance(s). As an example, the administration of antibodies in the
vaginal milieu can compromise their activity because of the acidic pH. Generally
monoclonal human antibodies are more stable at pH 4 – 7, losing binding and neutralizing
activity below pH 4 [187] . These fi ndings underline the importance of pH
buffering when delivering pH - sensible molecules such as antibodies.
5.12.5.4 Rheological Studies
Rheological properties of semisolid vaginal formulations are crucial to their suitability
as drug delivery systems. They experience in vivo a wide range of shear rates
(from less than 0.1 s . 1 to about 1000 s . 1 ), both steady and transient, while being
diluted with vaginal fl uids, which infl uences their rheological properties and hence
their spreading and retention. Events such as passive seeping, sliding, squeezing
between vaginal walls, and coitus, among others, infl uence the rheological performance
of vaginal formulations [188] . Thus, knowledge of rheological properties of
semisolid vaginal drug formulations may assist in improving their design, being
helpful in the process of predicting which formulations can retain their structural
stability over time, particularly in the physiological environment [189, 190] .
Qualitative and quantitative composition of a semisolid vaginal formulation can
strongly infl uence its rheological properties [191] . This fact is particularly important
when considering the optimization of a drug - containing formulation and its placebo
formulation. These two systems should only differ in the absence of the active
substance(s). However, this small difference can sometimes originate different
rheological properties that can greatly infl uence the formulation ’ s performance and
even the results of clinical trials [192] .
Rheological properties of a formulation can be studied either by simple fl ow
measurements or by dynamic oscillatory measurements, although the latter are
preferable as they allow a complete characterization of both elastic and viscous
components. Also, they are nondestructive and, if the strain is not too high, the
sample is not disturbed [193] . As already noticed, in vivo conditions, particularly
temperature and fl uids that may be present in the vagina (vaginal fl uid, cervical
mucus, and semen), can infl uence the rheology of pharmaceutical formulations.
Indeed, these factors have to be taken into account when formulating a vaginal drug
delivery system. Thus, optimization should not only focus upon the rheology of
undiluted material but also include mixtures of formulations and fl uids that may be
present in the vagina [194] . However, these biological fl uids are not always available
and considerable differences between individuals limit their use. In order to abbreviate
these limitations, some simulants of vaginal fl uid [21] , cervical mucus [26] , and
semen [195] have been used.
5.12.5.5 Textural Studies
Textural profi le analysis is a widely used analytical method based on the measurement
of the forces involved during the compression/decompression of a probe in a
sample of the product to be tested. From the obtained results, important parameters
can be calculated, including hardness (force required to attain a given deformation),
compressibility/spreadability (the work required to deform the product during the
fi rst compression cycle of the probe), and adhesiveness of the product. Besides their
infl uence in the ease of removal from a container (e.g., vaginal applicators), the ease
of application, or retention, among others, it is consensual that textural properties
of formulations will infl uence their clinical performance. Therefore, it is important
to fully characterize these properties during the formulation process [196] . Also,
these textural parameters can be converted into rheological properties, such as
shearing stress, shear rate, and viscosity, using dimensional analysis, allowing the
comparison of results generated by both techniques [197] .
PHARMACEUTICAL EVALUATION OF VAGINAL DRUG DELIVERY SYSTEMS 839
840 VAGINAL DRUG DELIVERY
5.12.5.6 Mucoadhesion
As previously discussed, development of mucoadhesive drug delivery systems is a
promising strategy in order to enhance vaginal drug administration. Several methods
for the in vitro evaluation of mucoadhesive properties of vaginal formulations may
be found in the literature. Although these tests present some limitations, they can
offer easy and valuable tools in the initial formulation and evaluation of vaginal
drug delivery systems. Most methods can be categorized as tensile strength or shear
stress tests, where the force needed to separate a model membrane attached to the
formulation measures mucoadhesion. Different results can be obtained for the same
sample because of the different types of forces involved in these two methods [198] .
For this reason, both types of tests should be performed in order to achieve a more
complete characterization of the mucoadhesive potential of formulations.
Alternative methods have also been shown to be useful. For example, mucoadhesive
properties of semisolid vaginal formulations can be assessed by the rheological
characterization of the mucoadhesive interface, based on the assumption that
the interpenetration extension between polymer gels and their mixtures with mucin
can be detected by measuring differences in rheological parameters. Also, the texture
analysis of these formulations/mucin interfaces was demonstrated to be a useful
technique in measuring bioadhesion, with results in the same rank as the ones
obtained with the rheological technique [199] . Kast et al. tested vaginal tablets for
their mucoadhesiveness by a simple method, comprising the use of a dissolution
apparatus [146] . In this procedure, the formulation to be tested is attached to a
vaginal mucosa that is fi xed on a cylinder; the cylinder is then immersed in the dissolution
vessel and rotated in an adequate testing solution at approximately 37 ° C.
The mucoadhesion is evaluated by the detachment time of the formulation. An
alternative method based on a modifi ed balance, in which the tablet is attached to
a vaginal mucosa fi xed to one side plate, has also been proposed [200] . In this case,
mucoadhesion is measured by the weight required to detach the formulation.
5.12.5.7 Vaginal Distribution and Retention
Vaginal mucosa coating by a formulation is an important parameter to be determined,
since its action may depend on the effectiveness of this phenomenon. Nonetheless,
knowledge about the distribution and retention of commercially available
products is limited. After being administered, liquid, semisolid, or solid (after liquefaction)
formulations can spread throughout the vagina, with part of the material
being able to exit this tube, either to the exterior or to the upper genital tract [201] .
This distribution is governed by physical forces that include gravity, normal forces
from contacting tissues, surface tension, and shearing. Formulation fl ow due to these
forces is affected by many factors, including formulation physical properties, amount
of formulation applied, surface interactions, surrounding tissue properties, vaginal
secretions, baseline dimensions of the vagina, ambulation and posture changes of
the user, and sexual intercourse [202, 203] . Drug delivery systems intended for local
effect should ideally spread evenly throughout the vaginal mucosa. Also, evaluation
of the possible erosion of a product coating during its residence in the vagina, particularly
during sexual intercourse, is extremely important. Thus, vaginal distribution
and retention studies may clarify some questions, such as the amount of product to
cover evenly the vaginal surface, the time required to distribute to all areas and to
be removed from the vagina, and the effect of daily activities such as ambulation
and sexual activity in these phenomena.
Simple in vitro tests can be used to quantify the vaginal coating of a vaginal formulation.
It is important that these tests model the natural history of a product in
the vagina, from initial application and contact with fl uids that may be present in
the vagina to the period during and following sexual intercourse [204] . The distribution
and retention of solid drug delivery systems can be evaluated by a simple
method proposed by Ceschel et al. [205] , where the formulations are placed in a
vertical thermostated cellophane tube, the discharged liquid collected and measured
throughout the time of the experiment. The amount of discharge liquid is related
to the retention while the distribution of formulations can be assessed by dosing
the amount of active substance(s) in different sections of the tube at the end of the
experiment. Also, the contribution of gravity to the vaginal coating fl ows of vaginal
semisolid drug delivery systems can be evaluated by a simple and objective method
proposed by Kieweg et al. [203] . The proposed technique is based on the measurement
of the fl ow behavior of a formulation sample after being placed in an inclined
plane surface. Obtained results, together with mathematical models, can help formulators
to select primary candidate formulations before in vivo tests commence.
In vivo assessment provides more reliable and complete information about the
vaginal distribution and retention of drug delivery systems, although ethical and
economical issues limit its applicability to routine evaluation. In recent years, imaging
techniques, which have been used for other purposes for a long time, emerged as
valuable tools for the evaluation of vaginal distribution and retention. Magnetic
resonance imaging proved to be a helpful method, providing cross - sectional images
of drug delivery systems that are administered in the vagina, both in animals and
in humans, allowing precise reproducible data regarding the spread of vaginal formulations
to be achieved [202] . As magnetic resonance imaging refl ects the images
of a chemical label, such as gadolinium, and not necessarily that of the drug delivery
systems or carried drugs, it is necessary to perform the association of the chemical
label with vaginal products and its validation [206] . Another interesting imaging
technique that has been used in this type of assessment is gamma scintigraphy,
shown to be a useful tool for evaluating and comparing the distribution, spreading,
and clearance of vaginal delivery systems [207, 208] .
Imaging methods, such as gamma scintigraphy and magnetic resonance imaging,
are useful but have some limitations concerning their resolution, being unable to
quantify or even identify the presence of vaginal coating layers of just a few hundred
micrometers. In order to overcome such limitations an optical instrument capable
of detecting coating layers as thick as 50 . m has been developed by Henderson and
co - workers [209] . The device is inserted and remains stationary in the vagina, where
both local video images and fl uorescence intensity measurements of fl uorescein -
labeled formulations are obtained. Since the tube that is inserted in the vagina is
shaped and sized like a phallus, the vaginal coating measured is analogous to that
observed during sexual intercourse. Nonetheless, in vivo fl uorescence - based methods
present limitations, particularly because of the diffusion of the dye out of the formulation
and photobleaching, limiting the interval over which measurements can
be performed accurately. A new technique based on low - coherence interferometry
that can overcome these diffi culties is being developed, allowing extended time
PHARMACEUTICAL EVALUATION OF VAGINAL DRUG DELIVERY SYSTEMS 841
842 VAGINAL DRUG DELIVERY
studies to be performed [210] . This easy, label - free, high - resolution method uses
broadband light in an interferometry scheme to achieve depth - resolved refl ection
measurements. Future studies focus on the development of an easy - to - use endoscopic
device that may be used in clinical studies.
5.12.5.8 Safety and Toxicology
It is accepted that a new pharmaceutical product should be assessed for its effects
on the vaginal mucosa before being approved by drug - licensing agencies. Nonetheless,
many of the older products that have been used for a long time by women all
over the world do not have this type of information available. Safety issues are
particularly important when a vaginal drug formulation is used repeatedly, as in the
case of microbicides, spermicides, and contraceptive vaginal rings. Also, it is important
to test vaginal applicators for their safety as they are considered an integral
part of vaginal products, being able to induce alterations in the mucosa [211] .
Local effects assessment should include not only short - term but also long - term
protocols, as some formulations are intended to be used for large periods, in order
to assess their real impact on vaginal health. Although systemic exposure to drugs
intended for topical action is expected to be minor, vaginal formulations should also
be assessed for systemic effects due to possible absorption. Alterations in blood
parameters and liver and renal function should be investigated [212] . Also, drugs
and formulations to be administered through the vaginal route must be assessed for
fertility and teratogenic effects in animal models, before entering clinical trialing
and human use [213] .
In vitro testing helps formulation scientists understand and predict the potential
harmful effects of formulations to the vaginal mucosa. Although animal testing still
needs to be performed, these in vitro methods can be of great interest in initial
screening of new products and formulations, reducing the amount of animal testing
required. Also and unlike animal testing, in vitro testing can often differentiate
products that are very mild in terms of toxicity potential. Thus, toxicity of vaginal
products can be assessed using simple tests with epithelial cell monolayers, where
maintenance of membrane integrity in the presence of testing formulation indicates
potential safety [214, 215] . Nonetheless, monolayer cell cultures lack histological
and functional resemblance with native ectocervical and vaginal tissues, which limits
the interpretation of the obtained results. In order to respond to this and other
problems, Ayehunie et al. recently proposed a fast and highly reproducible three -
dimensional organotypic vaginal – ectocervical tissue model that simulates the structure
of the vaginal epithelium [216] .
The standard preclinical test of local vaginal irritation and toxicity of pharmaceutical
products, and the only one recommended by the FDA, is the rabbit vaginal
irritation test [217, 218] . However, reproducibility problems and differences in
vaginal physiology when compared to women limit the interpretation of results.
Other animal models have also been used, namely primates, dogs, guinea pigs, pigs,
mice, and rats [219 – 223] . Classic animal testing is limited because of the number of
animals required, which makes testing burdensome, expensive, and ethically questionable,
and because of differences between species, which may jeopardize the
extrapolation of results to humans [224] . Thus, simpler toxicity tests performed
in nonvertebrate organisms, such as gastropods, are interesting alternatives to
vertebrate animal testing. These tests also proved to be superior than in vitro testing,
mainly because of the limitations that are intrinsic to simple cell culture models
[225] . Recently, a simple in vitro test using slugs ( Arion lusitanus ) has been proposed
as an alternative to vertebrates in order to screen new vaginal semisolid formulations
for local tolerance early in the development process. The irritation potential
is evaluated by mucus production, and protein and enzyme release (lactate dehydrogenase
and alkaline phosphatase). Experimental results showed that the slug
mucosa irritation test performance is comparable to the classically used rabbit
vaginal irritation test [226] .
When initiating human testing, symptoms and signs of genital irritation must be
assessed [227] . These investigations should be performed comparing results between
the formulation to be tested (vehicle plus active substance(s) and vehicle only) and
formulations that are well known for their irritative effects. In the late 1980s and
early 1990s colposcopy of the vagina and cervix began to be used in the in vivo
safety assessment of vaginal products, becoming a standard technique [228] . The
objective of this procedure is to detect epithelial changes, such as breaks in the epithelium,
infl ammation, or other not well characterized, that may be a consequence
of vaginal products usage. Although very important, it presents several limitations
such as costs, specifi c personnel training, and diffi culty in understanding which colposcopic
fi ndings indicate risk [229] . Also, other techniques, such as Papanicolau
stained smears or automated cytomorphometric analysis, have been used in order
to assess the effect of formulations on the vaginal mucosa [230] .
5.12.5.9 Other Characteristics
In addition to the discussed evaluation tests and methodologies, other characteristics
of vaginal formulations may be assessed according to their individual specifi cities.
For example, the compatibility of vaginal formulations with condoms is an
important parameter to be determined, particularly when they are used during
sexual intercourse. These studies are usually performed according to the American
Society for Testing and Materials (ASTM) norm D3492 - 89 (Standard Specifi cation
for Rubber Contraceptives), where condoms are tested by accelerated testing for
their tensile strength and elongation on break point after being exposed to vaginal
formulations [231, 232] . Also, it is important to consider the effects of vaginal formulations
that are used during sexual intercourse in the penis and the possibility
of drug penetration through this organ. Although the human penis is covered
with keratinized stratifi ed epithelium, and thus the expected absorption should
be less than that of the vaginal epithelium, it is always a possibility to be taken into
account [233] .
5.12.6 CLINICAL USAGE AND POTENTIAL OF
VAGINAL DRUG DELIVERY
5.12.6.1 Microbicides
Microbicides (initially termed “ virucides ” ) are anti - infective drugs formulated for
topical self - administration in the vagina before sexual intercourse in order to protect
against HIV and other sexually transmitted pathogens [234] . Once a neglected
CLINICAL USAGE AND POTENTIAL OF VAGINAL DRUG DELIVERY 843
844 VAGINAL DRUG DELIVERY
subject in the war against HIV and other sexually transmitted diseases, in the last
decade microbicide investigations have gained important boosting and interest by
the scientifi c community, being considered as a new approach for prevention [235] .
Despite the fact that several microbicides are already in clinical trialing (see Table
5 ), currently there are no available products on the market. Also, 100% effective
microbicides are not likely to be achieved, even though only partially effective,
microbicides can be a big help in reducing the spread of HIV infection. Investigators
estimate that the use of a 60% effective microbicide in only 20% of all coital acts
could prevent approximately 2.5 million infections over a period of 3 years [236] .
While waiting for the fi rst - generation microbicides, preclinical research in new and
improved microbicides is already in progress. Several candidates, such as PSC -
RANTES [237] , antimicrobial peptides [238] , monoclonal antibodies [239] , inhibitors
of virus – cell fusion [240] , and natural products [241] , are currently being
developed. Also, another strategy that seems to be gaining consensus among the
TABLE 5 Microbicides Currently Undergoing Clinical Trialing
Mechanism
of Action Active Substance(s)
Candidate
Product
Drug
Delivery
Systems Developers
Clinical
Trial
Status
Vaginal
defense
enhancer
— ACIDFORM Gel CONRAD/Instead Phase I
— BufferGel Gel Reprotect Phases
II/III
Membrane
disruptive
agent/
surfactant
C31G Savvy Gel CONRAD Phase
III
Entry/fusion
inhibitor
Carrageenan (PC - 515) Carraguard Gel Population Council Phase
III
Cellulose acetate 1,2 -
benzenedicarboxylate
Cellacefate/
CAP
Gel Lindsey F. Kimball
Research
Institute / Dow
Pharmaceuticals,
Inc.
Phase I
Cellulose sulfate Cellulose
Sulfate
Vaginal Gel
Gel CONRAD Phase
III
Sodium lauryl sulfate Invisible
Condom
Gel Laval University Phases
I/II
Naphthalene 2 -
sulfonate polymer
PRO 2000/5 Gel Indevus
Pharmaceuticals
Phase
III
SPL7013 VivaGel Gel Starpharma Phase I
Replication
inhibitor
Tenofovir PMPA Gel Gel Gilead Sciences Phases
II/IIb
Dapivirine (TMC120) TMC120
Vaginal Gel
Gel and
vaginal
ring
International
Partnership for
Microbicides
Phases
I/II
UC - 781 UC - 781Gel Gel CONRAD Phase I
Unknown
mechanism
Extracts of Azadirachta
indica, Sapindus
mukerossi , and
Mentha citrata
Praneem
polyherbal
Vaginal
tablet
Talwar Research
Foundation
Phases
II/IIb
scientifi c community is the synergistic association of microbicides with different
action mechanisms in order to improve protection [242] .
Early expectations created around the possibility of using nonoxynol - 9 (widely
used as a vaginal contraceptive) as an effective microbicide, based on its in vitro
effi cacy against HIV, were frustrated in clinical trials [243] . Several hypotheses for
this failure were brought up, an inadequate choice of drug delivery system being
one of them. These results confi rm that preformulation and formulation studies
play an important role in microbicide rational design and development, being a
big challenge to overcome. Although clinical development recommendations have
been extensively reviewed [244, 245] , pharmaceutical development algorithms for
microbicides are yet to be defi ned. Nonetheless, it is known that preformulation
parameters such as organoleptic characteristics, stability, permeability, inherent
bioadhesion and retention features, and compatibility with excipients and condoms
of candidate drugs, among others, play a crucial role when developing drug delivery
systems containing microbicides [246] . After collection of this information,
formulation studies are necessary in order to obtain a fi nal product that fulfi lls
microbicide objectives and requirements, namely safety, effi cacy, acceptability,
affordability, and regulatory duties [247] . As well as providing effective protection,
microbicide formulations must also be safe on multiple exposures over time, chemically
and physically stable, compatible with latex and other materials used in
barrier devices, and affordable and acceptable to the end user. Ideally, they should
be colorless, odorless, tasteless, and nonmessy [234, 248] . Tested microbicides have
been formulated mostly as gels, although creams, vaginal rings, foams, sponges,
vaginal suppositories, and fi lms may also be considered. Nonetheless, alternative
innovative options for the delivery of microbicides have been developed in recent
years. For instance, the formulation of a safe and inexpensive “ universal ” drug
carrier for microbicidal substances is an interesting strategy that may potentially
ensure the effi cacy of most currently researched molecules [249] . Another interesting
approach for the delivery of microbicides was proposed by Chang et al. These
researchers studied the possibility of using genetically modifi ed comensal vaginal
bacteria ( Lactobacillus jensenii ) to produce anti - HIV proteins [250] . In vitro experiments
showed that this strategy can be a new step toward an effective microbicide
formulation.
5.12.6.2 Antimicrobials
Vaginitis is a common condition in women which can be caused by bacteria, yeasts,
or protozoa. Treatment of vaginitis has been achieved by oral or vaginal administration
of antimicrobials, often with similar effi cacy rates [251] . Several drugs are
currently available for intravaginal treatment of bacterial (e.g., metronidazole,
clindamycin), fungal (e.g., azoles, boric acid, nystatin), and protozoal (e.g., metronidazole)
vaginitis [252] . Also, alternative vaginal therapies have been investigated
in order to treat vaginitis. For instance, herbal formulations, particularly those containing
essential oils, have been referred as potential antimicrobials for the treatment
of both fungal and bacterial infections [253, 254] . Several vaginal drug delivery
systems have been used in order to administer antimicrobial substances, particularly
gels, creams, tablets, and vaginal suppositories. Current research in antimicrobial
vaginal drug delivery systems is focused on more convenient single - dose
CLINICAL USAGE AND POTENTIAL OF VAGINAL DRUG DELIVERY 845
846 VAGINAL DRUG DELIVERY
formulations that can achieve clinical cure while improving patient compliance
[255 – 257] .
Vaginal treatment may be advantageous when compared with oral treatment, as
systemic adverse effects are less likely to occur [258] . For example, Cunningham
and co - workers conducted a study where systemic levels of metronidazole after
vaginal administration (5 g of a 0.75% metronidazole gel) were residual when compared
to the oral administration of a 500 - mg standard dose despite comparable
clinical outcome [259] . This low level of vaginal absorption may be attributed to
metronidazole ’ s poor lipid solubility. Similar results have been obtained for clindamycin
when comparing intravaginal and intravenous routes [260] . Also, contraindication
during pregnancy and possible interference with oral contraceptives are
situations that probably recommend vaginal administration of these therapeutic
agents over oral administration in women requiring treatment [261] .
As previously discussed, vaginal pH plays an important role in the normal physiology
of the vagina, being elevated in bacterial vaginitis. Vaginal acidifi ers, such
as vitamin C in the form of tablets, can be effective in the treatment of vaginitis,
with the advantage of maintaining or even improving the normal vaginal fl ora
equilibrium, particularly in diabetic and pregnant women or those with recurrent
bacterial vaginitis episodes [78, 262] . Also, other formulations such as acid - buffering
gels were demonstrated to be potential helpers in the maintenance of a healthy
vaginal milieu and as adjuvant of antimicrobials in the treatment of bacterial vaginosis
[263, 264] . In fact, some gels may even be used in the treatment of bacterial
vaginosis. For example, a mucoadhesive gel containing two polymers, polycarbophil
and Carbopol 974P, was demonstrated to be effective in the treatment of bacterial
vaginosis, even when compared with the clinical cure rate of vaginal metronidazole
or tinidazole [265] .
5.12.6.3 Hormonal Contraceptives and Hormonal Replacement Therapy
The vaginal route is one of the many available routes for estrogen and progestogen
delivery. When compared to the oral and transdermal route, vaginal administration
of these hormones presents the advantage of needing a smaller quantity of drug
to achieve similar systemic effects with less relative variability [266, 267] . Also,
the inconveniences associated with subcutaneous implants are circumvented. Thus,
vaginal hormonal contraception and replacement therapy were soon considered
as a possibility.
Hormonal contraception requires drug delivery systems that are able to achieve
sustained blood levels of these substances, either by multiple administrations, as in
the case of oral contraceptives, or by sustained drug release. Since daily vaginal
administration would not be feasible and acceptable by most women, sustained -
release systems were developed. This need led to the development of several
devices, including vaginal rings [109] . Progestogen - only rings were initially developed,
but menstrual bleeding problems and ineffi cacious control of ovulation led
to discontinuation in several studies. Thus, combined estrogen – progestogen rings
were the next natural step toward effective and acceptable formulations. Various
combined estrogen – progestogen rings have proven to be highly effective as contraceptives,
providing excellent inhibition of ovulation [41] . Also, serious vaginal
lesions were shown to be unlikely to occur with short - and medium - term exposure
to currently available rings, mainly due to their improved fl exibility and small
dimensions [268] . Although other alternatives have also been studied, the most
popular schedule for contraceptive vaginal rings is three weeks in, one week out.
With this regimen the ring is inserted in the vagina on day 5 of the menstrual cycle
and left in place for three weeks. On week 4 the ring is removed, allowing menstrual
bleeding. A new ring (one - month ring) or the same one (over - one - month ring) is
inserted after one week ring free [112] . Vaginal rings are easy to use, being self -
administered by women. Once inserted in the vagina, the ring fi ts in the upper
vagina and delivers the active substance(s) by contact with the vaginal mucosa (see
Figure 7 ).
Despite all the investigational work already performed, only one contraceptive
vaginal ring, Nuvaring (Organon), is available in the United States and Europe. This
three weeks in – one week out vaginal ring was the fi rst approved by the FDA, being
available in the market since 2002. It is a fl exible and transparent ring containing
etonogestrel and ethinyl estradiol in a poly(ethylene - co - vinylacetate) matrix, with
an outer diameter of 54 mm and a cross section of 4 mm [269] . When placed in the
vagina it releases 120 . g/day of etonogestrel and 15 . g/day of ethinyl estradiol.
Clinical studies showed that Nuvaring is as effective and reliable as commonly used
oral contraceptives, being well tolerated, convenient, and highly acceptable to most
women and their partners [270 – 272] . Moreover, this contraceptive ring is able to
provide lower and more stable systemic exposure to estrogens than other contraceptive
options, namely combined oral contraceptives or transdermal patches, thereby
reducing drug - related side effects (Figure 8 ) [267] . Other contraceptive vaginal rings
that are still in premarket research include a one - year contraceptive ring being
developed by the Population Council, New York. This nestorone and ethinyl estradiol
containing ring has been shown to be as effi cacious as oral contraceptives when
used up to 12 months on a 3 weeks in – 1 week out regimen [273, 274] .
FIGURE 7 X - ray image of vaginal ring after placement in human vagina. ( Reprinted with
permission from K. Malcolm et al., Journal of Controlled Release , 90, 217 – 225, 2003. Copyright
2003 by Elsevier .)
CLINICAL USAGE AND POTENTIAL OF VAGINAL DRUG DELIVERY 847
848 VAGINAL DRUG DELIVERY
Although conventional emergency contraception usually comprises the use of
orally administered levonorgestrel (either alone or in combination with ethinyl
estradiol), the vaginal route has been shown to be an effi cacious alternative. The
obtained hormonal plasma levels for the same oral dose are lower, but presumably
high enough to prevent pregnancy [275] . Nonetheless, increases in the administered
dose by the vaginal route have been suggested [276, 277] .
Hormone replacement therapy has been a common practice for a long time in
order to improve the quality of life of women suffering from acute symptoms related
to menopause. Several formulations for oral, buccal, subcutaneous, parenteral, intrauterine,
nasal, transdermal, or vaginal administration have been used for this purpose
[278] . Although in recent years hormone replacement therapy has been associated
with an increased risk of fatal breast cancer [279] , some postmenopausal women
may still benefi t from this treatment. Estrogens have been used through the vaginal
route for the treatment of vaginal symptoms associated with hormone decline in
menopausal woman, such as dryness, dyspareunia, pruritus, irritation, discomfort,
and atrophy and has been shown to be as effective as systemic therapy [280, 281] .
Creams and vaginal suppositories were the fi rst vaginal drug delivery systems to
be used to deliver estrogens. Recently, estradiol rings for urogenital symptoms
therapy (low - dose rings) or vasomotor plus vaginal symptoms relief (usually higher
dose rings) were approved in some countries, providing a new option for the
FIGURE 8 Mean serum ethinyl estradiol concentration versus time for subjects treated
during 21 days with Nuvaring ( n = 8), transdermal contraceptive patch (Evra, OrthoMcNeil
Pharmaceutical; releases 20 . g ethinyl estradiol and 150 . g norelgestromin daily; n = 6), and
combined oral contraceptive (COC) (Microgynon, Schering AG; contains 30 . g ethinyl estradiol
and 150 . g levonorgestrel; n = 8). Subject exposure to ethinyl estradiol in Nuvaring group
was on average 3.4 and 2 times lower than for subjects in patch and combined oral contraceptive
groups, respectively. ( Reprinted with permission from M. W. van den Heuvel et al., Contraception
, 72, 168 – 174, 2005. Copyright 2005 by Elsevier .)
administration of these drugs [282] . For instance, Estring (Pharmacia & Upjohn), a
currently marketed vaginal ring containing low - dose estradiol (releases 7.5 . g/day
when placed in the vagina), proved to be effi cacious, well tolerated, and safe when
used for up to a year in the treatment of urogenital symptoms in postmenopausal
women [283, 284] . This silicone polymer ring has a diameter of 55 mm and a cross -
sectional diameter of 9 mm and is used up to three months. Another similar ring
containing estradiol acetate (Femring, Warner Chilcott), with a diameter of 56 mm
and a cross - sectional diameter of 7.6 mm, is also currently available.
Estrogens and progestogens have also been administered through the vagina in
order to manage other conditions. In fact, vaginally administered progesterone is
commonly used for luteal - phase support in women undergoing assisted reproduction
treatment, allowing optimal uterine concentrations without the high serum
levels observed by other routes (oral and intramuscular), possibly due to the fi rst -
uterine - pass effect [285] . Several drug delivery systems such as capsules, tablets,
vaginal suppositories, or gels have been used and have been shown to be equally
effi cacious in increasing the chance of becoming pregnant. Nonetheless, sustained -
release formulations allow fewer administrations per day with lower doses [286] .
Also, progesterone vaginal rings have been successfully used for luteal - phase
support [287] .
5.12.6.4 Spermicides
The vaginal use of spermicidal substances during sexual intercourse is perhaps the
oldest method of contraception. However, the introduction of oral contraceptives
and the intrauterine device in the 1960s led to the decay in their use. Since many of
these substances also offer protection against sexually transmitted diseases, interest
and investigation in this fi eld have recently increased [288] . Also, the development
of new potential spermicides, namely antibodies [289] , contributed to further awareness.
Currently used spermicides include nonoxynol - 9, octoxynol, benzalkonium
chloride, and chlorhexidine.
Nonoxynol - 9 has been used for more than 30 years as a spermicide in over - the -
counter vaginal products, such as semisolid formulations, sponges, foams, fi lms, and
others, in order to prevent pregnancy. Although relatively safe and effective, nonoxynol
- 9 formulations are still not able to achieve the same decrease in pregnancy
risk obtained with hormonal methods [290] . Thus, some strategies have been used
to enhance nonoxynol - 9 effects while reducing its toxic effects. For instance, some
synergistic associations with chelating agents that have themselves little spermicide
activity, such as EDTA and ethylene glycol tetraacetic acid (EGTA), have been
shown to be promising in this matter [291] . Also, coprecipitation of nonoxynol - 9
with polyvinylpyrrolidone by a freeze - drying method can be useful, particularly
when the formulation of a solid system is desirable, as this process is necessary to
alter the chemical state (liquid to solid) of nonoxynol - 9 [292, 293] . Using these
coprecipitates, a tablet with an inner core that provides slow release of nonoxynol - 9
after its fast release of the outer core was shown to be an effi cient and safer way of
delivering this spermicide in rabbits [294] .
Semisolid formulations are often used as contraceptives, particularly gels. These
drug delivery systems were demonstrated to be useful, namely in reducing the toxicity
of nonoxynol - 9 [295] . Adequate formulation of these products has also been
CLINICAL USAGE AND POTENTIAL OF VAGINAL DRUG DELIVERY 849
850 VAGINAL DRUG DELIVERY
shown to be essential for their effi cacy. For example, the contraceptive effect of
spermicides can be enhanced by adequate consistency of the formulation. When the
viscosity of a formulation increases, contraceptive effi cacy may increase as a result
of becoming more tenacious and more resistant to sperm migration, consequently
decreasing its capacity of reaching the site of fertilization [296] . Other gel properties,
such as pH and osmolarity, may also infl uence its spermicidal effects [297] . Moreover,
vaginal rings and inserts have been proposed as adequate vehicles for the
delivery of nonhormonal contraceptives [298, 299] . These systems may be advantageous
due to their controlled and prolonged release properties.
5.12.6.5 Labor Inducers and Abortifacients
Cervical ripening for the induction of labor has been a common practice in modern
obstetrics. Several drugs have been tested with this purpose, misoprostol, dinoprostone,
and oxytocin the three most frequently used. These labor inducers are normally
administered by the vaginal route as gels, tablets, suppositories, or inserts, all
demonstrated to be effective and safe. Nonetheless, drug formulation and choice of
drug delivery are important factors to be considered. In fact, different times to
achieve cervical ripening have been observed in several studies due to different drug
release profi les [300, 301] .
Vaginal administration of prostaglandins can be useful in the termination of
pregnancy. Misoprostol has been used to terminate unwanted pregnancies, demonstrating
a relatively high effi cacy, even when different regimens have been tested
[302 – 304] . Indeed, improved results with vaginal administered misoprostol may be
expected, because of higher and prolonged serum drug concentrations obtained,
when compared with the oral route [305] . It is also noteworthy that since vaginal
formulations are usually not available, oral tablets containing misoprostol (Cytotec,
Pfi zer) have been routinely used to administer this drug by the vaginal route. As
these tablets are not specifi cally designed to be administered in the vagina, suboptimal
clinical results may occur.
5.12.6.6 Proteins and Peptides
A few proteins and peptides, such as insulin, leuprolide, and salmon calcitonin, have
been tested for their possible administration by the intravaginal route. In spite of
the rich blood supply, relatively large surface area, and good permeability, vaginal
absorption of peptides and proteins is infl uenced by hormonal - induced changes in
the mucosa histology and enzymatic activity, thus limiting their administration
through this route [306] . Nonetheless, rational design of adequate and innovative
drug delivery carriers allowed considerable progress in protein and peptide vaginal
delivery. For example, microspheres have shown good potential to deliver peptides
and proteins, such as calcitonin, being able to increase drug stability and absorption.
This enhancement of absorption is thought to be related with the intimate contact
between the microspheres and the mucosa, resulting in high local concentrations at
the site of absorption [307, 308] . Another tested approach to vaginal administration
of proteins and peptides has been polymeric matrices, identical to the ones used in
the design of vaginal rings. For instance, antibodies have been successfully administered
in mice using poly(ethylene - co - vinyl acetate) disks [309] . Also, these and other
polymeric matrices can provide long - term (up to several years), controlled, and
high - dose topical delivery of antibodies [310, 311] .
Although considered a route that offers little or no real opportunities for insulin
administration, mainly because of low and variable levels of absorption, efforts have
been made to systemically deliver insulin through the vaginal mucosa. In the early
1980s, studies by Morimoto et al. showed that polyacrylic acid gels containing insulin
were able to induce and maintain (up to 30 min) hypoglycemia when administered
to rats and rabbits. However, sustained release was necessary to achieve longer time
of hypoglycemia [312] . Later, Richardson et al. observed that insulin is almost not
absorbed by the vaginal mucosa of ovariectomized rats in the absence of permeation
enhancers, but the coadministration of substances such as sodium taurodihydrofusidate,
polyoxyethylene - 9 - laurylether, lysophosphatidyl choline, palmitoylcarnitine
chloride, and lysophosphatidyl glycerol signifi cantly increased its absorption and
consequently hypoglycemia [59] . Also, the use of mucoadhesive microspheres as
delivery systems for insulin improved the absorption rate of this drug in sheep,
particularly when associated with permeation enhancers [55] . Recently, Degim et
al. developed vaginal chitosan gels as carriers for insulin. Studies performed in
rabbits showed that chitosan gels containing 5% dimethyl - . - cyclodextrin as a penetration
enhancer may provide longer insulin release, offering a potential alternative
to the parenteral route [313] . Also, Ning et al. investigated the suitability of niosomes
as insulin carriers for vaginal administration in rats. Results demonstrated that the
bioavailability of insulin when administered through the vaginal route was comparable
to that of the subcutaneous route [158] .
5.12.6.7 Vaccines
Although once considered not to be a very promising approach, intravaginal vaccines
have emerged in recent years as a potential noninvasive immunization strategy,
particularly for the prevention of HIV transmission [314] . In fact, some animal
and human experiments suggest that the obtained female genital tract immunization
can be superior with vaginal administration of vaccines when compared to other
routes, such as oral, nasal, or rectal [315 – 317] . Also, it is known that effective immunization
against sexually transmitted diseases will require strong local genital tract
as well as strong systemic antibody responses [318] . Mucosal vaccination presents
several advantages over systemic immunization: improved safety profi le, minimization
of adverse effects, ease of administration, and potentially lower costs. However,
limitations such as epithelium changes with menstrual cycle, which leads to reduced
mucosa permeability and poor antigen presentation during certain stages, and inactivation
of antigens by exposure to the vaginal environment can modulate the
magnitude of immune response inducement [319] .
Phosphate - buffered saline has been conveniently used as a vehicle for the delivery
of antigens. Although these liquid formulations facilitate vaginal administration,
they allow poor retention, controlled release, and protection of the delivered antigens.
Thus, development of adequate vaginal drug delivery systems plays an important
role in the success of vaginal immunization. Thermosensitive delivery systems
with adequate mucoadhesiveness may be useful in enhancing the mucosal and systemic
immune responses, as they can increase the exposure and contact of antigens
to the vaginal mucosa [320] . The combination of thermosensitive polymers such as
CLINICAL USAGE AND POTENTIAL OF VAGINAL DRUG DELIVERY 851
852 VAGINAL DRUG DELIVERY
poloxamers and mucoadhesive polymers such as polycarbophil were shown to be a
good strategy in the development of mucoadhesive, thermosensitive, and controlled -
release formulations for vaginal delivery of vaccines [321] . Also, antigen susceptibility
to the vaginal environment can be circumvented by administering these molecules
in protective systems, such as the one described by Shen et al., which comprises
poly(ethylene - co - vinyl acetate) matrices containing plasmid DNA [322] . These disk -
shaped devices, produced by a solvent evaporation technique described by Luo
et al. [323] , were shown to be effective in inducing local immunity in mice, protecting
and providing controlled and sustained delivery of plasmid DNA. In addition, other
strategies for vaginal antigen administration have also been proposed, such as the
use of nonpathogenic bacterial vectors [324 – 326] and microspheres [327] .
5.12.6.8 Other Uses
Many other drugs have been administered in the vagina for the management of
either local or systemic conditions. Table 6 summarizes some of these reports.
5.12.7 ACCEPTABILITY AND PREFERENCES OF WOMEN RELATED
TO VAGINAL DRUG DELIVERY
Usage of vaginal products is intimately related to taboos and presumptions associated
with the knowledge and handling of genitalia. Results from a recent international
survey showed that, despite improvements in recent decades, society ’ s attitude
toward the vagina and its use as a drug delivery route is not very open and not as
open as women would like it to be. Also, this study revealed that the vagina is not
commonly recognized as a possible drug delivery route, with only approximately
35% of women acknowledging this fact [342] . Although frequently overlooked,
acceptability studies of vaginal products are important in predicting women ’ s compliance
and thus their effectiveness. Dedicated studies to women ’ s acceptability and
preferences toward vaginal drug delivery are not common, most information available
resulting from parallel research to clinical trials. Also, most of these studies
have focused on microbicides and spermicides, since their acceptance is decisive for
their consistent use [343 – 345] . In fact, there can be a sense of guilt or negative
feeling with this kind of vaginal product, contrasting with others that are prescribed
by a physician for a specifi c gynecological problem, which seem to be deprived of
moral issues due to the “ legitimacy ” of their use [346] . Acceptability studies should
also be extended to sexual partners, particularly when evaluating products meant
to be used during sexual intercourse [347] . Data collected from previous acceptability
studies must be considered during the development of formulations in order
to improve their suitability. For example, lubrication provided by a vaginal product
seems to be important in determining its acceptability, as this feature may be
regarded advantageous or not by different users [348] . Also, negative perceptions
regarding product characteristics should be identifi ed during clinical trials in order
to improve formulations.
Although results found in the literature related to women ’ s preferences toward
the vaginal route of drug administration may vary signifi cantly, particularly when
different locations and cultures are considered, some general statements can be
TABLE 6 Selected Drugs Administered in the Vagina
Drugs Intended Use
Drug Delivery
Systems Comments References
5 - Fluorouracil Treatment of intravaginal warts
Gel Demonstrated to be effective,
safe,
and tolerable 328
Maintenance therapy of cervical
dysplasias after standard excisional
or ablative therapy
Cream Reduction of recurrence was achieved
329
Bromocriptine Therapy of hyperprolactinemia
Oral tablet and
vaginal
suppository
Proved to be effective and safe, without the adverse
effects of oral administration; vaginal suppository
obtained higher reduction in serum prolactin
330
Cabergoline Therapy of hyperprolactinemia Oral tablet Proved to be effective and safe, without adverse
effects of oral administration
331
Chlorhexidine Prevention of peripartum infections of
newborn
Aqueous solution Proved to be useful 332
Danazol Treatment of pelvic endometriosis
Ring and vaginal
suppository
Effective without increased serum concentrations
observed during oral therapy
114
Etoposide Management of cervical dysplasias
associated with human papilloma
virus (HPV)
Vaginal
suppository
Demonstrated to be safe and tolerable 333
Imiquimod Treatment of high - grade vaginal
intraepithelial neoplasia
Cream Results suggest it can be an alternative conservative
therapy
334
Indomethacin Tocolysis in preterm labor
Rectal
suppository
Vaginal administration proved to be more effective
than conventional rectal plus oral administration
335
Lignocaine Cervical anesthesia during insertion of
tenaculum
Spray and gel No difference in pain management between spray
and gel
336
Morphine Pain control as alternative to
parenteral administration
Tablet and
vaginal
suppository
Requires close monitoring due to unpredictable
bioavailability
337
Oxybutinin Treatment of urge urinary incontinence Insert Proved to be effective and safe in rabbits 338
Propranolol Control of tachycardia Tablet Obtained serum levels were within . - blocking
range and comparable to those achieved by oral
route
339
Sildenafi
l Endometrial development for embryo
implantation
Vaginal
suppository
Enhanced endometrial development was achieved 340
Trichloroacetic
acid
Treatment of low - grade vaginal
intraepithelial neoplasia
Solution Proved to be effective as well as inexpensive and
easy to perform
341
853
854 VAGINAL DRUG DELIVERY
made. Ideally, formulations should be easy and comfortable to use, colorless, odorless,
and messiness free. Also, products that are better retained in the vagina seem
to be favored by women, since leakage is one of the most undesired feature of
vaginal formulations [349] . Concerning preferred vaginal dosage forms, gels and
creams seem to be the most popular among women. On the other hand, vaginal
suppositories and tablets are among the most disliked dosage forms. Others, such
as fi lms, present ambiguous results, mostly related with diffi culties during insertion
[350, 351] . Vaginal rings have been shown to be highly acceptable by both women
and their sexual partners, even during sexual intercourse. A trial conducted in North
America and Europe showed that couples rarely felt the device during penile penetration,
and when the ring was noticed, almost none of the partners seemed to
mind [352] . Furthermore, insertion and removal of vaginal rings are judged to be
easy by users [353] .
Packaging and applicators may also infl uence women ’ s choice. Products that are
placed in the vagina by means of an applicator seem to be preferred because it
facilitates administration and avoids direct touching of genitalia during insertion.
Applicator characteristics, namely length, width, color, fi lling features (single - use or
reusable applicators), and ease of usage, are also known to infl uence women ’ s
acceptability [351, 354] .
5.12.8 VETERINARY VAGINAL DRUG DELIVERY
As with humans, veterinary vaginal drug administration has been performed for a
long time, particularly for the treatment of local infections, traditionally involving
the use of vaginal suppositories, liquid formulations, or gels. Advantages of this route
include the avoidance of damage to the skin or to tissue that is associated with
injections, minor stress infl icted to the animals, and possibility of ceasing drug delivery
at will [355] . Nonetheless, the fi rst major studies on veterinary drug delivery
have been performed by Robinson in the 1960s with progestogen - impregnated
polyurethane sponges [356] . Since then, the major use of the vaginal route has been
the control of the estrus cycle in livestock by delivering progestogens and estrogens
in a controlled fashion. Also, the administration of these hormones showed good
results in treating reproductive disorders, such as ovarian quiescence, cystic ovary
or cystic corpus luteum [357] . Estrus synchronicity is advantageous as it allows
insemination of all or selected females in a herd or fl ock to occur during a single
period of several hours or days [358] . With this purpose, several drug delivery
systems have been developed, being generally based on polymeric matrices that
are able to control release of drug content. These devices also present two common
features: Retention is guaranteed by means of a gentle pressure applied to the
mucosa and the existence of a mechanism (e.g., an attached string) that allows their
easy removal at the end of the treatment. Table 7 presents a synopsis of these
systems, as reviewed by Rathbone et al. [358, 359] . Also, some of these devices are
shown in Figure 9 . In addition to the vaginal administration of progestogens and
estrogens, delivery of other therapeutic agents through this route for both local and
systemic effects has been investigated, namely 1,25 - dihydroxy vitamin D3 [360] ,
lactic acid – producing lactobacilli [361] , antimicrobials [362] , local anesthetics [363] ,
and vaccines [364] .
TABLE 7 Selected Veterinary Vaginal Drug Delivery Systems for Control of Estrus Cycle in Livestock
Drug Delivery
Systems
Active
Substance(s)
Matrix - Forming
Polymers Brief Description Comments
Sponges Progesterone,
estradiol, and
several
progestins
Polyurethane Polymeric devices impregnated with active
substance(s), usually cylindrical shaped;
alternative designs allow achieving zero -
order release profi le
Variable vaginal
retention; inexpensive
and simple to prepare
PRID
(Progesterone
Releasing
Intravaginal
Device, InterAg,
Hamilton, NZ)
Progesterone Poly(dimethylsiloxane) Spiral - shaped device obtained by molding and
curing polymer (containing active substance)
onto stainless steel spiral by high -
temperature ( . 190 ° C) injection molding;
also
may enclose hard gelatin capsule (containing
estradiol) glued to device
Excellent vaginal
retention
CIDR (Controlled
Internal Drug
Release, CEVA,
Libourne,
France)
Progesterone Poly(dimethylsiloxane) T - shaped nylon spine over which the polymer
(containing active substance) is molded and
cured by high - temperature injection molding;
several types developed: CIDR - S (rabbit
eared in shape), CIDR - G (slimmer, straight
T - shaped),
CIDR - B (similar to CIDR - G but
with increased dimensions)
Excellent vaginal
retention; only types G
and B currently
available
INVAS
(Intravaginal
Application
System)
Progesterone Poly(dimethylsiloxane) Similar to CIDR but obtained by lower
temperature ( < 120 ° C) method
Not commercially
available
Rings — Poly(dimethylsiloxane) Similar to human vaginal rings Not commercially
available; abandoned
because of poor
retention properties
Rajamehendran
intravaginal
device
17 . - estradiol/
progesterone
Poly(dimethylsiloxane) Two C - shaped polymer tubes (impregnated
with active substances) tied together, forming
“ umbrella - shaped ”
device
Excellent vaginal
retention; not
commercially available
IBD (Intelligent
Breeding Device,
Advanced
Animal
Technology,
Hamilton, NZ)
Progesterone/
estradiol/
prostaglandin
Poly(dimethylsiloxane) Composed by outer plastic sheath [designed to
protect circuit board (which controls drug
release) and two batteries, four polymeric
drug reservoirs (a large one at base and
three small ones at head of device)],
retention mechanism, and tail
Allows release of active
substances at different
rates and at specifi c
times
Source : From refs. 358 and 359 .
855
856 VAGINAL DRUG DELIVERY
In order to improve the performance of drug delivery systems, several strategies
have been tested. For example, improvement of currently available progestogen -
and estrogen - delivering devices and development of new ones have been a common
gold of vaginal drug delivery investigators [365] . In other therapeutic fi eld, Gavini
et al. used bioadhesive chitosan microspheres compressed into tablets with several
excipients to deliver acrifl avine [362] . In vitro results demonstrated that these
systems have good mucoadhesive properties, allowing increased residence time in
the vagina. Recently, Cross et al. proposed an interesting electronic device that
allows controlling drug release in response to in loco stimuli (e.g., vaginal temperature)
or external commanding via radio wireless link [366] . This device is placed
inside and behind the piston of a modifi ed syringe, being able to control the production
of hydrogen from a gas cell. The increased gas pressure behind the piston
propels a viscous pharmaceutical vehicle that fi lls the syringe. This drug release and
monitoring unit (DMU) have been shown to be a promising strategy in controlling
the delivery of active substances to cows while simultaneously collecting physiological
data in the vaginal environment.
5.12.9 CONCLUSIONS AND FUTURE TRENDS
As other routes of drug administration presented serious diffi culties to deliver some
active substances, the vagina emerged as a feasible alternative. Undoubtedly, the
vaginal route of drug delivery has attracted the interest of the scientifi c community,
particularly in the last few decades. The latest developments in this fi eld, namely
mucoadhesive formulations, vaginal rings, and other controlled - release drug delivery
systems, have boosted research and clinical use of this once - neglected route of
drug administration. Also, women ’ s emancipation in the last century has slowly led
to higher acceptability of vaginal formulations, as old fears and preconceived ideas
are being demystifi ed.
FIGURE 9 Some vaginal veterinary drug delivery systems used or investigated to control
estrus cycle in livestock: sponge (1), PRID (2), CIDR - S (3), CIDR - G (4), CIDR - B (5),
Rajamehendran intravaginal device (6), and IBD (7).
REFERENCES 857
Much work remains to be done, particularly in specifi c fi elds, such as the delivery
of macromolecular drugs (e.g., proteins and peptides) and other substances that
are poorly absorbed through the vaginal mucosa. Another promising area that
needs further investigation is vaginal administration of vaccines and microbicides.
Indeed, in an era where HIV and other sexually transmitted diseases are an increasing
concern, vaginal preventive strategies are required. Formulating scientists can
contribute decisively to these objectives, as optimization of drug delivery systems
seems to be essential. Issues such as poor vaginal distribution and retention, inadequate
drug release, limited drug protection from vaginal “ aggressors, ” and adverse
effects of currently available drug delivery systems still need to be solved.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Bruno Sarmento and Cl a udia
Carneiro for their kind review and useful comments on the manuscript.
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874 VAGINAL DRUG DELIVERY
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TABLET PRODUCTION
SECTION 6
881
6.1
PHARMACEUTICAL
PREFORMULATION:
PHYSICOCHEMICAL PROPERTIES OF
EXCIPIENTS AND POWDERS AND
TABLET CHARACTERIZATION
Beom - Jin Lee
Kangwon National University, Chuncheon, Korea
Contents
6.1.1 Introduction
6.1.2 Selection of Pharmaceutical Excipients
6.1.2.1 Defi nitions and Goals
6.1.2.2 Types of Pharmaceutical Excipients
6.1.2.3 Characteristics of Pharmaceutical Excipients
6.1.2.4 Selection Guideline of Pharmaceutical Excipients in Tablet Formulation
6.1.3 Drug – Excipient Compatibility
6.1.3.1 Experimental Studies for Drug – Excipient Compatibility
6.1.3.2 Analytical Methods for Drug – Excipient Compatibility
6.1.3.3 Reaction Types and Stabilization Guidelines
6.1.4 Powder Characteristics
6.1.4.1 Crystal Form and Habit
6.1.4.2 Particle Size Distribution
6.1.4.3 Flow Characteristics
6.1.4.4 Density and Bulkiness
6.1.4.5 Hygroscopicity
6.1.4.6 Mixing
6.1.4.7 Particle Size Reduction (Micronization and Milling)
6.1.4.8 Compaction (Compressibility)
6.1.4.9 Surface Area and Other Properties
6.1.5 Tablet Characterization
6.1.5.1 Disintegration
6.1.5.2 Dissolution
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
882 PHARMACEUTICAL PREFORMULATION
6.1.5.3 Weight Variation
6.1.5.4 Hardness or Breaking Strength
6.1.5.5 Friability
6.1.5.6 Content Uniformity
6.1.5.7 Tablet Thickness
6.1.5.8 Tablet Shape and Size
References
6.1.1 INTRODUCTION
Tablet is a major category of solid dosage forms which are widely used worldwide.
Extensive information is required to prepare tablets with good quality at high standards.
Based on preformulation studies, the optimal dosage forms are generally
decided. When given orally, the solid dosage form tablet undergoes in vitro disintegration
and dissolution followed by absorption through the gastrointestinal tract
(GI). The in vivo biodistribution of drug which enters the systemic circulation then
occurrs (Figure 1 ).
In this section, general preformulation approaches for tablet production are
described (Figure 2 ). The physicochemical properties of drug and excipients, which
are crucial factors at the beginning stages, are presented. The types and functions
of excipients used for tablet formulation and drug – excipient incompatibility are also
discussed. Because tablets are prepared by compression of the drug with powdered
excipients, the physicochemical properties of excipients and their multiple functions
under regulatory standards are very important. To prepare a drug substance for the
fi nal dosage forms, pharmaceutical excipients should be added. The guidelines for
selection of excipients are also given, based on the type and function of excipient,
drug – excipient compatibility, type of tablet, and manufacturing parameters. Drug –
excipient incompatibility is a very important issue before and after tablet prepara-
FIGURE 1 In vitro and in vivo pathways of drug which enters systemic circulation.
Preformulation
Formulation
(dosage forms)
Release/dissolution Absorption
Distribution
(blood concentration)
Metabolism
Elimination
Receptor sites
(efficacy)
In vitro In vivo
tion. According to global International Conferce on Harmonisation (ICH) guidelines,
we should cautiously choose the excipients in tablet formulations because the quality
and sources are variable by supplier and by batch.
In general, a tablet is prepared by mixing, milling, and compression the drug –
excipient mixtures using a tablet machine. Therefore, powder characteristics of drug
and excipients, including particle size, fl ow characteristics, bulk density, hygroscopicity,
mixing and milling, and compaction, are extensively discussed. If these physical
properties are not fully understood, tablets with good quality at high standards are
often impossible to produce.
The prepared tablet must be validated according to regulatory guidelines. In
general, disintegration, dissolution, friability, hardness, and weight are characterized
for quality validation of fi nished tablet. This biopharmaceutical preformulation
information for tablet production will guide us to design the optimal tablet very
effi ciently in the laboratory and industrial companies.
6.1.2 SELECTION OF PHARMACEUTICAL EXCIPIENTS
6.1.2.1 Defi nitions and Goals
A tablet contains active ingredients as well as other substances known as excipients,
which have specifi c functions. The types and functions of various excipients which
are incorporated into tablet formulations are discussed in many textbooks [1 – 3] .
A pharmaceutical excipient is defi ned as an inactive ingredient or any component
other than the active ingredient added intentionally to the medicinal formulation
or everything in the formulation except the active drug. Pharmaceutical excipients
are also called additives, pharmaceutical ingredients, or inactive pharmaceutical
ingredients. There are many reasons for selecting and adding these pharmaceutical
excipients in formulations. In the preparation of various dosage forms, it is essential
to combine pharmaceutical excipients with model drugs as adjuvants to prepare the
FIGURE 2 General preformulation approaches for tablet production.
Physicochemical properties of excipients Physicochemical properties of drug
Manufacturing process
Tablet production
Tablet validation
Drug–excipient compatibility
SELECTION OF PHARMACEUTICAL EXCIPIENTS 883
884 PHARMACEUTICAL PREFORMULATION
solid dosage forms, mainly tablets. The pharmaceutical excipients make the drug
into the fi nal dosage forms. Physicochemical properties such as solubility, stability,
metabolism, and even bioavailability of drugs can be varied by the pharmaceutical
excipients [4 – 6] . Figure 3 shows the correlation and functions of drugs combined
with pharmaceutical excipients in dosage from designs. Pharmaceutical excipients
are regarded as key ingredients not only to decide optimal dosage forms but also
to change the physicochemical and biological parameters of drugs. With an aid of
pharmaceutical excipients, drug effi cacy can be maintained. Changes of other types
of dosage forms for different routes of administration are also achieved. The excipients
can also function for the preparation of dosage formulation during the manufacturing
processes. Patient compliance and modifi ed releases of drugs can also be
achieved if the excipients are properly applied. The colorant makes the tablet distinguishable
after the coating process.
However, utilization of these pharmaceutical excipients is limited by the regulatory
guidelines to be satisfi ed in the dosage formulations [3, 7] . In general, the regulatory
guidelines require the following conditions for the use of excipients in the
dosage formulations: (a) no harmful or toxicological effect and listed GRAS (generally
recognized as safe), (b) good stability with no drug – excipient incompatibility
and by any impurities in the excipients, (c) no interference in quality validation and
analytical tests, (d) satisfaction of regulatory issues and guidelines in all countries
where the product is to be marketed, (e) no instability with primary packing materials,
(f) ease of accessibility, distribution, and economical cost, (g) satisfaction for
environmental issues, (h) be physiologically inert, (i) be physically and chemically
compatible with the active substance and the other excipients in the formulation,
and (j) no unacceptable microbiological burden.
The Handbook of Pharmaceutical Excipients [1] contains some details of functional
tests carried out on a wide range of excipients. The excipients all have pharmacopeial
monographs, but it is important to understand that compliance with a
monograph does not indicate equivalence between different grades or suppliers.
6.1.2.2 Types of Pharmaceutical Excipients
To prepare a drug substance into a fi nal dosage form, pharmaceutical excipients
should be added. The Handbook of Pharmaceutical Excipients presents more than
FIGURE 3 Correlation and functions of drug combined with pharmaceutical excipients in
dosage form design.
+
Efficacy,
Adverse effect
Multiple
Functionality
Bioavailability
Safety/Quality
drug Pharmaceutical
Excipients
TABLE 1 Pharmaceutical Excipients Used in Tablet Formulations
Excipient Type Defi nition Examples
Adsorbent Agent capable of holding other
molecules onto its surface by
physical or chemical
(chemisorption) means
Powdered cellulose, activated
charcoal
Antioxidant Agent that inhibits oxidation and
thus is used to prevent
deterioration of preparations by
oxidative process
Ascorbic acid, ascorbyl palmitate,
butylated hydroxyanisole,
butylated hydroxytoluene,
hypophosphorus acid,
monothioglycerol, propyl
gallate, sodium ascorbate,
sodium bisulfi te, sodium
formaldehyde, sulfoxylate,
sodium metabisulfi te
Colorant Used to impart color to tablet FD & C red no. 3, no. 20, FD & C
yellow no. 6, FD & C blue no. 2,
D & C green no. 5, D & C
orange no. 5, D & C red no. 8,
caramel, ferric oxide, red
Encapsulant Used to form thin shells for purpose
of enclosing drug substance or
drug formulation for ease of
administration
Gelatin, cellulose acetate
phthalate
Plasticizer Used as component of fi lm coating
solutions to enhance spread of
coat over tablets, beads, and
granules
Diethyl phthalate, glycerin
230 monographs of excipients used in the dosage formulations [1] . The amount and
type of pharmaceutical excipients are highly dependent on the fi nal dosage forms.
In the preparation of tablets, diluents or fi llers are commonly added to increase the
bulk of the formulation, binders to cause the adhesion of the powdered drug and
pharmaceutical substances, antiadherents or lubricants to assist in the smooth tableting
process, disintegrating agents to promote tablet break - up after administration,
and coating agents to improve stability, control disintegration, or enhance appearance.
Thus, the pharmaceutical excipients establish the primary features and physicochemical
properties of the tablet, such as the physical form, stability, dissolution,
taste, and overall appearance [3] . Table 1 presents examples of pharmaceutical
excipients used in tablet formulations according to principal categories.
6.1.2.3 Characteristics of Pharmaceutical Excipients
Filler (Diluent) Powder mixtures should be compacted to achieve the appropriate
strength at a low compaction pressure in the tablet preparation. The compaction
properties of a formulation will largely be governed by its major components. For
a high - dose drug the drug itself will strongly infl uence the compaction, while for
low - dose drugs the bulk size needs to be increased with an inactive ingredient
SELECTION OF PHARMACEUTICAL EXCIPIENTS 885
886 PHARMACEUTICAL PREFORMULATION
Excipient Type Defi nition Examples
Surfactant Substance that adsorbs to surfaces
or interfaces to reduce surface or
interfacial tension; may be used
as wetting agent, detergent, or
emulsifying agents
Benzalkonium chloride,
nonoxynol 10, oxtoxynol 9,
polysorbate 80, sodium
lauryl sulfate, sorbitan
monopalmitate
Tablet
antiadherent
Agent that prevents the sticking of
tablet formulation ingredients to
punches and dies during tablet
production
Magnesium stearate, talc
Tablet binder Substance used to cause adhesion
of powder particles in tablet
granulations
Acacia, alginic acid,
carboxymethylcellulose
sodium compressible sugar,
ethylcellulose gelatin, liquid
glucose, metylcellulose
povidone, pregelatinized starch
Tablet diluent Inert substance used as fi ller to
create desired bulk, fl ow
properties, and compression
characteristics in preparation of
tablets
Dibasic calcium phosphate,
kaolin, lactose, mannitol,
microcrystalline cellulose,
powdered cellulose,
precipitated calcim carbonate,
sorbitol, starch
Tablet sugar
and fi lm
coating
excipient
Used to coat a formed tablet for
purpose of protecting against drug
decomposition by atmospheric
oxygen or humidity, to provide
desired release pattern for drug
substance after administration,
to mask taste or odor of drug
substance, or for aesthetic
purposes
Sugar: liquid glucose,
sucroseFilm: hydroxyethyl
cellulose, hydroxypropyl
methylcellulose,
methylcellulose, ethylcellulose
Enteric: cellulose acetate
phthalate, shellac (35% in
alcohol)
Tablet direct -
compression
excipient
Used in direct - compression tablet
formulations
Dibasic calcium phosphate
Tablet
disintegrant
Used in solid dosage forms to
promote disruption of solid mass
into smaller particles which are
more readily dispersed or
dissolved
Alginic acid,
carboxymethylcellulose
calcium, microcrystalline
cellulose, polacrilin potassium,
sodium alginate, sodium starch
glycollate, starch
Tablet glidant Agent used in tablet and capsule
formulations to improve fl ow
properties of powder mixture
Colloidal silica, corn starch, talc
Tablet
lubricant
Substance used in tablet
formulations to reduce friction
during tablet compression
Calcium stearate, magnesium
stearate, mineral oil, stearic
acid, zinc stearate
Tablet
opaquant
Used to render tablet coating
opaque; may be used alone or in
combination with colorant
Titanium dioxide
Tablet -
polishing
agent
Used to impart an attractive sheen
to coated tablets
Carnauba wax, white wax
TABLE 1 Continued
termed a diluent (or fi ller). High - dose formulations may also use a diluent to overcome
compaction problems experienced with an active pharmaceutical substance.
There are a number of general rules for selecting a diluent. The selection of the
diluent will depend on the type of processing and plasticity of materials to be used.
A direct - compression formulation will require a diluent with good fl ow and compaction
properties. If the material is extremely plastic, it is appropriated to add a diluent
that compacts by brittle fracture; similarly, a brittle drug substance should be combined
with a plastic fi ller. The solubility of the drug substance should also be considered.
A soluble drug is normally formulated with an insoluble fi ller to optimize
the disintegration and dissolution process. The hydrophilic excipients added in the
formulation may also change drug solubility. Table 2 lists the more commonly used
diluents in tablet formulation.
Binder (Granulating Agent) Before tableting the powder mixture via direct compression,
generally powders are granulated simply by adding water or an organic
solvent to form liquid bridges followed by the drying process. This granulation
process can make powders of larger particle size and that are more free fl owing for
tablet production. The most common method of adding binders is as a solution in
the granulating fl uid. It is also possible add synthetic polymers such as PVP and
HPMC as powders and use water as the granulating agent. When the granulate dries,
the crystallization of any solids that had dissolved in the liquid will form solid bonds
between the particles [8] . Inclusion of granulating agents or binders to increase
granule strength is necessary. Granulating agents are usually hydrophilic polymers
that have cohesive properties that both aid the granulation process and impart
strength to the dried granulate.
For a granulating agent to be effective, it must form a fi lm on the particle surface
and be selected on the basis of its spreading coeffi cient. The spreading coeffi cient
is defi ned as the difference between the work of adhesion of the binder and the
substrate and the work of cohesion of the binder. Commonly used granulating
agents are listed in Table 3 . The binder may vary the disintegration and dissolution.
Binders form hydrophilic fi lms on the surface of the granules, which can aid in the
TABLE 2 Commonly Used Tablet Diluents
Diluent Comments
Lactose Available as anhydrous and monohydrate; anhydrous material
used for direct compression due to superior compressibility
Microcrystalline cellulose Originally direct - compression excipient, now often included in
granulations due to its excellent compressibility
Dextrose, glucose Direct - compression diluent, often used in chewable tablets
Sucrose Was widely used as sweetener/fi ller in effervescent tablets and
chewable tablets; less popular nowadays due to cariogenicity
Starch and derivatives Versatile material that can be used as diluent binder, and
disintegtant
Calcium carbonate Brittle material
Dicalcium phosphate Excellent fl ow properties; brittle material
Magnesium carbonate Direct - compression diluent
SELECTION OF PHARMACEUTICAL EXCIPIENTS 887
888 PHARMACEUTICAL PREFORMULATION
wetting of hydrophobic drugs. However, if added at too great concentrations, the
fi lms can form viscous gels on the granule surface and will retard dissolution.
Disintegrant Tablets must have suffi cient strength to withstand the stresses of
subsequent manufacturing operations, such as the coating, packaging, and distribution
process. However, once the tablet is taken by the patient, it must break up
rapidly to ensure rapid dissolution of the active ingredient in immediate - release
preparations. To overcome the cohesive strength produced by the compression
process and to break down the tablet into the primary particles as rapidly as possible,
the disintegrants are combined with other excipients during the tableting process.
Starch was the fi rst disintegrant used in tablet manufacture. Recently, so - called
superdisintegrants, including croscarmellose sodium, sodium starch glycolate, and
crospovidone, display excellent disintegration activity at low concentrations and
have better compression properties than starches. Traditionally, swelling and rate of
swelling have been regarded as the most important characteristics of disintegrants.
With the aid of these superdisintegrants, sustained - release acetaminophen tablets
with biphasic patterns were successfully established to mimic the bilayered Tylenol
ER tablet [9]. As a general rule, soluble drugs are formulated with insoluble fi llers
to maximize the effect of disintegrants. The positioning of disintegrants within the
intragranular and extragranular portions of granulated formulations can affect their
water uptake and disintegration time.
Commonly used disintegrants are listed in Table 4 . The greater the level of disintegrant,
the faster the tablet will disintegrate. The compaction properties of many
disintegrants, including starch, are not satisfactory and use of high concentration
could also reduce tablet strength. Disintegrants are hygroscopic materials and will
absorb moisture from the atmosphere, which could negatively affect the stability of
moisture - sensitive drugs if the packaging does not provide adequate protection
from the environment. Disintegrant activity can be affected by mixing with hydrophobic
lubricants so that care needs to be taken to optimize the manufacturing
TABLE 3 Commonly Used Granulating (Binding) Agents
Granulating Agent
Normal
Concentration
(%) Comments
Starch 5 – 25 Was once the most commonly used binder;
starch has to be prepared as paste, which
is time consuming
Pregelatinized starch 0.1 – 0.5 Cold - water soluble so easier to prepare
than starch
Acacia 1 – 5 Requires preparation of past prior to use;
can lead to prolonged disintegration
times if used at too high a concentration
Polyvinylpyrrolidone
(PVP)
2 – 8 Available in range of molecular weight/
viscosities; soluble in water and ethanol
Hydroxypropyl
methylcellulose (HPMC)
2 – 8 Low - viscosity grades most widely used
Methylcellulose (MC) 1 – 5 —
process as well as the formulation. If the tablet contains a high proportion of a
hydrophobic drug that has a high contact angle, a wetting agent or surfactant should
be added to the formulation to modify disintegration time and subsequent dissolution
of the drug from tablet. The most commonly used wetting agents are sodium
lauryl sulfate and the polysorbates.
Most pharmacopeias include a disintegration test which can be applied to tablets
and capsules and the detailed monograph is given in the pharmacopeias (see Section
6.1.5.1 ).
Lubricant The use of a lubricant is essential to increase the free fl ow of powders
and to prevent manufacturing disorders in the tablet production. The type and
amount of lubricant are cautiously selected in the formulation. The order of addition
and mixing time is also considered in the tableting process.
There are three types of lubricants employed in solid dosage form manufacture.
The fi rst class of lubricant is the glidant . The fl ow properties of a powder can be
enhanced by the inclusion of a glidant. These are added to overcome powder cohesiveness.
The two other classes of lubricant are antiadherent excipients, which reduce
the friction between the tablet punch faces and tablet punches, and die wall lubricant
excipients, which reduce the friction between the tablet surface and the die wall
during and after compaction to enable easy ejection of the tablet. The level of a
lubricant required in a tablet is formulation dependent and can be optimized using
an instrumented tableting machine.
Commonly used lubricants are listed in Table 5 . Talc is traditionally one of the
most commonly used glidants, having the additional benefi t of being an excellent
antiadherent. The level of talc that can be added to a formulation is restricted by
its hydrophobic nature, too high levels resulting in decreased wetting of the tablet
and a subsequent reduction in the rate of dissolution. Fumed silicon dioxides are
TABLE 4 Commonly Used Disintegrants
Disintegrant
Normal
Concentration
(%) Comments
Starch 5 – 10 Probably works by wicking; swelling
minimal at body temperature
Microcrystalline cellulose Strong wicking action; loses disintegrant
action when highly compressed
Insoluble ion exchange resins Strong wicking tendencies with some
swelling action
Sodium starch glycolate a 2 – 8 Free - fl owing powder that swells rapidly
on contact with water
Croscarmellose sodium a 1 – 5 Swells on contact with water
Gums — agar, guar, xanthan < 5 Swell on contact with water; form viscous
gels that can retard dissolution, thus
limiting concentration that can be used
Alginic acid, sodium alginate 4 – 6 Swell like gums but form less viscous gels
Crospovidone a 1 – 5 High wicking activity
a Often mentioned as superdisintegrant.
SELECTION OF PHARMACEUTICAL EXCIPIENTS 889
890 PHARMACEUTICAL PREFORMULATION
TABLE 5 Lubricants Commonly Used in Formulations
Lubricant
Typical
Percent Comments
Glidants
Talc 1–5 Fine, crystalline powder; Widely used as lubricant
and diluent
Fumed silicon dioxide:
Aerosil, Cab - O - Sil,
Syloid
0.1–0.5 Has small particle size and large surface area for
good fl owability; used for adsorbent, antitacking
agent disintegrant, and glidant
Starch 1 – 10 Mainly used for binder, disintegrant, and diluent but
also used for glidant
Sodium lauryl sulfate 0.2 – 2 Anionic surfactant, luricant and wetting agent
Boundary Lubricants
Magnesium stearate 0.2 – 2 Hydrophobic, variable properties between suppliers
Calcium silicate 0.5 – 4 Hydrophobic
Sodium stearyl fumarate 0.5 – 2 Less hydrophobic than metallic stearates, partially
soluble
Polyethylene glycol 4000
and 6000
2 – 20 Soluble, poorer lubricant activity than fatty acid
ester salts
Sodium lauryl sulfate 1 – 3 Soluble, also acts as wetting agent
Magnesium lauryl sulfate 1 – 3 Acts as wetting agent
Sodium benzoate 2 – 5 Soluble
Fluid Lubricants
Light mineral oil 1 – 3 Hydrophobic, can be applied to either formulation
or tooling
Hydrogenated vegetable
oil
1 – 5 Hydrophobic, used at higher concentrations as
controlled - release agents
Stearic acid 0.25 – 2 Hydrophobic
Glyceryl behenate 0.5 – 4 Hydrophobic, also used as controlled - release agent
perhaps the most effective glidants. These are materials with very small (10 - nm)
spherical particles that may achieve their glidant properties. They are available in a
number of grades with a range of hydrophobic and hydrophilic forms and also commercially
available under diverse brand names. Starch has also been used as a
glidant. The use of large amounts of starch has also aided the disintegration
properties.
Die - wall lubricants can be dived into two classes, fl uid and boundary lubricants .
Fluid lubricants work by separating moving surfaces completely with a layer of
lubricant. These are typically mineral oils or vegetable oils, and they may be either
added to the mix or applied directly to the die wall by means of wicked punches.
The oily lubricants may have a mottled appearance in the tablet due to uneven distribution,
poor powder fl ow due to their tacky nature, and reduced tablet strength.
Fluid lubricants include stearic acid, mineral oils, hydrogenated vegetable oils, glyceryl
behenate, paraffi ns, and waxes. Boundary lubricants work by forming a thin
solid fi lm at the interface of the die and the tablet. Metallic stearates are the most
widely used boundary lubricants. Such lubricants should have low shear strength
and form interparticulate fi lms.
Magnesium stearate is the most widely used lubricant. The magnesium stearate
used in the pharmaceutical industry is not a pure substance but a mixture of magnesium
salts of fatty acids, though predominantly magnesium stearate and magnesium
palmitate. Despite its popularity, which is a refl ection of its excellent lubricant
properties, it has some problems associated with product consistency: its effect on
tablet strength and its hydrophobicity. The U.S. Pharmacopeia (USP) requires that
the stearate content should account for not less than 40% of the fatty acid content
of the material, and the stearate and palmitate combined should account for not
less than 90%. Within this defi nition, there are a range of materials to be supplied
as magnesium stearate. For a given formulation, it is important that a single source
of magnesium stearate be used for all batches to get product reliability.
The lubricant activity of magnesium stearate is related to its readiness to form
fi lms on the die wall surface. As a result, it has two consequences: a reduction in the
ability of the powder to form strong compacts and, due to its hydrophobicity, a deleterious
effect on the dissolution rate of the tablets. The hydrophobic surfaces
created by magnesium stearate have been shown to reduce the rate of dissolution
and bioavailability of several tablet formulations. When both lubricant and disintegrant
are being added to a granulated formulation, the disintegrant should be
blended with the granules prior to the addition of the lubricant to minimize the risk
of forming a hydrophobic fi lm around the disintegrant.
The third class of lubricant activity is the antiadherent. Some materials have
adhesive properties and can adhere to the punch surfaces during compression. This
will induce tablet disorders: sticking, with a fi lm forming on the surface of the tablets,
or picking, where solid particles from the tablet stick to the punch surface. Most die
wall lubricants also have antiadherent actions, and in many formulations, the addition
of a specifi c antiadherent will not be required separately. The antiadherent
includes talc, maize starch, and microcrystalline cellulose.
Coating Materials The core compressed tablet can be used by itself, but are additional
coating process of the compressed tablet can be applied for several reasons:
(a) protection of the drug from the environment (moisture, air, light) for stability
reasons, (b) taste masking, (c) minimizing patient/operator contact with drug substance,
particularly for skin sensitizers, (d) improving product identity and appearance,
(e) improving ease of swallowing, (f) improving mechanical resistance and
reducing abrasion and attrition during handling, and mostly (g) modifying release
properties.
There are three main methods used to coat pharmaceutical tablets: sugar coating,
fi lm coating , and compression coating . Sugar coating has been the most commonly
used method and involves coating tablets with sucrose. A sugar - coated tablet is
water based and generally starts to break up in the stomach. This is a highly skilled
and multistep process that is very labour intensive. This coating process results in a
50% increase of the fi nal tablet weight and in a signifi cant increase in tablet size.
Traditionally, sugar coating has been performed in coating pans in which the tablets
are tumbled in a three - dimensional direction. The pan is supplied with a source of
warm air for drying and an extraction system to remove moist air and dust. The
coating solution is distributed around the tablets by their tumbling action. A dusting
SELECTION OF PHARMACEUTICAL EXCIPIENTS 891
892 PHARMACEUTICAL PREFORMULATION
powder may be sprinkled onto the surface of the tablets during the drying phase to
prevent the tablets from sticking together. The cycle of wetting and drying is continued
until the desired amount of coating has been applied to the tablets. Typically,
a sugar coating will consist of three types of coats: a sealing coat, a subcoat, and a
smoothing coat. Traditionally, a seal coating of shellac dissolved in ethanol or synthetic
water - resistant polymers such as cellulose acetate phthalate or polyvinylacetate
phthalate is used. The subcoat is an adhesive coat on which the smoothing coat
of the sharp corners of the tablet can be built. The subcoat is a mixture of a sucrose
solution and an adhesive gum, such as acacia or gelatin, which rapidly distributes
over the tablet surface. A dusting follows each application of solution with a subcoat
powder containing materials such as calcium carbonate, calcium sulfate, acacia, talc,
and kaolin. The smoothing coat consists of the majority of the tablet bulk and provides
the tablet with a smooth fi nish. A colorant is also applied if needed. The coat
consists of sucrose syrup which may contain starch or calcium carbonate. The coated
tablets are usually transferred to a polishing pan and coated with a beeswax –
carnauba wax mixture to provide a glossy fi nish to the surface.
Film coating involves the application of a polymer fi lm to the surface of the tablet,
gelatin capsules, and multiparticulate systems with a negligible increase in tablet
size. The method of application of the coat differs from the sugar coating in that the
coating suspension is sprayed directly onto the surface of the tablets, and drying
occurs as soon as the coat hits the tablet surface. The tablet only receives a small
quantity of coating solution at a time. The fi lm coat can generally be affected by the
following properties: a method of atomizing the coating suspension, the ability to
heat large volumes of air (which heat the tablets and facilitate the rapid drying of
the applied coat), and a method of moving the tablets that ensures all tablets are
evenly sprayed.
The main methods of coating are modifi ed conventional coating pans, side - vented
pans, and fl uid bed coating. The side - vented pan, now the most commonly used
equipment for fi lm coating, was designed to maximize the interaction between the
tablet bed and the drying air. The mixing effi ciency of the table, granules, or pellets
is achieved by using appropriately designed baffl es on the pan surface. Fluid - bed
coating offers an alternative to pan coating and is particularly popular for coating
multiparticulate systems. There are three methods by which the coating can be
applied: top spraying, bottom spraying, and tangential coating. The pellets, granules,
or tablets being coated are suspoended in an upward stream of air, maximizing the
surface available for coating. The coating is applied by an atomizer, and this is dried
by the fl uidizing air.
Table 6 gives commonly used polymers for fi lm coating of core tablet. With the
exception of HPMC, the polymers are rarely used alone but are combined with
other polymers to optimize the fi lm - forming properties. A polymer for fi lm coating
will ideally meet the following criteria:
1. Solubility in the solvent selected for application: Currently the organic solvent
are replaced with water as a suspension system, although certain types of fi lm
coatings may require organic solvents to be used. Commonly used solvents
include alcohols (methanol, ethanol, and isopropanol), esters (ethyl acetate
and ethyl lactate), ketones (acetone), and chlorinated hydrocarbons (dichloromethane
and trichloroethane).
2. Solubility in GI fl uids: The solubility of polymers is dependent on its physicochemical
nature and pH. Unless the coating is being applied for enteric coating,
it should ideally be soluble across the range of pH values encountered in the
GI tract.
3. Capacity to produce an elegant fi lm even in the presence of additives such as
plasticizers, pigments, and colorants.
4. Compatibility with fi lm - coating additives and the tablet being coated.
5. Stability in the environment under normal storage conditions.
6. Freedom from undesirable taste or odor.
7. Lack of toxicity.
Enteric coating materials are also used to prevent release of the drug substance
in the stomach if the drug is either an irritant to the gastric mucosa or unstable in
gastric juice. Table 7 lists enteric coating polymers commonly used in tablet formulations.
The choice of of enteric coating material depends on its solubility.
The third type of tablet coating is multiple - compression coating to make a bilayered,
multilayered tablet (a layered tablet of two drugs) or a tablet within a tablet
(a core of one drug and a shell of another). The multilayered tablet is prepared by
initial compaction of a portion of the fi ll materials in a die followed by additional
fi ll material and compression, depending on the number of fi ll materials. The layered
tablet can provide some advantages. Each layer has different drug in a separate
layer. The incompatible drug can be compressed simultaneously at different layers.
TABLE 6 Commonly Used Polymers for Film Coating of Core Tablet
Polymer Comments
Methylcellulose (MC) Soluble in cold water, GI fl uids, and a range of organic
solvents
Ethylcellulose (EC) Soluble in organic solvents, insoluble in water and GI
fl uids; used alone in modifi ed - release formulations
and in combination with water - soluble cellulose for
immediate - release formulations
Hydroxyethylcellulose (HEC) Soluble in water and GI fl uids
Methyl hydroxyethylcellulose
(MHEC)
Soluble in water and GI fl uids; has similar fi lm - forming
properties to HPMC but is less soluble in organic
solvents, which limited its popularity when solvent
coating was the norm
Hydroxypropyl cellulose (HPC) Soluble in cold water, GI fl uids, and polar solvents;
becomes tacky when dried, so is unsuitable for use
alone, often used in combination with other polymers
to optimize adhesion of coat
Hydroxypropyl methylcellulose
(HPMC)
Soluble in cold water, GI fl uids, alcohols, and
halogenated hydrocarbons; excellent fi lm former and
the most widely used polymer; can be used with
lactose to improve adhesiveness
Sodium carboxymethylcellulose
(NaCMC)
Soluble in water and polar solvents
SELECTION OF PHARMACEUTICAL EXCIPIENTS 893
894 PHARMACEUTICAL PREFORMULATION
The staged release and improved appearance from the layered tablet are also possible.
In the preparation of a tablet within a tablet, a special tableting machine is
required to place the intended core tablet precisely within the die, which already
contains some of the coating formulation to surround the fi ll materials. This multilayered
tablet or tablet within a tablet is also useful to get the modifi ed release,
either immediately or in a sustained manner.
Auxiliary Excipients Plasticizers are added during the tablet coating process. The
fi lm coating process involves two important stages: droplet formation and fi lm formation.
Film formation is a multistage process that involves wetting the tablet
surface followed by spreading the fi lm and eventually coalescence of the individual
fi lm particles into a continuous fi lm. Most fi lm - forming polymers have glass transition
temperatures in excess of the temperatures reached during the coating process
(typically 40 – 50 ° C), so it is necessary to add plasticizers to the formulations which
reduce the glass transition temperature. The choice of plasticizer is dependent on
the type of polymer and its permanence and compatibility. Permanence is the duration
of the plasticizer effect; the plasticizer should remain within the polymer fi lm
to retain its effect, so it should have a low vapor pressure and diffusion rate. Compatibility
requires the plasticizer to be miscible with the polymer. Commonly used
plasticizers include phthalate esters, citrate esters, triacetin, propylene glycol, polyethylene
glycols (PEGs), and glycerol.
The pigments or opacifi ers are also combined with the coating solution to get
colored tablets. Insoluble pigments are normally preferred to soluble dyes for a
number of reasons. Solid pigments produce a more opaque coat than dyes, protecting
the tablet from light. The presence of insoluble particles in the suspension allows
TABLE 7 Enteric Coating Polymers Commonly Used in Tablet Formulations
Polymer Solubility Profi le Comments
Shellac Above pH 7 Original enteric coating material, originally
used in sugar - coated tablets; high pH
required for dissolution may delay drug
release; natural product which exhibits
batch - to - batch variability
Cellulose acetate
phthalate (CAP)
Above pH 6 High pH required for dissolution a
disadvantage; forms brittle fi lms, so must be
combined with other polymers
Polyvinylacetate
phthalate (PVAP)
Above pH 5 —
Hydroxypropyl
methylcellulose
phthalate (HPMCP)
Above pH 4.5 Optimal dissolution profi le for enteric coating
Polymers of
methacrylic acid
and its esters
Various grades
available with
dissolution
occurring
above pH 6
—
the rate of solid application to the tablet to be increased without having an adverse
effect on the viscosity of the coating suspension, improving productivity.
6.1.2.4 Selection Guideline of Pharmaceutical Excipients in Tablet Formulation
In the proper selection of pharmaceutical excipients for the formulation, there are
numerous factors to be considered [7] . The type of excipient is highly dependent on
the model compound and its intended dosage form. The preparation method of the
dosage form and proper dosage regime are also factors.
The formulation parameters of the tablet are essential. The amount and type of
excipients relative to drug contents should be considered to determine the size of the
dosage form. The bulk density of excipients and drug and their fi lling doses in the die
are important factors to be considered in the tablet formulation . Uniform distribution
of drug or excipient is in trouble if its contents are low in the formulation, while the
use of high contents are diffi cult to fi ll in the die and the tablet size is also larger.
Excipients that are potentially incompatible with the drug should be avoided. The
amount and type of reactive impurities in the excipients should also be established.
Batch - to - batch and supplier - to - supplier variation in impurity levels is possible.
Excipients with potential adverse interaction and any unwanted incompatibility with
drug should be avoided in the formulation. For an ideal formulation, it is helpful to
consider these para meters as well as drug – excipient incompatibility.
The global harmonization and standardization of pharmaceutical excipients are
nowadays necessary in the formulation studies [3, 7] . Since all excipients in the market
worldwide are supplied from many countries or companies with facilities in more
than a single country, the quality of the excipients must be well documented and validated.
Otherwise, the quality of the excipients should be varied batch to batch, factory
to factory, and country to country. There are also hundreds of different brands and
grades of excipients available, but it would be unrealistic for the formulator to expect
to have a totally free choice. Most manufacturing companies select the excipients
used in their factory on the basis of cost, availability, and performance. To establish
the equivalency of excipients obtained from different sources, it is also necessary to
perform some kind of functionality testing. Since the sources, origin, and manufacturer
of the excipients are different, regulatory approval for the product is required
in each country. The standards for each drug substance and excipient are contained
in pharmacopeia. The four pharmacopeia with the largest international use are the
United States Pharmacopeia (USP) and National Formulary (NF), British Pharmacopoeia
(BP), European Pharmacopoeia (EP), and Japanese Pharmacopoeia (JP).
Unless global harmonization is established, analytical methods, testing criteria,
and specifi cation limits must be variable according to monographs of pharmacopeia
from the different countries. For example, Table 8 lists the different specifi cation
limits for the viscosity of cellulose ether among three pharmacopeias. If global harmonization
and standardization of pharmaceutical excipients are established, the
marketing and sales of a single formulation are more facilitated worldwide. The
manufacturing cost and research - and - development (R & D) cycles are also reduced.
Most of all, the quality and bioequivalence of the drug products with the same formulation
can be more validated since the regulatory approval of the pharmaceutical
product is enhanced. The global harmonization is an ongoing effort by corporate
representatives and international regulatory authorities.
SELECTION OF PHARMACEUTICAL EXCIPIENTS 895
896 PHARMACEUTICAL PREFORMULATION
In the selection of excipients, formulation scientists should also remember that
every excipient is not limited by a single function but rather can be used for many
pharmaceutical applications in dosage forms. For example, cellulose and its derivatives
(HPMC, EC, HPC) have been widely used as fi llers, binders, suspending agents,
and mainly controlled - release agents. Povidone, polymethacrylate, and cyclodextrins
are also multifunctional excipients in formulations. Table 9 gives the physical properties
of some directly compressible fi llers. The grades of physical properties are
variable among fi llers used in tablet formulation. The simple and optimal formulation
of drugs with multifunctional excipients can provide some advantages in tablet
preparations.
6.1.3 DRUG – EXCIPIENT COMPATIBILITY
The potential for excipients to cause chemical and physical instability in drugs has
been recognized for over 30 years. Drug compatibility studies have been used as an
TABLE 8 Specifi cation Limits for Viscosity of Cellulose Ether
Parameter EP USP XXII JP XII
Concentration of
solution, %
2.0 2.0 2.0
Temperature, ° C 20 ± 0.1 20 ± 0.1 20 ± 0.1
Type of viscometer Rotating
viscometer
Capillary type,
Ubbelohde
Capillary type,
Ubbelohde
Shear rate, S . 1 10 — —
Unit cP (Pa · s) cP (Pa · s) cSt
Sample preparation a
a Very similar in all three pharmacopeia.
TABLE 9 Comparative Properties of Some Directly Compressible Fillers a
Filler Compactibility Flowability Solubility Disintegration Hygroscopicity Lubricity Stability
Dextrose 3 2 4 2 1 2 3
Spray dried
lactose
3 5 4 3 1 2 4
Fast - Flo
lactose
4 4 4 4 1 2 4
Anhydrous
lactose
2 3 4 4 5 2 4
Emdex
(dextrates)
5 4 5 3 1 2 3
Sucrose 4 3 5 4 4 1 4
Starch 2 1 0 4 3 3 3
Starch 1500 3 2 2 4 3 2 4
Dicalcium
phosphate
3 4 1 2 1 2 5
Avicel
(MCC)
5 1 0 2 2 4 5
a Graded on a scale from 5 (good/high) to 1 (poor/low); 0 means none.
approach for accepting/rejecting excipients for use in pharmaceutical formulations
[1, 10 – 12] . In general, drug stability can be investigated under the stress condition
according to the guideline of accelerated stability testing. The factors for stress
condition usually include temperature, pH, light, moisture, agitation, gravity, packaging,
and method of manufacture [13, 14] . Despite this fact, approaches for excipient
selection in drug formulation are often empirical. The stability issues in the development
of a drug must be considered at the early and late formulation stages. Nowadays,
advanced analytical instrumentation makes it possible to more rapidly identify
potential excipient - induced instability to select excipients. Excipients that exhibit
incompatibility with the drug are “ rejected ” and not included in subsequent tablet
formulation studies.
6.1.3.1 Experimental Studies for Drug – Excipients Compatibility
Compatibility studies are carried out by mixing drug with one or more excipients
under some type of stress condition. It has also been suggested that aqueous suspensions
of the drug and excipients or drug – excipient complexes provide a better model
for tablet formulations. It has been recommended that small - scale formulations
using the selected excipients (this may eventually be used for the eventual formulation)
be prepared using experimental processes.
Table 10 provides examples of binary and factorial design for drug – excipient
compatibility studies. The two conditions of each potential parameter can be included
as a binary system. All potential parameters can also be combined for factorial
design. For example, water is added to the drug – excipient mixture at reasonable
temperatures (50 ° C or less) and then both intact drug and degradation products
are measured. The high - temperature and high - humidity conditions are usually used
to obtain more rapid stability assessment of drug and excipients. Generally, physicochemical
properties such as drug content, color, taste, related substances, thermal
analysis, and high - performance liquid chromatography (PLC) studies of evaluated
for drug – excipient compatibility studies using conventional analytical techniques.
6.1.3.2 Analytical Methods for Drug – Excipient Incompatibility
The key to the early assessment of instability in formulations is the availability of
analytical methods to detect low levels of degradation products, generally less than
2%. With the aid of thermal analysis and chromatographic methods [HPLC and
TABLE 10 Examples of Binary and Factorial Designs for Drug-Excipient Compatibility
Studies
Binary Design Factorial Design
Drug – excipient ratio 1 : 1 or 1 : 10 Total number of formulation = a . b . c . d . e
Water addition = yes/no Type of drug – excipient ( a )
Storing temperature 25 ° C or 40 ° C/75% Amount of drug – excipient ( b )
Storing time 1 or 4 weeks Water addition ( c )
Storing temperature ( d )
Storing time ( e )
DRUG–EXCIPIENT COMPATIBILITY 897
898 PHARMACEUTICAL PREFORMULATION
liquid chromatography/mass spectrometry (LC/MS)], it can assign at least tentative
structures for most degradation products as well as the intact drug contents [15] .
Excipient compatibility screening must provide more rapid identifi cation of excipient
- mediated instability that is detected in complete formulations.
Early compatibility studies relied on color change as an indication of incompatibility.
Subsequently, there were numerous reports on the use of thermal analysis
techniques such as differential thermal analysis (DTA) or differential scanning calorimetry
(DSC) to detect drug – excipient incompatibilities. DTA and DSC have the
advantage of rapid analysis. Generally, formation of new peaks and disappearance
of drug peak by the endothermic or exothermic reaction are carefully investigated.
Diffuse refl ectance spectroscopy (DRS) can be also used to determine the ultraviolet
(UV) absorption of drug on the surface of drug – excipient mixtures.
6.1.3.3 Reaction Types and Stabilization Guidelines
Understanding the degradation chemistry of drug with excipients is essential to
select proper excipients in the formulation stages [16, pp 101 – 151]. Drug - excipient
compatibility studies are crucial to decide optimal tablet formulation and to understand
the possible mechanism in many cases [10, 12, 14] . Drug instability occurs by
three types of reactions: hydrolysis, oxidation, and aldehyde – amine addition. Table
11 gives reaction types of chemical and physical instability.
Hydrolysis is the most common mechanism to induce drug – excipient incompatibility.
Most hydrolysis reactions are catalyzed by acids and/or bases. The microscopic
pH between drug and excipients is critical. The pH degradation rate profi les are
good predictors for hydrolysis reactions in solid dosage forms. The pH of optimal
stability in solution is similar to the “ microscopic pH ” in solid dosage forms. A shift
TABLE 11 Reaction Types of Chemical and Physical Instability
Type of Instability Order of Frequency
Chemical instability
pH - dependent hydrolysis 12
Oxidation
Air oxidation 5
Metal - catalyzed oxidation 3
Peroxides in excipients 2
Aldehyde – amine addition
Aldose excipient 2
Formaldehyde from excipients 1
Michael addition with maleate salt 1
Intramolecular cyclization 1
Dimerization (Diels – Alder) 1
Racemization 1
Addition of ammonia residue from gelatin capsule 1
Esterifi cation 1
Physical instability
Evaporation of volatile free base 2
Reaction of methanesulfonic acid with disintegrants 1
Gelatin cross - linking by excipient impurities 1
in only one pH unit can increase or decrease the reaction rate by a factor of 10.
Although solid - state compatibility studies are commonly used, solution kinetic
studies are generally conducted to select excipients since it is possible to study all
three of these reactions in solution. Often solution kinetic studies allow the identi-
fi cation of more potential degradation products and hence the development of
better stability - indicating assays. The pH of optimal stability is also important for
selecting the appropriate salt form of the compound and excipients in the solid
formulation. Acetylsalicylic acid (ASA) is a readily hydrolyzable drug and there are
literature references to its rate of hydrolysis in aqueous solution.
The moisture content of the drug and excipients plays a critical role in their
incompatibility by hydrolysis. Excipients such as starch and povidone have particularly
high water contents (povidone contains about 28% equilibrium moisture at
75% relative humidity), which can increase the possibility of drug contact. Magnesium
trisilicate causes increased hydrolysis of aspirin in tablet form because, it is
thought, of its high water content. For these reasons, some scientists recommend
inclusion of water in the samples for compatibility studies. Depending on the degree
of hydrolytic susceptibility, different approaches for tablet formulation can be used
to minimize hydrolysis. For compounds such as ASA that are readily hydrolyzable,
direct compression or dry granulation is preferable rather than wet granulation.
However, drug – excipient incompatibility still occurrs. Chemical interaction between
moieties of drug and excipients may lead to increased decomposition. The transacetylation
reaction between aspirin and paracetamol and also possible direct
hydrolysis of the paracetamol can occur. The amount of free salicyclic acid at 37 ° C
in the tablets containing paracetamol increases by the addition of talc (0.5 – 1%). The
stearate salts should be avoided as tablet lubricants if the active component is
subject to hydroxide ion – catalyzed degradation. The degradative effect of the alkali
stearates is inhibited in the presence of malic, hexamic, or maleic acid.
As a general rule, in selecting excipients, it is probably best to avoid hygroscopic
excipients when formulating hydrolytically labile compounds. One of the most effective
ways to stabilize a pH - sensitive drug is through adjustment of the microscopic
pH of the formulation. Excipients with high pH stability and buffering agents are
recommended. The equilibrium moisture content (hygroscopicity) at different relative
humidities for a variety of drug and excipients would be a clue to selecting
optimal formulations. If dry processing and the use of nonhygroscopic excipients
still result in unacceptable rates of hydrolysis, use of a dessicant and/or moisture
protective packaging can further increase stability against drug hydrolysis. The
manufacturing process should be conducted under low - humidity conditions and not
during the hot summer season to improve drug – excipient compatibility.
Oxidation reactions are complex and it is diffi cult to understand the reaction
mechanism. The best approach is to avoid excipients containing oxidative reactants
such as peroxides and metal ions. The air oxidation or metal ion – catalyzed oxidation
can be tested after storing the samples in the solutions. The impurities in excipients
such as povidone or as degradation products in PEGs are organic peroxides and
are typically more reactive than hydrogen peroxide. A commercially available
organic peroxide such as tert - butyl hydroperoxide is better to evaluate the susceptibility
of a compound to peroxide oxidation rather than the hydrogen peroxide.
Reactive impurities such as peroxides and ionic chemicals (talc and titanium oxide)
in the excipients commonly may act as catalysts for oxidation of the drug.
DRUG–EXCIPIENT COMPATIBILITY 899
900 PHARMACEUTICAL PREFORMULATION
Therefore, it is essential to remove any oxidative peroxides and ionic chemicals.
Use of free - radical scavengers such as butylated hydroxyanisole (BHA) or butylated
hydroxytoluene (BHT) can stabilize the oxidation reaction of the drug with
excipients via a free - radical mechanism. However, uniform distribution of the low
levels of these antioxidants in solid formulations is quite diffi cult. The BHA is added
to both lovastatin and simvastatin tablets. If an oxidation reaction is catalyzed by
metal ions or excipients with high transition metal ion contents (talc), chelating
agents can also be used to bind trace metal ions. However, citrate is often the agent
of choice because of the potential toxicity of many chelating agents. Oxidation is
less likely to occur if the oxidizable group of phenols/catechols and secondary and
tertiary amines is protonated. If some oxidation reactions are catalyzed by light, the
formulation can be stabilized by a light - absorbing coating.
Aldehyde – amine addition is also a typical reaction type. The potential aldehyde
interaction also makes sense to screen out a compound as part of the preformulation
evaluation. All aldoses such as lactose or excipients such as starch and microcrystalline
cellulose that have terminal aldose groups should be avoided with the excipients
having primary/secondary amines. 5 - (Hydroxymethyl) - 2 - furaldehyde (HMF) would
be a good choice since it is a degradation product of many sugars (lactose) and celluloses,
at least in trace levels. The Schiff ’ s base[ – CH=N – ] is formed when the sugar
aldehyde (lactone) and the primary/secondary amines are mixed. The isoniazide
and trace level of HMF from lactose can also readily form the Schiff ’ s base. It has
been reported that the reaction of hydrazine hydrochloride and starch to form
high - molecular - weight addition products gives high - molecular - weight products.
The reaction of fl uoxetine hydrochloride is more rapid with spray - dried lactose
monohydrate.
Many excipients are acids or bases or have acidic or basic impurities. For this
reason, the reaction of amines with aldehydes requires that the amine exist in the
nucleophilic - free base, rather than the protonated cationic form. For example, the
reaction of fl uoxetine hydrochloride with lactose was much more rapid as potassium
hydroxide was added to neutralize the hydrochloride salt. Michael addition between
seproxetine and maleic acid from a tablet formulation following dissociation of the
hydrochloride salt occurs because of the conversion of a salt to a free acid/base.
Finally, the physical instability that can occur is the cross - linking of gelatin or its
capsules. Low levels of aldehyde impurities in excipients (starch, polysorbate 80,
PEG, and rayon coils) and packaging materials have been reported to cause cross -
linking of gelatin. Dissolution slowing is also more pronounced for wet granulation
tablets than direct - compression tablets. Dissolution slowing appears to be due to
hardening of the water - soluble excipients and a reduction of disintegration time.
Once suspect excipients have been eliminated from consideration, small - scale
formulations with manufacturing processes such as granulation, drying, and compression
can be used to assess whether interactions between excipients or processing
conditions result in any unpredicted instability. The ratio of drug and excipients is
also a critical factor to consider in incompatibility studies. In addition, the lower the
drug dose, the greater the possibility of degradation by low - level impurities in the
excipients.
In conclusion, drug – excipient compatibility studies have a key role at the early
preformulation stages to select excipients or after formulation to help identify the
mechanism of any detected instability [14] . An understanding of the potential physicochemical
interactions of drug with known chemical reactivities of excipients and
POWDER CHARACTERISTICS 901
their impurities will aid in the proper selection of excipients. Knowledge of trace
impurities/additives in excipients and the consistency of these levels from batch to
batch and supplies to supplier is also essential to select proper pharmaceutical
excipients. Although the best solution kinetics and drug – excipient compatibility
studies may be established, the possibility of unexpected instability because tablets
and capsules are complex multicomponent systems must be remembered.
6.1.4 POWDER CHARACTERISTICS
Most drug and inactive excipients used in tablet formulation are in the solid state
as amorphous powder or crystals of various morphological structures. There may
be substantial differences in particle size, surface area, crystal morphology, wetting,
and fl owability as well as many physical properties of drug, excipients, and their
blends [16] . Table 12 describes common micromeritic topics important to pharmaceutical
preformulation.
Before their use in the solid dosage forms, it is necessary to understand and characterize
the physical and chemical properties of drug, excipient, and their powder
mixtures , including crystal habit, particles size, shape, fl ow characteristics, density,
hygroscopicity, and compressibility and compaction [2, 3] . Hiestand noted that successful
tableting operations require the selection of excipients that balance desirable
physical, fl ow, and mechanical properties for tablet manufacturing [17] . The quantifi -
cation of these properies using a unifi ed approach is essential to the design and potimization
of solid dosage formulations [1] . Instrumental analyses such as scanning
electron microscopy (SEM), DSC, and powder X - ray diffraction (PXRD) can be very
useful to characterize powder properties such as purity, polymorphism, salvation,
degradation, drug – excipient compatibility, and other desirable characteristics.
6.1.4.1 Crystal Form and Habit
The morphology of a pharmaceutical solid is of importance since this property can
infl uence the bulk powder properties. The six crystal systems are cubic, hexagonal,
tetragonal, orthorhombic, monoclinic, and triclinic crystals. The observed overall
TABLE 12 Micromeritic Topics Important to
Pharmaceutical Preformulation
Particle shape
Particle size distribution
Solid geometry (packing, density, porosity, void)
Surface characteristics (adsorption, area, surface energy,
solubility)
Methods of determination
Chemical stability
Dynamics (fl ow rate, transport)
Particle separation
Processing (sieving, sedimentation, grinding, mixing,
compaction)
Sampling
Drug release
902 PHARMACEUTICAL PREFORMULATION
shape of crystal habits comprises plate, tabular, equant, columnar, blade, and avicular
as well as dendrites, treelike pattern, and spherulites, tiny crystals radiating from
a center [2, 18, 19] . Crystal form (crystal habit) as well as a noncrystalline amorphous
form may affect drug stability, dissolution rate, fl ow, mechanical properties, and
ability to mix with excipients. The amorphous form of a drug has the lowest melting
point and usually the fastest dissolution rate, but it is most likely to react or degrade.
Mechanical properties such as fl owability, miscibility, particle strength, and cohesiveness
often vary among the polymorphs, crystal shapes, or habits. Cohesiveness, the
surface free energy effect that results in particle aggregation, may also be different
for various polymorphs and habits.
6.1.4.2 Particle Size Distribution
The determination and control of particle size distribution are often very important
in pharmaceutical preformlation since the drug safety, stability, and viability of the
dosage form and manufacturing process can be signifi cantly infl uenced [19] . Particle
shape is also important in determining particle size. The particle size of materials is
readily expressed in terms of its diameter according to the defi nition of particle size.
As the degree of asymmetry of particles increases, however, so does the diffi culty
of expressing size in terms of a meaningful diameter [20] . Figure 4 shows different
ways of defi ning a diameter. The “ diameter ” can be simply the longest or the shortest
linear dimension of the crystal. If one can calculate the area of the particle, one
may obtain the diameter d a , which is the diameter of a circle with the same area as
the particle.
If all the particles in a sample are of the same size, then the powder is monodisperse.
Truly, the particles have more than one size in polydisperse samples. The
monodisperse particle size distribution is more desirable than the polydisperse one.
Therefore, the shape and surface area of the individual particles, the size ranges
based on number or weight of particles, as well as the total surface area are variable.
The commonly illustrated particle forms are sphere, rod, fi ber, granular, cubical,
fl ake, condensation fl oc, and aggregate.
The fi neness of the powder is characterized by a number (e.g., a diameter d ).
Particles, of course, will have different shapes so that there are different ways of
defi ning a diameter. The technique for obtaining d a given above has been used
microscopically. More conventional is the so - called surface mean diameter, which,
is the diameter of a sphere that has the same surface area as the particle. The so -
called single - particle volume mean diameter is possible if there are instruments that
can measure the volume of an odd - shaped particle. If the shape factor is indepen-
FIGURE 4 Different ways of defi ning a diameter.
Irregular shape Sphere with area (A) and volume (V)
d (small)
d (A), d(V) d (large)
POWDER CHARACTERISTICS 903
dent of the size of the particle, then the shape is called isometric. Examples of isometric
shapes are cube, sphere, and cylinder.
Suppose a sample of 12 particles of corn starch with fairly narrow particle size
distribution (25 – 35 . m) were measured. In this case, the distribution of particles is
very narrow and approximately distributed as normal or Gaussian pattern. To
convert these numbers into frequencies, it is noted that there are 12 particles in
total; that is, dividing each number by 12 and multiplying this by 100 will give the
percent frequency, as shown in Table 13 .
If the frequency is plotted as a function of the midpoint of the diameter ranges,
then a frequency histogram is obtained. It is noted that for the noncumulative curve
the midpoints of the intervals are used, but for the cumulative curve the interval
endpoint is used. When the number or weight of particles lying within a certain size
range is plotted against the size range or mean particle size, a frequency distribution
curve is obtained However, due to the deviation from the normal distribution of
particles, a lognormal distribution of the particle size is statistically plotted against
the cumulative percent frequency on a probability scale, and a linear relationship is
observed (Figure 5 ). Probability paper is a type of paper that straightens out this
type of S - shaped curve. The logarithm of the particle size is equivalent to 50% on
the probability scale, that is, the 50% size is known as the geometric mean diameter
and the slope is given by the geometric standard deviation .g , which is also the quotient
of the ratio: (84% undersize or 16% oversize)/(50% size) or (50% size)/(16%
undersize or 84% oversize).
Many methods are available for determining particle size in pharmaceutical
practice, including microscopy, sieving, sedimentation, and determination of particle
volume [19] . Sieve analysis with U.S. standard sieves is widely used to determine the
particle size distribution based on powder weight. Sieves are classifi ed according to
the number of openings (Table 14 ) and are generally made of wire cloth woven
from brass, bronze, or other suitable wire.
The USP uses descriptive terms to characterize the particle size of a given
powder, which are related to the proportion of powder that is capable of passing
through the openings of standardized sieves of varying dimensions in a specifi ed
time period under the mechanical sieve shaker as follows:
Coarse (or a no. 20) powder: All particles pass through a no. 20 sieve and not
more than 40% through a no. 60 sieve.
Moderately coarse (or a no. 40) powder: All particles pass through a no. 40 sieve
and not more than 40% through a no. 60 sieve.
TABLE 13 Particle Size Distribution of Powdered Cornstarch Samples
Particle Size
Range ( . m)
Number of
Occurrences ( n )
Percent
Frequency
Cumulative
Frequency (%)
25 – 27 1 8.3 8.3
27 – 29 3 25.0 33.3
29 – 31 4 33.3 (33.4) 66.7
31 – 33 3 25 91.7
33 – 35 1 8.3 100
Total 12 99.9 (100) —
904 PHARMACEUTICAL PREFORMULATION
Fine (or a no. 80) powder: All particles pass through a no. 80 sieve. There is no
limit as to greater fi neness.
Very fi ne (or a no. 120) powder: All particles pass through a no. 120 sieve. There
is no limit as to greater fi neness.
1 10 100
0.01
0.1
1
10
30
50
70
90
99
99.9
Weight
Number
Cumulative % undersize
Particle size (.m)
FIGURE 5 Typical lognormal distribution of particles based on weight and number.
TABLE 14 Openings of Standard Sieves
Sieve Number Sieve Opening
2 9.5 mm
3.5 5.6 mm
4 4.75 mm
8 2.36 mm
10 2.00 mm
20 850 . m
30 600 . m
40 425 . m
50 300 . m
60 250 . m
70 212 . m
80 180 . m
100 150 . m
120 125 . m
200 75 . m
230 63 . m
270 53 . m
325 45 . m
400 38 . m
POWDER CHARACTERISTICS 905
Microscopy, in which the particles are sized through the use of a calibrated grid
background or other measuring devices (range 0.2 – 100 . m). SEM can also readily
measure the smallest particle size. The microscope allows the observer to view the
actual particles, but it gives two - dimensional views. With the sedimentation rate,
particle size is determined by measuring the terminal settling velocity of particles
through a liquid medium in a gravitational or centrifugal environment (range
0.8 – 300 . m). Sedimentation rate may be calculated from the well - known Stokes
equation. The sedimentation methods yield a particle size relative to the rate at
which particles settle through a suspending medium, a measurement important in
the development of emulsions and suspensions. With light energy diffraction (light
scattering), particle size is determined by the reduction in light reaching the sensor
as the particle, dispersed in a liquid or gas, passes through the sensing zone (range
0.2 – 500 . m). On the other hand, laser scattering utilizes a H 3 – Ne laser, silicon
photodiode detectors, and an ultrasonic probe for particle dispersion (range 0.02 –
2000 . m). The measurement of particle volume using a Coulter counter allows one
to calculate an equivalent volume diameter, but no information on shape of the
particles is available. Laser holography, in which a pulsed laser is fi red through an
aerosolized particle spray and photographed in three dimensions with a holographic
camera, allows the particles to be individually imaged and sized (range
1.4 – 100 . m). The above methods and others may be combined to provide greater
assurance of size and shape parameters. Automated particle size analyzers linked
with computers are commercially available for data processing, distribution analysis,
and printout.
Determination and control of particle size are often prerequisites in preformulation
stages because the size distribution of excipients, drug, and their mixtures can
infl uence safety, effi cacy, stability, viability of dosage form, and manufacturing processes.
Furthermore, the particle size of pharmaceuticals can affect uniform mixing,
fl ow characteristics, formulation characteristics, dose - to - dose content uniformity,
dissolution rate, and bioavailability of drug. Tablet characteristics such as porosity
and fl owability are highly affected by the particle size as well. The smallest particles
induce electrostatic forces and aggregations while the larger particles show greater
weight variations. The ideal size ranges of particles are usually 10 – 150 . m. Therefore,
detailed information of the particle size of drug, excipients, and their blends should
be required in tablet formulation as well as regulatory issues. Particle size is also
important in the tableting fi eld, since it can be very important for good homogeneity
in the fi nal tablet. The particle size should be consistent throughout the production
to satisfy table formulation and regulatory demands.
6.1.4.3 Flow Characteristics
Good fl ow properties are a prerequisite for the successful manufacture of both
tablets and powder - fi lled hard gelatin capsules. Proper fl uidity of materials is
required to be transported through the hopper of a tableting machine. The elongated
particle shape and small particle size could cause high tablet weight variation,
strength, unacceptable blend uniformity, and diffi culty in fi lling containers and
dies. Excipients with good fl ow characteristics and low cohesive powders
should be more preferable in tablet production. Powder fl ow is affected by the
numerous parameters, including purity, crystallinity, electrostatic forces, mechanical
906 PHARMACEUTICAL PREFORMULATION
properties (brittleness, elasticity), density, size, shape, surface area, moisture content,
direction and rate of shear, storage container dimension, and particle – wall interaction
[19] .
It is a property of all powders to resist the differential movement between particles
when subjected to external stresses. A bulk powder is somewhat analogous to
a non - Newtonian liquid, which exhibits plastic fl ow and sometimes dilatancy if the
particles being infl uenced by attractive forces. Accordingly, powders may be free
fl owing or cohesive ( “ sticky ” ). The resistance is to free fl ow is due to the cohesive
forces between particles [18] . Three principal types of interparticular forces are
forces due to electrostatic changing, van der Waals forces, and forces due to moisture.
Electrostatic forces are dependent on the nature of the particles, in particular
their conductivity. Van der Waals forces are the most important forces for most
pharmaceutical powders. These forces are inversely proportional to the square of
the distance between the two particles and hence diminish rapidly as particle size
and separation increase. Powders with particles below 50 . m will generally exhibit
irregular or no fl ow due to van der Waals forces. Particle shape is also important;
for example, the force between a sphere and a plane surface is about twice that
between two equal - sized spheres. At low relative humidity, moisture produces a
layer of adsorbed vapor on the surface of particles. Above a critical humidity, typically
in the range 65 – 80%, it will form water liquid bridges between particles. Where
a liquid bridge forms, it will give rise to an attractive force between the particles
due to surface tension or capillary forces. The role of the formulator is to ensure
that the fl ow properties of the powder are suffi cient to enable its use on modern
pharmaceutical equipment, powder hoppers, and fl ow through orifi ces in the tablet
production.
It is important that the powder fl ows from the hopper to the fi lling station of the
tablet machine at an appropriate rate and without segregation occurring. There are
two types of fl ow that can occur from a powder hopper: core fl ow and mass fl ow [2] .
Figure 6 shows the two different powder fl ow patterns in hoppers. When a small
amount of powder is allowed to leave the hopper, there is a defi ned region in which
downward movement takes place and the top surface begins to fall in the center. A
core fl ow hopper is characterized by the existence of dead spaces during discharge.
A mass fl ow hopper is one in which all the material is in motion during discharge,
in particular the areas adjacent to the hopper wall. As a small amount of powder is
discharged, the whole bulk of the powder will move downward. Whether core fl ow
or mass fl ow is achieved is dependent on the design of the hopper (geometry and
wall material) and the fl ow properties of the powder.
Powder fl ow into orifi ces is also important when fi lling dies in tablet machines
and in certain types of capsule - fi lling machines. For a given material, the fl ow into
or through an orifi ce is dependent on the particle size (Figure 7 ). In general, as the
particle size increases, the powder fl ow rate also increases. However, there is practically
no fl ow if the particle size is below 50 . m or above 1200 . m. The Carr index
gives us the guidance for powder fl owability. A lower Carr index of excipients is
more desirable for acceptable powder fl ow. At the lower end of the particle size
range, cohesive forces will result in poor fl ow. Powders with particles below 50 . m
will generally exhibit irregular or no fl ow due to van der Waals forces. As the particle
size increases, the fl ow rate increases until a maximum is achieved, at an orifi ce
diameter – particle diameter ratio of 20 – 30. As the particle size continues to increase,
POWDER CHARACTERISTICS 907
FIGURE 6 Powder fl ow patterns in hoppers.
(a) Core flow
(b) Massflow
FIGURE 7 Effect of particle size on rate of powder fl ow through orifi ce.
Particle size
Powder flow rate
No flow below 50 .m
No flow above 1200 .m
the rate decreases due to mechanical blocking or obstruction of the orifi ce. Flow
will stop completely when the orifi ce – particle ratio falls below 6 and if the size is
above 1200 . m.
There are several different methods available for determining the fl ow properties
of powders. Shear cell methods provide an assessment of powder fl ow properties as
a function of consolidation load and time. There are a number of types of shear cells
available, the most common being the Jenike shear cell [21] .
908 PHARMACEUTICAL PREFORMULATION
Common indices of fl owability are the Hausner ratio and the Carr index (compressibility).
The increase in bulk density of a powder is related to the cohesiveness
of a powder. So measurement of the bulk density of a powder is essential to defi ne
the fl ow characteristics. Ratios of poured - to - tapped bulk densities are expressed in
two ways to give indices of fl owability:
Hausner ratio
tapped bulk density
poured bulk density
=
Carr index (compressibility)
(tapped bulkdensity poured bu =
. . 100 lk density)
poured bulk density
The Hausner ratio varies from about 1.2 for a free - fl owing powder to 1.6
for cohesive powders. Carr index classifi cations for fl owability [2] are listed in
Table 15 .
Compressibility indices are a measure of the tendency for arch formation and
the ease with which the arches will fail and, as such, is a useful measure of fl ow. A
limitation of the bulk density indices for fl ow characteristics is that they only
measure the degree of consolidation; they do not describe how rapidly consolidation
occurs. Angle of repose is a common method used to measure powder fl ow with
small sample quantity. If powder is poured from a funnel onto a horizontal surface,
it will form a cone due to gravitational forces. The angle between the sides of the
cone and the horizontal is referred to as the angle of repose. So there is a correlation
between powder fl ow and angle of repose. The relationship between the Carr
index and angle of repose is now discussed. The angle of repose is a measure of the
cohesiveness of the powder, as it represents the point at which the interparticle
attraction exceeds the gravitational pull on a particle. A free - following powder will
form a cone with shallow sides, and hence a low angle of repose, while a cohesive
powder will form a cone with steeper sides. As a rough guide, angles less than 30 °
are usually indicative of good fl ow, while powders with angles grater than 40 ° are
likely to be problematic.
The avalanching behavior of powder is also a measure of fl owability. If a powder
is rotated in a vertical disc, the cohesion between the particles and the adhesion of
the powder to the surface of the disc will lead to the powder following the direction
of rotation until it reaches an unstable situation where an avalanche will occur. After
the avalanche, the powder will again follow the disc prior to a further avalanche.
TABLE 15 Carr Index Classifi cation and Powder
Flowability
Carr Index (%) Flow
5 – 12 Free fl owing
12 – 16 Good
18 – 21 Fair
23 – 35 Poor
33 – 38 Very poor
> 40 Extremely poor
POWDER CHARACTERISTICS 909
Measurement of the time between avalanches and the variability in time is a measure
of the fl ow properties of the powder.
If a powder fl ows poorly, the vibrator can be used, but it also causes powder segregation
and stratifi cation. The addition of glidant (occasionally lubricant) in the
powder mixtures can readily increase fl owability at the low concerntration. Talc or
fumed silicon dioxide is an example of a glidant. If this is not suffi cient to improve
the fl ow, other means of fl ow improvement are necessary. There are two main factors
that affect powder fl ow: particle size and particle shape. The more spherical a particle
is, the better it fl ows. Small particles are very cohesive and hence do not fl ow
well, but increasing the particle size will improve fl ow. With the aid of spray drying
or spheronizers , particles become spherical.
In general, powder below 50 . m is not very free fl owing because the cohesive
forces below this size become stronger than the gravitational force, and fl ow through
the orifi ce is prevented. This, of course, is a function of the size of the orifi ce, and
fl ow might be possible in a larger orifi ce.
Particle size enlargement of the drug substance can be brought about by manipulation
of the recrystallization step in the synthesis of the drug. To increase powder
fl ow, particle size enlargement by slugging, roller compaction, and wet granulation
can be used. If a powder is compressible but does not fl ow well, then slugging may
be employed. In slugging, tablets are made of the poorly fl owing substance on a
high - duty, slowly operating machine into large dies. The dies are large so that the
fl ow is suffi ciently increased, but now the compression forces must be increased
because the larger area dictates a larger force to attain a given pressure (the elastic
limit being in stress units). In roller compacting , the powder is processed between
two heavy - duty rollers, compacted between the rolls, and emerges as a compressed
sheet, which is then milled. These two methods are necessary if the drug substance
(e.g., aspirin) is suffi ciently moisture sensitive and there are stability issues so it
cannot be wet granulated. Otherwise, wet granulation is a frequently used method
of particle enlargement for free - fl owing powder.
6.1.4.4 Density and Bulkiness
Density When a powder is poured into a container, the volume that it occupies
depends on a number of factors, such as particle size, particle shape, and surface
properties. In normal circumstances, it will consist of solid particles and interparticlulate
air spaces (voids or pores). The particles themselves may also contain
enclosed or intraparticulate pores. If the powder bed is subjected to vibration or
pressure, the particles will move relative to one another to improve their packing
arrangement. Ultimately, a condition is reached where further densifi cation is not
possible without particle deformation. The density of a powder is therefore dependent
on the handling conditions to which it has been subjected, and there are several
defi nitions that can be applied either to the powder as a whole or to individual
particles.
Because particles may be hard and smooth in one case and rough and spongy in
another, one must express densities with great care. Density is universally defi ned
as weight per unit volume. Three types of densities — true density, particle density,
and bulk density — can be defi ned, depending on the volume of particles containing
microscopic cracks, internal pores, and capillary spaces.
910 PHARMACEUTICAL PREFORMULATION
The true density is the material itself exclusive of the voids and interparticular
pores larger than molecular or atomic dimension in the crystal lattice. Particle
(granular) density, determined by the displacement of mercury, which does not
penetrate at ordinary pressure into pores smaller than 10 . m, is the mass of the
particle divided by its volume. The different terms arise from the defi nition of
volume: True particle density is when the volume measured excludes both open and
closed pores and is a fundamental property of a material; apparent particle density
is when the volume measured includes intraparticulate pores; effective particle
density is the volume “ seen ” by a fl uid moving past the particles. Bulk density
(powder density) is the volume in a graduated cyclinder including both the particulate
volume and the pore volume. The bulk density will vary depending on the
packing of the powder. Based on the defi nition of volume, minimum bulk density is
when the volume of the powder is at a maximum, caused by aeration, just prior to
complete breakup of the bulk. Poured bulk density is when the volume is measured
after pouring powder into a cylinder, creating a relatively loose structure. Tapped
bulk density is, in theory, the maximum bulk density that can be achieved without
deformation of the particles.
Based on the defi nition of density, two new terms are defi ned. Porosity is defi ned
as the proportion of a powder bed or compact that is occupied by pores and is a
measure of the packing effi ciency of a powder and relative density is the ratio of the
measured bulk density and the true density:
Porosity
bulk density
true density
= . 1
Relative density
bulk density
true density
=
Bulkiness The specifi c bulk volume, the reciprocal of bulk density, is often called
bulkiness or bulk . It is an important consideration in the packaging and fi lling of
powders for tablet production. The bulk density of calcium carbonate can vary from
0.1 to 1.3, and the lightest or bulkiest type would require a container about 13 times
larger than the heaviest type. Bulkiness increases with a decrease in particle size. In
a mixture of materials of different sizes, however, the smaller particles sift between
the larger ones and tend to reduce the bulkiness.
To defi ne bulkiness in detail, the porosity and density of powders should be
understood. Suppose a powder, such as zinc oxide, is placed in a graduated cylinder
and the total volume is noted. The volume occupied is known as the bulk volume
V b . If the powder is nonporous, that is, has no internal pores or capillary spaces, the
bulk volume of the powder consists of the true volume of the solid particles plus
the volume of the spaces between the particles. The volume of the spaces, known
as the void volume v , is given by equation
v V V = . b p
where V p is the true volume of the particles.
The porosity or voids ( . ) of a powder is also defi ned as the ratio of the void
volume to the bulk volume of the packing given below. Porosity is frequently
expressed in percent, . . 100:
POWDER CHARACTERISTICS 911
. =
.
= .
V V
V
V
V
b p
b
p
b
1
6.1.4.5 Hygroscopicity
The hygroscopicity of a drug and pharmaceutical substances is a potential parameter
to be considered in tablet formulation. The moisture uptake rate is quite variable
depending on the type of drug and excipients as well as the environmental conditions.
So, a concise defi nition of hygroscopicity is not possible. Powders can absorb
moisture by both capillary imbibition and swelling. The instantaneous water absorption
prosperties of pharmaceutical excipients correlate with total surface area while
the total absorption capacity correlates with powder porosity [22] .
If drug and excipients are so hygroscopic, they can readily adsorb water until
they deliquesce, or begin to dissolve. Moisture adsorption is important because
adsorbed water can cause incorrect weighing and degradation of drug and/or excipients.
The drug, excipients, and water reaction will continue as the amount of water
increases. When a solid is placed in a room, moisture will condense onto it. If this
occurs simply as a limited amount of adsorbed moisture, then the substance is not
hygroscopic under these conditions. These conditions exist if the water vapor pressure
in the surrounding atmosphere is lower than the water vapor pressure over a
saturated solution of the solid in question. However, if the water vapor pressure in
the atmosphere is higher than that of the saturated solution, there will be a thermodynamic
tendency for water to condense upon the solid materials (drug and
excipients).
The drug and pharmaceutical excipients adsorb or lose the moisture depending
on the relative humidity in the atmosphere. The nonhygroscopic materials are not
affected by the moisture and are in a equilibrium state. In general, solid dosage
forms such as tablets or capsules should be hydrophilic because the solid materials
must dissolve after swallowing. However, solid dosage forms must also be stable
against physical and chemical factors.
The moisture uptake rates (MUR) can simply be obtained by weighing the
sample after a given time (six days), but in such a case it is assumed that the moisture
uptake is still in the linear phase. If, for instance, the weight gain is 5 mg per 10 - g
sample in six days, then the MUR is 5/(10 . 6) = 0.083 mg/g/day. If the MUR values
are plotted versus relative humidity (RH) and the curve that intercepts the x axis
at 20% RH is obtained from a straight line, the compound can be stored without
moisture pickup in atmospheres of less than 20% RH. In addition, the hygroscopicity
of materials is indicated as follows:
Loss of drying (LOD%)
weight of water in sample
total weight of wet sa
=
mple
. 100
Moisture content (MC%)
weight of water in sample
weight of dry sampl
=
e
. 100
Equilibrium moisture contents (EMC)
%LOD
LOD
=
+
.
% 100
100
Relative humidity (RH)
water vapor pressure in atmosphere
saturat
=
ed water vapor pressure
. 100
912 PHARMACEUTICAL PREFORMULATION
Depending on the hygroscopicity based on the EMC, various drug and excipients
are classifi ed in four groups (Table 16 ).
Figure 8 also shows an example of moisture uptake for four selected excipients
as a function of relative humidity. Depending on the hygroscopicity of the exscipients,
the uptake behaviors are quite variable. Excipients such as microcrystalline
cellulose (MCC) and starch can pick up signifi cant amounts of water at relatively
low relative humidity. Since this water is not present as a hydrate, it is potentially
free to interact with a drug.
TABLE 16 Classifi cation of Hygroscopicity and Example Pharmaceutical Excipients
Type II: Type II:
Type I:
Nonhygroscopic
Slightly
Hygrocopic
Moderately
Hygroscopic
Type IV:
Very Hygroscopic
No MC change
below RH 90% or
less than 20% MC
changes at RH
90% after 1 week
storage
No MC change
below RH
80% or less
than 40% MC
changes at RH
80% after 1
week storage
Not more than 5%
MC change below
RH 60% or less
than 50% MC
changes at RH
70% after 1 week
storage
Increase of MC at RH
40 – 50% or more
than 70% MC
changes above RH
40% after 1 week
storage
Examples
Lactose USP MCC HPC Povidone
Dicalcium phosphate Sucrose, dextrose HPMC Sodium starch glycolate
Ethylcellulose Poloxamer 188 Bentonite Polyplasdone XL
Magnesium stearate PEG 3350 Pregelatinized starch Sorbitol
CAP Starch USP, Corn CMC Na
Gelatin USP
FIGURE 8 Profi les of moisture uptake for four selected excipients as function of relative
humidity.
Relative humidity(%)
0 20 40 60 80 100
Moisture content (%) 0
10
20
30
40
50
60
Lactose USP, anhydrous
Cellulose acetate phtahlate NF
Magnesium aluminum silicate NF
Povidone USP
POWDER CHARACTERISTICS 913
The moisture content of drug can affect the cohesiveness of particles through
hydrogen bonding or by changing surface energy effects. Water also can act as a
plasticizing agent and can lower the glass transition temperature of amorphous
polymorphs, thus allowing a rubbery state to exist at a lower temperature. Water
also can take up intra - and intergranular pore spaces (capillaries) of powders by
acting as an interparticle bridge through surface adsorption mechanisms.
The hygroscopicity of materials infl uences the fl owing characteristics of drug and
excipients by forming adsorption fi lm with an aid of water as a solvent. Dissolution
of drug and excipients from solid dosage forms can also occur. The crystallinity of
troglitazone - PVP K30 solid disperisions can be changed by the water content [23] .
During manufacturing operations, water ’ s ability to act as a bridge between particles
can improve the compression capabilities of powder masses and affect the tensile
strength and hardness of the tablet. The milling or blending is also changed. Most
of all, the chemical hydrolysis of labile materials is more facilitated, resulting in low
stability during storage. Adsorbed water also can act locally as a solvent, and in
many cases drug – excipient incompatibility occurs by dissolving drug or excipients
in granulation or blended powder mass, as observed when aspirin tablets liberate a
strong odor of free salicyclic acid. Any free, unbound water in the mass can migrate
throughout the material mass and act as a reagent. A waterproof packaging or
container is used for many moisture - sensitive materials.
6.1.4.6 Mixing
The mixing of powders is a key step in the manufacture of virtually all solid dosage
forms. In general, particle size, shape, and surface energy are important factors for
blending of drug and excipients in unit operations. The perfect mixing of powder is
desirable, that is, a mixture in which the probability of fi nding a particle of a given
component is the same at all positions in the mixture, but the powder mixing has a
maximum degree of randomness (Figure 9 ). To determine the degree of mixing
obtained in a pharmaceutical operation, it is necessary to reasonably sample the
mixture and determine the variation within the mix statistically [2] .
Uniform mixing of powdered materials occurs if they have similar particle size
distributions and particle shapes. Spherical particles mix least well while plate and
fi ber shapes also do not mix well because they tend to clump. The more cohesive
the material, the more diffi cult it is to mix that material with other materials. Similarly,
cohesiveness between drug and excipient or among excipients may hinder the
FIGURE 9 Comparison of powder mixing: perfect mixing and random mixing.
914 PHARMACEUTICAL PREFORMULATION
successful blending process. Ideally, the uniform mixing of powders should be such
that the weight of sample taken is similar to the weight that the powder mix contributes
to the fi nal dosage form.
Figure 10 shows the effect of mixing time on the mechanical strength of powders.
As the mixing time increases, the tensile strength of powders (or curshing strength
of the tablet) gradually decreases [24, 25] . While the strength decreases with increasing
mixing time for all materials tested, the effect is far more marked for materials
that deform plastically. For example, the glidant tensile strength invariably decreases
as the mixing time (2 min vs. 3 min) increases (Figure 11 ). There is no direct correlation
of tensile strength with primary particle size of glidant (Figure 12 ). However,
FIGURE 10 Effect of mixing time on mechanical strength of excipients.
Mixing time
Crushing strength or tensile strength
FIGURE 11 Effect of mixing time glidant tensile strength.
Mixing time (min)
Aerosil
R 812
Aerosil 300
Aerosil R 805
Aerosil 200
Aerosil OX 50
Aluminum oxide C
Printex 95
Printex G
Printex 25
Titanium dioxide P 25
Titanium dioxide T 805
Tensile strength (Pa)
0
2
4
6
8
10
12
14
2 min
30 min
POWDER CHARACTERISTICS 915
this correlation was further improved when some outlying glidant (Aserosil 200 and
300 and Printex 25) were excluded ( R 2 values of 0.1287 – 0.8616).
If a powder consisting of two materials both having identical physical properties
is mixed for a suffi cient time, random mixing will eventually be achieved. Unfortunately,
most pharmaceutical powders consist of mixtures of materials with differing
physical properties, such as size, shape, density, and surface area, leading to segregation
among particles, where particles of similar properties tend to collect together
in part of the powder. When segregating powders are mixed, as the mixing time is
extended, the powders appear to unmix. The differences in particle size are the most
important for segregation in pharmaceutical powders. One exception to overcome
segregation is ordered mixing rather than random mixing. When one component of
a powder mix has a very small particle size (less than 5 . m) and the other is relatively
large, the fi ne powder may coat the surface of the larger particles, and the adhesive
forces will prevent segregation, known as ordered mixing. This ordered mixing
makes the powders produce greater homogeneity than by random mixing. The percolation
of fi ne particles is also a factor. If the particles sizes are quite different, the
smaller particles can drop easily and move to the bottom of powder, resulting in
segregation. This segregation process can occur whenever movement of particles by
vibration, shaking, and pouring takes place.
6.1.4.7 Particle Size Reduction (Micronization and Milling)
Mechanical attrition, that is , high - energy ball milling of powders, is a nonequilibrium
processing method that has generated the reduced particle size and the formation
of physically metastable materials. It can be used to modify materials by refi ning
the microstructure, homogenizing the composition, extending solid solubility, creating
matastable crystalline phases, or producing metallic glass. High - energy ball
milling is both a processing method to reduce particle size and a route to the physical
synthesis of metastable materials. Early in product development, when only
small amounts of drug are available, comminution (grinding/mixing) may be carried
out with a mortar and pestle. For lager batches, ball milling or micronization can be
FIGURE 12 Correlation of tensile strength with primary particle size of glidant.
Primary particle diameter (nm)
0 10 20 30 40 50 60
Tensile strength (Pa)
0
2
4
6
8
10
12
14
Aerosil 300
Aerosil R 812
Printex 95
Aerosil OX 50
Aerosil 200
Aerosil R 805
Printex G
Printex 25
Titanium dioxide P 25
Titanium dioxide T 805
Aluminum oxide C
916 PHARMACEUTICAL PREFORMULATION
used to reduce the particle size because comminution or grinding are not practical
due to the length of time required [18] .
There are numerous methods of particle size reduction, but their application is
dependent on the intended particle size, particle distribution, cleaning convenience,
operating cost, dust containment, temperature, and fl exibility. The type of milling
machine includes slurry, fl uid energy (jet), universal, cone, and hammer. This size
reduction process may reduce the risk of dissolution rate - limited bioavailability of
drugs. Particle size reduction can be accomplished by using a hammer mall or a
similar mill, but this process may only break up the larger crystal aggregates without
signifi cantly changing the distribution of smaller particle sizes. On the other hand,
air jet mills, which impinge two streams of particles at a right angle to each other
in high - velocity air streams, reduce particle size signifi cantly within microsized
ranges.
Ball milling was the most commonly used at the preformulation stage to reduce
the particle size of small amounts of a compound. Ball mills reduce the size of particles
through a combined process of impact and attrition. Usually they consist of a
hollow cylinder that contains balls of various sizes which is rotated to initiate the
grinding process. Micronized particles are typically less than 10 . m in diameter. The
effi ciency of the milling process is affected by rotation speed, number of balls, mill
size, wet or dry milling, amount of powder, and length of time of milling. Although
ball milling can effectively reduce the particle size of compounds, prolonged milling
may be detrimental in terms of changes of compound crystal form from crystalline
to polymorphic or amorphous form and stability.
Although ball milling on a large scale is possible, hammer milling is more preferable
in the pharmaceutical industry. Powder is bled into the mill house via the
hopper, and the rotating hammers impact with the powder. When this is fi ne
enough to pass the screen, the powder will exit. The powder exiting will have a
maximum particle size of that of the screen. The average particle size of the milled
powder will be smaller, the smaller the feed rate, the more rapid the milling
speed (rpm ’ s), and the fi ner the screen. The knife has a blunt edge on one side and
a knife edge on the other. Milling with the blunt edge forward gives rise to a smaller
average particle size. The usual minimum particle range is about 50 . m. Large,
heavy - duty hammer mills (micropulverizers) give much smaller particle size further
down, typically to 20 . m. If particle sizes less than 5 . m are desired, micronizers are
used.
If particle sizes in the micrometer range are required, then the attrition mill can
be used. Here particles are bled into a chamber in which great turbulence has been
created by two inlets of air at different pressure. The particles hit one another and
are removed by centrifugal means and collected in a cyclone setup or in a bag above
the cyclone. Such particles become highly electrically charged during the operation
and also become very cohesive.
The milling process provides energy to the powders so that melting occurs if their
melting point is suffi ciently low. There is no guarantee that the original polymorph
and habit will be regained upon resolidifi cation. The milling and micronization
process can also reduce the particle size of poorly soluble drugs so that the maximum
surface area is exposed to enhance the solubility and dissolution properties. Although
micronization of the drug offers the advantage of a small particle size and a lager
surface area, it can also result in processing problems due to high dust, low density,
POWDER CHARACTERISTICS 917
and poor fl ow properties. Indeed, micronization may be counterproductive, since
the micronized particles may aggregate, which may decrease the surface area. Milling
and micronization process also induce the changes of crystallinity of drug into
amorphous form. It has been shown that the amorphous change of the crystalline
structure can be achieved by vapor condensation, supercooling of melt, rapid precipitation
from solution, and mechanical applications of a crystalline mass by milling
or compaction [26] .
Particle size reduction of the powder has produced defects on the surface that,
if enough energy is imparted, leads to amorphous regions on the surface. In turn,
these regions are found to have a greater propensity to adsorb water. The dissolution
rate increases as the particle size of drug powder decreases due to its greater
surface area for wetting.
It is known that ball milling and other types of milling can change the morphology
of a solid, for example, make a crystalline compound amorphous, increase the surface
energy of a solid, and distort the crystal lattice. For example, the crystalline solid state
of sorbitol exhibits a complex polymorphism made of fi ve different forms called A - ,
B - , . - , . - , and E - sorbitol [27] . The structures of these polymorphs have been identifi ed
by single - crystal X - ray diffraction. The structure of the . form is also the most stable
and the most common polymorph of sorbitol. It was reported that the . form of sorbitol
underwent a complete gransformation toward the A form upon ball milling and
also was affected by milling time. The DSC and XRPD patterns are useful in identifying
these phenomena, althought the low level of amorphous character cannot be
detected by techniques such as XRPD and DSC. Crystal structures by the milling
process as well as compaction forces have been shown [2] .
6.1.4.8 Compaction(Compressibility)
Compressibility is the property of forming a stable and intact mass when pressure
is applied. The manufacture of tablets involves the process of powder compaction
or compressibility, the purpose of which is to convert a loose incoherent mass of
powder into a single solid object. A protocol to examine the compression properties
of fl owing powder should be considered by the formulation scientist when selecting
the excipient, the formulation type, and the manufacturing process, for example,
direct compression or granulation for the intended solid dosage form. Acetaminophen
is poorly compressible whereas lactose compresses well. In general, drug and
excipients, including lubricant, are blended in a tumbler mixer for a period of time
and then compressed into tablets in a hydraulic press . The crushing strength is also
determined to test the compressibility of tablet at room temperature.
The compression of drug powder may change the crystal structure into a polymorphic
form — likewise in the milling process. The PXRD patterns and DSC of a
drug can detect these phenomena. Knowledge of the crystal structure of a drug is
a prerequisite for compaction.
To fully understand the compaction behavior of a material, it is necessary to be
able to quantify of its elasticity, plasticity, and brittleness. A powder in a container
subjected to compressive force will undergo particle rearrangement [28] . The density
of the bed will increase with increasing pressure at a characteristic rate. Brittle
materials will undergo fragmentation, and the fi ne particles formed will percolate
through the bed to give secondary packing. Plastically deforming materials will
918 PHARMACEUTICAL PREFORMULATION
distort to fi ll voids and may also exhibit void fi lling by percolation. When the limit
of plastic deformation is reached , fracture occurs.
Many of the basic principles of compaction and the test methodologies are currently
employed in pharmaceutical formulation. To characterize the compaction
properties of a material or formulation, it must be possible to measure the relationship
between the force applied to a powder bed and the volume of the powder bed.
There are two principal types of compaction studies used to characterize materials:
pressure – volume relationships and pressure – strength relationships. While ultimately
it is the strength of a tablet that is important, the pressure – volume relationships
provide information about the compaction properties of a material that allows an
appropriate formulation to be developed. The instrumented tableting machine provides
information for compaction that is directly relevant to production conditions.
The compression profi les differ from those of rotary tableting machines used for
commercial production. The profi le of a single punch involves the powder bed being
compressed between a moving upper punch and a stationary lower punch, while on
a rotary machine, both punches move together simultaneously. A major advantage
of instrumented machines is that they provide information not only on the compaction
properties but also on fl ow and lubrication. The disadvantage of using instrumented
rotary machines is the large quantity of materials required.
A large number of equations have been proposed to describe the relationship
between pressure and volume reduction during the compaction process. The most
widely used equation to describe the compaction of powders is the Heckel equation.
Pharmaceutical powders do not produce perfect straight lines, and the type of deviation
provides information about the compaction behavior of the material. A typical
Heckel plot for a pharmaceutical powder is obtained showing a straight - line portion
over a certain pressure range with a negative deviation at low pressures and a positive
deviation at high pressures. The strength of tablets has traditionally been determined
in terms of the force required to fracture a specimen across its diameter. The
fracture load obtained is usually reported as a hardness value. Initially, most materials
will demonstrate an increase in tensile strength proportional to the compaction
pressure applied. As the compaction pressure increases, the tablet approaches zero
porosity, and large increases in pressure are required to achieve small volume reductions
[28] . Some materials will attain a maximum strength, and subsequent increases
in pressure will produce weaker tablets. Other materials will also display an initial
increase in strength proportional to the applied pressure, but the strength reaches
a maximum before falling off sharply, resulting in capping or lamination in tablet
production [28] . Figure 13 shows the correlation of hardness with compression pressure.
Capping is the partial or complete removal of the crown of a tablet from
the main body, while lamination is the separation of a tablet into two or more distinct
layers. If the compressibility and fl ow of powders are good, it is possible to
directly compress the powders into tablet. If either compressibility or fl ow is not
satisfactory, roller compact, slugging, or we granulation can be utilized to improve
compressibility.
6.1.4.9 Surface Area and Other Properties
The surface area of particles is related to particles size, as discussed previously.
The surface area of powders affects the drug dissolution rate, powder fl ow, cohesiveness,
and adsorption. Furthermore, the surface area of solid materials may also
infl uence the physicochemical properties, adsorption, dissolution, and bioavailability
of drugs.
The particle size and surface area distributions of pharmaceutical powders can be
obtained by microcomputerized mercury porosimetry. Mercury porosimetry gives
the volume of the pores of a powder, which is penetrated by mercury at each successive
pressure; the pore volume is converted into a pore size distribution. Two other
methods, adsorption and air permeability, are also available that permit direct calculation
of surface area. In the adsorption method, the amount of a gas or liquid solute
that is adsorbed onto the sample of powder to form a monolayer is a direct function
of the surface area of the sample. The air permeability method depends on the fact
that the rate at which a gas or liquid permeates a bed of powder is related, among
other factors, to the surface area exposed to the permeant. The determination of
surface area is well described by the BET (Brunauer, Emmett, and Teller) equation.
The wetting behavior of powders is an also important factor for drug dissolution.
If the wetting is not satisfactory, hydrophilic excipients (lactose) and surfactant
(sodium lauryl sulfate or polysorbate) are combined in the powder mixtures. The
contact angle is used as an index of wetting. The lower the contact angle, the better
the wetting occurs.
The general appearance of a tablet with good visual identity and overall
“ elegance ” are essential for consumer acceptance, quality control of lot - to - lot uniformity,
general tablet - to - tablet uniformity, and monitoring trouble - free manufacturing.
Control of the general appearance of a tablet involves the measurement
of a number of attributes, such as a tablet ’ s size, shape, color, presence or absence
of and odor, taste, surface texture, physical fl aws and consistency, and legibility of
any identifying markings.
6.1.5 TABLET CHARACTERIZATION
In order to formulate the optimal tablet, various properties should be considered,
including drug – excipient compatibility, fl owability, lubricity, appearance, dissolution,
and disintegration [2] . The prepared tablet must also meet physical specifi cation and
FIGURE 13 Correlation of compression pressure with hardness.
Pressure (kP)
Hardness (kP)
Capping pressure (kP)
5000 7000 2000 2
12
6
TABLET CHARACTERIZATION 919
920 PHARMACEUTICAL PREFORMULATION
quality standards according to the monograph of the pharmacopeia. In general,
weight and its variation, content uniformity, thickness, hardness, friability disintegration,
and dissolution should be considered for tablet validation [3] . These factors
must be controlled during tablet production (in - process control) and are validated
after the production to ensure the quality standards.
6.1.5.1 Disintegration
Immediate - release tablets should be readily disintegrated in the stomach when
swallowed. This disintegration involves bursting apart the compact masses by
aqueous fl uid penetrating the fi ne pore structure of tablet. Disintegration testing is
an important part of in - process control testing during production to ensure batch -
to - batch uniformity, but its role in end - product testing has largely been superseded
by dissolution testing because recently modifi ed - release preparations are getting
popular. It was recognized in the 1940s that tablets had to disintegrate in order for
them to be bioavailable due to lack of biopharmaceutical information and primarily
analytical limitations. Later, of course, in the 1950s and 1960s, the pharmaceutical
scientist became aware of the importance of dissolution rates as well.
In general, for the medicinal agent in a tablet to become fully available for
absorption, the tablet must fi rst disintegrate and discharge the drug to body fl uids
for dissolution. The general manner in which a tablet disintegrates is as follows:
(a) the tablet wets down, (b) the dissolution liquid penetrates the pore space, (c)
the disintegrant absorbs water and swells, and (d) this swelling causes the tablet to
break down into granules. Figure 14 shows the disintegration pathways of solid
dosage forms for the dissolution and absorption of drugs. After the disintegration
process, the solid dosage forms change into granules or smaller and fi ne particles
ready for dissolution and absorption in the fl uid.
FIGURE 14 Disintegration and dissolution pathways of solid dosage forms for absorption
of drug.
Solid
dosage form
Drug in solution
Granules
or
aggregates
Primary
drug particles
Disintegration Deaggregation
Dissolution
(major) Dissolution
(major)
Dissolution
(major)
Absorption
Drug in body fluids and tissues
Tablet disintegration is the important fi rst step in the dissolution of the drug
substance contained in immediate - release tablets but dissolution is more meaningful
in case of many modifi ed release products rather than disintegration . A number
of formulation and manufacturing factors can affect the disintegration and dissolution
of a tablet, including the particle size of the drug substance in the formulation;
the solubility and hygroscopicity of the formulation; the type and concentration of
the disintegrant, binder, and lubricant used; the manufacturing method, particularly
the compactness of the granulation and the compression force used in tableting; and
the in - process variables which may occur. Therefore, it is vitally important for batch -
to - batch consistency to establish disintegration and dissolution standards and controls
for both materials and processes.
Tablet disintegration also is important for those tablets containing medicinal
agents (such as antacids and antidiarrheals) that are not intended to be absorbed
but rather to act locally within the GI tract. In these instances, tablet disintegration
provides drug particles with an increased surface area for localized activity within
the GI tract.
It is evident that there are some correlations of physical parameters with tablet
disintegration time. Figure 15 shows the correlation of water penetration force, disintegration
force, disintegrant contents, and compression forces of the tablet with
disintegration time of the tablet. As the water penetration force increases, the tablet
disintegration force also increases, resulting in shorter disintegration time [29, 30] .
The amount of disintegrant in the tablet also decreases the disintegration time.
The disintegration time increases as tableting pressure increases below the critical
capping pressure [18] . At very low pressures the penetration of liquid into the tablet
is virtually unhindered (almost like pouring water into a breaker) but the pores will
be too large to allow disintegrant swelling to cause stress and the disintegration time
will decrease. Once the pores are suffi ciently small, penetration of the liquid into
the disintegrant becomes the limiting step, and the disintegration time will increase
FIGURE 15 Correlation of physical parameters on tablet disintegration time.
Water penetration force or disintegration force Disintegration time
Tablet compression force
Amount of disintegrants
TABLET CHARACTERIZATION 921
922 PHARMACEUTICAL PREFORMULATION
with increasing pressure. Disintegrants and lubricants are added to wet - granulated
products after the granulation has been dried. Disintegration time increases as the
amount of hydrophobic lubricant increases. The mixing time for the lubricant must
be kept short because otherwise the lubricant may fl uidize during the mixing
step and lose part of the lubricant properties that are necessary for fl ow in the
tablet die. If the disintegration is not satisfactory, numerous types of disintegrants
are added in the tablet formulations, including starch, croscarmellose sodium, sodium
starch glycolate and crospovidone known as superdisintegrants. In general, the
swelling rate and water uptake are the most important properties of disintegrants.
All USP tablets must pass a test for disintegration, which is conducted in vitro
using a testing apparatus. The detailed monograph for disintegration testing is
described in the many pharmacopeias [2, 3] .
The apparatus consists of a basket - rack assembly containing dimensions held
vertically upon a 10 - mesh stainless steel wire screen. During testing, a tablet is
placed in each of the six tubes of the basket, and the mechanical device raises and
lowers the basket in the immersion fl uid at a frequency of between 29 and 32 cycles
per minute, the wire screen always maintained below the level of the fl uid. For
uncoated tablets, buccal tablets, and sublingual tablets, water maintained at about
37 ° C is used as the immersion fl uid unless another fl uid is specifi ed in the individual
monograph. For these tests, complete disintegration is defi ned as that state in which
any residue of the unit, except fragments of insoluble coating or capsule shell,
remaining on the screen of the test apparatus is a soft mass having no palpably fi rm
core. Buccal tablets must disintegrate within the time set forth in the individual
monograph, usually 30 min, but varying from about 2 min for nitroglycerin tablets
to up to 4 h. If one or more tablets fail to disintegrate, additional tests prescribed
by the USP must be performed. Enteric - coated tablets are also similarly tested,
except that the tablets are permitted to be tested in simulated gastric fl uid for 1 h
with no sign of disintegration, cracking, or softening. They are then switched to the
simulated intestinal fl uid for the time stated in the individual monograph during
which time the tablets disintegrate completely. If 1 or 2 of the 6 tablets fails to disintegrate
completely, disintegration testing is repeated on 12 additional tablets, and
not less than 16 of the total 18 tablets tested must disintegrate to meet the
standards.
6.1.5.2 Dissolution
Defi nitions Dissolution is the dynamic process by which drug is dissolved in a
solvent (water) and is characterized by a rate (amount dissolved per unit time). In
vitro dissolution testing of a tablet is very important for many reasons: It guides the
formulation and product development process toward optimization of dosage forms
for quality control and reliability. By conducting dissolution studies in the early
stages of a product ’ s development, the formulation compositions and manufacturing
parameters are also tuned and monitored about the old and newly advanced tablet.
The U.S. Food and Drug Administration (FDA) allows manufacturers to examine
scale - up batches of 10% of the proposed size of the actual production batch or
100,000 dosage units, whichever is greater, by performing in vitro dissolution testing
to assure bioequivalence from batch to batch and processing parameters. New drug
applications (NDAs) submitted to the FDA contain in vitro dissolution data generally
obtained from batches that have been used in pivotal clinical and/or bioavail
ability studies and from human studies conducted during product development.
Once the specifi cations are established in an approved NDA, they become offi cial
(USP) specifi cations for all subsequent batches and bioequivalent products. The
dissolution testing is also used as a tool of SUPAC (scale - up postapproval change)
and variation of equipment, location, and processing factors. In addition, dissolution
testing is used as a tool to examine the short - and long - term stability of dosage
forms. Release mechanism and parameters which change the dissolution are also
studied. Most of all, one of main goals of in vitro dissolution testing is to provide
reasonable prediction and correlation with the product ’ s in vivo bioavailability. Differentiations
in the formulations and other related variables may cause deviations
from in vivo bioavailability data.
A system has been developed which relates combinations of a drug ’ s solubility
(high or low) and its intestinal permeability (high or low) as a possible basis for
predicting the likelihood of achieving a successful in vivo – vitro correlation (IVIVC).
The four classes based on BCS are (I): high solubility and high permeability, (II):
low solubility and high permeability, (III): high solubility and low permeability, and
(IV): low solubility and low permeability. In class I, dissolution testing can be used
as a prognostic tool to predict in vivo biovailability.
Equations Many dissolution equations are well described in the text. Most of the
equation are based on the well - known Fick ’ s law. Figure 16 shows a diagram of the
concentration gradient between the matrix tablet and bulk fl uid for dissolution.
From these gradient situations, the well - known Noyes – Whitney equation is
given as
dc
dt
DAK
Vh
C C = . ( ) s t
where dc / dt = rate of drug dissolution, where dQ / dt = V dc / dt
V = volume of dissolution fl uid
D = diffusion rate constant
A = surface area of dosage forms
C s = concentration of drug in stagnant layer
FIGURE 16 Concentration gradient from the tablet between matrix and bulk fl uid for
dissolution. Cs, drug solubility; C, uniform concentration; h, thickness of stagnant fi lm; X =
diffusional path length.
Tablet Stagnant layer Bulk fluid
Concentration
Cs
C
Matrix
X = 0 X = h
TABLET CHARACTERIZATION 923
924 PHARMACEUTICAL PREFORMULATION
C t = concentration of drug in bulk fl uid at given time
K = partition coeffi cient
h = thickness of stagnant layer
If the bulk volume is large and the concentration of drug in the fl uid is much
lower than the drug solubility ( C s > > C t ), it is regarded as a sink condition. In this
case, the equation is much simpler and the dissolution behaviors continuously occur
because the chemical potential ( C s . C t ) approximates drug solubility ( C s ).
In a matrix tablet, the following Higuchi equations are given depending on the
polymeric structures of the homogenous and porous matrix [20] :
Q
D A C C
D A C C
=
. [ ]
. [ ]
( )
( )
2
2
1
1
S S
/2
S S
/2
t for homogenousmatrix
t / f . . . or porousmatrix
...
In the dissolution of granular powders, the Hixson – Crowell equation is also
established as
Q Q kt 0
1 1/3 /3 for granular powders . =
Testing Method In addition to formulation and manufacturing controls, the method
of dissolution testing also must be controlled to minimize important variables such
as paddle rotational speed, vibration, and disturbances by sampling probes. The
USP includes seven apparatus designs for drug release and dissolution testing of
immediate - release oral dosage forms, extended - release products, enteric - coated
products, and transdermal drug delivery devices:
Apparatus I: rotating basket method, 25 – 150 rpm (100 rpm)
Apparatus II: rotating paddle method, 25 – 150 rpm (50 rpm)
Apparatus III: reciprocating cylinder method, inner tube (5 – 40 dips/min), outer
tube (300 mL)
Apparatus IV: fl ow - through method: 4 – 16 mL/min
Apparatus V: paddle over disc
Apparatus VI: cylinder method
Apparatus VII: reciprocating holder method
Detailed guidelines for dissolution testing are described in monographs of many
pharmacopeias. The USP apparatus I and USP apparatus II are used principally for
tablet dissolution testing. In USP apparatus I, the dosage unit is placed inside the
basket. In USP apparatus II, the dosage unit is placed on the bottom in the vessel.
In each test, a volume of the dissolution medium (500 – 900 mL in general) is placed
in the vessel and allowed to come to 37 ± 0.5 ° C. Then the stirrer is rotated at the
specifi ed speed (50 – 200 rpm). The samples of the medium are withdrawn for analysis
of the proportion of drug dissolved. The tablet or capsule must meet the stated
monograph requirement for rate of dissolution. For example, “ not less than 85% of
the labeled amount is dissolved in 30 minutes in case of immediate release tablet. ”
In a fl oating tablet, the sinker can be used in the paddle method.
Variables Affecting Dissolution In general, the dissolution profi les are highly
dependent on the physicochemical properties, formulation, processing parameters,
and testing conditions [2] . The physicochemical properties include particle size,
surface area, crystal habit and polymorphism, solubility, molecular size, salt formation,
p K a , hydration, wetting, and surface tension. Physical factors such as viscosity,
density, fl occulation, and agglomeration are also considered. The formulation factors
are also of importance. The amount and type of excipients and type of dosage forms
play a key role in modifying dissolution behaviors. For example, Figure 17 gives the
effect of lubricant and its mixing time on the dissolution rate of drugs. The presence
of lubricant and its mixing time signifi cantly changed tablet dissolution [31] . For
poorly soluble drug, numerous pharmaceutical methods have been utilized to
increase the dissolution rate of drug, including micronization, amorphous crystallization,
spray drying, inclusion complex, microemulsion, and solid dispersion.
The processing parameters in the tablet preparation also change the dissolution,
including temperature, mixing, milling, rotation speed, solvent, hardness, and surface
area. The testing conditions are also important in modifying the dissolution of tablet.
Therefore, the testing conditions are well defi ned by the regulations in the many
pharmacopeias. The testing conditions include pH of the fl uid, temperature, ionic
strength, common ion effect, type of apparatus, rotation speed, volume size of
dissolution fl uid, analytical conditions, aeration, sample treatment, and mainly
composition of dissolution media. Table 17 provides some of the media compositions
suggested for in vitro dissolution testing of the tablet. These modifi ed
dissolution media can be used to achieve dissolution of drug under simulated in
vivo conditions.
In Vitro – In Vivo Correlation In vitro dissolution testing can provide a reasonable
prediction of the product ’ s in vivo bioavailability. For a high - solubility and high -
permeability drug (class I), an IVIVC may be expected if the dissolution rate is
slower than the rate of gastric emptying (the rate - limiting factor). In a low - solubility
and high - permeability drug, drug dissolution may be the rate - limiting step for
drug absorption and an IVIVC may be expected. In a high - solubility and low -
FIGURE 17 Effect of lubricant and its mixing time on dissolution rate of drugs.
Time
Percent dissolved
No lubricant
Lubricant, 2 min
Lubricant, 30 min
TABLET CHARACTERIZATION 925
926 PHARMACEUTICAL PREFORMULATION
permeability drug, permeability is the rate - controlling step and only a limited IVIVC
may be possible. In a drug with low solubility and low permeability, signifi cant
problems would be likely for oral drug delivery.
However, the in vivo GI condition is very complicated in terms of complex physiology
and absorption process and is not simulated by the simple in vitro dissolution
conditions [32] . Moreover, in vivo conditions are also complicated by food composition,
type and composition of dosage formulations, relative rates of permeation, GI
transit time, site of absorption, complexity of GI fl uids such as pH, enzymes, bile
and mucin, rate and capacity of metabolism by intestinal and hepatic enzymes,
ethical difference, and patient conditions such as mood, disease, bed rest, and fasting
volume of fl uid given. For these reasons, in vitro dissolution is not always correlated
with in vivo absorption, especially low - soluble and low - bioavailable drugs. To use
dissolution testing as a prognostic tool for in vivo bioavailability, the dissolution
fl uid is simulated with in vivo GI condition as possible, compromising the biorelevant
composition of dissolution fl uid (see Table 17 ), gradient pH, proper stirring
rate, and addition of lipid, enzyme, and surfactants.
For bioequivalence, dissolution profi les of two tablets are often compared. In
this case, the difference factor and similarity factor are considered, defi ned by the
equations
f
R T
R
t t
t
1 100 =
.
. .
.
f
n
R T t t 2
2
0 5
50
1
100 = . ...
...
. ...
...
.
.
log 1+ ( )
.
where R t and T t are the cumulative percents dissolved at each of the selected n time
points.
TABLE 17 Suggested Media Compositions for In Vitro Dissolution Testing
Medium Type Codes Dissolution compositions
Water W Purifi ed water
Gastric fl uid
(pH 1.2)
SGF HCl – NaCl buffer
SGF/TW HCl – NaCl buffer/Tween 80 (1%)
SGF/SLS HCl – NaCl buffer/sodiun lauryl sulfate (1%)
SGF/PEP HCl – NaCl buffer/pepsin (0.32%)
Intestinal fl uid
(pH 6.8)
SIF KH 2 PO 4 buffer
SIF/TW KH 2 PO 4 buffer/Tween 80 (1%)
SIF/SLS KH 2 PO 4 buffer/sodiun lauryl sulfate (1%)
SIF/Pan KH 2 PO 4 buffer/pancreatin (1%)
SIF/fasted 0.2 M KH 2 PO 4 buffer (3.9 g)/3 m M Na - taurocholate, 75 m M
lecithin/KCl (7.7 g)/NaOH (qs)/water (qs)
Intestinal fl uid
(pH 5.0)
SIF/fed Acetic acid (8.65 g)/15 m M Na - taurocholate/3.75 m M , lecithin/
KCl (15.2 g)/NaOH (qs)/water (qs)
The difference factor f1 is proportional to the average difference between the two
profi les while the similarity factor f2 is inversely proportional to the average squared
difference between the two profi les and measures the closeness between the two
profi les. The two dissolution profi les are identical if f2 = 100. An average difference
of 10% at all measured time points results in a f2 value of 50. The FDA guideline
states that f2 values of 50 – 100 indicate similarity between two dissolution profi les.
6.1.5.3 Weight Variation
With a tablet designed to contain a specifi c amount of drug in a specifi c amount of
tablet formula, the weight of the tablet being made is routinely measured to help
ensure that the tablet contains the proper amount of drug. The quantity of fi ll placed
in the die of a tableting press determines the weight of the resulting tablet. The
volume of fi ll is adjusted to yield tablets of desired weight and content. The depth
of fi ll in the tablet die must be adjusted to hold a predetermined volume of powder
or granulation. Each tablet weight should be calculated if the amount of drug and
other excipients such as diluent, disintegrant, and binder are decided.
During production, sample tablets are periodically removed for visual inspection
and automated physical measurement known as in - process control (IPC). The USP
provides some guidelines for weight variation. In the test, 10 uncoated tablets are
weighed individually and the average weight is calculated. The tablets are assayed
and the content of active ingredient in each of the 10 tablets is calculated assuming
homogeneous drug distribution.
6.1.5.4 Hardness or Breaking Strength
Tablets require a certain amount of strength, or hardness and resistance to friability,
to withstand mechanical shocks of handling in manufacture, packaging, and shipping.
Adequate tablet hardness and resistance to powdering and friability are necessary
requisites for consumer acceptance while immediate - release tablets should
readily disintegrate in the stomach as quick possible. For this reason, the relationship
of harness to tablet disintegration and drug dissolution has been described.
Tablet hardness has been defi ned as the force required to break a tablet in a
diametric compression test. To perform this test, a tablet is placed between two
anvils, and the crushing strength that just causes the tablet to break is recorded.
Hardness is thus sometimes termed the tablet crushing strength. It is not unusual
for a tableting press to exert as little as 3000 lb and as much as 40,000 lb of force in
the production of tablets. Generally, the greater the pressure applied, the harder the
tablets, although the formulation composition and manufacturing process may also
change tablet hardness. In general, tablets should be suffi ciently hard to resist breaking
during normal handling or transportation but have no problem in disintegrating
and/or dissolving after swallowing.
The hardness of a tablet, like its thickness, is a function of the die fi ll and compression
force. At a constant die fi ll, the hardness values increase and thickness
decreases as additional compression force is applied. This relationship holds up to
a maximum value for hardness and a minimum value for thickness beyond which
increases in pressure cause the tablet to laminate or cap, thus destroying the integrity
of the tablet. At a constant compression force (fi xed distance between upper
TABLET CHARACTERIZATION 927
928 PHARMACEUTICAL PREFORMULATION
and lower punches), hardness increases with increasing die fi lls and decreases with
lower die fi lls. The amount and mixing time of lubricants and excipients can affect
tablet hardness. Large tablets require a greater force to cause fracture and are
therefore “ harder ” than small tablets.
Special dedicated hardness testers or multifunctional systems are used to measure
the degree of force (in kilograms, pounds, or arbitrary units) required to break a
tablet. Devices to test tablet hardness include the Monsanto tester, the Strong - Cobb
tester, the Pfi zer tester, the Erweka tester, and the Schleuniger tester. A force of
about 4 kg is considered the minimum requirement for a satisfactory tablet. Multifunctional
automated equipment can determine tablet weight, hardness, thickness,
and diameter. Unfortunately, these testers do not produce uniform results for the
same tablet due to operator variation, lack of calibration, spring fatigue, and manufacturer
variation.
6.1.5.5 Friability
Tablet hardness is not an absolute indicator of tablet strength since some formulations,
when compressed into very hard tablets, tend to “ cap ” on attrition, losing
their crown portions. Another measure of a tablet ’ s strength is tablet friability.
More powders , chips, and fragments can be produced during friability test if the
tablet lacks proper strength and is manufactured in dirty processes during coating
and packaging. The high friability causes lacks of elegance and consumer acceptance
and even weight variation or content uniformity problems.
A tablet ’ s durability may be determined using friability tester like Varian Friabilator
testng apparatus . This apparatus determines the tablet ’ s friability, or its tendency
to crumble, by allowing it to roll and fall within the rotating apparatus.
Normally, a preweighed tablet sample is placed in the friabilator, which is then operated
for 100 revolutions. The tablets are then dusted and reweighed. Any loss in
weight is determined. Resistance to loss of weight indicates the tablet ’ s ability to
withstand abrasion in handling, packaging, coating, and shipment. Compressed
tablets that lose a maximum of not more than 0.5 – 1% of their weight are generally
considered acceptable [3] .
6.1.5.6 Content Uniformity
Tablet weight cannot be used as a potency indicator of its potency, except perhaps
when the active ingredient is 90 – 95% of the total tablet weight. In tablets with
smaller dosages, a good weight variation does not ensure good content uniformity,
but a large weight variation precludes good content uniformity. The weight variation
test would be a satisfactory method of determining the drug content uniformity of
tablets. The content uniformity of the tablet is more important since the potency of
tables is expressed on labels in terms of grams, milligrams, or micrograms. The
content uniformity of tablets can be varied by three factors: (1) nonuniform distribution
of the drug substance throughout the powder mixture or granulation,
(2) segregation of the powder mixture or granulation during the various manufacturing
processes, and (3) tablet weight variation.
By the USP method, 10 dosage units are individually assayed for their content
uniformity according to the assay method described in the individual monograph.
The requirements for content uniformity are met if the amount of active ingredient
in each dosage unit lies within the range of 85 – 115% of the label claim and the relative
standard deviation is less than 6.0%. If one or more dosage units do not meet
these criteria, additional tests as prescribed in the USP are required. Offi cial compendia
or other standards provide an acceptable potency range around the label
potency. For highly potent, low - dose drugs such as digoxin, this range is usually not
less than 90% and not more than 110% of the labeled amount. For most other larger
dose drugs in tablet form, the offi cial potency range that is permitted is not less than
95% and not more than 105% of the labeled amount.
6.1.5.7 Tablet Thickness
At a constant compressive load, tablet thickness varies with changes in die fi ll, particle
size distribution and packing of the particle mix being compressed, and tablet
weight, while with a constant die fi ll, thickness varies with variations in compression
forces. The thickness of individual tablets may be measured with a micrometer,
which permits accurate measurement and provides information on the variation
between tablets. Other techniques employed in production control involve placing
5 or 10 tablets in a holding tray, where their total crown thickness may be measured
with a sliding caliper scale. Tablet thickness should be controlled within ± 5% variation
of a standard value. Any variation in tablet thickness within a particular lot of
tablets or between manufacturer ’ s lots is not appropriate for consumer acceptance
of the product. In addition, tablet thickness must be also controlled to facilitate
packaging.
6.1.5.8 Tablet Shape and Size
The shape of the tablet alone can infl uence the choice of tableting machine used.
Figure 18 shows some representative tablet shapes. The size, shape, and thickness
FIGURE 18 Example tablet shapes.
TABLET CHARACTERIZATION 929
930 PHARMACEUTICAL PREFORMULATION
are very changeable. Shaped tablets requiring slotted and sophisticated punches
must be run at slower speeds to avoid manufacturing disorder as compared with
round tablets using conventional punches. Because of the nonuniform forces
involved during compression, the more convex the tablet surface, the more likely it
is to cause capping or laminating problems. The more complicated shaped tablet
requires the use of a slower tableting machine or one with precompression capabilities.
The size and shape of the tablet are governed by the choice of tableting
machine, the best particle size for the granulation, production lot sizes, and the best
type of tablet processing, packaging operation, and cost to produce the tablet.
REFERENCES
1. Kibbe , A. H. ( 2000 ), Handbook of Pharmaceutical Excipients , 3rd ed. , American Pharmaceutical
Association and Pharmaceutical Press , London .
2. Gibson , M. ( 2001 ), Pharmaceutical Preformulation and Formulation, A Practical Guide
from Candidate Drug Selection to Commercial Dosage Form , HIS Health Group, Denver,
CO .
3. Allen , L. V. , Popovich , N. G. , and Angel , H. C. ( 2005 ), Pharmaceutical dosage forms and
drug delivery systems , Lippincott Williams & Wilkins , Baltimore, MD, USA.
4. Mountfi eld , R. J. , Senepin , S. , Schleimer , M. , Walter , I. , and Bittner , B. ( 2000 ), Potential
inhibitory effects of formulation ingredients on intestinal cytochrome P450 , Int. J. Pharm. ,
211 , 89 – 92 .
5. Cornaire , G. , Woodley , J. , Hermann , P. , Cloarec , A. , Arellano , C. , and Houin , G. ( 2004 ),
Impact of excipients on the absorption of P - glycoprotein substrates in vitro and in vivo ,
Int. J. Pharm. , 278 , 119 – 131 .
6. Wang , S. - W. , Monagle , J. , McNulty , C. , Putnam , D. , and Chen , H. ( 2004 ), Determination
of P - glycoprotein inhibition by excipients and their combinations using an integrated
high - throughput process , J. Pharm. Sci. , 93 , 2755 – 2767 .
7. Karsa , D. R. , and Stephenson , R. A. ( 1995 ), Excipients and Delivery Systems for Pharmaceutical
Formulations , The Royal Society of Chemistry , Cambridge , pp. 1 – 34 .
8. Farber , L. , Tardos , G. I. , and Michaels , J. N. ( 2003 ), Evolution and structure of drying
material bridges of pharmaceutical excipients: Studies on a microscope slide , Chem. Eng.
Sci. , 58 , 4515 – 4525 .
9. Cao , Q. - R. , Choi , Y. - W. , Cui , J. - H. , and Lee , B. - J. ( 2005 ), Formulation, release characteristics
and bioavailability of novel monolithic hydroxypropylmethylcellulose matrix
tablets containing acetaminophen , J. Controlled Release , 108 , 351 – 361 .
10. Jackson , K. , Young , D. , and Pant , S. ( 2000 ), Drug - excipient interactions and their affect
on absorption , PSTT , 3 ( 10 ), 336 – 345 .
11. Patel , H. , Stalcup , A. , Dansereau , R. , and Sakr , A. ( 2003 ), The effect of excipients on the
stability of levothyroxine sodium pentahydrate tablets , Int. J. Pharm. , 264 , 35 – 43 .
12. Verma , R. K. , and Garg , S. ( 2004 ), Compatibility studies between isosorbide mononitrate
and selected excipients used in the development of extended release formulations ,
J. Pharm. Biomed. Anal. , 35 , 449 – 458 .
13. Young , W. R. ( 1990 ), Accelerated temperature pharmaceutical product stability determinations
, Drug Dev. Ind. Pharm. , 16 ( 4 ), 551 – 569 .
14. Waterman , K. C. , and Adami , R. C. ( 2005 ), Accelerated aging: Prediction of chemical
stability of pharmaceuticals , Int. J. Pharm. , 293 , 101 – 125 .
15. Simon , P. , Veverka , M. , and Okuliar , J. ( 2004 ), New screening method for the determination
of stability of pharmaceuticals , Int. J. Pharm. , 270 , 21 – 26 .
16. Florence , A. T. and Attwood , D. ( 1998 ), Physicochemical principles of pharmacy , 3rd ed. ,
Macmillan , London , pp. 5 – 35 , 101 – 151.
17. Hiestand , E. N. ( 1989 ), The basis for practical applications of the tableting indices , Pharm.
Technol. Int. , 8 , 54 – 66 .
18. Carstensen , J. T. ( 1993 ), Pharmaceutical Principles of Solid Dosage Forms , Technomic
Publishing , Lancaster, PA .
19. Brittain , H. G. ( 1995 ), Physical Characterization of Pharmaceutical Solids , Marcel Dekker ,
New York .
20. Sinko , P. J. ( 2006 ), Physical Pharmacy and Pharmaceutical Sciences , Lippincott Williams
and Wilkins , Baltimore, MD .
21. Carson , J. W. and Wilms , H. ( 2006 ), Development of an international standard for shear
testing , Powder Technol. , 167 , 1 – 9 .
22. Hedenus , P. , Mattsson , M. S. , Niklasson , G. A. , Camber , O. , and Ek , R. ( 2000 ), Characterization
of instantaneous water absorption properties of pharmaceutical excipients , Int. J.
Pharm. , 141 , 141 – 149 .
23. Hasegawa , S. , Hamaura , T. , Furuyama , N. , Kusai , A. , Yonemochi , E. , and Terada , K. ( 2005 ),
Effects of water content in physical mixture and heating temperature on crystallinity of
troglitazone - PVP K30 solid dispersions prepared by closed melting method , Int. J. Pharm. ,
302 , 103 – 112 .
24. Mullarney , M. P. , Hancock , B. C. , Carlson , G. T. , Ladipo D. D. , and Langdon , B. A. ( 2003 ),
The powder fl ow and compact mechanical properties of sucrose and three high - intensity
sweeteners used in chewable tablets , Int. J. Pharm. , 257 , 227 – 236 .
25. Meyer , K. , and Zimmermann , I. ( 2004 ), Effect of glidants in binary powder mixtures ,
Powder Technol. , 139 , 40 – 54 .
26. Hancock , B. C. , and Zografi , G. ( 1997 ), Characteristics and signifi cance of the amorphous
state in pharmaceutical systems , J. Pharm. Sci. , 86 ( 1 ), 1 – 12 .
27. Willart , J. , Lefebvre , J. , Dan e de , F. , Comini , S. , Looten , P. , and Descamps , M. ( 2005 ), Polymorphic
transformation of the G - form of d - sorbitol upon milling: Structural and nanostructural
analyses , Solid State Communi. , 135 ( 8 ), 519 – 524 .
28. Adolfsson , A. , and Nystrom , C. ( 1996 ), Tablet strength, porosity, elasticity and solid state
structure of tablets compressed at high loads , Int. J. Pharm. , 132 , 95 – 106 .
29. Caramella , C. , Colombo , P. , Conte , U. , Ferrari , F. , and Manna , A. L. ( 1986 ), Water uptake
and disintegration force measurements: Towards a general understanding of disintegration
mechanism , Drug Dev. Ind. Pharm. , 12 , 1749 – 1766 .
30. Pourkavoos , N. , and Peck , G. E. ( 1993 ), The effect of swelling characteristics of superdisintegrants
on the aqueous coating solution penetration into the tablet matrix during the
fi lm coating process , Pharm. Res. , 10 ( 9 ), 1363 – 1371 .
31. Lerk , C. F. , Bolhuis , G. K. , Smallenbroek , A. J. , and Zuurman , K. ( 1982 ), Interaction of
tablet disintegrations and magnesium stearate during mixing II. Effect on dissolution rate ,
Pharm. Acta Helv , 57 , 282 – 286 .
32. Gundert - Remy , U. , and Moller , H. ( 1990 ), Oral Controlled Release Products, Therapeutic
and Biopharmaceutical Assessment , Wissenschaftliche Verlagsgesellschaft mbH , Stuttgart,
Germany , pp. 155 – 173 .
REFERENCES 931
933
6.2
ROLE OF PREFORMULATION IN
DEVELOPMENT OF SOLID
DOSAGE FORMS
Omathanu P. Perumal and Satheesh K. Podaralla
South Dakota State University, Brookings, South Dakota
Contents
6.2.1 Introduction
6.2.2 Physical/Bulk Characteristics
6.2.2.1 Crystallinity and Polymorphism
6.2.2.2 Hydrates/Solvates
6.2.2.3 Amorphates
6.2.2.4 Hygroscopicity
6.2.2.5 Particle Characteristics
6.2.2.6 Powder Flow and Compressibility
6.2.3 Solubility Characteristics
6.2.3.1 p Ka and Salt Selection
6.2.3.2 Partition Coeffi cient
6.2.3.3 Drug Dissolution
6.2.3.4 Absorption Potential
6.2.4 Stability Characteristics
6.2.4.1 Solid - State Stability
6.2.4.2 Solution - State Stability
6.2.4.3 Drug – Excipient Compatibility
6.2.5 Conclusions
References
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
934 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
6.2.1 INTRODUCTION
The advent of combinatorial chemistry and high - throughput screening (HTS) has
exponentially increased the number of compounds synthesized and screened during
the drug discovery phase. However, the overall effi ciency of the drug discovery
process is still exceedingly low (only 1 in 10,000 makes it to the market). Drug discovery
is mostly driven by “ activity screens ” with little emphasis on “ property
screens. ” This is exemplifi ed by the fact that 40% of attrition in drug discovery and
development is attributed to poor biopharmaceutics and pharmacokinetic properties
[1] , which in turn are related to poor physicochemical properties. As a result,
pharmaceutical companies have started to redesign their strategies by including
property screens quite early in the discovery stage [2] . Preformulation is the study
of fundamental properties and derived properties of drug substances. In other
words, preformulation is the fi rst opportunity to learn about the drug ’ s physicochemical
properties from the perspective of transforming a biologically active molecule
to a “ druggable ” molecule. The type and extent of preformulation activities
vary in a discovery and generic setting.
The main goal of a drug discovery program is to develop an orally deliverable
molecule for obvious reasons of ease of manufacture and convenience of drug
administration. More than 75% of the drug products in the market are oral formulations,
of which more than half are solid dosage forms [3] . The “ rule of fi ve ” developed
by Christopher Lipinski [4] is one of the “ physicochemical screens ” to weed
out molecules with poor physicochemical properties very early in the drug discovery
process. According to Lipinski ’ s rule, a drug will show poor oral absorption if it does
not conform to any of the two physicochemical requirements listed in Table 1 . The
rule of fi ve is applicable only to small molecules and it relates the chemical properties
of the drug to its solubility and permeability characteristics. During the initial
stages of drug discovery, the preformulation activities are mainly focused on developing
a water - soluble compound for early activity studies and preclinical testing in
animals. At this stage, the preformulation scientist is faced with the challenge of
working with a limited quantity of compound (few milligrams) for testing a long list
of physicochemical parameters. On the other hand, developing preclinical formulations
can be quite a daunting task given the fact that toxicological studies require
a high dose of drug (10 – 100 times above the effective dose) to be delivered in a
small volume of the formulation. Preformulation activities increase as the molecule
proceeds through the development phase. The “ discovery and development phar-
TABLE 1 Lipinski Rule of Five for Orally Active Compounds
Physicochemical Parameter Lipinski rule
Molecular weight Not more than 500 Da
log P Not more than 5
Hydrogen bond donors Not more than 5 hydrogen bond donors expressed as the
sum of OH ’ s and NH ’ s
Hydrogen bond acceptors Not more than 10 expressed as the sum of OH ’ s and NH ’ s
maceutics ” documentation forms a signifi cant portion of the investigational new
drug application (IND) application and new drug application (NDA) fi led to the
U.S. Food and Drug Administration (FDA). In a generic setting, preformulation
studies are mainly focused on developing a formulation that is bioequivalent to the
innovator ’ s product with the main objective of fi ling an abbreviated new drug application
(ANDA). A strong preformulation team can generate intellectual property
in the form of new salts, solid - state forms, or new stabilized formulations of the drug
for an innovator and/or a generic manufacturer.
In the present chapter, the discussion is mainly focused on preformulation testing
for oral solid dosage forms in a drug discovery setting. The chapter address the following
goals of preformulation: (i) to gain knowledge about the physicochemical
characteristics of the drug, (ii) to defi ne the physical characteristics of the drug, (iii)
to understand the stability characteristics of the drug, and (iv) to determine the
compatibility of excipients with the drug. In this chapter, we have grouped all
the parameters under three sections and discussed in a logical sequence for the
convenience of the reader.
6.2.2 PHYSICAL/BULK CHARACTERISTICS
The bulk or physical characteristics of a drug substance are mainly dictated by its
solid - state properties. Purity of the drug substance is a fundamental property that
is characterized at the beginning of preformulation studies. In the initial stages of
drug development, the drug is usually not very pure. Nevertheless, it is essential to
know the purity of the material at hand using simple measurements such as melting
point. This would serve to set drug specifi cations during later stages of drug development.
Differential scanning calorimetry (DSC) requires very little sample (1 – 5 mg)
and is a useful tool to estimate the purity of the compound. The drug sample is
heated in a crucible, where the difference in heat between the sample and a reference
crucible is seen as an endotherm or exotherm in the thermogram depending
on whether heat is taken up or given up, respectively, by the sample. The integrated
area under the endotherm or exotherm gives a measure of the heat or enthalpy
involved in this process. Melting is seen as an endothermic event and the purity of
the sample will govern the peak position, shape, sharpness, and heat of fusion ( .Hf ).
DSC is sensitive in detecting impurities to the extent of 0.002 mol % [5] . The DSC
fi ndings should be substantiated by a stability - indicating high - performance liquid
chromatography (HPLC) assay. On the other hand, thin - layer chromato graphy
(TLC) may be used to qualitatively detect the number of impurities in the drug
sample. Impurity profi ling is an important aspect of the drug development process,
particularly for optimizing the synthetic process. The impurities can originate from
many sources, including starting materials, intermediates, synthetic processes, or
degradation reactions [6] . The regulatory guidelines stipulate that any impurity
> 0.05% of total daily dose (for drugs with a dose < 2 g/day) or > 0.15% of total daily
dose (for drugs with a dose > 2 g/day) should be evaluated for its safety [6] . Organoleptic
properties such as color, taste, and odor are assessed qualitatively to set bulk
drug specifi cations. If the drug has an unacceptable taste or odor, the chemistry
group is advised to make a suitable salt form of the drug.
PHYSICAL/BULK CHARACTERISTICS 935
936 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
6.2.2.1 Crystallinity and Polymorphism
The majority of the drugs exist in crystalline form and are characterized by their
crystal habit and crystal lattice. The crystal habit describes the external morphology
of the crystal, including shape and size, while the crystal lattice describes the internal
arrangement of molecules in the crystal (Figures 1 a and b ). Drug molecules arrange
in more than one way in a crystal, and this difference in the internal arrangement
of crystals is known as polymorphism. The polymorphs have the same elemental
composition but differ in their physical, chemical, thermodynamic, stability, and
spectroscopic properties. A crystal lattice represents the space in which molecules
arrange in different ways. Organic molecules arrange in one or more of the seven
possible crystal systems: triclinic, monoclinic, orthorhombic, tetragonal, rhombohedral,
hexagonal, and cubic [7] . Each crystal system is characterized by its three -
dimensional geometry and angles between the different crystal faces. The crystal
lattice geometry is obtained using single - crystal X - ray diffractometry (XRD) and
the details can be found elsewhere [7] . The difference in the crystal lattice of a drug
arises as a result of the difference in packing of the molecules if the molecules are
conformationally rigid (e.g., chlordiazepoxide) or due to the differences in conformation
for fl exible drug molecules (e.g., piroxicam). Although polymorphs differ in
their internal crystal lattice, it may not be necessarily refl ected in their external
crystal habit (Figure 1 b ). In other words, a drug can exist in different crystal habits
without any change in the internal crystal lattice (isomorphs).
Crystal habit is mainly dependent on crystal growth conditions [8] . For example,
Figure 1 a shows two different crystal habits for a given crystal lattice. A prismatic
crystal habit will result if the growth is equal in all directions, while plates are formed
if the growth is slow in one direction. Alternatively, needle - shaped crystals (acicular)
are formed when the growth is slow in two directions. Thus, the crystal habits can
FIGURE 1 Schematic of crystal habits, polymorphs and amorphous drug forms. ( a ) Two
crystal habits are shown. The internal crystal lattice is the same while the external morphology
is different. ( b ) In a crystal the molecules are arranged in a regular fashion. However,
the arrangement may vary depending on how the molecules orient themselves in the internal
crystal lattice. The internal crystal lattice is different in all the three polymorphic forms. The
polymorphs may or may not differ in their external morphology. ( c ) Random arrangement
of molecules in amorphous form.
(a)
(c)
(b)
be altered without any change in the internal crystal lattice by varying the crystallization
conditions. The polarity of the crystallizing solvent mainly infl uences the
crystal habit by preferentially adsorbing to one surface of the crystal face. Similarly,
surfactants or additives are added to the crystallization medium to prevent or
promote the growth of a specifi c crystal habit [8] . Crystal habits mainly differ in
physicomechanical properties such as packing, fl ow property, compressibility, and
tablettability. Acetaminophen crystallizes as polyhedral crystals when crystallized
from water and as plates when crystallized from ethanol – water (Figure 2 a ). Both
these crystal habits are isomorphic [9] , that is, have the same internal crystal arrangement,
since their melting points and heats of fusion were similar (melting point
178 ° C and . H f = 177 kcal/mol). The polyhedral crystals have better fl ow and
FIGURE 2 Difference in crystal habit of acetaminophen and resultant difference in compressibility
( a ) Acetaminophen crystallizes as either platy crystals or polyhedral crystals
depending on the solvent of crystallization. Both crystal habits have the same internal crystal
lattice since they showed the same melting point. ( b ) Difference in compression behavior of
two crystal habits. The x axis represents the compression pressure while the y axis represents
the densifi cation of the drug sample on compression. This plot is known as Heckel plot. The
polyhedral crystal habit shows a higher densifi cation implying better compressibility than
plate like crystals. [From Garekani, H. A., Ford, J. L., Rubinstein, M. H., and Rajabi - Sahboomi,
A. R., International Journal of Pharmaceutics , 187, 77 – 89, 1999. Reproduced with permission
from Elsevier. )
(a)
(b)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
ln [1/(1–D)]
Polyhedral crystals
Thin platelike erystals
0 10 20 30 40 50 60 70 80
Compression pressure (MPa)
PHYSICAL/BULK CHARACTERISTICS 937
938 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
compression properties than platy crystals, which were brittle and fragmented
during tableting (Figure 2 b ). Crystal habits are characterized using optical and
electron microscopy, but their internal crystal lattice should be confi rmed using
DSC, XRD, and spectroscopic techniques.
Polymorphs are generated by crystallizing the drug from various solvents. The
solvents are usually those that are used in the synthesis and purifi cation of the bulk
drug but may also include solvents used in drug formulations [10] . By convention,
polymorphs are named based on their order of discovery, such as forms I, II or A,
B or . , . . In general form I is considered the most stable and least soluble form,
while form II is considered the more soluble and least stable form. The least stable
and more soluble polymorphic form is usually called the metastable form. They are
not “ unstable ” but “ metastable, ” because the least stable form can remain stable
provided the conditions are controlled to prevent its conversion to the more stable
polymorphic form. Polymorphs are characterized by their solubility and stability
differences with respect to temperature (Figure 3 ). Thermodynamically, polymorphs
are classifi ed as enantiotropic or monotropic depending on their thermal reversibility
from one form to another [11] . Enantiotropic polymorphs are reversible
polymorphs, where one form (form I) is more stable at higher temperature, while
the other form (form II) is stable at lower temperature. They are characterized by
a transition point ( Ts ) below the melting points of both forms (Figure 3 a ). The
transition point represents the temperature at which one form converts to another.
In the temperature – solubility curve, this is represented by the intersection of the
solubility curves of both forms; that is, at the transition temperature, both polymorphs
have the same solubility. As shown in Figure 3 a , form II can convert to form
I at a temperature above Ts , while form I can convert to form II at a temperature
below Ts . On the other hand, monotropic polymorphs are not reversible but can
only convert from the metastable form to the stable form. Here, Ts is higher than
the melting point of both forms (Figure 3 c ). Both forms are stable in the entire
temperature range below Ts .
The different polymorphs are generated based on their solubility differences in
a given solvent. According to Ostwald ’ s rule [12] , the least stable or highly energetic
form (form II, or metastable) will precipitate out fi rst from a supersaturated solution
followed by the stable or less energetic form (form I). Supersaturation is
achieved by antisolvent addition or by altering the temperature. So, if the initial
precipitate is separated rapidly, it would have predominantly the metastable form.
Alternatively, the stable form can be melted and rapidly cooled to crystallize the
metastable form. A stable or metastable polymorphic form is also used as a “ seed ”
to preferentially grow and isolate the desired form during drug crystallization [13] .
Several rules have been proposed to differentiate enantiotropic and monotropic
polymorphs [11, 13] . A simple way to differentiate enantiotropic and monotropic
polymorphs is the use of the heat – cool cycle in DSC [11] . As shown in Figure 3 b ,
the enantiotropic polymorph is characterized by the appearance of solid – solid
endothermic transition of form II to I followed by melting of form I. On cooling
the melt of form I followed by reheating, the same thermogram is regenerated,
proving the reversibility of the polymorphs. In monotropic polymorphs (Figure 3 d ),
the thermogram is characterized by melting of metastable (form II) and recrystallization
to form I followed by melting of form I. On cooling and reheating the
sample, the transition and recrystallization peaks are not seen, indicating the irre
versible nature of these polymorphs. The heating rate in DSC is critical for characterizing
the polymorphs, as a faster heating rate may not be able to identify the
transition temperature, while a lower heating rate may lead to lower resolution of
peaks. Therefore, it is a usual practice to generate DSC thermograms under different
heating rates during polymorph characterization [11] . Also it is important to
note that the sample preparation, particle size, and crucible type can affect the
quality of the thermogram [5] . XRD is also another indispensable tool in identifying
polymorphs. This is based on the differential scattering of X rays when passed
FIGURE 3 Difference between enantiotropic and monotropic polymorphs. ( a ) Solubility
of enantiotropic polymorphs as function of temperature. The dotted line indicates the melting
curve. Form I is less soluble below the transition temperature ( T s), while form II is more
soluble above T s. Form I has a higher melting point ( T m,I) than form II ( T m,II). Below T s,
form I is converted to form II and above T s form II coverts to form I. ( b ) Thermogram generated
from heat – cool – heat cycle in DSC. In the fi rst heating cycle two endotherms are seen
corresponding to conversion of form II to form I and melting of form I, respectively. On
cooling both events show up as exotherms and on second heating cycle both endotherms
reappear, indicating thermal reversibility of enantiotropic polymorphic pairs. ( c ). Solubility
of monotropic pairs as a function of temperature. The T s is above melting point of both forms.
Forms I and II are stable in entire temperature range and their corresponding melting points
are shown. ( d ) On heating in DSC, form II melts ( T m,II) followed by recrystallization ( T crys)
and subsequent melting of form I ( T m,I). In cooling cycle only melting of form I is seen as
an exotherm and on reheating only one endotherm corresponding to form I is seen. This is
typical of monotropic polymorphs which converts from form II to stable form I and not vice
versa.
Second heat
cycle
First heat cycle
Endotherm
Temperature (°C)
Temperature (°C)
Solubility Heat flow
Temperature (°C)
Temperature (°C)
Solubility Heat flow
Ts Ts
Ts
Tm,I
Tm,II
Tm,I Tm,I Tm,II
Tm,II
Tm,I
I
I II
II
Cool cycle
Second heat
cycle
First heat cycle
Cool cycle
Endotherm
Tcrys
(a) (c)
(b) (d)
PHYSICAL/BULK CHARACTERISTICS 939
940 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
through a powder sample. Typically, on passing through a powder sample, X rays
will tend to get diffracted at various angles, and at some angle of detection, the X
rays diffracted from the different planes of the crystal converge to form an ampli-
fi ed signal, which is detected by a photomultiplier tube. The angles at which the
XRD peaks are obtained are characteristic for a polymorph (Figures 4 a & b ). The
sample should be uniformly spread to get a good X - ray diffractogram, as an
improper sample preparation may lead to variation in intensities due to the preferred
orientation of a crystal in the XRD sample holder [14] . Other techniques,
such as infrared (IR) spectroscopy and solid - state nuclear magnetic resonauce
(NMR), are also used to characterize the polymorphs and are listed in Table 2 .
FIGURE 4 Schematic X - ray diffractograms of two polymorphic forms of a hypothetical
drug. The x axis represents the detection angle and the y axis represents the intensity of the
peak. As can be seen, there is a difference in the diffractograms due to the difference in the
internal crystal lattice of polymorphs. The different internal arrangement in a crystal defl ects
the X ray at different angles.
10 20 30 30 40 50
50
100
2 .
2 .
10 20 30 30 40 50
Intensity
50
100
Intensity
(b)
(a)
Polymorphism has signifi cant implications in the solubility, bioavailability stability,
processing, packaging, and storage of solid drug substances [15 – 17] . The metstable
polymorphic form may be used to improve the solubility of drug substances.
Many drugs are known to exhibit polymorphism, particularly, steroids, barbiturates,
anti - infl ammatory drugs, and sulfonamides, which have a high probability of exhibiting
polymorphism [15] . The existing knowledge on drug polymorphism is a good
starting point for a preformulation scientist to anticipate polymorphs based on the
drug chemistry. In some cases, polymorphism may provide an opportunity to improve
the solubility of a drug. For example, form II of chloramphenicol palmitate has a
higher dissolution rate resulting in signifi cantly higher plasma concentration than
form I when administered orally [15] . However, in many cases [16] the difference
in solubility may not be signifi cant enough to cause differences in oral bioavailability
(Table 3 ). Although the polymorphs differ in their dissolution rates, it should be
realized that once the drug goes into solution, they do not differ in their properties.
If a drug ’ s absorption is limited by its poor membrane permeability, then the difference
in solubility of polymorphs may not impact its bioavailability. Similarly, if the
drug dissolution is rapid in comparison to the gastrointestinal (GI) transit time, then
the difference in polymorph solubility will not infl uence its bioavailability [16] .
TABLE 2 Techniques to Characterize Different Crystalline Forms
Technique Applications
Thermal analysis
Differential scanning
calorimetry
Melting point, enthalpy of fusion, and crystallization; solid - state
transformations
Thermogravimetric
analysis
Stoichiometry of solvates and hydrates; identifying vaporization
and volatilization
Hot - stage microscopy Visualization of solid - state transformations and desolvation
events
X - ray diffractometry Identifying polymorphs; quantifi cation of degree of crystallinity;
crystal lattice geometry and solid - state transformations
Spectroscopy
Infrared Characterization of polymorphs based on functional groups;
characterization of hydrates and solvates
Near infrared In situ analysis of solid - state conversions; identifi cation and
quantifi cation of polymorphs in dosage forms
Nuclear magnetic
resonance
Useful to understand difference in molecular arrangement of
polymorphs, hydrates, and solvates
TABLE 3 Difference in Solubilities of Polymorphs
Drug Melting Point ( ° C) Solubility Ratio a
Indomethacin 157, 163 1.1
Sulfathiazole 177, 202 1.7
Piroxicam 136, 154 1.3
a Indicates ratio of solubility of low - melting polymorphic form to solubility
of high - melting polymorphic form of drug.
PHYSICAL/BULK CHARACTERISTICS 941
942 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
More than the presence of metastable polymorph, it is the conversion of the
metastable to the stable form during processing, storage, or use that is of great
concern to the pharmaceutical scientist [17] . The unpredictability in “ conditions ”
that result in the generation and conversion of one polymorphic form to another
mainly aggravates such a situation [18] . This is exemplifi ed by ritonavir, which is a
classical case of appearance of a “ new polymorphic form ” after the drug was marketed
[19] . Ritonavir, an anti - retroviral, drug was introduced in the market as a
polymorphic form I in soft gelatin capsule in 1996. Two years later, a new polymorphic
form II appeared in the formulation due to some unknown reasons causing the
drug to be less soluble. The drug manufacturer withdrew the product due to failure
of the batches in dissolution tests. After extensive investigation and reformulation,
the drug was reintroduced in the market in 1999.
Nonetheless, in spite of their unpredictability, a good preformulation team will
be able to anticipate the different polymorphs during drug development. It should
not be an impossible task given the recent advancements in high - throughput generation
and characterization of polymorphs [20] . From an innovator ’ s perspective, the
identifi cation and thorough characterization of multiple polymorphs during drug
discovery can extend the patent life and delay the market entry of generic manufacturers.
On the other hand, it is also an opportunity for the generic manufacturers
to generate new polymorphs with better solubility and stability for gaining market
entry. The patent dispute on ranitidine polymorphs is a good example in this regard
[21] . Two polymorphic forms of ranitidine were patented by the innovator company
and the generic manufacturers had to fi nd an appropriate method to manufacture
the desired polymorph without the accompanying impurity of the other polymorph.
This provided an edge for the innovator to extend the drug ’ s market exclusivity for
a little longer than they would otherwise have had. If a pure polymorph cannot be
generated, the extent of polymorphic impurity should be quantifi ed and ensured
from batch to batch. The preformulation scientist closely works with the synthetic
chemist in setting specifi cations for polymorphs.
6.2.2.2 Hydrates/Solvates
In addition to drug molecules, solvent molecules also get incorporated in the crystal
lattice, resulting in altered physicochemical properties. When the solvent is water,
they are known as hydrates, while if it is any other solvent, they are known as
solvates. They are also known as pseudopolymorphs or solvatomorphs. Hydrates
are important in this regard as one - third of all marketed drugs are hydrates [13] .
Depending on how the water is arranged inside the crystals, they are classifi ed as
isolated hydrates, channel hydrates, and ion - associated hydrates [13] . In isolated
hydrates, the water molecules are separated from each other by the intervening drug
molecules in the crystal lattice (e.g., cephadrine dihydrate). Channel hydrates result
when water molecules are linked to one another forming a channel (e.g., theophylline
monohydrate). The water molecules may be present either stoichometrically or
nonstoichometrically within the crystal lattice. Ion - associated hydrates are typically
seen when the water is metal ion coordinated (e.g., nedocromil zinc). Nonstoichometric
channel hydrates are problematic due to the presence of diffusible water,
which can easily move in and out of the crystal lattices [13, 22] .
Hydrates or solvates are formed by crystallizing the drug in the presence of water
or solvents. The hydrate formation is dictated by water activity in a given solvent
[22] . Hydrates are characterized using gravimetric methods such as thermogravimetric
analysis (TGA) or by Karl Fischer titrometry [23] . In TGA, the loss of
water/solvate on heating a sample is recorded as a thermogram (Figure 5 ). The mass
change due to dehydration is seen as a step loss in the TGA thermogram. Based on
the weight of the initial sample and its elemental composition, the number of water
molecules can be calculated. The TGA curve in combination with a DSC thermogram
helps to differentiate hydrates from other thermal transitions. In Figure 5 ,
the endotherm in the DSC thermogram corresponds to water loss as indicated
by the TGA curve. The TGA can be coupled to an IR or mass spectro meter
to characterize solvates. Thermal microscopy is a useful qualitative tool to
visualize the release of water from the drug crystals as a function of temperature
[23] .
Hydrate formation and dehydration signifi cantly infl uences the processing and
storage of drug products [17] . Hydrates may take up further water or dehydrate to
lose water. Dehydration of hydrates leads to several possibilities [24] , as shown in
Figure 6 . Hydrates on dehydration can form isomorphic desolvates retaining the
same crystal lattice as the hydrate but without the water. Alternatively, hydrates can
lose water and become anhydrous crystals. They can also lose water, forming amorphates
with the loss of crystal lattice. Higher hydrates can lose water to form lower
hydrates, for example, pentahydrate converting to di - or monohydrate. The hydrates
also can exhibit polymorphism or on dehydration can convert to a different polymorphic
form [13, 17] . Such solid - state transformations are possible during processing,
such as granulation, tableting, and storage [17] . In general, hydrates are less
soluble than anhydrous forms while solvates are more soluble than ansolvates in
water. Ampicillin trihydrate is a classical example which shows lower solubility and
lower plasma concentration than anhydrous ampicillin [15] . Preformulation studies
FIGURE 5 Characterization of hydrate. ( a ) TGA thermogram of monohydrate. The thermogram
shows weight loss as a function of temperature. The step in the thermogram shows
weight loss due to dehydration of a hydrate. ( b ) DSC thermogram showing endotherm at
corresponding temperature ( T dehyd). The second endotherm indicates the melting point of
the hydrate ( T m).
Percent weight loss Heat flow
Tm Tdehyd
Temperature (°C)
(a)
(b)
PHYSICAL/BULK CHARACTERISTICS 943
944 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
provide valuable inputs to the formulator in selecting a suitable form of the drug.
For example, ampicillin is hygroscopic and hence can be used in suspension dosage
forms, while ampicillin trihydrate, which is non - hygroscopic, is used in solid dosage
forms.
6.2.2.3 Amorphate
Unlike a crystalline drug, an amorphous form of the drug does not have a regular
crystal lattice arrangement and the molecules are arranged in random order (Figure
1 c ). Glass is a typical amorphous substance and so amorphous drugs are also known
as glasses [25] . Amorphous form is prepared through milling, rapid cooling of a melt,
rapid precipitation using an antisolvent, rapid dehydration of a hydrate, spray drying,
or freeze drying [26] . Some of the above methods may also unintentionally produce
an amorphous form during processing of the crystalline form of the drug [17] . For
instance, milling during dosage form manufacture may produce an amorphous form
unintentionally, as in the case of indomethacin. The amorphous form does not show
a melting point but is characterized by a glass transition temperature ( T g ). This
temperature indicates the conversion of the amorphous form from a rigid glassy
state to a more mobile rubbery state. Above T g the amorphous form will tend to
recrystallize and convert to the crystalline form, which then undergoes melting, as
shown in Figure 7 a . The T g for an amorphous drug can vary depending on the
storage conditions and thermal history of the sample and is sensitive to moisture,
pressure, and temperature [26] . The T g is seen only as a slight shift in the baseline
due to a change in the specifi c heat capacity of the sample and is infl uenced by the
heating rate in DSC [25] . In XRD, the amorphous form shows a shallow peak
or halo, as opposed to sharp and intense peaks for a crystalline drug compound
(Figure 7 b ).
The main advantage of amorphous form of the drug substance state is its signifi -
cantly higher solubility than the crystalline form of the drug, primarily due to the
excess surface energy [16, 27] . Therefore, conversion of a crystalline drug into an
amorphous form is one of the strategies to increase drug solubility. Table 4 compares
FIGURE 6 Various possibilities that arise from dehydration of hydrate. A hydrate can
dehydrate reversibly into various solid - state forms. It can dehydrate to form an anhydrous
form of the drug or to a lower hydrate. Hydrate can also dehydrate to form an isomorphic
desolvate where the crystal lattice is retained except for the absence of water. The crystal
structure may also collapse on dehydration to form an amorphous form. Hydrates on dehydration
can also result in different polymorphs.
Amorphous Lower hydrate
Polymorphs
Anhydrous Isomorphic
desolvate
Hydrate
FIGURE 7 Characterization of amorphous form. ( a ) DSC thermogram of amorphous
substance. Thermogram is characterized by a glass transition temperature ( T g) above which
the amorphous form is mobile and recrystallizes ( T crys) into a crystalline form which fi nally
melts ( T m). ( b ) Amorphous form that does not show any peaks in XRD as it does not have
regular arrangement of molecules. Shallow peaks are indicative of an amorphous drug
substance.
(a)
(b)
10 30 20 30 40 50
100
50
Temperature (°C)
Tm Tg Tcrys
Heat flow
Endotherm
2 .
Intensity
the solubility of amorphous and crystalline forms of a few drugs. However, the
biggest challenge lies in the stabilization of the amorphous form to prevent it from
converting to the less soluble crystalline form during storage and use. It should be
noted that they can take up moisture to convert to a crystalline form or to a hydrate,
resulting in decreased drug solubility. The moisture uptake can also lead to chemical
degradation [26] . The stabilization of drug amorphates is usually accomplished by
increasing the T g using polymers and thus restricting their molecular mobility and
PHYSICAL/BULK CHARACTERISTICS 945
946 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
chemical reactivity [26] . Particle size reduction, which is a very common processing
step in dosage form manufacturing, can result in varying degrees of amorphous and
crystalline forms in the drug. In such cases it is essential to quantify the degree of
crystallinity using various analytical techniques such as DSC and XRD [28] .
6.2.2.4 Hygroscopicity
Some solid drug substances have a tendency to absorb moisture from the atmosphere
leading to physical and/or chemical instabilities. Hygroscopicity is the rate
and extent of moisture adsorbed/absorbed by a solid substance. Solid drug substances
may vary in their behavior to moisture and are classifi ed as deliquescent,
effl orescent, and effervescent. Deliquescent materials such as hydrochloride salts
absorb moisture and become a liquid. Effervescent substances (e.g., a mixture of
citric/tartaric acid and sodium bicarbonate) absorb moisture and release carbon
dioxide. On the other hand, effl orescent substances such as hydrates may lose moisture
depending on the relative humidity (RH). Therefore, it is important to study
the moisture absorption behavior of drugs to choose the processing and storage
conditions for the drug.
Hygroscopicity is measured by exposing the drug sample to various RH in a dessicator.
The RH is maintained at a constant level by using salt solutions of varying
concentrations (e.g., KNO 3 , KCl) and the humidity is expressed with respect to the
humidity of a saturated salt solution. Moisture sorption and desorption curves are
generated to study the moisture uptake. The moisture absorption profi le is generated
by noting the increase in mass on exposure to varying RH and the desorption
profi le is generated by recording the change in weight with decreasing RH [23, 29] .
This can be measured using a dynamic vapor sorption instrument. A typical sorption/
desorption profi le is shown in Figure 8 for an anhydrous and hydrate form of
a hypothetical drug. As can been, for a hydrate the sorption and desorption profi le
is superimposable and is hence non - hygroscopic. On the other hand, the anhydrous
form of the drug is hygroscopic and shows hysteresis on the sorption and desorption
profi le, indicating signifi cant moisture uptake. Such profi les give useful clues to the
preformulation scientist. Signifi cant hysteresis is indicative of hydrate formation and
can be used as a guide to evaluate the potential of hydrate formation. Further the
profi le also helps to differentiate hygroscopic and non - hygroscopic salts [29] . The
profi le also gives information on processing and storage conditions that can overcome
solid - state transformations. For example, in Figure 8 , it is seen that the anhydrous
form does not take up moisture if RH is below 80% and it retains the moisture
TABLE 4 Comparative Solubilities of Amorphous and
Crystalline Forms of Drugs
Drug Solubility Ratio a
Carbamazepine 1.5 – 1.7
Griseofulvin 38 – 441
Glibenclamide 14
a Indicates ratio of solubility of amorphous form to solubility of crystalline
form of drug.
FIGURE 8 Sorption and desorption profi le of hydrate and anhydrous form of hypothetical
drug. The solid lines represent the profi le for an anhydrous form of the drug, while the broken
lines represent the profi le for a hydrate form of the drug. Anhydrous form of the drug does
not take up moisture until it reaches 80% RH, and on reducing the RH, it does not lose
moisture until it reaches RH of 20%. The hysteresis is indicative of hygroscopicity and
signifi cant moisture uptake. The hydrate form of the drug does not show hysteresis but
both the sorption and desorption curve superimpose on each other indicating that it is
non - hygroscopic.
20 40 60 80
Change in mass (%)
Relative humidity (%)
until the RH is reduced below 20%. The study can also be used to extract kinetic
and temperature information on moisture uptake by drug substances. The studies
conducted during preformulation testing should be representative of the anticipated
processing and storage conditions of the drug.
6.2.2.5 Particle Characteristics
Drug particle characteristics such as size, shape, and surface area impact the drug ’ s
processability and product performance. Particle size is the most infl uential among
these as the other two properties can be related to it. When suffi cient drug is available,
the preformulation scientist characterizes the particle size and size distribution
to set specifi cations for formulation and future drug lots. Table 5 lists the various
methods used to measure particle size. The methods differ in their principle of
operation and also in the range of particle sizes they can measure [30] . Usually, the
gross particle morphology is characterized using a simple optical microscope and if
required is further characterized using a scanning electron microscope. Light -
scattering methods are commonly used to measure particle size due to their low
sample requirements and ease of measurement. The instrument readout is in the
form of a graph where the particle size is plotted against the percent frequency of
particles in different particular size ranges. The results are used to set particle size
specifi cations and understand polydispersity or multimodal particle size distribution
in powders. Especially if the drug is potent, a narrow size distribution is desired to
ensure drug homogeneity during formulation. The surface area of a powder bed is
determined using the Brunauer, Emmett, and Teller (BET) method. In this method,
PHYSICAL/BULK CHARACTERISTICS 947
948 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
N2 gas is passed through a powder bed and the surface area is calculated based on
the volume of gas coming out in the absence and presence of the powder [31] .
Particle size reduction is an initial step in the development of any dosage form.
The data generated during preformulation testing guides the formulator in deciding
about size reduction. Particles with size > 100 . m generally require size reduction
and particles in the size range of 10 – 40 . m are generally acceptable for solid dosage
forms [32] . As mentioned in the earlier section, particle size reduction may lead to
partial or complete amorphization of a powder, and this factor should be taken into
consideration. Also worth mentioning are the other solid - state transformations that
may take place during milling, such as the conversion of one polymorphic form to
another or the desolvation of a hydrate [17] . It is important to maintain the particle
size distribution within a narrow range to avoid powder stratifi cation and avoid
fl owability issues during capsule fi lling or tablet compression. Particle size reduction
increases drug solubility due to the enormous increase in surface area with
decreasing particle size. Griseofulvin is often widely quoted in the literature in this
regard, where the bioavailability of this water - insoluble drug is increased 10 times
on reducing the particle size [32] . On the other hand, particle size reduction may
be counterproductive for some drugs such as nitrofurantoin. Particle size reduction
of nitrofurantoin causes rapid drug absorption with an associated increase in its
adverse effects. In contrast, the slowly dissolving macrocrystals of nitrofurantoin do
not cause adverse effects [32] . Excessive particle size reduction ( < 30 . m) may also
lead to static charge buildup on the surface, resulting in agglomeration and reduced
powder fl ow or reduced drug solubility. If the drug is sensitive to moisture or
oxygen, then the increased surface area associated with particle size reduction
may accelerate degradative reactions [32] . Therefore, it is important to study the
infl uence of particle size on drug solubility and stability during preformualtion
testing.
TABLE 5 Methods for Particle Size Analysis
Method Principle
Sieve Sieve analysis utilizes a series or stack, or nest of electro brass or
stainless steel sieves that have smaller mesh at the bottom followed by
meshes that become progressively coarser toward the top of the series.
Useful for measuring particles in size range 10 – 50,000 . m
Microscopy Analysis is carried out on two - dimensional images (projected diameter)
of particles which are assumed to be randomly oriented in three
dimensions. Can measure particles in the size range 1 – 1000 . m.
Electron microscopy is useful for analysis of particles in
submicrometer range (0.01 – 100 . m). It also gives information on
surface morphology and shape of the powder.
Sedimentation Size analysis is based on sedimentation of particles as a function of their
size due to gravitational pull or by using a centrifugal force. Can
measure particles in the size range 0.01 – 100 . m.
Light
scattering
This is based on the principle of light scattering of particles as a function
of their hydrodynamic radius. Commonly used, as it requires a small
sample size and is a rapid method for particle size measurement. It can
be used to measure particles varying in size from 0.001 to 100 . m.
6.2.2.6 Powder Flow and Compressibility
The derived properties of a drug substance play a critical role in deciding about the
feasibility of a solid dosage form. These include the bulk density, fl ow properties,
and compressibility of the drug powder. Powder fl ow is infl uenced by many solid -
state properties, including crystal habit, bulk density, particle size, and shape. Bulk
density is an important determinant of powder fl ow. It is the ratio of a known weight
of the powder and its bulk volume. This is determined by pouring a weighed amount
of the powder into a graduated cylinder and measuring its volume. Bulk density is
particularly important for high - dose drugs, where the drug would occupy a major
portion of the tablet or capsule dosage form. The true density of a powder is determined
using a helium densitometer [31] . The volume of helium gas that passes
through an empty tube is compared with the volume of helium passing through the
tube when fi lled with a defi ned weight of the powder. The difference in the volume
gives the true volume of the powder, which is then used to calculate the true density
of the powder. This information can be used to calculate the porosity:
Porosity
bulk volume true volume
bulk volume
= . . 100
(1)
The porosity of a powder depends on particle size and shape; pharmaceutical
powders vary in porosity from 30 to 50% [31] . Powder with varying particle size will
give a porosity of less than 30%, where the small particles may occupy the pores in
between the larger particles. On the other hand, powder aggregates lead to increased
porosity and poor fl ow properties.
The powder fl ow is determined using the angle of repose or Carr ’ s index (Figures
9 a and b ). The angle of repose is the angle that the powder makes with the horizontal
surface when allowed to fl ow through a funnel (Figure 9 a ). This is based on the
principle that powder fl ow is infl uenced by the relative infl uence of interparticle
friction and the gravitational pull on the powder. Pharmaceutical powders have an
angle of repose of 25 ° – 40 ° [33] , and in general, a good fl owing powder will have a
lower angle of repose (Figure 9 c ). The angle of repose using the funnel provides a
good estimate of the infl uence of particle size, shape, and electrostatic interaction
between the particles when the powder fl ows through the hopper in a tableting
machine [34] . When there is a limited drug sample, Carr ’ s index is used to estimate
powder fl ow and compressibility. In this method, a small quantity of the powder is
used to determine its bulk density and this is followed by determining the tapped
density of the powder. After fi lling, the graduated cylinder is tapped on a hard
surface (3 – 30 taps) until the powder consolidates and gives a constant volume
(Figure 9 b ). Carr ’ s index is calculated using the equation
Carr s index
tapped poured density
tapped density
’ = . . 100
(2)
This index is a good measure of powder consolidation and compressibility for predicting
the feasibility of developing a tablet dosage form. A lower Carr ’ s index is
indicative of a good fl owing powder. There is a good correlation between angle of
repose and Carr ’ s index [Figure 9 c ], and depending on the quantity of the drug
PHYSICAL/BULK CHARACTERISTICS 949
950 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
available for preformulation testing, either method may be used to estimate the
powder fl ow property. Poor fl owing powders may require glidants to improve their
fl ow property. Compressibility is studied by compressing the drug in a hydraulic or
IR press, and such experiments give early warnings to the formulator about capping
and lamination issues in tablets. Altogether, these preformulation tests give valuable
information to the formulator in deciding excipients and processes.
6.2.3 SOLUBILITY CHARACTERISTICS
Drug solubility is one of the physicochemical parameters that receives lots of attention
during preformulation testing. In the initial stages, solubility studies are usually
kinetically determined, where the drug is placed in contact with the solvents and
then the solubility is assessed using turbidometric methods almost instantaneously.
FIGURE 9 Measurement techniques for powder fl ow. ( a ) Angle of repose is determined
by pouring a powder through a funnel and noting the angle that the powder heap makes with
the horizontal surface. ( b ) Carr ’ s index is measured by pouring a known weight of the powder
into a graduated cylinder and tapping it on a hard surface until the powder is consolidated.
( c ) Carr ’ s index is measured as percent, while angle of repose is measured in degrees,
and both methods show good correlation. Powder fl ow is classifi ed based on either of the
measurement methods.
Poor
Good
Bulk volume
Volume after tapping
(a) (b)
(c)
Carr’s index (%)
5–15
12–16
18–21
23–35
33–38
>40
<20
20–30
30–34
—
>40
—
Angle of repose (deg) Powder flow
Excellent
Fair to
passable
Very poor
Extremely
poor
SOLUBILITY CHARACTERISTICS 951
A high - throughput solubility screen consists of dissolving the drug in a minimal
volume (few microliters) of dimethyl sulfoxide (DMSO) and then adding different
volumes of water until turbidity is observed. The appearance of turbidity is considered
as a rough estimate of the drug ’ s water solubility and turbidity is measured in
a 96 - well plate format in a turbidometer [35] . Solubility is qualitatively described in
terms of how much solvent is required to solubilize 1 g of the drug and is shown in
Figure 10 . Kinetic solubility measurements are only rough estimates, as they do not
take into account the solid - state transitions and should be followed up with equilibrium
solubility studies later when more drug is available.
Equilibrium solubility is determined by placing an excess solid drug in a few
milliliters of the solvent and shaking at 37 ° C for 60 – 72 h until equilibrium is reached.
One to three samples are withdrawn, fi ltered, and assayed for drug content. Equilibrium
solubility helps to identify polymorphic or amorphous forms of the drug,
which shows an apparently higher solubility in kinetic studies. Intrinsic solubility
measurements are measured for ionic compounds to determine the inherent solubility
of the un - ionized form of the drug. This would mean that the solubility of a
weakly acidic drug is tested in an acid medium and a weakly basic drug is tested in
an alkaline medium, where the drug would remain completely un - ionized. The
studies are followed by determining drug solubility at different pH and are discussed
later in this section.
FIGURE 10 Terminologies for drug solubility. The drug solubility is qualitatively described
depending on how much solvent is required to solubilize 1 g of the drug.
Practically insoluble
>1000 mL
Very slightly soluble
100–1000 mL
Slightly soluble
100–300 mL
Sparingly soluble
30–100 mL
Soluble
10–30 mL
Freely soluble
1–10 mL
Very soluble
<1 mL
Solvent
required for
1 gm of solid
952 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
A drug solubility of 1 mg/mL is usually considered acceptable to avoid dissolution
rate – limited absorption in vivo. If the drug solubility is < 1 mg/mL, salts should be
considered, if the drug is ionic and cosolvents should be considered if the drug is
nonionic [33] . This brings us to a discussion of the factors that infl uence drug solubility:
temperature, pH, crystal form, and solvents. The effect of temperature on drug
solubility depends on the heat of solution ( . H s ), which is the amount of heat given
up or taken up when a drug goes into solution. The relationship between drug solubility
S and temperature T is given by the van ’ t Hoff equation, where R and C are
constants:
ln S
H
RT
C = + . s
(3)
Drugs which have . ve . H s (exothermic) generally decrease in solubility with
increase in temperature (e.g., lithium salts and hydrochloride salts), while drugs
which have + ve . H s (endothermic) usually increase in solubility with increase
in temperature (e.g., most organic drugs). Nonelectrolytes have a . H s of 4 – 8 kcal/
mol and show signifi cant increase in solubility when the temperature is increased.
On the other hand, salts have a . H s of . 2 – 2 kcal/mol and are therefore less
sensitive to temperature [34] . Solubility studies are conducted at 25, 37,
and 50 ° C. In addition to providing information on the drug solubility at the
body or processing temperatures, it is also useful to understand polymorphic
interconversions.
With respect to selecting cosolvents, one should consider drug polarity and
solvent polarity. Usually the solvents include glycerol, propylene glycol, and ethanol.
Other solubilization techniques such as complexation and surfactants can also be
used to enhance the solubility of the drug [36] . However, the solubilization techniques
used in preclinical testing may not be same as the fi nal formulation used in
clinical studies and marketing.
For ionic substances, salt formation is the preferred strategy for drug solubilization.
The salt selection is crucial during early discovery, as any change will require
repeating some of the earlier preclinical studies. Salts provide wider fl exibility in
modulating the drug properties without changing its activity. The salt formation is
used to address drug solubility, stability, and processing issues [37] . Sometimes, salt
formation may be used to deliberately reduce the solubility of drug to overcome
unpleasant taste or stability of the drug. For example, clindamycin pamoate is used
in place of hydrochloride salt to overcome the unpleasant taste of the latter. The
various factors to be considered in salt selection are discussed in the next section.
In addition to improving the solubility of the drug, solubility data guides the formulator
to choose a suitable granulating solvent for a tablet dosage form.
6.2.3.1 p K a and Salt Selection
The majority of the pharmaceutical drugs are weak bases or weak acids. Among the
marketed drugs, more than 75% are weak bases, 25% are weak acids, and 5% are
nonionic [38] . Therefore, knowledge of p K a is useful for enhancing drug solubility
and stability. The Henderson – Hasselbalch equation is used to describe the ionization
of a weak acid or base:
SOLUBILITY CHARACTERISTICS 953
Percent ionized
100
1+10
for weak acid
fo
(p pH
pH p
a
a
=
+
.
.
K
K
)
( )
100
1 10
r weak base
.
. .
. .
(5)
The equations theoretically predict the ionization and solubility of a drug at a given
pH. The p K a , which is a characteristic property of an ionizable drug, defi nes the pH
at which the drug is half ionized and half un - ionized. A weakly acidic drug will be
predominantly in the un - ionized form two pH units below its p K a and predominantly
ionized at two pH units above its p K a . In a weakly basic drug, it is exactly
the opposite of what is seen with an acidic drug. The p K a of a drug can be measured
using potentiometry, solubility, conductometry, and spectroscopic techniques [35] .
Potentiometry measures the change in potential when the drug is titrated with an
acid or base and is suitable for drugs which have p K a of 3 – 10 [33] . If the drug is not
water soluble, it is usually dissolved in a water - miscible solvent such as DMSO or
methanol. In order to nullify the effect of cosolvents, measurements are made with
various cosolvent concentrations and are plotted against p K a . The intercept on the
y axis gives the p K a at zero cosolvent concentration. Some drugs have more than
one ionizable group, such as ampholytes, and are characterized by more than one
p K a value. They are classifi ed as ordinary or zwitterionic ampholytes [39] . In ordinary
ampholytes, p K a,acidic > p K a,basic (e.g., chlorambucil), while in zwitterionic ampholytes,
p K a,acidic < p K a,basic ampholytes (e.g., amino acids and proteins). The amino acids
and proteins are characterized by their isoelectric point (pI), which is the pH at
which the net charge is zero and is calculated using the formula
pI p p a,acid a,base = + 1
2
( ) K K
(6)
Drugs may also have more than two p K a values, such as polyprotic or polybasic
compounds (e.g., minocycline), and such drugs exhibit a complex pH solubility
profi le. It is essential to know per se pH of the drug solution during preformulation
studies. The pH is measured or theoretically calculated if the p K a and drug concentration
C are known. The pH of a weak acid or the salt of a weak base and a strong
acid can be calculated using the equation
pH
p a = . ( log ) K C
2
(7)
Similarly, the pH of a weak base or a salt of weak acid – strong base can be calculated
using the following equation, where p K w is the ionization constant of water:
pH
p p w a = + + ( log ) K K C
2
(8)
Solubility of a drug is directly proportional to the extent of ionization of a drug in
water. Therefore, p K a of the drug may also be determined by noting the change in
solubility of a drug as a function of pH. It is customary to check the drug solubility
(4)
954 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
in the pH range usually encountered in the gastrointestinal tract (GIT; pH 1 – 8). The
infl uence of pH on drug solubility can be estimated from the equations
S
S K
S K
=
+ .
+ . {u a
u a
pH p for weak acid
p pH for weak base
[ ( )]
[ ( )]
1 10
1 10
(10)
In this equation S represents the total solubility of ionized and un - ionized forms of
the drug, while S u represents the intrinsic solubility of un - ionized form of the drug.
The pH solubility profi le and the drug p K a are important for salt selection. In addition,
the salt selection is governed by various factors as outlined in Table 6 . As a
rule of thumb, a strong acid is used to form a salt with a weakly basic drug and a
strong base is used to form a salt with a weakly acidic drug. The probability of salt
formation can be predicted from the relative p K a values of the drug and the counterions
by using the equation [40]
.p p p a a,drug a,counterion K K K = . (11)
The probability of salt formation is high for a weak acid if . p K a is negative, that is,
p K a,drug < p K a,counterion . Sodium phenytoin is an example, where phenytoin has a p K a
of 8.3 and sodium has a p K a of 16. Similarly, a weak base will form a salt with acid
counterion if . p K a is positive, that is, p K a,drug > p K a,counterion . In atropine sulfate, atropine
has a p K a of 9.9 and sulfuric acid has a p K a of . 3. It is important to mention
that salts do not alter the intrinsic p K a of the drug but increase drug solubility by
keeping pH on the ionization side of the drug ’ s p K a (Figures 11 a and b ). The salt
formation is a futile exercise if the p K a of a drug is < 3 or > 10 and other solubilization
strategies have to be pursued [33] .
Inorganic ions such as hydrochloride and sodium are the most frequently encountered
species in pharmaceutical salts, primarily because of the ease of salt formation
and their low molecular weights. They provide signifi cant increase in drug solubility
and at the same time strong acids/bases may also be hygroscopic, posing problems
during processing and storage. In those cases, salts are formed using weaker organic
anions or cations such as mesylate, besylate, and choline. Table 7 lists some of the
commonly used inorganic and organic counterions used in pharmaceutical salts. The
pH provided by the salts signifi cantly infl uences the drug dissolution and subsequent
drug absorption from the GIT. Though salts increase drug ionization and aqueous
drug solubility, it is the un - ionized form of the drug that is absorbed through the
membrane. According to the well - known pH partition hypothesis [33] , a weakly
TABLE 6 Factors to Consider during Salt Selection
Relative p K a of the drug and the counterion
Common - ion effects
Crystal habit and crystallinity
Polymorphic conversions
Hygroscopicity
Chemical stability
Manufacturability
Toxicity
(9)
SOLUBILITY CHARACTERISTICS 955
TABLE 7 Various Counterions Used to Form Drug Salts
Chemical class Salt - Forming Counterions
Inorganic Hydrochloride, hydrobromide, sulfate, nitrate, phosphate, sodium,
potassium, calcium, and zinc
Organic Triethanolamine, ethanolamine, lactic acid, maleic acid, citric acid, acetic
acid, choline, ethanesulfonic acid, oleate, and stearate
FIGURE 11 Relative p K a of drug and salt - forming counterion. ( a ) For a weak acid, the
p K a of the salt - forming counterion should be higher than the drug p K a to keep the pH on
the ionization side. ( b ) For a weak base, the salt - forming counterion should have a p K a less
than the drug ’ s p K a to keep the pH in the ionization side.
(a)
(b)
pH
pKa of counterion pKa of drug
Percent Ionization Percent Ionization
pH
pKa of
counterion
pKa of drug
956 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
acidic drug is primarily absorbed from the stomach (pH 1 – 3), where it remains in
un - ionized form. A basic drug is expected to be absorbed from the intestines (pH
6.5 – 8), where it would be in the un - ionized form. But in many cases, the absorption
of drugs cannot be satisfactorily explained by this theory. This is understandable if
one considers the fact that drug dissolution from a salt is mainly infl uenced by the
surface pH of the dissolving drug (microenvironment) rather than the bulk pH of
the GI fl uids. For instance, the surface pH of weakly acidic drug is 1.5 times higher
than the bulk pH in the stomach, and therefore the dissolution of the salt of a weak
acid will be 100 times faster than the free - acid form of the drug in stomach [40] .
Similarly, it is the free base rather than its salt form which will dissolve faster in the
gastric pH. So it is possible to modulate the drug solubility of a pharmaceutical salt
irrespective of its location in the GIT. Considering the fact that most of the drugs
are absorbed from the intestine (due to large surface area), it is desirable to select
a salt that is not completely ionized or unionized at the intestinal pH to have optimal
dissolution and absorption.
An important phenomenon that is often overlooked during the salt selection
process is the suppression of salt ionization in GI fl uids due to the common - ion
effect. This is particularly important with inorganic salts, where salt ionization can
be suppressed by ions such as chloride and sodium which are abundant in GI fl uids.
A hydrochloride salt will ionize in solution, as shown in the Equation (12) , but in
gastric fl uid, the presence of chloride ions suppresses the drug ionization [as shown
by the thicker arrow in the reverse direction in Equation (13) ] to maintain an equilibrium
between the ionized and un - ionized form of the drug:
DH Cl DH Cl + . + . + (12)
DH Cl DH > Cl + . + . + (13)
Hence it is important to study this effect during preformulation by testing the solubility
of the salt in the presence and absence of sodium chloride. Although salts do
not alter the pharmacological activity of the drug, safety is an important consideration
in the selection of salts. From this perspective, salts are treated as a new
molecule by the FDA. The safety of the salt is evaluated with respect to its route
of administration and dose of the drug [37] .
6.2.3.2 Partition Coeffi cient
In simple terms, the partition coeffi cient represents the relative solubility of a drug
in a hydrophobic and a hydrophilic solvent. The hydrophilic solvent is usually water
or buffer (pH 7.4), while the hydrophobic phase is usually n -octanol. The partition
coeffi cient ( K o/w ) is defi ned as the ratio of concentration of the drug in the organic
phase ( C o ) to drug concentration in the aqueous phase ( C w ):
K
C
C o/w
o
w
=
(14)
The choice of n - octanol is based on its ability to mimic the lipophilicity of the biological
membranes [33] and further its solubility parameter ( . = 10.24; solubility
SOLUBILITY CHARACTERISTICS 957
parameter is a measure of internal cohesive energy) falls within the solubility
parameter range of most drugs (8 – 12.4). The partition coeffi cient is determined by
dissolving the drug in one of the phases and shaking both the phases together in a
fl ask for 30 min to achieve equilibration. Then the drug concentration is determined
from one of the phases, usually the aqueous phase, and the drug concentration in
the oil phase is determined by subtracting the drug concentration in the aqueous
phase from the total drug that was added. This value when expressed in logarithmic
form is known as log P . The phase volume of the two phases is 1 : 1 but, if the drug
is less soluble in the aqueous phase, the ratio (water – octanol) is changed to 1 : 10 or
1 : 20 to have a measurable drug concentration in the aqueous phase [33] . It is important
to saturate the phases with respect to the other solvent before starting the
experiment to rule out the infl uence of solvent partitioning on drug distribution
between the two phases.
Another important factor is the infl uence of drug ionization on the partition
coeffi cient and this is particularly important when a buffer is used instead of water.
The partition coeffi cient determined from Equation (14) is an apparent value rather
than a true partition coeffi cient for ionic drugs. However, the true partition coeffi -
cient can be calculated from the apparent partition coeffi cient if the drug ’ s p K a and
the pH of the drug solution are known [39] , as shown in the equations
log
log log
log log
( )
P
P
P
K
=
.
+ ( )
.
+
. app pH p
app
a
for weak acid
1
1 10
1
1 10( ) p pH a
for weak base K . ( )
.
. .
. .
(16)
During the initial stages of drug screening, the partition coeffi cient is calculated
based on the chemical structure ( C log P ). This is done by assigning values to different
fragments in the chemical structure [41] . The calculated values are only estimates,
but they are useful to rank order a homologous series of compounds based
on their lipophilicity for further lead optimization. Given the fact that drugs have
to cross many biological membranes before reaching the site of action, the log P
value has a signifi cant infl uence on drug absorption, drug pharmacokinetics, and
pharmacology. This is exemplifi ed from the numerous structure – activity and structure
– property relationships using log P [41] . The log P is important in assessing the
oral absorption potential of a drug. If a drug has a low log P ( < 1), it is expected to
have poor membrane permeability, while if a drug has a large log P ( > 5), it will be
trapped in the lipophilic membrane. A log P of 1 – 5 is usually considered optimal
for oral drug absorption [3] . For an ionic drug, the un - ionized form of the drug will
be more lipophilic than the ionized form of the drug. Therefore, at any given pH in
vivo, the relative proportion of ionized versus un - ionized form of the drug dictates
drug dissolution and absorption through the membrane.
6.2.3.3 Drug Dissolution
Dissolution is the rate at which a solid drug goes into solution and is a critical
determinant in the absorption of drugs from solid dosage forms. A drug has to go
into solution before it can be absorbed. In vitro dissolution studies are a valuable
(15)
958 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
tool for determining the infl uence of different solid - state properties on drug dissolution
and vis - a - vis predict in vivo drug dissolution and absorption. It is used to screen
drugs that show dissolution rate – limited absorption. The factors that infl uence drug
dissolution rate ( dc / dt ) can be understood from the well - established Noyes – Whitney
equation [42]
dC
dt
kSC
V
= s
(17)
where k is the dissolution rate constant (cm/s), C s is the saturated solubility of the
drug, S is surface area of solid exposed to the solvent, and V is the volume of the
dissolution medium. The dissolution rate constant is a function of the diffusion coef-
fi cient D of the drug through a stagnant aqueous layer adjacent to the dissolving
surface and thickness h . Powder or particulate dissolution is carried out in a dissolution
vessel in a specifi c volume of dissolution fl uid (900 mL) which is stirred and
maintained at 37 ° C. Several confi gurations are available to study the dissolution of
various dosage forms (Figures 12 a and 12 b ). Various simulated physiological media
are used to understand the in vivo behavior of drug and dosage forms [43] . The
usual dissolution media include water, 0.1 N HCl, and pH 7.4 buffers. It is important
to maintain sink conditions in the dissolution medium by keeping the drug concentration
at 10% of saturated solubility of drug (to mimic in vivo). For poorly water
soluble drugs, surfactants are often added to the dissolution medium for this purpose.
During preformulation testing, particulate dissolution studies reveal the infl uence
FIGURE 12 Various drug dissolution methodologies. ( a ) In the paddle method, the tablet
is placed in the dissolution vessel containing dissolution medium and the paddle is rotated
at defi ned rpm, while the dissolution vessel is maintained at body temperature. ( b ) In the
basket method, the tablet is kept inside a meshed basket and rotated. ( c ) For IDR studies,
the tablet is kept inside a die cavity and only one face of the tablet is exposed to the dissolution
medium.
(a) (b) (c)
Tablet
Tablet
Tablet
SOLUBILITY CHARACTERISTICS 959
of particle size, crystal habit, and wettability of a drug substance. A formulator, on
the other hand, routinely uses dissolution testing as a quality control tool in the
design of dosage forms. Various models have been developed to describe the release
kinetics of conventional and modifi ed release dosage forms [44] .
Since powder dissolution is infl uenced by changing surface area, it is not useful
for delineating the effects of polymorphs, hydrates, and pharmaceutical salts. Instead,
the intrinsic dissolution rate (IDR) is used. The IDR studies are conducted at a
constant surface area and hence the dissolution rate observed is only a function of
the intrinsic solubility of the drug. The Noyes – Whitney equation is modifi ed for
IDR, where the surface area is kept constant, and Equation (17) reduces to
d
d
k C
C
t s = 1
(18)
where k 1 is the intrinsic dissolution rate constant and dC / dt is the intrinsic dissolution
rate (mg · cm 2 /s). For IDR studies, the drug (500 mg) is compressed in a hydraulic
press (at 500 mPa) to a 13 - mm disc. This disc is then loaded onto a holder which
exposes only one surface of the disc to the dissolution medium (Figure 12 c ). The
IDR is obtained by dividing the slope of the plot between the amount of drug dissolved
and time by the area of solid exposed to the dissolution medium. The IDR
predicts the infl uence of drug solubility on in vivo drug dissolution and absorption.
A drug which has an IDR of 1 mg · cm 2 /s will not generally show dissolution rate –
limited absorption in vivo. However, if the IDR falls between 0.1 and 1 mg · cm 2 /s,
then further studies may be required to make a decision. Drugs with IDR <
0.1 mg · cm 2 /s show dissolution rate – limited absorption in vivo, necessitating drug
solubilization strategies [45] . With respect to pharmaceutical salts, IDR is used to
understand the infl uence of surface pH on drug dissolution and absorption. The
common - ion effect can be studied by including 0.1 – 0.15 M NaCl in the dissolution
medium. Also IDR is useful to understand the difference in solubility of polymorph
and amorphous forms. However, in some cases, the compression force used in
making the IDR disk may by itself induce solid - state transformations [45] . DSC, IR,
and XRD must be used to identify the drug ’ s solid state before and after compression
as well as at the end of the dissolution studies. A well - designed dissolution study
is used as an early warning for drug molecules that would pose absorption problems
in vivo.
6.2.3.4 Absorption Potential
The ultimate goal of any drug development program is to develop an orally absorbable
compound. Solubility and permeability are the two most critical parameters
that dictate oral absorbability of a molecule. All other parameters are directly or
indirectly related to these two physicochemical properties. As can be seen from
Figure 13 , there are several barriers that a drug needs to overcome before reaching
the systemic circulation. The oral absorption of a drug is mainly limited by drug
dissolution and/or by the drug permeation across the GI membrane. Considering
their importance, drug solubility and permeability are screened very early in the
drug discovery process. Solubility studies are typically run in a high - throughput
format using a turbidometric method as discussed earlier. Based on this primary
960 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
screen, detailed solubility and dissolution studies are carried out as the compound
moves through different development phases (Figure 14 a ). A usual target for drug
solubility during lead selection is 1 mg/mL for avoiding dissolution rate – limited
absorption [46] .
Drug permeability is commonly screened in the early discovery phase using
CaCo - 2 cell lines, which is a human colon carcinoma cell line. Using a 96 - well format,
the cell lines are used as a primary screen to rank order compounds based on permeability
[4] . If designed properly, the cell culture studies can be used to understand
the drug absorption mechanisms [47] . To avoid permeability - limited absorption in
vivo, a drug should have a permeability coeffi cient of 2 . 10 . 4 – 4 . 10 . 4 cm/s [48] .
Inputs from the drug metabolism team (based on liver microsomal studies) can give
clues on the drug ’ s susceptibility to fi rst - pass metabolism. Following cell culture
studies, a select group of compounds are studied using isolated rat intestine and
segmental absorption studies to understand the drug absorption mechanism and the
site of drug absorption in the GIT (Figure 15 b and 15 a ). This is further substantiated
using in situ perfusion experiments in rats [49] . The details of the studies are
depicted in Figure 15 b . Some of these compounds are studied in whole animals of
which a few may make it to clinical trials in humans.
FIGURE 13 Barriers to drug absorption. Drug from the dosage form should be soluble and
dissolve in GI fl uids before it can be absorbed. Drug dissolution is one of the major rate -
limiting steps in drug absorption. Drug absorption may be affected if the drug is unstable in
GI fl uids. Furthermore, drug absorption will also depend on how long the drug resides in a
particular region of the GIT. The drug has to diffuse through the highly viscous mucous layer
before getting absorbed through the membrane. Membrane permeability is one of major the
rate - limiting steps in absorption. After absorption the drug may be subject to fi rst - pass
metabolism (FPM) in the liver before reaching the systemic circulation. Dark arrows indicate
that solubility and permeability are the most infl uential factors.
Permeability
FPM
Systemic
circulation
GI
membrane
GI transit time
Dosage form Drug in
solution
Solubility and
dissolution
Drug stability
Mucous layer
SOLUBILITY CHARACTERISTICS 961
FIGURE 14 Different methods to determine solubility and permeability during various
stages of drug discovery and development. ( a ) In the initial stages the drugs are screened
using kinetic solubility studies, which are later followed by equilibrium solubility studies, pH
solubility profi le, and dissolution studies during development phase. ( b ) Drug permeability
is initially screened using CaCo - 2 cell lines followed by rat intestinal studies. This is followed
by pharmacokinetic studies in animals and fi nally the potential drug molecules are tested in
humans in clinical trials.
(a) (b)
Drug discovery and development
Human studies
Whole-animal studies
Isolated rat
intestine and In Situ
perfusion in rats
CaCo-2
cells
Kinetic
solubility
Equilibrium
solubility
pH solubility profile
Dissolution studies
Thus, the preformulation team in coordination with other discovery teams gets
useful estimates on in vivo drug absorption. In addition to drug solubility and permeability,
it is also important to consider the anticipated dose while assessing the
absorption potential. A useful tool to optimize the drug ’ s physicochemical properties
is the maximum absorbed dose (MAD) model [46] . The model predicts the dose
that would be absorbed based on the drug ’ s solubility ( C s ; solubility in intestinal pH
of 6.5), absorption rate constant ( k a ; usually obtained from rat permeability studies),
physiological factors such as gastric transit time ( T i ; approximated as 4.5 h), and
intestinal fl uid volume ( V int ; approximated as 250 mL):
MAD s a i =C k V T int (19)
Using this model, the required solubility or permeability can be estimated for a
given dose of the drug. For example, a drug with an anticipated human dose of
1 mg/kg (70 mg for a normal 70 - kg subject) will require a solubility of 0.05 mg/mL
provided the drug shows good permeability. Similarly, the absorption rate required
to achieve the same dose for a drug with good solubility (1 mg/mL) is 0.001 min . 1 .
The MAD model is helpful in guiding development teams on optimizing drug solubility
and/or permeability.
A further refi nement of this model led to the evolution of the biopharmaceutics
classifi cation system (BCS), which classifi es the drugs into four classes depending
on their solubility and permeability (Figure 16 ). The BCS is applicable only to the
oral route of administration, and according to this classifi cation, a drug is considered
to be highly soluble if the highest dose of the drug is soluble in a glass of water
(250 mL) covering the entire pH range in GIT from 1 to 7.5, and a drug is considered
to be highly permeable if the drug has more than 90% oral bioavailability [48] . The
model has been developed based on the solubility and permeability characteristics
962 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
of marketed drugs. However, BCS is bound to become an important biopharmaceutical
tool in lead optimization in drug discovery setting and also at the same time
serving as a useful guide to develop new formulations in a generic setting. From a
regulatory perspective, BCS provides a scientifi c basis to grant biowaivers. As per
BCS, the dissolution test can serve as a surrogate tool for costly and time - intensive
bioequivalence studies for generic drugs which are highly soluble. This requires the
establishment of a good in vitro – in vivo correlation. At present, the FDA grants
biowaivers to generic manufacturers of immediate - release dosage forms for drugs
in class I (highly soluble and highly permeable) provided they can prove the “ dissolution
equivalence ” of their product to that of the innovator ’ s drug product [50] .
FIGURE 15 Intestinal permeability studies. ( a ) Everted rat intestine is used to study the
mechanism of drug absorption. Isolated rat intestine is fi lled with drug solution and tied at
both ends and the drug permeation into the external medium is measured (left). In another
set of experiment, the rat intestine is everted with the internal mucosal membrane facing
outside (right) and the serosal side facing inward. If the drug is passively absorbed, then there
would not be any differences in permeability in these two experiments. If the drug is transported
by carriers, then drug permeation would be seen only in the fi rst experiment, as the
carriers are present only on the mucosal side. ( b ) In situ intestinal perfusion studies are conducted
in an anesthetized animal and the drug solution is pumped through a tube and drug
coming out on the other side of the intestine is measured. The drug is also measured by
sampling from the jugular vein in the animal. This is useful to measure the dynamic drug
absorption into systemic circulation.
(b)
(a)
Passive
transport
Drug solution
Carriermediated
transport
pH 7.4 buffer
Passive
transport
Drug solution
pH 7.4 buffer
Rat intestine
Drug out
Drug in
SOLUBILITY CHARACTERISTICS 963
The dissolution equivalence is tested using the statistical dissolution model, termed
f 2 or similarity factor, and is described by the equation
f
N
R T t t
t n
2
2
1
0 5
50 1
1
100 = +( ) + ...
...
. ...
...
= .
.
. log ( )
.
(20)
where N is the number of dosage form units (12 units are tested), t is dissolution
time points from 1 to n, R t is the percent drug dissolved for the reference drug
product, and T t is the percent drug dissolved for the test product. Two dissolution
profi les are considered similar, if f 2 . 50 and if the coeffi cient of variation does not
exceed 20% at early dissolution time points (usually 10 min) and 10% at other time
points in the pH range 1 – 7.5. However, if . 85% of drug is dissolved in . 15 min, then
no comparison is required and the dissolution is based on a single time point determination
[50] .
The BCS paradigm can be used to develop strategies for enhancing drug solubility
and/or permeability (Tables 8 and 9 ). Solubility enhancement may involve only
FIGURE 16 Biopharmaceutics classifi cation system. The drugs are classifi ed based on drug
solubility and drug permeability. A drug is considered to be highly soluble if the highest dose
of the drug is soluble in 250 mL of water varying in pH from 1 to 7.8 (GIT pH range). A drug
is considered highly permeable if more than 90% of the drug is bioavailable by oral route.
Class I drugs are highly soluble and highly permeable, class II drugs are poorly soluble but
highly permeable, class III drugs are highly soluble but poorly permeable, and class IV drugs
have poor solubility and permeability.
e.g., Metronidazole, propranolol
Class I Class II
Class III Class IV
e.g., Amhotericin B, taxol e.g., Atenolol, cimetidine
e.g., Ibuprofen, griseofulvin
Permeability Low
Low
High
High Solubility
964 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
TABLE 8 Solubility Enhancement Methods
Technique Principle
Salt formation Increases drug solubility by keeping the pH at which the drug is ionized.
Particle size
reduction
Increase in surface area increases drug solubility. Particle size is reduced
to nanodimensions (nanosuspensions) for increasing drug solubility.
Change of
form
Crystalline drugs are converted to amorphous forms which show higher
solubility than crystalline forms of the drug.
Cosolvents Various water miscible cosolvents are used to increase the water solubility
of drug. The cosolvents are selected based on the polarity of the drug.
Common cosolvents include glycerol, ethanol, and propylene glycol.
Complexation Drug is entrapped or complexed with excipients that can mask the
lipophilic groups of the drug and enhance drug solubility in water.
Cyclodextrins are commonly used to entrap hydrophobic drugs in the
core, while the hydrophilic groups on the periphery help to solubilize
the drug.
Surfactants Surfactants are characterized by the presence of lipophilic and hydrophilic
groups and form spherical structures known as micelles in water at
a certain concentration. The hydrophobic drug is entrapped in the
hydrophobic core of the micelle while the hydrophilic groups on the
periphery help to solubilize the drug.
Disperse
systems
The hydrophobic drug is dissolved in an organic solvent and in addition
may also contain an emulsifi er. On contact with the intestinal fl uids,
the drug is emulsifi ed (microemulsions) by bile salts and is absorbed
through the intestine.
TABLE 9 Permeability Enhancement Methods
Method Mechanism
Transcellular
transport
Sorption promoters can be used to enhance the transcellular transport
in the intestine, including bile salts and fatty acid esters. They tend to
fl uidize the lipid bilayer and enhance drug permeation across the
membrane.
Paracellular
transport
Enhancement is achieved by modulating the tight junctions between the
cells. Chelating agents such as ethylenediamine tetraacetic acid can
chelate calcium ions and transiently open the tight junctions for drug
transport.
Carrier -
mediated
transport
Nutrient transport carriers are utilized for drug transport. Prodrugs are
designed to meet the structural requirements for carrier - mediated
transport.
Blocking
effl ux
pump
P - glycoprotein is a major effl ux mechanism that pumps out drug from the
intestinal cells back into the intestinal fl uid. Several drugs and food
substances are known to inhibit p - glycoprotein and enhance drug
permeation.
a physical intervention, as opposed to molecular modifi cation for permeability
enhancement. Generally [46] , the permeability range of drugs varies by only 50 - fold
(0.001 – 0.5 min . 1 ) in comparison to drug solubility, which varies by six orders of
magnitude (0.1 . g/mL – 100 mg/mL). Hence, the formulator has greater fl exibility in
altering the drug ’ s solubility in comparison to altering the drug ’ s permeability. This
is evident form the fact [51] that the majority of the marketed drugs are highly
soluble ( > 55% in classes I and III). Sometimes, enhancing the permeability by altering
the drug ’ s chemical structure may be counterproductive. This because of the
associated increase in molecular weight that attenuates the permeability enhancement
gained with structural modifi cation. On the other hand, optimization of drug
solubility may be more fruitful if the poor permeability is overcome by increasing
drug solubility to provide a high drug concentration at the absorption site. However,
this may be a diffi cult strategy if the dose is very high. The preformulation team
should use the “ appropriate tools ” at every stage of the drug discovery and development
process to guide or alert other development teams about drug solubility and
permeability issues (Figure 17 ).
6.2.4 STABILITY CHARACTERISTICS
Drug stability is an essential component of preformulation testing. Establishing
the stability of the drug under a variety of conditions is an expensive and time -
consuming process. It cannot be overemphasized that the availability of a stability -
indicating assay is the key to stability studies. The preformulation scientist works
closely with the analytical method development team in developing a stability -
indicating assay. During the early stages, a foolproof stability - indicating assay may
FIGURE 17 Flow chart for determining absorption potential of a drug during drug discovery
and development. Solubility and permeability studies from preclinical phase are used to
calculate the maximum absorbable dose and, when correlated to BCS, this can provide information
on its biopharmaceutics class. This is useful to estimate if the drug absorption would
be dissolution and/or permeability limited for developing appropriate drug delivery
strategies.
Early drug discovery
Exploratory studies
Solubility/permeability
Maximum absorbable dose
BCS classification
Ora ldosage form
development
Clinical development
Drug development
STABILITY CHARACTERISTICS 965
966 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
not be available. This is understandable considering the fact that the initial drug lots
are not pure and the purity is improved as the molecule progresses to subsequent
development stages. Therefore, the intention of the preformulation scientist is not
to generate a thorough kinetic rate profi le of the drug but to broadly defi ne the
conditions under which the drug would be stable. Only relevant stability data are
generated during various phases for developing preclinical and clinical formulations
[52] . The stability data are evaluated and conveyed to the formulation development
team upfront if stabilization and additional packaging requirements are needed.
Often the chemical structure of the drug can give clues on the drug ’ s degradation
pathway and its stability characteristics [53] . Table 10 lists some of the functional
groups that are susceptible to common degradation pathways.
Stability test conditions are chosen keeping in mind the environment that the
drug will encounter during drug development, processing, storage, and use (Table
11 ). One parameter is studied at a time while keeping all other parameters constant.
Apart from classical stability studies, such as hydrolysis, oxidation, and photolysis,
the preformulation scientist has to determine the stability of drugs in unconventional
matrices such as animal feed used for toxicological studies [34] . The stability
of a drug in the animal feed is complicated by the feed composition, including
enzymes and vitamins among others. The moisture content in the feed may also vary
with storage temperature. In such cases, it is appropriate to study the stability of the
drug under the storage conditions encountered in the toxicological laboratory. Sensitive
techniques such as liquid chromatography/mass spectrometry (LC/MS) are
used to evaluate drug stability in such complex mixtures.
In general, most of the drugs undergo fi rst - order degradation, while some drugs
may follow zero - order kinetics and only a few drugs undergo second - order degradation
[34] . The fi rst - and zero - order reactions are readily differentiated by studying
drug stability at two different initial drug concentrations. First - order kinetics
will depend on initial drug concentration, while a zero - order reaction will be
TABLE 10 Groups Susceptible to Common Degradation Pathways
Degradation
Pathway Functional Groups
Hydrolysis Esters, lactones, amides, lactams, oximes, imides, and malonic ureas
Oxidation Amines, sulfi des, disulfi des, sulfoxides, phenol anions, thiols, nitriles, and
catechols
Photolysis Aromatic hydrocarbons, aromatic heterocyclics, aldehydes, and ketones
TABLE 11 Stability Testing Conditions
Parameters Conditions
Temperature 5, 25, 30, 37, 40, and 60 ° C
Moisture 30, 45, 60, 75, and 90% RH
pH 1, 3, 5, 7, and 9 at room and body temperature
Oxygen Sparging with 40% oxygen or adding 100 – 200 ppm of hydrogen peroxide
Light 1.2 million lux hours of exposure to visible light and 200 h/m 2 exposure to
UV light (360 – 400 nm)
independent of initial drug concentration. Accelerated stability studies are conducted
to expedite the degradative reactions, where temperature is the commonly
used accelerant. The infl uence of temperature on drug stability kinetics is described
by the Arrhenius equation:
k Ae E RT = . a/ (21)
where k is a reaction rate constant, A is a frequency factor, E a is activation energy,
R is the gas constant, and T is absolute temperature. According to the Arrhenius
equation, every 10 ° C rise in temperature increases the reaction rate by two - to fi vefold
[31] . The usual temperatures selected for early stability studies include 5, 25, 37,
40, and 60 ° C to cover the temperatures encountered in processing, use, and storage
of the drug product. Using the Arrhenius equation, the rate constant from higher
temperatures can be extrapolated to determine the stability at room temperature
[31] . The slope of the plot of the reciprocal of temperature and the rate constant
gives the activation energy. The activation energy usually varies between 15 and
60 kcal/mol with a mean value of 19.8 kcal/mol [33] . A break in the line is usually
indicative of change in the activation energy due either to change in the reaction
order or the mechanism of degradation at higher temperature. In such cases, it
becomes imperative to conduct detailed studies to understand the drug degradation
mechanism. Some of the reactions seen at higher temperature may not be representative
of the reactions at room temperature. Hence, short - term high - temperature
studies should be supplemented with long - term real - time stability testing at room
temperature. Additionally, the drug is also exposed to moisture, oxygen, and UV light
(250 – 360 nm). The conditions used for stress studies may vary depending on the drug
type and the drug development stage [54] . The stability studies in this chapter are
discussed with respect to a solid dosage form which includes solid - state stability,
limited liquid state stability, and drug – excipient compatibility.
6.2.4.1 Solid - State Stability
In general, solid - state reactions are slow, complex, and at times diffi cult to quantify.
They may manifest as either physical and/or chemical instabilities. Physical instabilities
include solid – solid transformations, desolvation of hydrates, and change in color
[34] . On the other hand, chemical instabilities may involve a change in drug content
as a result of hydrolysis, oxidation, or light - induced degradations [32] . The infl uence
of temperature is studied by exposing the solid drug to increasing temperatures and
also exposing the drug to various relative humidities at room temperature for two
to eight weeks (Table 11 ). If substantial change is observed at higher temperatures,
the drug samples stored at 5 ° C are analyzed. If no degradation is seen at higher
temperature, then none can be expected at room temperature. The results from
higher temperature should be carefully interpreted. For instance, a hydrate may lose
moisture at higher temperatures and make a drug unstable which otherwise would
be stable at room temperature. Similarly, chlortetracycline hydrochloride converts
from the . form to the . form at above 65% relative humidity, in contrast to < 65%
relative humidity, where no transformations are observed [32] .
Oxidative degradation is studied by exposing the sample to an atmosphere of
40%. The oxygen is combined with heat to accelerate the reaction. The results
STABILITY CHARACTERISTICS 967
968 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
should be compared with samples stored in an inert atmosphere. Photolytic degradation
is studied by exposing the sample to UV light (254 and 360 nm) for two to
four weeks and observed for color fading and/or darkening [33] . Color fading may
not always mean drug decomposition. It may be just a physical change which can
be overcome by including dyes in the formulation. As indicated earlier, the solid -
state reactions may be slow, sometimes generating only 1 – 5% of degradation products,
which may be below the detection limit of HPLC [34] . A combination of
qualitative and quantitative analytical tools is helpful to detect drug degradation.
Nevertheless, accelerated stability studies provide an early warning of potential drug
degradation and the preformulation scientist should use discretion in interpreting
and sharing the results with other development teams.
6.2.4.2 Solution - State Stability
Detailed solution - state stability is of limited value if the fi nal anticipated dosage
form is a solid. However, solution - state stability studies can predict the stability of
the drug in the granulating fl uid and the GI fl uids. Solution - state reactions are
faster than the solid - state reactions. This is helpful in generating degradation products
through so - called forced - degradation studies for toxicological screening and
analytical method development. The studies are conducted by exposing the drug
to extreme conditions such as 0.1 N HCl, 0.1 N NaOH, and water at 90 ° C [34] .
Forced - degradation studies are useful to qualify the safety of the degradation
products if it exceeds 0.1 or 0.05% of total daily dose for drugs with < 1 g or > 1 g
dose/day, respectively [6] .
The pH rate profi le is an important parameter that is studied in the solution state.
In early preformulation studies, an approximate pH rate profi le is generated, usually
including the pH encountered in salt selection and in vivo [55] . The studies are later
followed with a detailed pH profi le in the whole pH range of 1 – 10. A typical pH
rate profi le is shown in Figure 18 , which is useful to extract useful information on
FIGURE 18 Representative pH kinetic rate profi le. The drug shows a minimal degradation
at around 2 – 4 and the degradation rate is high in alkaline pH.
pH
2 4 6 8 10
K (day–1)
drug stability. The profi le is used to predict if the drug degradation is catalyzed by
hydronium or hydroxyl species (specifi c acid – base catalysis). The minimal point in
the profi le is indicative of the pH at which the drug is relatively stable. However,
the buffer used in the study can by itself accelerate the reaction, which is referred
to as general acid – base catalysis [39] . The infl uence of buffer is nullifi ed by conducting
the study at one to three buffer concentrations and then plotting the rate constant
against the buffer concentration. The intercept on the y axis gives the rate
constant at zero buffer concentration.
Oxidation reactions are studied by passing oxygen in the head space of the drug
solution and comparing the drug degradation with a drug solution (Table 11 ) fi lled
with an inert gas in the head space. The reduced solubility of oxygen at higher temperatures
may lead to an apparently reduced reaction rate in comparison to lower
temperature [34] . Light sensitivity is studied by exposing the drug solution in a
clear - fl int bottle to UV radiation and comparing the results with the drug solution
in an amber - colored container [55] .
The Solution - state studies should be extrapolated to the solid state with caution.
The reaction in the solution state is usually done in a dilute drug solution, in contrast
to reactions in the solid state, where the saturated drug solution at the surface
undergoes a multiphase reaction [33] . Moreover, the reaction order may be different
in the solution and solid states. Due to the excess solvent, reactions in the
solution state are usually pseudo – fi rst order as opposed to fi rst - order or zero - order
reactions in the solid state. In spite of these limitations, solution - state studies
provide clues in selecting appropriate granulating solvent and in predicting in vivo
drug stability. The pH – rate profi le data are also useful to predict the solid - state
stability of salt forms or the stability of a drug in the presence of acidic and basic
excipients [55] . Further, the pH – rate studies predict the stability of drug and its salt
in the gastrointestinal pH. This is illustrated by the example of erythromycin and
its salts [32] . Erythromycin is rapidly inactivated in the acidic environment of the
stomach. This is overcome by using insoluble erythromycin estolate, which is stable
in the gastric pH, unlike the other salts, which are easily displaced by hydrochloric
acid in the stomach.
6.2.4.3 Drug – Excipient Compatibility
Excipients are the backbone of a solid dosage form, and they function as diluents,
binders, disintegrants, and fi llers. The excipients are in intimate contact with the
drug in a solid dosage form, therefore necessating the need for drug – excipient
incompatibility testing. Since the formulator has a wide selection of excipients to
choose from, it would be a daunting task for the preformulation scientist to screen
all possible excipients. A general practice is to select those excipients that are
routinely used in in - house manufacturing of dosage forms. At least two are selected
from each class of functional excipients. Excipients that are known to cause potential
incompatibilities (e.g., glucose and amines) with drugs are excluded from the
study [34] . The excipients are intimately mixed with the drug in the ratio that is
realistic for the desired solid dosage form. For example, diluents are mixed in the
ratio 20 : 1, while other excipients are used in the ratio 1 : 5 with drug [32] . The
drug – excipient mixtures are then stored in a tightly sealed container (ampoule or
in a bottle, where the cap is sealed with wax) in the presence and absence of 5%
STABILITY CHARACTERISTICS 969
970 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
w/v moisture. The mixture is stored at 40 and 25 ° C and analyzed for three months.
The mixture is physically observed for caking, liquefaction, and gas formation and
chemically analyzed for drug degradation. A variety of techniques are used for
studying drug – excipient compatibility. DSC is a useful primary screen which can
rapidly detect potential drug – excipient incompatibilities [33] . For DSC studies, the
drug and excipient are mixed together without moisture and are analyzed within
a few hours of preparation. Moisture is avoided, as it complicates the interpretation
of thermograms. If there is no interaction, then the thermogram should be the
sum of thermogram of drug and excipient. The thermogram is observed for peak
position, peak appearance, peak shape, appearance of new peaks, disappearance
of drug peaks, peak shift, and change in enthalpy values. It is obvious that a mixture
of two substances will result in depression of melting point, but if the change is
signifi cant, then it may be indicative of eutectic formation. Some of the reactions
seen at high temperatures in DSC may not be refl ective of room temperature.
Therefore, excipients that are suspected to show incompatibilities are screened
further through stability studies using TLC and HPLC (Figure 19 ). TLC is used to
qualitatively detect any new spots with the drug – excipient mixture. The spots
should be compared with the control drug sample. This is important since in the
early discovery stage the drug is not pure and contains impurities. The fi ndings
from TLC should be corroborated using HPLC. The studies may be further followed
- up with stability studies using multicomponent mixtures. In addition, the
drug and excipient may be tested by compressing in a hydraulic press or fi lling in
a capsule to simulate the actual dosage form [32] . A well - designed drug – excipient
preformulation study can thus help the formulator in judicious choice of excipients
for the fi nal dosage form.
FIGURE 19 Flow chart for studying drug – excipient compatibility.
Use in formulation development
Mix in suitable ratio
Excipient Drug
Screen
for interactions
using DSC
Interaction
detected
Short-term stability
Long-term stability
Characterize
interaction
by TLC and HPLC
Yes
No
Excipient
excluded
Interaction
confirmed
6.2.5 CONCLUSIONS
Preformulation testing has a signifi cant role in a drug discovery and development
program, as it provides valuable feedback to the various discovery and development
teams in enabling druggability during lead identifi cation and optimization.
The preformulation data may mean different things to different groups in the discovery/
development phase (Figure 20 ). For the chemistry team, the feedback from
preformulation testing provides clues to optimize the chemical structure with
respect to solubility, permeability, and stability. Preformulation studies give inputs
to the biology group for ensuring optimal drug exposure based on solubility, permeability,
and stability data, in addition to developing preclinical formulations for
phar macokinetic and toxicological studies. The analytical team gets inputs from the
preformulation group on developing stability - indicating assays and setting drug
specifi cations. Once the lead is selected and as the molecule moves to the development
phase, the preformulation group provides guidance to the bulk manufacturing,
formulation, and clinical evaluation teams. The bulk manufacturing team uses
the data generated from the preformulation studies on salts, polymorphic purity,
and particle size specifi cations. It is the formulation team that utilizes the maximum
data from the preformulation testing to design an appropriate dosage form. The
physicochemical properties are utilized for improving the drug ’ s solubility, improving
the drug ’ s permeability, developing stabilization strategies, selecting appropriate
excipients, selecting processing conditions to design, and evaluating the fi nal dosage
form. The clinical evaluation team utilizes the preformulation data along with the
preclinical animal studies to understand the drug ’ s pharmacokinetics in humans
through the MAD and BCS paradigms. Therefore, a strong preformulation team in
a drug discovery setting is critical for optimizing the pharmaceutical properties of
the drug. This can signifi cantly reduce the attrition rate, time, and cost of discovering
a new drug. On the other hand, the preformulation team in a generic setting is
valuable to optimize or further enhance the effi cacy of an existing drug by designing
a new drug delivery system and thus giving a new life to an old drug. As opposed
FIGURE 20 Role of preformulation in supporting other discovery and development
teams.
Manufacturing
Drug discovery
Biology
Chemistry
Preformulation
Stability-indicating assay
pH rate profile
Impurity profile
Solid-state stability
Salt selectionPolymorphism, amorphous/crystallineHydrates/solvates, log P
Preclinical formulation
Solubility
log P, permeability
Analytical development
Polymorphic purity
Hydrate/anhydrate interconversions
Particle size specifications
Preformulation
Clinical
Formulation
Drug development
BCS
MAD
Solubility enhancement, permeability enhancementExcipient selectionProcessing conditions
CONCLUSIONS 971
972 ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS
to discovering a new drug, a new drug delivery system can be designed at one - third
of the cost and money involved in a new drug discovery process [56] . The availability
of high - throughput property screens and predictive models is expected to
improve the effi ciency and maximize the output of preformulation data in the
coming years. However, the main challenge lies in intelligent use of such tools to
make sense from loads of data and communicate the appropriate information to
the relevant discovery/development groups for getting the drug to the marketplace
in time.
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977
6.3
TABLET DESIGN
Eddy Castellanos Gil , 1,2,3 Isidoro Caraballo , 2 and
Bernard Bataille 3
1 Center of Pharmaceutical Chemistry and University of Havana, Havana, Cuba
2 University of Sevilla, Seville, Spain
3 University of Montpellier 1, Montpellier, France
Contents
6.3.1 Introduction
6.3.2 Tableting Cycle
6.3.3 Limitations for Direct Compression
6.3.4 Previous Granulation: Biopharmaceutical Versus Technological Properties
6.3.5 Tablet Design for Matrix System
6.3.5.1 Controlled - Release Tablet by Direct Compression and Wet Granulation
6.3.6 Tablet Design with Natural Products
6.3.6.1 Tablet Design from Aqueous Plant Extract
6.3.6.2 Natural Product as Vehicle for Manufactured Tablets
6.3.6.3 Natural Product as Vehicle for Controlled - Release System
6.3.6.4 Mechanism of Soluble Principle Active Propranolol Hydrochloride and
Lobenzarit Disodium from Dextran Tablets
6.3.7 Design Tools of Tablet Formulation
6.3.7.1 MODDE 4.0
6.3.7.2 iTAB
6.3.7.3 Percolation Theory
6.3.7.4 Artifi cial Neural Networks
6.3.8 Coating Systems
6.3.8.1 Subcoating of Tablet Cores as a Barrier to Water
6.3.8.2 Kollidon VA 64
6.3.8.3 SEPIFILM
6.3.9 Development of Pharmaceutical Tablets Using Percolation Theory
6.3.9.1 Case Study: Optimization of Inert Matrix Tablets for Controlled Release of
Dextromethorphan Hydrobromide
6.3.9.2 Critical Points of Hydrophilic Matrix Tablets
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
978 TABLET DESIGN
6.3.9.3 Case Study: Estimation of Percolation Thresholds in Acyclovir Hydrophilic
Matrix Tablets
6.3.10 Ultrasound - Assisted Tableting (a New Perspective)
References
6.3.1 INTRODUCTION
The compression of powders — the fourth state of matter in the words of Hans
Leuenberger — is a complex task due to the variability in particle size and shape,
even for particles of the same component, the unknown distribution of the particles
in the die, their different ability to fl ow, and the forces needed to create bonds
between them.
Science - based formulation is nowadays a good strategy for the development of
a pharmaceutical formulation. The new regulatory environment (process analytical
technology) will transform science - based formulation in the next years.
Therefore, there is a need for statistical tools able to predict the behavior of a
powder mixture when it is subjected to compression forces. The purpose of this
chapter is to provide a brief description of the main theoretical aspects of tablet
design and formulation together with practical examples of tablet development and
characterization using different techniques.
6.3.2 TABLETING CYCLE
Compressing powder or granule into a tablet is one of the simplest and oldest
ways of forming a product known to humans. Nowadays, as technology has
advanced, more complex machines are used with different procedures, but the basic
principle, the tabletting cycle, remains the same. In tablet design many factors have
to be taken into account, such as the physicolchemical properties of active compound
and excipients. An important role also has to be attributed to tableting
machines.
Tablet press subclasses primarily are distinguished from one another by how the
powder blend is delivered to the die cavity. Tablet presses can deliver powders
without mechanical assistance (gravity) (e.g., Ronchii, Manesty, Stokes, and Colton
machines), with mechanical assistance (power assisted) (e.g., Ronchii, Courtoy,
Kilian, Manesty, Kikusui, Fette, and Hata machines), by rotational forces (centrifugal)
(e.g., the Comprima machine), and in two different locations where a tablet
core is formed and subsequently an outer layer of coating material is applied (compression
coating) (e.g., Kilian Manesty, and Kikusui machines).
The basic unit of any tablet press is tooling consisting of two punches and a die,
called a station. The upper and lower punches come together in the die that contains
the tablet formulation. Principally, two different types of machines are used, the
eccentric and the rotary press. The eccentric press produces about 50 – 130 tablets
per minute. The rotary press has a multiplicity of stations arranged on a rotating
table with the dies. A few or many thousands of tablets can be produced per minute.
There are numerous models of presses, manufactured by a number of companies,
ranging in size, speed, and capacity.
The eccentric press is widely used early in the development stage, because the
tabletting machine and the tooling are inexpensive, it can be easily instrumented,
little material is needed, and setting, servicing, cleaning, and tool changeover of the
machine are easy . During the manufacturing process the tablet mixture is dosed by
a hopper into the die. The position of the lower punch defi nes the volume of the
subsequent tablet mass. The compression force is given by the position of the upper
punch, which defi nes its immersion depth into the die, and the reagent force that is
built up during the densifi cation of the material. The compressed tablet is ejected
by the lower punch.
During the compression process on an eccentric press, there are other pressure
ratios at the upper and lower punches. The pressure at the upper punch is usually
higher than the pressure at the lower punch. A part of the pressure is lost in the
material and in the resulting radial friction force against the die wall during the
compression [1, 2] .
Figure 1 shows tablets (300 mg) of native dextran obtained from Leuconostoc
mesenteroides B - 512F (Sigma) with a 10% water (w/w) punch in an eccentric
machine (Manesty) at 15 kN with tablet side (fl at - fl ace diameter) 10 mm. Axial displacement
of water was observed according to the change in color.
On a rotary press the fi lling of the die and the following compression process is
done at the same time at different stations. The compression is carried out in the
simplest case with two rolls touching the upper and lower punches and compressing
the powder mixture. In contrast to the eccentric press, the upper and lower punches
exert pressure on the tablet mixture from both sides at the same time.
Tablets compressed on a rotary press generally show a more consistent hardness
when the upper and lower sides of the tablets are compared, where the upper
side of tablets compressed on an eccentric press is usually harder than the lower
side [3] .
Figure 2 illustrates the difference in compression profi les of the upper and lower
punches and the punch movement with fi ctitious rotary and eccentric presses.
FIGURE 1 Native dextran tablet press in eccentric machine shows variability of color due
to different pressure between upper and lower punches.
TABLETING CYCLE 979
980 TABLET DESIGN
Another reason, to use a rotary press rather than an eccentric press for tablet compression
is the dwell time is usually shorter on a rotary press (see Figure 3 ).
All compression mixes have an optimum compressing speed. This is why tablet
press manufacturers install variable speed controls for the rotor or turret and the
powder feeding mechanism. Many compression mixes are speed sensitive and will
not produce satisfactory tablets at inappropriate speeds. The dwell time, where
maximum pressure is applied to the mix, is relative to the peripheral speed of the
turret and the diameter of the punch head fl at. Any air in the compression mix must
be expelled to avoid laminating or splitting of the tablet. If air is compressed within
the tablet, when the pressure applied by the punch is released, the compressed air
expands and breaks the tablet. Precompression or dies with a taper in the bore will
FIGURE 2 Punch movements and compression profi les of upper and lower punches of
rotary and eccentric presses.
Rotary press
Eccentric press
Lower compression force
Upper compression force
FIGURE 3 Dwell time as function of tableting machine.
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.2
0.4
0.6
0.8
1.0
Effective contact time
Consolidation time
Maximum UP force
Dwell time
Residence time
FIGURE 4 Stages of tableting process: ( a ) fi lling; ( b ) compression; ( c ) ejection.
(a)
(b)
(c)
often reduce this problem. Rotary presses sometimes have two pairs of compression
rolls (for precompression and compression) . During precompression additional
compression can take place and the absolute dwell time can be prolonged. In other
words , there are numerous different tablet presses with various possibilities to carry
out the compression process.
A direct correlation between the results of an eccentric press and a rotary press
cannot always be drawn. In addition there are many different tablet presses with
different settings and possibilities. These problems can be overcome by using a
compaction simulator early in the development stage. An advantage of such a simulator
is its versatility, that is, all types of presses can be simulated with small amounts
of solid. The problem, however, is that such a simulator is very expensive.
The tableting cycle is well explained in the literature and is broken down into
three stages (Figure 4 ):
1. Filling The volume of the granule is measured (Figure 4 a ):
A: The lower punch is allowed to descend to its lowest point.
B: The bore of the die is fi lled completely with powder.
C: The lower punch is raised to a predetermined point so that excess.
D: The powder is leveled by passing under a blade.
TABLETING CYCLE 981
982 TABLET DESIGN
E: This ensures that the bore of the die is fi lled with as exact volume of the
material to be used, and the next stage can begin.
2. Compression Pressure is applied to form the granule into a solid (Figure
4 b ):
F: The upper punch is lowered into the bore of the die.
G: Precompression gives the powder an initial “ punch ” to remove excess
air.
H: The powder is fully compressed.
I: The correct pressure is reached.
J: The upper punch is lifted out of the way ready for tablet ejection.
3. Ejection The tablet is ejected and the next tablet will be formed (Figure
4 c ):
K: The lower punch begins to rise in the bore of the die lifting the tablet until
step L is reached.
L: Its base is level with the tap of the die.
M: The tablet is pushed aside into the take - off chute by passing under a static
blade.
N: The lower punch moves to its lowest position ready for fi lling ( O ), similar
to A and the entire cycle is repeated.
Guidance on compression levels for each tablet type (series 1, 2, and 3) and maximum
punch pressures are given in Table 1 :
Series 1 : Flat fl ace, normal concave, shallow concave tablets
Series 2 : Double radius, bevel and concave tablets
Series 3 : Flat beveled edge, deep concave, ball or pill
In Figure 4 the tablets progress from start to fi nish from left to right. On an actual
machine this will be determined by the direction in which the entire turret rotates
in relation to the fi xed item, such as the fi ll hopper precompressions and compression
rollers. It is important to note that this direction may vary from machine to
machine, but as a general rule, British, American, and some Asian machines
rotate in a clockwise direction while European machines rotate anticlockwise
(see Figure 5 ).
6.3.3 LIMITATIONS FOR DIRECT COMPRESSION
In tablet formulation, a range of excipient materials are normally required along
with the active ingredient in order to give the tablet the desired properties. For
example, the reproducibility and dose homogeneity of tablets are dependent on the
properties of the powder mass. The tablet should also be suffi ciently strong to
withstand handling but should disintegrate after intake to facilitate drug release.
The choice of excipients will affect all these properties:
1. Filler illers are used to make tablets of suffi cient size for easy handling by
the patient and to facilitate production. Tablets containing a very potent active
LIMITATIONS FOR DIRECT COMPRESSION 983
FIGURE 5 Tableting machine (anticlockwise).
TABLE 1 Guide Punch Pressures for Series 1, 2, and 3
Tablet Size Pressure (kN)
mm in. Series 1 Series 2 Series 3
3
1
8
5 3 2
4
5
32
10 5.6 3.7
5
3
16
15 8.8 5.8
6
7
32
22 12 8.5
7
9
32
30 17 11
8
5
16
40 22 15
9
11
32
50 28 19
10
13
32
60 35 23
11
7
16
70 42 27
12
15
32
90 50 33
13 1
2 100 59 39
14
9
16
120 67 46
15
19
32
130 78 53
16 5
8 160 90 60
17
21
32
180 102 68
18
23
32
203 114 74
19 3
4 226 127 85
20
25
32
251 141 94
25 1 393 221 147
984 TABLET DESIGN
substance would be very small without excipients. A good fi ller will have good
compactability and fl ow properties and acceptable taste and be non - hygroscopic
and preferably chemically inert. It may also be advantageous to have a fi ller that
fragments easily, since this counteracts the negative effects of lubricant additions
to the formula [4] .
2. Binder A material with a high bonding ability can be used as a binder to
increase the mechanical strength of the tablet. A binder is usually a ductile material
prone to undergo plastic (irreversible) deformation. Typically, binders are polymeric
materials, often with disordered solid - state structures. Of special importance
is the deformability of the peripheral parts (asperities and protrusions) of the binder
particles [5] .
This group of materials has the capacity of reducing interparticulate distances
within the tablet, improving bond formation. If the entire bulk of the binder particles
undergoes extensive plastic deformation during compression, the interparticular
voids will, at least partly, be fi lled and tablet porosity will decrease. This increases
contact area between the particles, which promotes the creation of interparticular
bonds and subsequently increases tablet strength [6] . However, the effect of the
binder depends on both its own properties and those of the other compounds within
the tablet. A binder is often added to the granulation liquid during wet granulation
to improve the cohesiveness and compactability of the powder particles, which
assists the formation of agglomerates or granules. It is commonly accepted that
binders added in dissolved form, during a granulation process, are more effective
than those added in dry powder form during direct compression.
3. Disintegrating Agent A disintegrant is normally added to facilitate the
rupture of bonds and subsequent disintegration of the tablets. This increases the
surface area of the drug exposed to the gastrointestinal fl uid; incomplete disintegration
can result in incomplete absorption or a delay in the onset of action of the drug.
There are several types of disintegrants, acting with different mechanisms: (a) promotion
of the uptake of aqueous liquids by capillary forces, (b) swelling in contact
with water, (c) release of gases when in contact with water, and (d) destruction of
the binder by enzymatic action [7] . Starch is a traditional disintegrant; the concentration
of starch in a conventional tablet formulation is normally up to 10% w/w.
The starch particles swell moderately in contact with water, and the tablet disrupts.
So - called superdisintegrants are now commonly used; since these act primarily by
extensive swelling, they are effective only in small quantities [8] cross - linked sodium
carboxymethyl cellulose (e.g., Ac - Di - Sol), which is effective in concentrations of
2 – 4%, is a commonly used superdisintegrant. Larger particles of disintegrants have
been found to swell to a greater extent and with a faster rate than fi ner particles,
resulting in more effective disintegration [9] .
4. Glidant, Antiadherent, and Lubricant Glidants are added to increase the
fl owability of the powder mass, reduce interparticulate friction, and improve powder
fl ow in the hopper shoe and die of the tableting machine. An antiadherent can be
added to decrease sticking of the powder to the faces of the punches and the die
walls during compaction, and a lubricant is added to decrease friction between
powder and die, facilitating ejection of the tablet from the die. However, addition
of lubricants (here used as a collective term and including glidants and antiadherents)
can have negative effects on tablet strength, since the lubricant often reduces
LIMITATIONS FOR DIRECT COMPRESSION 985
the creation of interparticulate bonds [e.g., 4] . Further, lubricants can also slow the
drug dissolution process by introducing hydrophobic fi lms around drug and excipient
particles [e.g., 10] . These negative effects are especially signifi cant when long
mixing times are required [11] . Therefore, the amount of lubricants should be kept
relatively low and the mixing procedure kept short to avoid a homogenous distribution
of lubricant throughout the powder mass. An alternative approach could then
be to admix granulated qualities of lubricant [12] .
5. Flavor, Sweetener, and Colorant Flavor and sweeteners are primarily used
to improve or mask the taste of the drug, with subsequent substantial improvement
in patient compliance. Coloring tablets also has aesthetic value and can improve
tablet identifi cation, especially when patients are taking a number of different
tablets.
General instructions for the determination of tablet properties (e.g., hardness,
disintegration, friability, dissolution profi le, and stability) are contained in pharmacopeia
[e.g., European Pharmacopoeia (Eur. Ph.) and U.S. Pharmacopeia
(USP)].
In the manufacture of tablets it is important to defi ne and appreciate the
physical properties of the active substance, in particular particle size and
fl owability. The technology involved in direct compression assumes great importance
in tablet formulations because it is often the least expensive, particularly
in the production of generics that the active substance permits. The limiting
factors are the physical properties of the active substance and its concentration
in the tablets. Even substances such as ascorbic acid, which are not generally
suitable for direct compression owing to the friability of the crystals, can normally
be directly pressed into tablets at concentrations of 30 – 40%. However, this technique
is not as suitable if the content of ascorbic acid is higher. This limit may be
shifted upward by special direct - compression auxiliaries, for example, Ludipress
(BASF).
Ludipress is derived from lactose, Kollidon 30, and Kollidon CL. It thus combines
the properties of a fi ller, binder, disintegrant, and fl ow agent and also often
acts as a release accelerator. By virtue of its versatility, formulations containing it
are usually very simple. It can also be combined with almost all active substances
with the exception of those that enter into a chemical interaction with lactose (Maillard
reaction). Active substances (e.g., many analgetics) behave very differently
with Ludipress when the dosage is extremely high. Acetylsalicylic acid (ASA) and
metamizole can be pressed when little Ludipress has been added; ibuprofen requires
a larger amount; and the fraction of Ludipress required in the tablets is too large
for paracetamol (acetaminophen).
An alternative to the Ludipress grades is the outstanding dry binder Kollidon
VA 64 together with excipients (e.g., calcium phosphate, microcrystalline cellulose,
lactose, or starch) and a disintegrant (e.g., Kollidon CL). This combination even
allows 500 mg of paracetamol to be pressed into good tablets with a weight of
700 mg.
No other dry binder has binding power and plasticity comparable to Kollidon
VA 64. Plasticity, in particular, is an important parameter in direct compression.
As can be seen in Figure 6 (99.5% binder + 0.5% magnesium stearate), this property
of Kollidon VA 64 is not adversely affected by increasing the pressure. The benefi cial
986 TABLET DESIGN
FIGURE 6 Plasticity of dry binders in tablets.
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Plasticity
HPMC 11,000 Microcrystaline
cellulose
Povidone K 30 Kollidon VA 64
Compression force 25 kN
Compression force 18 kN
properties of Kollidon VA 64 can also be exploited for the production of concentrated
active substance that is subsequently used for direct tableting. Kollidon VA
64 and Ludipress can also be combined with one another.
Acetylsalicylic Acid – Acetaminophen – Caffeine Tablet (250 mg + 250 mg +
50 mg )
Formulation
Acetaminophen powder 39.25%
Caffeine powder 7.85%
ASA powder 39.25%
Kollidon VA 64 9.4%
Kollidon CL 3.15%
Aerosil 200 0.5%
Magnesium stearate 0.6%
Manufacture by Roller Compaction The active ingredients and Kollidon VA 64
were granulated in a Gerteis Roller compactor.
Process Parameters
Force 5.0 kN/cm
Crack 2.9 mm
Looser 5.0 rpm
Dosing screw 90.5 rpm
Plug screw 108.6 rpm
Granulator 50 rpm
Tableting The granules were passed together with magnesium stearate, Aerosil
200, and Kollidon CL through an 800 - . m sieve, blended for 10 min in a Turbula
LIMITATIONS FOR DIRECT COMPRESSION 987
FIGURE 7 Dissolution profi le for acetylsalicylic acid – acetaminophen – caffeine tablet
(250 mg + 250 mg + 50 mg): paddle 50 rpm, 37 ° C deionized water.
0
20
40
60
80
100
120
0
Time (min)
Drug release (%)
20 40 60 80 100 120 140
mixer, and compressed into tablets with a force of about 12 kN (tablet press
Korsch PH106, 30 rpm, compression force 11.5 kN)
Tablet Properties
Weight 629.7 mg
S rel 1.4%
Diameter 12 mm
Form Biplanar
Hardness 72 N
Friability 2.75%
Dissolution See Figure 7 .
Enteric Film Coating of Tablets (Organic Solution)
Formulation of Cores
Component Percent/Tablet (w/w)
ASA 33.4
Ludipress 49.5
Avicel PH 102 16.6
Magnesium stearate 0.5
Total 100.0
Manufacture by Direct Compression All the components were mixed for 10 min,
passed through an 0.8 - mm sieve, and compressed into tablets on a rotary press
with a rate of 40,000 tablets/h at a compression force of 15 - kN. Core shape
was convex with a diameter of 9 mm and the engraving BASF . Hardness of
the tablets was about 60 N.
Formulations of Coating Suspension The formulation is for 5 - kg cores (diameter
9 mm; 300 mg):
988 TABLET DESIGN
Component
Parts by Weight [g]
No. 1 No. 2
Kollicoat MAE 100 P 344.52 344.52
Triethyl citrate 34.45 34.45
Ethanol 4214.60 —
Isopropanol/Wasser (70/30) — 4214.60
Total 4593.57 4593.57
Polymer applied 10.0 mg/cm 2
Content of polymer 7.5%
Preparation of Spray Suspension Kollicoat MAE 100P and triethyl citrate were
stirred into the solvent until complete dissolution.
Process Parameters
Coating pan 24 Accela Cota
Size of batch 5 kg
Inlet air temperature 50 ° C
Outlet air temperature 35 – 38 ° C
Product temperature 30 – 35 ° C
Inlet air rate 70 m
Outlet air rate 140 m
Spraying pressure 2.0 bars
Nozzle diameter 1.0 mm
Rate of spraying 30 g/min
Time of spraying 2.5 h
Preheating 3 min
Final drying 5 min
Dissolution: (See Figure 8 ). Dissolution was done according to USP monograph
“ Aspirin Delayed - Release Tablets. ”
FIGURE 8 Enteric fi lm coating of tablets, acetylsalicylic acid (organic solution): paddle
100 rpm, 37 ° C; 0 – 2 h: 0.08 M HCl, 2 h+: phosphate buffer Ph 6.8.
0
20
40
60
80
100
120
0 50 100 150 200
Time (min)
Drug release (%)
Ethanol
Isopropanol/wasser
(70/30)
LIMITATIONS FOR DIRECT COMPRESSION 989
Beta Carotene Tablets (15 mg )
Formulation
Formulation 1 Formulation 2
Beta carotene dry powder 10% 160.0 g 150.0 g
Ludipress 240.0 g —
Dicalcium phosphate, granulated
with 5% Kollidon 30
— 175.0 g
Avicel PH 101 — 100.0 g
Kollidon CL 6.0 g 5.0 g
Aerosil 200 — 2.5 g
Talc — 20.0 g
Calcium arachinate — 2.5 g
Magnesium stearate 2.0 g —
Manufacturing (Direct Compression) All components were mixed, passed
through a 0.8 - mm sieve, and pressed with a medium compression force.
Tablet Properties
Formulation 1 Formulation 2
Weight 400 mg 502 mg
Diameter 12 mm 12 mm
Form Biplanar Biplanar
Hardness 59 N 57 N
Disintegration 12 min 1 min
Friability 0.1% 0%
Chemical and Physical Stability (20 – 25 ° C)
6 Months 12 Months
Formulation 1
Loss of beta carotene 3% 4%
Hardness 60 N 59 N
Disintegration 9 min 7 min
Friability 0.15% 0.16%
Formulation 2
Loss of beta carotene 8% 9%
Diclofenac Na – Dispersion – Tablet (50 mg )
Formulation
Diclofenac Na 50.0 mg
Avicel PH 102 143.8 mg
Kollidon CL 50.0 mg
Aerosil 200 5.0 mg
Magnesium stearate 1.0 mg
990 TABLET DESIGN
Procedure The ingredients were mixed, passed through a 0.8 - mm sieve, and
compressed into tablets with a force of about 10 kN. (The tablet press was
Korsch PH106, 30 rpm, compression force was 11.8 kN.
Tablet Properties
Weight 248.0 mg
S rel 1.7%
Diameter 10 mm
Form bipla Nar
Hard Ness 93 N
Friability < 0.1%
Dissolution See Figure 9 .
In direct – compression formulation, there is a wide particle size distribution.
Usually, the active drug is at the fi ne end of the range. Such a wide particle size
range can easily result in signifi cant segregation. Five primary mechanisms are
responsible for most particle segregation problems [13] . Of these, only three typically
occur with pharmaceutical powders: sifting, entrainment of air, and entrainment
of particles in an air stream.
Sifting is a process by which smaller particles move through a matrix of larger
ones. It is by far the most common method of segregation. Sifting has been found
to occur with particle size ratios as low as 1.3 : 1 or with a suffi ciently large mean
particle size (the tendency to segregate by sifting decreases substantially with particle
size < 500 . m. Free - fl owing material and interparticle motion also caused segregation
by sifting.
Two techniques can be used to decrease a material ’ s segregation tendencies:
change the material or change the design of the equipment.
Lisinopril (5 mg ) Reducing the ratio of excipient, lisinopril (2 : 1) tablets for direct
compression can be obtained:
FIGURE 9 Dissolution profi le for diclofenac – Na dispersion tablet (50 mg): paddle 50 rpm,
37 ° C phosphate buffer, pH 7.2.
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Time (min)
Drug release (%)
Formulation
Voltaren dispersion
FIGURE 10 Disolution profi le for lisinopril 5 mg: paddle 50 rpm, 37 ° C, HCl 0.1 N .
0
20
40
60
80
100
120
0 4 8 12 16 20 24 28
Time (min)
Lisinopril release (%)
Formulation %
Lisinopril 5.0
Aerosil 0.5
Calcium phosphate dibasic 19.8
Starch 1500 2.0
Magnesium stearate 0.7
Cellulose microcrystalline (PH - 250) 72.0
Lisinopril and Aerosil ( < 150 . m) were mixed for around 10 min. All other components
were added and mixed for 15 min, passed through a 0.150 - mm sieve, and
pressed with 10 kN compression force.
Properties of 5 - mg lisinopril tablets are as follows:
Weight 130 mg
Diameter 8 mm
Form Biplanar
Hardness 98 N
Disintegration 2 – 3 min
Friability 0.05
The content uniformity of the formulation was measured at the beginning, middle,
and end of the batch (50 kg) (Table 2 ).
Special attention should be given to the physical stability of the tablets manufactured
by direct compression because some fi llers/binders are known to soften or
harden on storage.
6.3.4 PREVIOUS GRANULATION: BIOPHARMACEUTICAL VERSUS
TECHNOLOGICAL PROPERTIES
Granulation is the process by which primary powder particles are made to adhere
to form larger, multiparticle entities called granules. Pharmaceutical granules
BIOPHARMACEUTICAL VERSUS TECHNOLOGICAL PROPERTIES 991
992 TABLET DESIGN
typically have a size range between 0.2 and 4.0 mm, depending on their subsequent
use. In the majority of cases this will be in the production of tablets or capsules,
when granules will be made as intermediate products and have a typical size range
between 0.2 and 0.5 mm, but larger granules are used as a dosage form in their own
right.
Granulation normally commences after initial dry mixing of the necessary powdered
ingredients so that a uniform distribution of each ingredient through the mix
is achieved. After granulation the granules either will be packed or may be mixed
with other excipients prior to tablet compaction.
The principal reasons for granulation are as follows:
1. To prevent segregation of the constituents of the powder mix.
2. To improve the fl ow properties of the mix.
3. To improve the compaction characteristics of the mix
Methods of Granulation Granulation methods can be divided into two types:
wet methods , which use a liquid in the process, and dry methods , in which no
liquid is used. In a suitable formulation a number of different excipients will be
needed in addition to the drug. The common types used are diluents, to produce a
unit dose weight of suitable size, and disintegrating agents, which are added to
aid the break - up of the granule when it reaches a liquid medium (e.g., on ingestion
by the patient). Adhesives in the form of a dry powder may also be added, particularly
if dry granulation is employed. These ingredients will be mixed before
granulation.
1. Dry Granulation In the dry methods of granulation the primary powder
particles are aggregated under high pressure. There are two main processes:
Either a large tablet ( slug ) is produced in a heavy - duty tableting press ( slugging )
or the powder is squeezed between two rollers to produce a sheet of material
( roller compaction ). In both cases these intermediate products are broken down
using a suitable milling technique to produce granular material, which is usually
sieved to separate the desired size fraction. This dry method may be used for drugs
TABLE 2 Study of Uniformity for Formulation Lisinopril 5 mg (Batch 50 kg)
Number 0 – 5 kg 5 – 10 kg 10 – 15 kg 15 – 20 kg 20 – 25 kg 25 – 30 kg 35 – 40 kg 40 – 45 kg 45 – 50 kg
1 4.99 5.00 5.00 4.95 5.02 5.08 5.00 4.96 5.00
2 5.00 5.06 5.00 4.92 5.00 4.99 4.83 5.11 5.17
3 5.00 4.81 4.90 5.19 4.92 5.03 4.92 5.00 5.03
4 5.05 5.00 5.00 4.99 4.82 4.96 5.00 4.93 4.99
5 4.89 4.99 4.90 5.05 5.13 5.04 4.98 4.93 4.87
6 5.02 5.06 4.85 5.10 5.13 5.00 5.18 4.81 4.93
7 5.00 5.08 5.11 5.00 5.00 4.83 4.89 5.00 5.00
8 5.00 5.00 5.03 5.03 5.00 5.06 5.00 5.18 5.00
9 4.97 4.99 5.01 5.04 5.05 4.85 5.00 5.00 4.99
10 4.91 5.00 5.01 4.98 4.97 5.00 5.00 5.11 5.05
that do not compress well after wet granulation or those which are sensitive to
moisture.
2. Wet Granulation Wet granulation involves the massing of a mix of dry
primary powder particles using a granulating fl uid . The fl uid contains a solvent which
must be volatile so that it can be removed by drying and be nontoxic. Typical liquids
include water, ethanol, and isopropanol, either alone or in combination. The granulation
liquid may be used alone or, more usually, as a solvent containing a dissolved
adhesive (also referred to as a binder or binding agent ) which is used to ensure
particle adhesion once the granule is dry. Water is commonly used for economical
and ecological reasons. Its disadvantages as a solvent are that it may adversely affect
drug stability, causing hydrolysis of susceptible products, and it needs a longer drying
time than do organic solvents. This increases the length of the process and again
may affect stability because of the extended exposure to heat.
Captopril (25 mg ) + Hydrochlorothiazide (25 mg )
Formulation
Formulation for 500 mg
Captopril 5%
Hydrochlorothiazide 5%
Lactose 65%
Carboxyethylcellulose sodium 10%
Ac - Di - Sol 3%
Starch 10%
Stearic acid 2%
Manufacturing (Wet Granulation) A mixture of all compounds (with 1.5%
stearic acid) is granulated with solution 2 - propanol (around 8% v/w), passed
through a 0.8 - mm sieve, and the rest (0.5% stearic acid) added and pressed
with low compression force.
Tablet Properties (Initial Time)
Weight 500 mg
Diameter 12 mm
Form Normal concave
Hardness 60 N
Disintegration < 4 min
Friability < 0.3%
Dissolution (captopril + hydrochlorothiazide)
30 min 90.00%
60 min 100%
Stability of Three Batches (5 kg each) at 25 ° C and 70% Relative Humidity (RH)
during 12 Months
BIOPHARMACEUTICAL VERSUS TECHNOLOGICAL PROPERTIES 993
994 TABLET DESIGN
Formulation
6 months 12 months
Batch 1 Batch 2 Batch 3 Batch 1 Batch 2 Batch 3
Hydrochlorothiazide (%) 98.91 99.47 100.65 99.06 100.40 102.88
Captopril (assay) (%) 101.02 100.89 100.99 100.65 100.03 100.30
Captopril disulfuric a 0.62 0.71 0.69 0.69 0.79 0.72
Weight 500.12 501.23 499.65 499.00 500.33 500.14
Hardness 63 N 67 N 61 N 60 N 63 N 61 N
Disintegration (min) 3 3 4 4 4 4
Friability 1.98 1.15 1.12 1.71 1.12 1.12
Dissolution
30 min 89.45 90.54 88.77 88.00 91.00 88.05
60 min 100 100 100 100 100 100
a Captopril degradation product (%).
a - Methyldopa Tablet (250 mg )
Manufacturing (Wet Granulation) A mixture of . - methyldopa with lactose or
calcium phosphate (for formulations F1 or F2 , respectively) is granulated with
isopropanol solution of Kollidon 30 and passed through a sieve, the dry granules
are mixed with Kollidon CL and magnesium stearate, and pressed with
medium compression force.
Tablet Properties
F1 F2
Weight 361 mg 362 mg
Diameter 11 mm 11 mm
Hardness 118 N 156 N
Disintegration 5 min 4 min
Friability < 0.1% < 0.1%
Dissolution
10 min 45% 55%
20 min 82% 90%
30 min 90% 98%
TABLE 3 Comparative Study of Lactose Monohydrate and Calcium Phosphate, Dibasic
Formulation F1 Formulation F2
. - Methyldopa 275 g (78%) 275 g (78%)
Lactose monohydrate 15.5% —
Calcium phosphate, dibasic — 15.5%
Kollidon 30 4% 4%
Isopropanol 80 mL 80 mL
Kollidon CL 2% 2%
Magnesium stearate 0.5% 0.5%
Formulation Table 3 presents a comparison of lactose monohydrate and calcium
phosphate.
Calcium phosphate, dibasic offers high hardness and faster dissolution profi le
than lactose for . - methyldopa tablets in wet granulation.
6.3.5 TABLET DESIGN FOR MATRIX SYSTEM
The advantages of controlled - release systems include maintenance of drug levels
within a desired range, the need for fewer administrations, optimal use of the drug
in question, and increased patient compliance. Evaluation of matrix tablets is the
same as for conventional formulations but the dissolution profi le and stability have
to be carefully studied. Numerous methods for development of matrix tablets can
be used, such as direct compression, wet granulation, pelletization, and spheronization
exclusion. Nevertheless, the potential disadvantages cannot be ignored: the
possible toxicity or no biocompatibility of the materials used, undesirable by -
products of degradation, any surgery required to implant or remove the system,
the chance of patient discomfort from the delivery device, and the higher cost of
controlled - release systems compared to traditional pharmaceutical formulations.
The importance of matrix systems that they release bioactive component over an
extended period of time has long been recognized in the pharmaceutical fi eld.
Matrix systems can be divided into three groups depending on the type of polymer
formed:
1. Inert Matrices Polymers that after compression form an indigestible and
insoluble porous skeleton [14] constitute the inert matrices. The main challenge in
the preparation of these systems is to achieve, by means of a suitable design, total
drug release from the device as well as adequate and precise drug release, guaranteeing
the integrity of the matrix.
2. Hydrophilic Matrices Cellulose derivatives have been widely used in the
formulation of hydrogel matrices for controlled drug delivery. Among them hydroxypropyl
methylcellulose (HPMC) is the most extensively employed because of its
ease of use, availability, and very low toxicity [15] . Drug release from these systems
is controlled by the hydration of HPMC, which forms a gelatinous layer at the
surface of the matrix through which the included drug diffuses.
Drug release from swellable matrix tablets is based on the glassy – rubbery transition
of the polymer which occurs as a result of water penetration into the matrix.
Therefore, the gel layer is physically delimited by the erosion (swollen matrix –
solvent boundary) and swelling (glassy – rubbery polymer boundary) fronts.
Water - soluble drugs are released primarily by diffusion of dissolved drug molecules
across the gel layer, while poorly water soluble drugs are released predominantly
by erosion mechanisms.
The factors infl uencing the release of drugs from hydrophilic matrices include
viscosity of the polymer, ratio of the polymer to drug, mixtures of polymers, compression
pressure, thickness of the tablet, particle size, pH of the matrix, entrapped
air in the tablet, solubility of the drug, the presence of excipients or additives, and
the mode of incorporation of these substances.
3. Lipid Matrices These matrix tablets are formed with lipid polymers with low
melting point. The drug is dissolved or solubilized in the melted lipid, such as cetyl
TABLET DESIGN FOR MATRIX SYSTEM 995
996 TABLET DESIGN
TABLE 4 Theophylline Formulation by Direct Compression and Wet Granulation
(mg/tablet)
Direct Compression Wet Granulation
Granulated theophylline 264 264
90SH - 4000SR HPMC (Metolose SR) 64.5 64.5
Mg stearate 1.5 1.5
Total 330 330
alcohol, ceto - stearilic alcohol, and stearic acid. Solid lipid nanoparticles are an
example of an innovative lipid matrix system.
6.3.5.1 Controlled - Release Tablet by Direct Compression and
Wet Granulation
Theophylline is granulated in a fl uid bed with Pharmacoat 606 3% (Shin Etsu,
Metolose SR) as shown in Figure 11 . Table 4 gives a comparison of direct compression
(DC) and wet granulation (WG) using theophylline:
Diret compression: using a twin - cell mixer, theophylline and HPMC are mixed
for 10 min; then Mg stearate is added and mixed for 2 min.
Tableting conditions for DC and WG: A rotary tableting machine (KIKUSUI) is
used with 12 punches (punch size 10 mm diameter, 12 mm radius, compression
force 98, 147, and 196 MPa; tableting speed 20, 40, and 60 min . 1 ).
Condition for WG: Granulation machine, vertical granulator FM - VG - 05; charge
300 g; binder solution ethanol – water 8 : 2; agitation 600 (blade)/1000 (chopper)
min . 1 ; granulation time 5 min.
Powder properties for compression: Bulk density 0.35 g/mL, tapped density 0.48,
average particle size 122 . m.
Theophylline tablets made by both direct compression and wet granulation have
been assessed. There is almost no difference between direct compression and wet
granulation methods (see Figures 12 – 14 ) under the following conditions: appropriate
formulation (suffi cient level of HPMC in the tablet) and precise control of the
wet granulation process. Direct compression using Metolose SR is recognized as a
suitable process for matrix tablets.
6.3.6 TABLET DESIGN WITH NATURAL PRODUCTS
The development and production of tablets containing a high dose of active ingredients
is a complex and extensive technological challenge. Dried plant extracts are
often used as therapeutically active material in the manufacture of tablets. They are
FIGURE 11 Granulation process: ( a ) before 90SH - 4000SR; ( b ) after 90SH - 4000SR.
(a) (b)
FIGURE 12 Dissolution profi le for theophylline tablets (DC and WG).
0
20
40
60
80
100
0 3 6 9 12 15 18
Time (h)
Theophylline release
(%)
WG
DC
FIGURE 13 Hardness of theophylline tablets (Dc and WG).
0
5
10
15
20
25
75 100 125 150 175 200 225
Compression force (MPa)
Tablet hardness
(kgf)
WG
DC
FIGURE 14 Weight deviation for theophylline tablets (DC and WG).
0
1
2
0 20 40 60 80
Compression speed (min–1)
Tablet weight
deviation (CV %)
WG
DC
TABLET DESIGN WITH NATURAL PRODUCTS 997
998 TABLET DESIGN
often very fi ne, poorly compressible, and very hygroscopic powders. Tablets containing
a high amount of spray - dried extract show prolonged disintegration times,
affecting the release of active constituents [19] . Some alternatives have been proposed
to minimize these problems. Granulation is the technique most often used to
improve the technological properties of these products. However, because of the
products ’ high hygroscopicity, extracts sometimes cannot be granulated using
aqueous systems. Some reports have shown that the use of lubricants during direct
compression of vegetable dried extracts increased the disintegration time. According
to our experience and some previous work, tablets with high amounts of some
lubricant such as aerosil (up to 25% w/w) and magnesium [16] stearate incorporated
into the granules had shorter disintegration time than did tablets containing the
powdered mixture.
Natural products can be used as plant extracts with pharmacological activity
(e.g., Mangifera indica L., vallerian, aloe, Cratoxylum prunifl orum , microporous
zeolite), excipient for direct compression or granulation (chitin, chitosan, and
dextran), and controlled - release systems (cellulose and native dextran). For natural
products the most important factor is the standardization of the extract because
properties such as the amount of active substance can be changed from batch to
batch. Factors such as the origin of the extract, geographic zone, and age of the tree
could affect the properties of natural extracts.
6.3.6.1 Tablet Design from Aqueous Plant Extract
A bioactive product of natural origin has been developed from folk knowledge
of Asian, Latin - American, European, and U.S. ethnic medicine. We developed
an extract of the M. i ndica L. (mango) stem bark, obtained by decoction of some
varieties grown in tropical and subtropical climates, that is used at present as an
antioxidant nutritional supplement (Vimang). The aqueous extract was dried by
atomization in a spray dryer until a brown solid with 10.5% (RSD = 0.9%) water
content (measured by Karl Fischer) was achieved. Tablets obtained by wet granulation
(plant extract, 300 mg/unit) were used for the applications. The product is a
fi ne brown powder that has provend to be useful in the treatment of a large population
sample presenting physical stress due to age or deteriorated physiological
conditions caused by chronic diseases such as cancer, diabetes, or cardiovascular
disorders [17] . Recent studies have shown that treatment with the extract provided
signifi cant protection against 12 - O - tetradecanoylphorbol - 13 - acetate (TPA) –
induced oxidative damage and better protection when compared with other
antioxidants (Vitamin C, E and beta – carotene) [18] . Furthermore, the results
indicate that this extract is bioavailable for some vital target organs, including
liver and brain tissues, peritoneal cell exudates, and serum. Therefore, it was
concluded that it could be useful to prevent the production of reactive oxygen
species (ROS) and oxidative tissue damage in vivo. All these effects are likely due
to the synergic action of several compounds, such as polyphenols, terpenoids, steroids,
fatty acids, and microelements, which have been reported to be present in
the extract [17] .
Mangiferin (1,3,6,7 - tetrahydroxyxanthone - 2 - C - . - d - glucopyranoside), a C - glucosylxanthone,
which was fi rst isolated from the bark, branches, and leaves of M. indica
L., has been found to be the major component of this extract. Mangiferin is a naturally
occurring chemopreventive agent in rat colon carcinogenesis [19] ; exerts antidiabetic
activity by increasing insulin sensitivity; shows signifi cant inhibitory effect
on bone resorption; appears to act as a potential biological response modifi er with
antitumor, immunomodulatory, and anti – human immunodefi ciency virus (HIV)
effect; is capable of providing cellular protection as an antioxidant and a radical
scavenger agent; is useful as an analgesic without adverse effects; and inhibits the
late event in herpes simplex virus - 2 replication [20 – 24] .
The quality control of 16 batches of Vimang active ingredient [by high -
performance liquid chromatography (HPLC) and the ultraviolet (UV) method]
obtained from different regions of the country (batches 1 – 8 from the west and
batches 9 – 16 from the east) and its pharmaceuticals (optimum formula) were investigated
and are demonstrated in Table 5 . Each sample was analyzed in triplicate and
the average values are listed. All assay results fell between 100 and 300 . g of mangiferin
per milligram of Vimang powder, except samples 12 – 16, which were rejected.
The differences found are probably due to the fact that the mangiferin content in
the plant varies with the season of the year and the zone where it was grown. The
claimed contents of this natural product required by our producers are 85 – 115%
for tablets.
Different tablet formulations were tested, but even superdisintegrants such as
Ac - Di - Sol and CMCNa up to concentrations of 5% were not suffi cient to disintegrate
the tablets. With the use of pH - modifi ed product such as NaCO 2 or canalling
as NaCl the release of extract from the tablets changed dramatically. Use of lactose
TABLET DESIGN WITH NATURAL PRODUCTS 999
TABLE 5 Content of Mangiferin ( m g/mg) in Natural Product Samples from Mangifera
indica L. and assay Result of Pharmaceuticals
Sample
Content, . g/mg
(RSD, %) Sample
Content, . g/mg
(RSD, %)
No.1 (batch 901) 254 (0.7) No.9 (batch 0201) 125 (0.1)
No.2 (batch 903) 195 (2.0) No.10 (batch 0202) 109 (1.5)
No.3 (batch E - 1923) 187 (1.6) No.11 (batch 0203) 116 (1.2)
No.4 (batch E - 1924) 180 (1.4) No.12 (batch 0204) 79 (2.4) *
No.5 (batch E - 2032) 206 (1.6) No.13 (batch 0205) 56 (0.5) *
No.6 (batch 0103) 149 (5.7) No.14 (batch 0206) 55 (2.3) *
No.7 (batch 0104) 162 (0.4) No.15 (batch 0207) 66 (1.8) *
No.8 (batch 0112) 159 (0.3) No.16 (batch 0208) 49 (0.2) *
Pharmaceuticals from
batch No.8
Amount of Vimang ®
(mg)
Percentage of claimed content %,
(RSD, %)
Tablets (batch A) 299.91 99.97 (5.47)
Tablets (batch B) 291.36 97.12 (3.14)
Tablets (batch C) 310.51 103.51 (1.42)
* RSD = relative standard deviation.
TABLE 6 Formulations by Wet Granulation
F15 F16 F17 F18 F19 F20 F21 F22 F23 F24 F25 F26 F27 F28 F29 F30
Extract a
50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50
Lactose — — 20 a
16
a
16
a
22
a
27
a
29
a
32
a
42
a
32 a
+
10 35 a
35
a
35
a
35
a
30
a
CMM 20 a
20
a
NaCl 20 a
15 a
+
5
10 a
5
a
5
a
5
5
5
5
—
—
—
5
PEG6000 — — — — — — — — — — — — — 5 5 5
PEG400 a
—
—
— —
—
—
—
—
—
—
—
—
—
4
Na 2 CO 3
—
—
—
5 a
+
5
10 a
5
5
5
5
—
—
5
—
—
—
5
CMCNa — — 5
PVP a
5
5
5
4 4
4
4
4
4
4
4
4
4
4
4
SDS 2 a
1 a
+
1
1
a
+
1
1
1
2
2
2
2
2
2
2
2
2
Talc 1 1 — 1 1 2 2 2 2 4 4 4 4 4 4 4
MgEs 1 1 1 1 1
Aerosil 1 1 5 a
+
2
5
a
+
2
5
a
+
2 5 a
Acdisol — — — 5 5 5 a
5
3
% 62% 65% 71.5% 89% 85% 88% 100% 100% 100% 46% nF 80% 70% 71% 72% 100%
Release
IN 31 IN 34 IN 23
In 30 Min
MIN MIN MIN
Abbreviations: CMM, cellulose microcrystalline; PEG, polyethylene glycal; CMCNa, sodium carboxymethyl cellulose; PVP, Kollidon K30; SDS, sodium docecyl
sulfate; MgEs, magnesium stearate; NF, no fl ow ability.
a Internal phase.
1000
TABLE 7 Two Formulations for mangifera indica L . (Mango)
Formulation F Formulation F31
Internal Phase
Extract M. indica L. 50.0% a 50.0% a
Lactose 31.0% a
Povidone (PVP K30) 4.0% a 4.0% a
Starch 10.0% a
CMM 28.0% a
Ac - Di - Sol 5.0% a
External Phase
SDS — 2.0%
Na 2 CO 3 — 5.0%
PEG6000 — 5.0%
Talc 2.5% 2.5%
Magnesium stearate 0.5% 0.5%
a Product added to internal phase.
FIGURE 15 Formulation F31.
was better than CMM, especially for fl ow properties. In all cases tablets always
eroded and never disintegrated. All formulations studied are shown in Table 6 .
F23 and F30 are shown in Figure 15 . Two formulations, F31 (obtained from the
result of F23 and F30 ) and F, were compared for water uptake and dissolution
profi le, as shown in Table 7 .
The properties of formulations F31 and F were measured in ENSLIN equipment
in order to compare the kinetics of water uptake and to study the dissolution profi le,
as shown in Figures 16 – 19 . As observed in Figure 19 , the dissolution of plant extract
after 60 min is very slow when pH - modifi ed ( NA 2 CO 3 ) and sodium dodecyl sulfate
(SDS) are not used (almost not change in color for formulation F ).
The properties of formulation F31 are as follows: diameter 12 mm, normal
concave; stability studies (see Table 8 ) under tropical condition, 30 ° C and 75% RH
with desiccant (silice 1 g for plastic bottle of polyethylene no. 8 for 60 tablets).
TABLET DESIGN WITH NATURAL PRODUCTS 1001
1002 TABLET DESIGN
FIGURE 16 Formulation F31 in ENSLIN equipment at initial time.
FIGURE 17 Formulation F31 in ENSLIN equipment after 60 min.
6.3.6.2 Natural Product as Vehicle for Manufactured Tablets
Chitin was evaluated as a direct - compression vehicle using powder fl ow properties
and the physicomechanical properties of the manufactured tablets, and it was proven
that this natural polymer has suitable characteristics for being used for this end.
A comparative study of chitin obtained from lobsters, starch, and carboxymethyl
chitosan as disintegrating agents was conducted. The infl uence of the method in the
preparation of tablets on the disintegrating activity of both polymers was evaluated.
Chitin proved to have good characteristics as a disintegrating agent independently
FIGURE 18 Formulation F in ENSLIN equipment at initial time.
FIGURE 19 Formulation F in ENSLIN equipment after 60 min.
TABLE 8 Stability Studies for F31 under Tropical Condition
Initial Time 3 months 6 months 9 months 12 months
Weight 600.7 601.47 601.30 602.09 601.33
Assay 99.85 100.98 100.64 99.10 101.07
Disintegration, min 27 29 30 30 30
Hardness, N 59 60 64 60 60
Friability, % 0.89 0.74 0.80 0.77 0.81
Humidity, % 5.71 5.11 5.45 5.21 5.64
TABLET DESIGN WITH NATURAL PRODUCTS 1003
1004 TABLET DESIGN
TABLE 9 Formulation with Chitin, Chitosan, and Croscarmelose Sodium
I II III IV V VI VII
Papaverin 19.25 18.25 17.50 18.25 17.50 18.25 17.50
Binder 10.00 10.00 10.00 10.00 10.00 10.00 10.00
Chitin — 1.00 1.50
Chitosan — — — — — 1 1.50
Croscarmelose sodium — — — 1.00 1.50
Dibasic calcium
phosphate
70.00 70.00 70.00 70.00 70.00 70.00 70.00
Magnesium stearate 0.75 0.75 0.75 0.75 0.75 0.75 0.75
TABLE 10 Results for Direct Compression
I II III IV V VI VII
Weight, mg 348 347 345 347 348 346 347
Release at 30 min, % < 40 92.6 92.4 94.2 96.6 92.1 94.8
Disintegration, min > 60 2.30 1.40 0.28 0.22 1.30 1.10
Hardness, kgf - Erw 3.92 3.99 3.97 3.97 4.03 3.99 3.92
Friability 2.44 2.24 2.10 2.01 1.97 1.89 1.94
TABLE 11 Results for Wet Granulation
FI FII FIII
Weight, mg 346.50 348.20 349.00
Release at 30 min, % 81.00 86.00 < 70.00
Disintegration, min 23.70 23.00 45.30
Hardness, kgf - Erw 6.90 8.20 6.30
Friability 0.14 0.08 0.13
of the method used to make tablets (Tables 9 – 11 ) . The disintegrating activity of
carboxymethyl chitosan was affected by the granulation process.
Three formulas were prepared by wet granulation comparing starch, chitin, and
chitosan as disintegrant (10%) formulations FI (10% starch), FII (10% chitin), and
FIII (10% chitosan) (Table 11 ) . Chitin has good properties as disintegrant. This
product can be used for direct compression and wet granulation. The method for
development (direct compresion or wet granulation) of tablets infl uences chitosan
disintegrant properties.
6.3.6.3 Natural Product as Vehicle for Controlled - Release System
Dextrans are composed of chains of d - glucan (1 – 20 . 10 6 ) with . - 1,6 as the main -
chain linkage and variable numbers of . - 1,2, . - 1,3, or . - 1,4 branched - chain linkages.
Dextran is synthesized from sucrose by dextransucrases, glucansucrases, and glucosyltransferases
produced by Leuconostoc or Streptococcus . These bacteria growing
in sugar juices produce dextran. High concentrations of dextran on solids ( > 1000 ppm)
can result in severe fi nancial losses to the sugar industry [25] .
Dextran fractions obtained from enzymatic hydrolysis of native dextrans are
supplied in molecular weights from 1000 to 2 . 10 6 Da. The molecular weight of the
fraction is in most cases a key property and is defi ned in terms of the average
molecular weight ( M w ) and the number average molecular weight ( M n ). The functionality
of this raw material for controlling drug release is studied as a function of
molecular weight. Fractions 43,000 ( F3 ), 71,000 ( F2 ), and 170,000 ( F1 ) as native
dextran 2 . 10 6 and 20 . 10 6 M w are used [26] .
Wet - and Dry - Weight Studies The method used was based on that of Tahara [15]
and Jamzad [27] . The swelling and erosion of dextran polymers of differing molecular
weights were examined by measuring the wet and subsequent dry weights of
matrices. The experiment consisted of allowing the tablet (dextran alone ) to dissolve
in the medium (at the same condition described in drug release studies) for
certain time periods (15, 30, 60 and 90 minutes) before being removed into a preweighed
weighing boat. The excess dissolution medium was drained and blotted
from around the tablet without touching it. The tablet and boat were then weighed
to establish the wet weight of the tablet. The tablets were then dried to a constant
weight in an oven at 105 ° C. Each determination at each time point was performed
in triplicate and mean values were expressed. The dissolution medium uptake per
weight of dextran remaining was calculated at each time point for a particular matrix
to correct for the effect of erosion and dissolution in the measurement of degree of
dissolution medium uptake [Equation (1) ]. Erode dextran was measured according
to the equation described by Jamzad et al. [30] [ Equation (2) ]:
Water uptake per unit
polymer remaining
wet weight dry weight
d (%) = .
ry weight
. 100 (1)
Mass polymer loss (%)
original weight remaining (dry) weight
orig
=
.
inal weight
. 100 (2)
The Davidson and Peppas model [Equation (3) ] was applied to these data to study
the mechanism and the rate of water uptake.
w Ktn = s (3)
where w is the weight gain of the swelled matrix (water/dry polymer), K s the kinetic
constant of water penetration, t the penetration time, and n the exponent which
depends on the water penetration mechanism.
Swelling and Erosion The change in wet weight, refl ecting swelling, over time for
the fi ve polymer types is shown in Table 12 . The higher molecular weight polymers
showed the highest maximum average relative swelling, which occurred since the
initial time with little erosion. In contrast, the lower molecular weight polymers
(fractions F1 , F2 , and F3 ) exhibited minimal swelling and the erosion mechanism
predominated. Consequently, tablets were dissolved very fast (100% before 45 min).
These polymers and fractions show a wide range of viscosities, which cause differences
in their swelling and erosion behaviors [26] . These results agree with results
obtained by Sakar [C] and Walker [D] for HPMC polymer.
TABLET DESIGN WITH NATURAL PRODUCTS 1005
1006 TABLET DESIGN
TABLE 12 Swelling and Erosion Properties of Dextran Tablets as Funtion of Molecular
Weight
Hardness (N)
Mass Polymer Loss (%)
15 min 30 min 60 min 90 min
DT, MW 2 . 10 6 431 11.48 14.59 27.473 36.224
DT, MW 20 . 10 6 482 5.29 7.33 11.835 16.153
F1 431 52.89 87.38
F2 420 62.79 89.21
F3 460 66.3 95.85
Percentage of Water Uptake
15 min 30 min 60 min 90 min
DT, MW 2 . 10 6 59.80 84.68 103.73 110.15
DT, MW 20 . 10 6 110.03 142.94 178.50 210.02
F1 31.61 33.14
F2 17.61 26.89
F3 16.82 17.65
Note: All values are referred to applied force, 14 kN, particle size for dextran 150 – 200 . m, tablet weight
300 ± 11 mg, dissolution media 1000 mL distilled water, temperature 37 ° C, 100 - rpm paddle. Abbreviations:
DT, dextran; MW, molecular weight.
For native dextran a linear relationship was seen between mass polymer loss and
initial dissolution time. Native dextrans also showed the highest maximum dissolution
medium uptake. Here, an increase in the molecular weight of dextran resulted
in an increase in water uptake (native polymer with 10 times more than fraction F1 ,
13 times more than F2 , and 15 times more than F3 for the fi rst 30 min) and less
erosion. Anywhere the rate of water uptake per unit weight of polymer started
to decline with last initial time and in consequence for longer periods of time, nonlinear
dependence could be expected. Applying the Davidson – Peppas model [Equation
(3) ], a value of n = 0.356 ( r 2 = 0.998) for native dextran was obtained
( r 2 = 0.984). An inverse relationship between erosion rate constant and molecular
weight was reported by Reynolds et al. [28] . Tahara et al. [18] reported that the
lower viscosity HPMC (50 - cps) polymer eroded faster than the 4000 - cps polymer,
consistent with the current work. Thus, the higher molecular weight native dextran
polymers have higher intrinsic water - holding capacity and the matrices formed
from such polymers are less prone to erosion than the lower molecular weight
fractions.
6.3.6.4 Mechanism of Soluble Principle Active Propranolol Hydrochloride and
Lobenzarit Disodium from Dextran Tablets
Soluble drugs are considered to be released by diffusion through the matrix and
poorly soluble drugs are released by erosion of the matrix. Moreover, it is considered
that factors affecting swelling and erosion of these polymers may account for
differences between in vitro dissolution results and subsequent in vivo performance
when hydrophilic matrix tablets are compared [15] .
Lobenzarit disodium (LBZ) is a drug conceived for the treatment of rheumatoid
arthritis. This drug produces an improvement of immunological abnormalities and
has a regulatory effect upon the antibody - producing system. Propranolol hydrochloride
(PPL) is a . - adrenergic blocking agent, that is, a competitive inhibitor of the
effects of catecholamines at . - adrenergic receptor sites. It is widely used in therapeutics
for its antihypertensive, antiangorous, and antiarrhythmic properties. These
two drugs are suitable candidates for the design of controlled - release delivery
systems [25, 29] . According to their solubility in water they can be considered as
high soluble (PPL) and soluble (LBZ) drugs.
A comparative study of the dissolution profi le for PPL and LBZ was established
as the analysis of a similarity factor defi ned as
f
n
R T
t
n
t t 2
1
2
0 5
50 1
1
100 = + . ( ) ...
...
. ...
...
=
.
. log
.
(4)
In the above equation f 2 is the similarity factor, n is the number of time points, R t
is the mean percent drug dissolved of the reference formulation, and T t is the mean
percent drug dissolved of the tested formulation.
The evaluation of similarity is based on the following conditions:
• A minimum of three time points
• Twelve individual values for every time point
• Not more than one mean value of > 85% dissolved
• Standard deviation of the mean that is less than 10% from the second to last
time point
An f 2 value between 50 and 100 suggests that two dissolution profi les are similar
[30] . In this study experimental data corresponding to 30, 60, 90, 120, 180, 240, 300,
360, 420, and 480 min were considered.
Figure 20 shows dissolution profi les for tablets of PPL or LBZ from the native
dextran DTB110 - 1 - 2 matrix system, respectively 1 : 1 (w/w). The value for relative
standard deviation (CV) was less than 5% for all points measured ( n = 12).
The Higuchi and Hixson Crowell model as well as the nonlinear regression of
Peppas and Peppas - Sahlin were employed to study the release data. Higuchi ’ s slope
FIGURE 20 Disolution profi le for PPL and LBZ dextran tablets (direct compression):
paddle 100 rpm, 37 ° C, deionized water.
0 60 120 180 240 300 360 420 480
0
20
40
60
80
100
LBZ
PPL
Amount released (%)
Time (min)
TABLET DESIGN WITH NATURAL PRODUCTS 1007
1008 TABLET DESIGN
(3.179 and 4.500% min . 1/2 for LBZ and PPL, respectively), Korsmeyer ’ s rate constant
(1.195% min . 0.697 and 4.125% min . 0.540 for LBZ and PPL, respectively), the low
relaxational constant K r (0.101% min . 0.898 for LBL and . 0.040% min . 0.898 for PPL),
compared with K d values (2.941 and 6.518% min . 0.449 , respectively) of Peppas - Sahlin
indicated the diffusional mechanism as predominant for soluble drugs since native
dextran tablets. The infl uence of solubility of the drug can be observed for the
hydrophilic matrix (release of PPL is faster than LBZ in correspondence with its
solubility in water). The value of the diffusional exponent, 0.697 (by the Korsmeyer
equation), for the less soluble drug corresponds to the increment of the infl uence of
the erosion mechanism, in agreement with other authors. This can be also observed
in the Peppas y Salhin equation where the negative value obtained for K r for the
dissolution profi le of PPL from dextran tablets should be interpreted in terms of a
relaxation mechanism, which is insignifi cant compared to the diffusion process.
The dissolution profi les for LBZ:B110 - 1 - 2 and PPL:B110 - 1 - 2 tablets were also
compared using similarity factor f 2 . A value obtained for f 2 that is below 50 (37.48)
indicates the infl uence of drug solubility in the dissolution profi le. Furthermore,
other parameters, such as dextran – drug ratio, particle size of polymer and drug,
and infl uence of pH, have to be studied to obtain an optimum and robust
formulation.
The mechanisms of drug release from dextran matrix occur in the early stage by
polymer swelling, and the tablet thickness increases. Soon thereafter, polymer (and
drug) dissolution starts occurring. The polymer dissolves because of chain disentanglement.
Thus, there is a slow diminution of the thickness because of erosion
until, fi nally, the tablet disappears (time > 480 min).
6.3.7 DESIGN TOOLS OF TABLET FORMULATION
Nowadays, most experimentation on tablet formulation development is still performed
by changing the levels of each variable (factor) at a time, in an unsystematic
way, keeping all other variables constant in order to study the effects of that specifi c
variable on the selected response or to fi nd the optimal conditions of a complete
system. This methodology (trial and error) is based on a large number of experiments
and often relies merely on the analyst ’ s experience [31] .
Statistical experimental design, also called design of experiments (DoE), is a
well - established concept for planning and execution of informative experiments.
DoE can be used in many applications. An important type of DoE application refers
to the preparation and modifi cation of mixtures. It involves the use of “ mixture
designs ” for changing mixture composition and exploring how such changes will
affect the properties of the mixture [32] .
In the DoE approach, fi rst process variables are “ screened ” to determine which
are important to the outcome (excipient type, percentage, mixture time, etc.). The
next step is optimization , when the best settings for the important variables are
determined. In particular, response surface methodologies have been successfully
applied in both drug discovery and development [33] . Advances in supporting software,
automated synthesis instrumentation, and high - throughput analytical techniques
have led to the broader adoption of this approach in pharmaceutical discovery
and chemical development laboratories [34] .
The benefi ts of using experimental design together with software to facilitate the
formulation of a tablet for specifi c purposes, from screening to robustness testing,
are well known. This technique has some advantages compared to the trial - and -
error method. By applying a multivariate design for the screening experiments,
many excipients are evaluated using comparatively few experiments.
The formulation work is generally based on designed experiments. Most of the
experiments are fractional or full - factorial designs and are generated and evaluated
in some cases with the center point replicated. The robustness of the formulation
and batch - to - batch variation of the excipients and the active pharmaceutical ingredient
can be evaluated with experimental designs on different occasions. Experimental
design and optimization of the formulation can be performed with the use
of software. Some of them have been useful in tablet design. MODDE (version 4.0
and 5.0, Umetri, Ume a , Sweden), iTAB [35] , and TabletCAD are some
examples.
6.3.7.1 MODDE 4.0
Tablet design for controlled - release propranolol hydrochloride was performed with
the use of MODDE software [25] . A central composite design (one of the most
used designs in pharmaceuticals) was applied to the optimization. This experimental
design required 17 experiments (2 k + 2 k + 3, where k is the number of variables)
including three center points. Three variables and fi ve responses (according to USP
25 tolerances for the dissolution profi le for propranolol hydrochloride extended -
release capsule) were involved in the experimental design. The variables and their
ranges studied are summarized in Table 13 . The high and low values of each variable
were defi ned based on preliminary experiments. The critical responses were
t 100% and t 30% corresponding to the time when 100 and 30% of drug contained in the
tablets is delivered to the dissolution medium because this system was developed
to release drug in 24 h ( t 100% . 24 h) and to prevent an overdose for the fi rst minutes
( t 30% > 1.5 h). The other responses were in the amount of PPL dissolved at 4, 8, and
14 h.
Table 14 shows results obtained for every formula development according to
MODDE 4.0 software. The collected experimental data were fi tted by a multilinear
regression (MLR) model with which several responses can be dealt with simultaneously
to provide an overview of how all the factors affect all the responses. The
responses of the model, R 2 and Q 2 values, were over 0.99 and 0.93 for t 100% and 0.98
and 0.89 for t 30% , respectively, implying that the data fi tted well with the model.
Here, R 2 is the fraction of the variation of the response that can be modeled and
Q 2 is the fraction of the variation of the response that can be predicted by the model.
The relationship between a response y and the variables x i , x j , . . . can be described
by the polynomial:
TABLE 13 Levels of Formulation Variables (Central Composite Design)
Parameter Low Value ( . 1) Central Value (0) High Value ( + 1)
Ratio DT – HPMC (w/w) 1 : 1 4 : 1 7 : 1
Cetyl alcohol (% w/w) 10 15 20
Ratio excipients – PPL (% w/w) 30 50 70
DESIGN TOOLS OF TABLET FORMULATION 1009
1010 TABLET DESIGN
TABLE 14 Matrix of Central Composite Design and Results
Run Order CEx – PPL DT – HPMC ce t 100% * t 30% * t 2 * t 3 * t 4 *
10 30 1 : 1 10 10 0.5 a 71 a 90 a — a
7 70 1 : 1 10 15 2.1 b 44 b 73 b 97 a
1 30 7 : 1 10 11 0.5 a 63 a 82 a — a
11 70 7 : 1 10 14 2.2 b 40 b 76 b 99 a
9 30 1 : 1 20 14 1.3 a 49 b 70 b 99 a
8 70 1 : 1 20 16 2.2 b 46 b 62 b 96 a
5 30 7 : 1 20 14 1.6 b 49 b 70 b 99 a
15 70 7 : 1 20 16 2.4 b 43 b 60 b 96 a
3 30 4 : 1 15 20 1.2 a 54 b 79 b 94 b
16 70 4 : 1 15 26 2.6 b 39 b 56 b 72 b
17 50 1 : 1 15 15 1.6 b 45 b 63 b 98 a
4 50 7 : 1 15 16 1.7 b 43 b 65 b 96 a
2 50 4 : 1 10 21 1.4 a 52 b 71 b 93 b
12 50 4 : 1 20 24 2.2 b 45 b 63 b 82 b
6 50 4 : 1 15 24 2.1 b 48 b 65 b 85 b
14 50 4 : 1 15 23.5 2.2 b 49 b 66 b 86 b
13 50 4 : 1 15 24 2.1 b 48 b 66 b 86 b
Note: cEx – PPL, ratio of excipients to propranolol; DT – HPMC, ratio of native dextran to hydroxypropylmethylcellulose;
ce, percentage of cetyl alcohol (w/w) in the tablets. Time t 100% is time (hours) when
100% of PPL is dissolved in dissolution medium and t 30% is time (hours) when 30% of PPL is dissolved
in dissolution medium; t 2 is percent of PPL dissolved at 4 h, t 3 percent of PPL dissolved at 8 h, and t 4
percent of PPL dissolved at 14 h. Values presented are the average of eighteen replicates for each batch.
a Inside the USP range. b Outside USP range.
y x x x x x x E i i j j ij i j ii i jj j = + + + + + + . . . + . . . . . . 0
2 2
where . j are coeffi cients to be determined and E is the overall experimental error.
Figure 21 presents the dissolution profi les of all 17 trials generated from the central
composite design.
The response surface plots formed by plotting the values for t 30% and t 100% as a
function of the most important variables are shown in Figure 22 , where the optimum
condition obtained by the model can be seen. The optimum dextran (DT) – HPMC
ratio of 4 : 1 (w/w) gave t 100% equal to 24 h.
With tablet formulations composed of a matrix excipient and PPL at a ratio
ranging from 40 : 60 to 70 : 30 (w/w), the values for t 100% were satisfactory (around
24 h). However, the respective values for t 30% increased according to the ratio of
matrix excipient and PPL ranging from 40 : 60 to 70 : 30 (w/w), showing that early
drug release was demanded and the initial dose required for pharmacological effect
could not be suffi cient.
Sustained - release matrix tablets with good properties were obtained with a
dextran – HPMC ratio of 4 : 1 (w/w), with a matrix excipient – PPL ratio of 60 : 40 (w/w),
and with a cetyl alcohol amount of 15% (w/w). The hydrophilic polymers – PPL ratio
of 60 : 40 (w/w) is more robust for any manufactured variability than 50 : 50 (w/w),
because the central point of the design is near the lowest desired area (Figure 22 ).
Under the optimal conditions, the mean value of hardness was 106 ± 3 N and the
friability was less than 1% (0.2%).
FIGURE 21 Dissolution profi le generated from central composite design.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
110
Exp.10
Exp.7
Exp.1
Exp.11
Exp.9
Exp.8
Exp.5
Exp.15
Exp.3
Exp.16
Exp.17
Exp.4
Exp.2
Exp.12
Exp.6
Exp.14
Exp.13
Amount released PPL (%)
Time (h)
FIGURE 22 Response surface plots formed by plotting values for t 30% and t 100% .
30
40
50
60
70
10
12
14
1
6
182
0
1.0
1.5
2.0
2.5
Ratio DT:HPMC (4:1 w/w)
t
30%
ce
cE
xc:P
P
L
30
40
50
60
70
1
2
3
4
5
6
7
12
14
16
18
20
22
24
Cetyl alcohol 15% w/w
t
100%
DT:HPMC
cEx
c:P
PL
DESIGN TOOLS OF TABLET FORMULATION 1011
1012 TABLET DESIGN
Cetyl alcohol (ce in Figure 23 ) has a signifi cant positive effect on both responses
in the range studied. This may be because the hydrophobic polymer prevents the
fast release of PPL for the fi rst few hours with an increase in the diffusional pathlength
of the drug because the swelling of hydrophilic polymers (DT and HPMC)
retards the rate of release. Interaction of cetyl alcohol and hydrophilic polymers –
PPL was observed for t 30% . If a prolonged release rate is desired during this period,
the ratio of hydrophilic polymers to cetyl alcohol can be increased, resulting in a
decreased interspace volume after erosion of cetyl alcohol. In contrast to other
products, such as lactose [15] , cetyl alcohol as hydrophobic polymer can be increased.
The viscosity and texture of the gel layer and some modifi cations in polymer –
polymer and polymer – solvent interaction are present.
6.3.7.2 i TAB
iTAB is I Holland ’ s new tablet design aid that calculates basic tablet parameters
and stress analysis for “ rounds ” and “ shapes ” in three easy steps. iTAB is Windows
FIGURE 23 Regression coeffi cients.
cEx
DT
ce
cEx*cEx
DT*DT
ce*ce
cEx*DT
cEx*ce
DT*ce
-0.4
-0.2
0.0
0.2
0.4
0.6
Variables and interactions, t30%
Regression coefficients
cEx
DT
ce
cEx*cEx
DT*DT
ce*ce
cEx*DT
cEx*ce
DT*ce
-8
-6
-4
-2
0
2
Variables and interactions, t100%
Regression coefficients
based, saving users from any extra set - up costs and making software upgrades
easy.
Users are not required to possess any technical expertise or have any formal
training, as iTAB is very user friendly and intuitive throughout. Using the drag - and -
click feature, iTAB results in 2D drawings ready for production and automatically
generated design reports:
Step 1 involves the selection of a specifi c tablet profi le and modifi cation of key
parameters such as diameter, mass, volume, and surface area in real time. The
iTAB safety zone ensures that unfeasible designs cannot be output from the
system, meaning that only quality tablets can be produced.
Step 2 runs a real - time fi nite - element analysis (FEA) simulation against the
design to give an indicative punch tip maximum force calculation.
Step 3 automatically produces a report summarizing key design data, for example,
cup depth and surface area, that can be emailed directly to the I Holland team
for further analysis, design work, and tooling production.
iTAB allows non – computer - aided design (CAD) and non - FEA users to simply and
quickly design nonembossed tablets and obtain detailed information with the click
of a button.
Core benefi ts include the avoidance of tablet manufacturing problems such as
punch tip breakage from the outset. Immediate and accurate punch tip load calculations
and 3D dynamic rotation of punch tip stress allow for instant decision making
on design issues. iTAB is also unique in that it incorporates tooling and tablet
design.
6.3.7.3 Percolation Theory
Leuenberger et al. introduced percolation theory in the pharmaceutical fi eld in 1987
to explain the mechanical properties of compacts and the mechanisms of the formation
of a tablet [36, 37] . Knowledge of the percolation thresholds of a system results
in a clear improvement of the design of controlled - release dosage forms such as
inert or hydrophilic matrices.
Percolation theory is a statistical theory that studies disordered or chaotic systems
where the components are randomly distributed in a lattice. A cluster is defi ned as
a group of neighboring occupied sites in the lattice, being considered an infi nite or
percolating cluster when it extends from one side to the rest of the sides of the
lattice, that is, percolates the whole system [38] .
Thus, a tablet is regarded simply as a heterogeneous binary system formed by
the active principle and an excipient. As a function of their relative volume ratio,
one or both components constitute a percolating cluster formed by particles of the
same component that contact each other from one side to the other sides of the
tablet.
In a binary pharmaceutical tablet (cylindrical lattice), the sites can be occupied
by the component drug or excipient. The percolation threshold of the drug indicates
at which concentration this substance dominates the drug/excipient system. The
concept is very similar to the point where a component passes from being the inner
DESIGN TOOLS OF TABLET FORMULATION 1013
1014 TABLET DESIGN
to being the outer phase of an emulsion. It is not surprising that the component
becoming the “ outer phase ” or percolating phase will have more infl uence on the
properties of the system.
Furthermore, the concentration point at which a component is starting to percolate
the system is usually related to a change in the properties of the system, which
will now be more affected by this component. This is known as a critical point. Close
to the critical point important changes can take place, for example, changes in the
release mechanism of the active agent and modifi cation of the tablet structure
(e.g., monolith versus a desegregating device).
An important difference between particulate solids and emulsions is that in the
solids two components can percolate the system at the same time, that is, two components
can act as the outer phase simultaneously. In this case the system is known
as a bicoherent system.
Study of Ternary Tablets Percolation theory has been developed for binary
systems, however, drug delivery systems usually contain more than two components.
The existence and behavior of the percolation thresholds in ternary pharmaceutical
dosage forms have been studied [39] employing mixtures of three substances with
very different hydrophilicity and aqueous solubility (Polyvinylpyrrolidone (PVP)
cross - linked, Eudragit RS - PM, and potassium chloride).
After evaluation of the technological parameters and in vitro release behavior
of the tablets, no sharp percolation thresholds were found in these ternary systems
for the employed components separately. Nevertheless, a combined percolation
threshold of the hydrophilic components was found, demonstrating that a multicomponent
system can be reduced to a binary one using a discriminating property
[39] .
Matrix Systems with Different Particle Sizes Another disadvantage in the application
of percolation theory to the rationalization of the pharmaceutical design was
the prerequisite of an underlying regular lattice. Usually, drug delivery systems
contain substances with different particle sizes. Therefore, the particles cannot be
considered as each occupying one lattice site.
This problem can be initially overcome using a volume ratio instead of a lattice
site ratio, expressing the percolation thresholds as critical volume fractions [36,
40 – 42] . Nevertheless, the infl uence of the particle size of the components on the
percolation threshold cannot be explained using a volume fraction; that is, from this
point of view, tablets with the same excipient volume are equivalent independent
of their particle size. A fi rst qualitative study of the infl uence of particle size on the
percolation threshold [43] demonstrated that this is in clear disagreement with
experimental data.
According to percolation theory, the effect of a reduction in the drug particle size
should be similar to an increase in the excipient particle size in a binary system: It
may be expected that the relative particle size of the component, but not its absolute
particle size, will determine the properties of the system.
A quantitative study of the infl uence of particle size on the percolation threshold
employing inert matrix tablets prepared with KCl and Eudragit RS - PM as matrix -
forming material [44 – 46] showed that experimental data are in agreement with this
hypothesis.
As Figure 24 shows, a linear relationship was found in this study [44] between
the mean drug particle size and the corresponding drug percolation threshold
(Figure 24 , line A ). Furthermore, the excipient particle size exerts a contrary effect
than the drug particle size (Figure 24 , line B ); that is, the larger the excipient particle
size, the lower the drug percolation threshold [46] .
In addition, when the obtained drug percolation thresholds were plotted as a
function of the drug – excipient particle size ratio of the matrices (see Figure 25 ), a
linear relationship was found between the drug percolation threshold and the relative
drug particle size [46] . These results are in agreement with the above exposed
theoretical model based on percolation theory.
One of the advantages of the proposed model versus classical theories is its ability
to explain the changes in the release behavior of the matrices by means of a change
in the critical points of the system (related to the drug and excipient percolation
thresholds), which can be experimentally calculated, providing a scientifi c basis for
the optimization of these dosage forms.
FIGURE 24 Drug percolation threshold (mean ± SE) as function of mean particle size of
drug (line A ) and excipient (line B ) employed.
Percolation threshold (.c)
A
B
Mean size (.m)
0.6
0.4
0.2
0
0 50 100 150 200 250 300 350 400
FIGURE 25 Drug percolation threshold (mean ± SE) as function of drug – excipient particle
size ratio employed.
Percolation threshold (.c)
Drug–excipient particle size ratio
0.6
0.4
0.2
0
0 0.5 1 1.5 2 2.5 3 3.5
DESIGN TOOLS OF TABLET FORMULATION 1015
1016 TABLET DESIGN
Mechanical Properties The percolation approach was also employed to model the
tensile strength of tablets [47, 48] . A critical tablet density was here understood as
a minimal solid fraction needed to build a network of relevant contact points spanning
the entire tablet. A rising tablet density led to a power law increase of the
tensile strength showing an universal exponent T f = 2.7.
It was shown that a power law based on percolation theory was suitable to fi t
the obtained tensile strength data of the binary matrix tablets studied. The best
fi tting was observed for a model where an initial tensile strength . 0 was supposed
[49] :
. . . . t c = . + k( ). 2 7
0
The observed critical relative densities are understood as threshold values for the
tensile strengths of the tablets. One practical consequence of these works is to avoid
the manufacture of matrix tablets close to these critical densities. The formulation
may not be robust in this critical range from the viewpoint of mechanical tablet
stability.
6.3.7.4 Artifi cial Neural Networks
Artifi cial neural networks (ANNs) are computer programs designed to model the
relationships between independent and dependent variables. They are based on the
attempt to model the neural networks of the brain [50] . Functions are performed
collectively and in parallel by the units, rather than there being a clear delineation
of subtasks to which various units are assigned.
This methodology represents an alternative modeling technique that has been
applied to pharmaceutical technology data sets, including tableting parameters [51] .
The main advantage with respect to classical statistical techniques, such as response
surface methodology, is that ANNs do not require the prior assumption of the nature
of the relationships between input and output parameters, nor do they require the
raw data to be transformed prior to model generation [51] . ANNs are capable of
modeling complex, nonlinear relationships directly from the raw data.
The functional unit of ANNs is the perceptron. This is a basic unit able to generate
a response as a funtion of a number of inputs received from others perceptrons.
For example, the response value can be obtained as follows:
Y
W I WI W
W I WI W
=
+ + >
+ + . {1 0
0 0
0 0 1 1
0 0 1 1
if
if
b
b
where I x is the input received from perceptron x and W x the weight assigned to this
input by the perceptron. The weights can be changed to adapt the answer to the
desired one using a learning algorithm.
Usually complex structures with more than 15 layers are employed, called the
multilayer perceptron (MLP). Some of the commercial programs which have been
used to fi t tableting parameters are INForm (Intelligensys, Billingham Teesside),
CAD/Chem (AI Ware, Cleveland, OH), which is no longer commercially available,
and the Neural Network Toolbox of MATLAB (MathWorks, Natick, MA).
6.3.8 COATING SYSTEMS
Coating processes have come to play an important role for the protection of substances
prior to application or for their sustained release. Coatings on cores usually
consist of a mixture of substances. The matrix formers are responsible for the stability
of the coating structure, and they also determine the coating process. Depending
on the type of matrix former on binder used, three coating categories can be
distinguished:
Coatings with Sucrose and Other Sugars Permit application of copious amounts
of mass to the core and are widely used in the manufacture of pharmaceuticals
and confectionery.
Hot Melts Add a considerable amount of mass and are applied hot and solidify
while cooling on the core. They are mainly used for confectionery. The most
important raw materials are fats, mostly cocoa fat, polyethylene glycol (PEG)
[52] , and the sugar – alcohol mixture xylitol – sorbitol.
Film Coatings Require less material, forming thin membranes which largely
follow the contours of the substrate, for example, scores and engravings. The
partly pH - dependent solubility and selective permeability of coatings are
affected by the fi lm formers. Such fi lms are sometimes also used as intermediate
layers in sugar coatings.
Whether or not a core is suitable for coating depends on its hardness, shape,
surface, size, heat sensitivity, and tendency to interact with the coating material.
Moreover, since sugar and fi lm coating processes involve very different techniques,
they place different demands on the cores to be used [53] . Tablets used as cores
must be biconvex in shape to prevent them from sticking together like coins in a
roll.
The ideal tablets for sugar coating will have a pronounced convex curvature and
a narrow band. The consumption of coating material depends very much on the
tablet shape and increases sharply if the tablets are not round. Film coating supplies
coated products in which the core surface (e.g., with notches, engravings, and
defects) is faithfully reproduced.The fi lms tend to chip at sharp edges or are particularly
thin in these areas. For this reason slightly curved tablet cores are preferred
for fi lm coating [54] .
Film coating of pharmaceuticals is a common manufacturing stage for the following
reasons: (i) to provide physical and chemical protection for the drug, (ii) to
mask the taste or color of the drug, or (iii) to control the release rate or site of the
drug from the tablet. When a coating composition is applied to a batch of tablets
or granules (or to a batch of liquid drops or even gas bubbles), the core surfaces
become covered with a polymeric fi lm that is formed as the surfaces dries. The major
component in a coating formulation is a fi lm - forming agent which ideally is a high -
molecular - weight polymer that is soluble in the proper solvent (today, most preferably
in aqueous - based media). The polymer forms a gel and produces an elastic,
cohesive, and adhesive fi lm coating.
In the pharmaceutical industry, organic - solvent - based fi lm coatings have been
used for over 40 years. In the 1990s, however, interest and demands in the use of
aqueous - based fi lm coating systems rapidly increased owing to the well - documented
COATING SYSTEMS 1017
1018 TABLET DESIGN
drawbacks (unsafe, toxic, pollutive, and uneconomic) associated with organic -
solvent - based coating systems. Consequently, and for the reasons mentioned above,
much effort has been focused on the research and development of new aqueous -
based fi lm coating formulations. Nowadays, aqueous - soluble/dispersable polymers
available on the market consist primarily of either cellulose polymers, PEGs, or
acrylate copolymers. There are, however, some material - related limitations in using
these polymers in aqueous - based fi lm coatings. Consequently, application of new
fi lm formers such as chitosan, native starches, and special types of proteins for
pharmaceuticals and foodstuffs has been increasingly studied.
The choice between sugars of fi lm coatings depends not only on the desired coatings
quality but also on the technical requirements, that is, on the economy of the
process. The fi nancial outlay for a selected technology often commits the manufacturer
to this technology for a prolonged period of time. Film and sugar differ
substantially in thickness and therefore also in the necessary mass of coating
material.
The most important coating raw material is the fi lm - forming polymer, which must
be able to produce a coherent fi lm on the substrate under the given process conditions
[55, 56] . Second in importance is the solvent or dispersing system in which the
polymer is applied to the surface and introduced to form a fi lm. Other frequently
used raw materials are plasticizers, glidants, fi llers, and colorants. All these substances
act together and infl uence the properties of the fi lm.
The fi rst decision to be made when developing a formulation concerns the desired
function of the fi lm. Depending on the requisite dissolution performance in physiological
media, the fi lm former to be tested — or several of them — is then selected
from the available polymers. The economy of the coating process and the quality of
the coated product depend on the correct calculation of the required amount of
coating material. Empirical adjustment of new developments to existing products is
therefore not recommended. The coating quantity needed for fi lm coatings is directly
related to the surface area of the core. Table 15 shows a simplifi ed calculation of the
surface area of tablets. It is based on the surface area of a cylinder circumscribing
these shapes. All these data are taken from the literature. For sugar coating processes,
the shape of the cores and end products have to be studied stereometrically.
The sugar coating process balances irregularities, since the coating buildup does not
follow the structure of the surface. Areas which require a high degree of rounding,
such as high bands and very fl at curvatures, attract more coating mass. Where the
band height varies owing to manufacturing technique, this results in weight differ-
TABLE 15 Calculation of Surface Area ( mm 2 ) for Different Types of Cores
Height (mm)
Diameter (mm)
3 4 5 6 7 8 9 10 12 14
2 33 56 70 95 120 150
3 42 62 85 115 145 175 210 250 340
4 75 100 130 165 200 240 280 380 485
5 185 225 270 315 415 530
6 300 345 450 570
7 505 615
8 660
ences between the individual sugar - coated tablets. This is one of the reasons why
sugar - coated products always show a much wider weight distribution than fi lm -
coated tablets.
Several authors have made proposals for the most convenient size of tablet for
sugar coating. A further rule has been established according to which the convex
radius should be between 0.7 and 0.75 times the tablet diameter and the band height
between 0.07 and 0.12 times that diameter. However, for calculation of the band
height only a factor of 0.12 is advisable, since, otherwise, the minimum value of
1 mm will be fallen short of by far [53, 57 – 60] . For fi lm coating the recommended
convex radius is 1.5 times the tablet diameter (less curved tablets are preferred).
Numerous formulation are available for all fi lm - forming polymers offered in the
market. Thus, it is possible to dispense with many of the preliminary tests and
concentrate development work on the special problems of the formulation in question.
In this chapter, we will focus on SEPIFILM and Kollidon VA 64. Table 16
presents proven basic formulations reported on in the literature [61] and polymers
used for the most important commercially available fi lm formers, which also can be
used as a basis for further tablet coating.
The major operational coating process parameters related to fi lm coating are able
to be measured and monitored continuously in some pan - type coaters. The inlet
airfl ow rates infl uence the coating process and the subsequent quality of the coated
tablets. Increasing the inlet airfl ow rate accelerated the drying of the tablet surface.
At high inlet airfl ow rate, obvious fi lm coating defects, that is, unacceptable surface
roughness of the coated tablets, are observed and the loss of coating material
increased.
Today advantages of aqueous fi lm coating are well recognized and fi lm coating
technology is much developed to successfully perform these types of coatings.
Process automation and monitoring of critical process parameters can be utilized
to increase the overall process effi ciency and predictability and to improve the
homogeneity and reproducibility of the tablet batches. This will ensure high quality
and safety of the fi nal coated products, which are mandatory requirements of tablet
manufacturing.
6.3.8.1 Subcoating of Tablet Cores as a Barrier to Water
As tablets are nowadays coated mostly with aqueous solutions or dispersions, it has
become increasingly necessary to provide the tablet cores with a barrier layer prior
to sugar or fi lm coating. This is mainly to protect water - sensitive drugs against
hydrolysis and chemical interactions, for example, between different vitamins, and
to prevent the swelling of high - performance tablet desintegrants that are very sensitive
even to small quantities of water. It can be especially useful when controlled -
release systems with hydrophilic polymer are studied and the water contained can
change the dissolution profi le. Kollidon VA 64 also can be used to improve the
adhesion of subsequent coatings by hydrophilization of the surface.
6.3.8.2 Kollidon VA 64
We are studying the ability of Kollidon VA 64 as a subcoating in a combined hydrophilic
(dextran – HPMC) – hydrophobic (cetyl alcohol) matrix core prior to sugar
COATING SYSTEMS 1019
1020 TABLET DESIGN
TABLE 16 Basic Formulations for Film - Coated Tablets
Formulation with Hydroxypropyl Methylcellulose (HPMC)
A B
Oprady 73.0% —
Pharmacoat — 80.0%
PEG 6000 — 8.0%
Talc 20.0% 5.0%
Pigments included, TiO 2 7.0% 7.0%
Solid content 20.0% 12.0%
Coating quantity, mg/cm 2 1 – 5 1 – 5
Formulation with Methacrylic Acid Copolymers
A B C
Eudragit L100 5.0% — —
Eudragit S100 — 7.5% —
Eudragit L30D - 55 — — 16.5%
PEG 6000 0.7% 1.0% 1.6%
Talc 6.0% 2.0% 4.0%
Pigments included, TiO 2 3.3% — —
Isopropyl alcohol 41.0% 86.5% —
Acetone 41.0% — —
Water 3.0% 3.0% 77.9%
Solid content 14.0% 10.5% 22.1%
Coating quantity, mg/cm 2 2 – 4 2 – 4 3 – 5
Formulation with Hydroxypropyl Cellulose Acetate Succinate (HPMCAS)
Aqoat AS - MF 10.0%
Triethyl citrate 2.8%
Talc 3.0%
Sorbitan sesquioleate 0.0025%
Water 84.2%
Formulation with Ethylcellulose (EC)
A B
Ethylcellulose 5.0% —
Aquacoat (30% solids) — 30.0%
PEG 6000 — 2.0%
Glycerol triacetate 1.0% —
Ethanol 94.0% —
Water — 68.0%
Formulation with Carboxymethyl Ethylcellulose (CMEC)
Duodcell 8.0%
Trisodium citrate 0.70%
Tween 80 0.04%
Glycerol monocaprylate 2.40%
Water 88.86%
Coating quantity 7 mg/cm 2
Formulation with Polyvinyl Acetate Phthalate (PVAP)
PVAP 11.0%
PEG 400 1.0%
Ethanol 66.0%
Water 22.0%
coating. The copovidone (i.e., Kollidon VA 64) not only increases the mechanical
properties of the tablet (less friability) but also prevents the amount of water
absorbed from the air in tropical and subtropical stability conditions (25 and 75%
relative humidity).
Figure 26 shows a comparative study of uncoated tablet and sugar - coated tablet
after a barrier to water with copovidone 0.5 mg/cm 2 of the warm tablet cores using
a 10% solution in ethanol. During two years the coating tablets (with initial humidity
2.5%) remained stable and dissolution profi les were similar to the initial time
with similarity f 2 = 82 observed and friability decreased from 0.25% (uncoated
cores) to 0.02% (coated tablets).
Similar results are reported in the literature when Kollidon VA 64 is compared
to povidone (Kollidon K 25, K 30, and 90 F). Copovidone absorbs about three times
less water than the other soluble Kollidon K 25, 30, and 90F after seven days at
25 ° C up to 80% relative humidity [62] . Kollidon VA 64 is manufactured by free -
radical polymetization of 6 parts of vinylpyrrolidone and 4 parts of vinyl acetate in
2 - propanol. A water - soluble copolymer with a chain structure is obtained. In contrast
to the soluble grades of Kollidon, the number 64 is not a K value but the mass
ratio of the two monomers, vinylpyrrolidone and vinyl acetate. The K value of
Kollidon VA 64 is of the same order of magnitude as that of Kollidon 30. Synonyms
for Kollidon VA 64 are copovidone, copovidonum, copolyvidone, copovidon, and
PVP - VAc - copolymer [Eur. Ph., Japanese Pharmaceutical Excipients, and USP
National Formulary (NF)] [63] .
Copovidone forms soluble fi lms independently of the pH value, regardless of
whether it is processed as a solution in water or in organic solvents. He offers better
plasticity and elasticity than other povidones. On the other hand, fi lms are also less
tacky. Kollidon VA 64 usually absorbs water, and it is seldom used as the sole fi lm -
forming agent in a formulation. Normally it is better to combine it with less hygroscopic
substances such as cellulose derivates [54] , shellac, polyvinyl alcohol (PVA),
FIGURE 26 Water absorption of precoating and uncoated cores of combinated dextran –
HPMC matrix tables.
0 100 200 300 400 500 600 700 800
0
1
2
3
4
5
6
7
8
Water absorption, %
Days
Precoating cores with Kollidon VA 64
Uncoated cores
COATING SYSTEMS 1021
1022 TABLET DESIGN
PEG (e.g., Macrogol 6000), or sucrose. Others plasticizers such as triethyl citrate,
triacetin, or phtalates are not required. The properties of coatings can be improved
with combination copovidone – cellulose derivates [64, 65] . Cellulose polymers of
high viscosity, such as HPMC 2910, are used in fi lm coating. Spray suspension at
12% HPMC K4M offers values of viscosity above 700 mPa · s and can not be normally
used because 250 mPa · s is considered the limit for spraying of a coating suspension.
This value of viscosity can be reduced signifi cantly up to 250 mPa · s if 60%
HPMC is substituted by Kollidon VA 64 [66] and this leads us to apply this polymer
concentration and therefore to economize the spraying procedure.
6.3.8.3 SEPIFILM
SEPIFILM and SEPISPERSE Dry are ready - to - use, immediate - release, fi lm coating
compositions designed for pharmaceutical and nutritional supplement applications.
Based on a unique technology, SEPIFILM/SEPISPERSE Dry are granular forms
offering the following benefi ts:
Easy handling: no dust
Easy mixing: no lumps, no foam
Homogeneous composition: no segregation
Most coating compositions are based on hypromellose (HPMC) as fi lm - forming
polymer and contains microcrystalline cellulose (MCC) (see Table 17 ).
SEPPIC was the fi rst company to introduce MCC in coating formulations some
20 years ago. The use of MCC allows higher solid content and enhances the adhesion
of the fi lm to the tablet core, consequently improving logo defi nition. Different
formulas are provided by Sepifi lm, such as SEPIFILM LP, SEPIFILM 003 and 752,
and SEPISPERSE Dry. The last one can be provided with SEPIFILM and Kollicoat
IR.
Moisture Protection of SEPIFILM LP Water vapor transmission rates were
measured on free fi lms, including titanium dioxide (Figure 27 ) . SEPIFILM LP
shows signifi cantly lower moisture permeability compared to regular or PVA - based
coating formulations. Removal of titanium dioxide (SEPIFILM LP clear) improves
moisture resistance.
Like many other herbal extracts, valerian extract is very hygroscopic. Inclusion
into tablets raises stability issues. Coatings reduce moisture absorption but often
lead to tablet explosion or visual deterioration (black specs). Tablets formulated
with 250 mg valerian extract, spray - dried lactose, and compressible starch were
TABLE 17 General Formulation for SEPIFILM Coating
Film
Film - forming agent Hypromellose (former HPMC)
Binder Microcrystalline cellulose
Hydrophobic plasticizer Stearic acid (vegetable origin)
Colors Pigments, lakes
FIGURE 27 Water vapor transmission rates of free fi lms.
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
mg/h·m
Sepifilm
LP clear
Sepifilm
LP
HPMC +
Macrogol
stearate
HPMC +
PEG 400
PVA
FIGURE 28 Tablets coated with ( a ) conventional hypromellose and ( b ) Sepifi lm LP 770
white.
(a) (b)
stored at 40 ° C and 90% RH (relative humidity). Pictures were taken after one
month storage (Figure 28 ).
Tetrazepam Tetrazepam, a well - known muscular relaxant, undergoes chemical
degradation when exposed to moisture and oxygen. 3 - Ketotetrazepam is one of the
main degradation substances. The amount of 3 - ketotetrazepam, measured by HPLC,
has been monitored under 25 ° C/60 RH and 40 ° C/75% RH aging conditions.
SEPIFILM LP 770 signifi cantly improves the stability of tetrazepam. Uncoated
tablets and tablets coated with a PVA - based formulation show a strong increase in
the amount of degradation substance (Figure 29 ).
Dissolution Profi le SEPIFILM LP can effi ciently improve the moisture barrier
on moisture - sensitive active pharmaceutical ingredients (API) or hygroscopic cores.
The breakthrough in this technology is that SEPIFILM LP does not modify the
dissolution profi le when compared to conventional coating (Figure 30 ).
COATING SYSTEMS 1023
1024 TABLET DESIGN
FIGURE 29 Stability of tetrazepam: uncoated tablets and different tablets coated. Dosage
of 3 - ketotetrazepam in 50 mg tetrazepam tablets stored at 25 ° C and 60% RH leads to partial
degradation of tetrazepam at T 0 . Initial amount of 3 - ketotetrazepam is lower in uncoated
tablets: cores are likely to absorb water during the aqueous coating process.
0.45%
0.40%
0.35%
0.30%
0.25%
0.20%
0.15%
0.10%
0.05%
0.00%
0 1
month
2
months
3
months
4
months
Uncoated
PVA based
Sepifilm LP 770
FIGURE 30 Dissolution profi le of tetrazepam, 3 months storage at 40 ° C and 75% RH.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
% Released
0 5 10 15 20
min
25 30 35 40 60
PVA based
Uncoated
Sepifilm
LP 770
HPMC/
PEG based
SEPIFILM 003 and 752 The association of cellulose with a fi lm coating agent
was originally patented by SEPPIC. Microcrystalline cellulose is probably one of
the most extensively used excipients in pharmaceutical and nutritional products.
Unlike other fi llers, such as lactose, cellulose is inert, vegetable derived, and accepted
worldwide and its shelf life is unlimited. Figure 31 shows the advantage of cellulose
microcrystalline in fi lm coating.
Faster Film Coating Operations Cellulose is insoluble and does not increase viscosity.
Dispersions with higher solid content can therefore be used and total spraying
time is signifi cantly decreased.
The maximum acceptable viscosity of a typical coating dispersion is 500 mPa · s,
which corresponds to an 11% hypromellose 6 mPa · s dispersion. SEPIFILM can be
dispersed up to 15% in order to reach the same viscosity (Figure 32 ).
FIGURE 31 ( a ) Tablet coated with HPMC smooth fi lm, medium discontinuity between fi lm
and core. ( b ) Tablet coated with SEPIFILM 752 white: clear edge perfectly coated, good
adhesion of fi lm to core and continuity between fi lm and core.
(a) (b)
FIGURE 32 Viscosity for deferments HPMC coating dispersion.
1400
1200
1000
800
600
400
200
0
0 5% 10% 15% 20%
Sepifilm 003 HPMC 6 cPs HPMC 15 cPs
Viscosity, cPs
Enhanced Film Adhesion Comparative adhesion values were measured on 3%
coated placebos using a modifi ed crushing strength tester: Regular fi lm formulation
exhibited an adhesion value of 4 N. Addition of cellulose signifi cantly improved fi lm
adhesion as it became impossible to remove fi lm without disrupting tablets or fi lm.
Breakage occurred for an applied force of 6 N, which can be considered a minimum
adhesion value (Figure 33 ).
Ingredient Segregation Avoided Powders such as hypromellose and titanium
dioxide exhibit dramatically different particle size distribution and density. Such
particle heterogeneity may result in constituent segregation inducing fi lm imperfections
or color deviations on tablets.
FIGURE 33 Tablet surface and edges are smoothly covered whereas no logo bridging is
observed.
COATING SYSTEMS 1025
1026 TABLET DESIGN
As seen in (Figure 34 ), SEPIFILM rules out this weak feature of powdered
coating agents as all constituents are closely bound together. Ingredient segregation
is avoided and batch - to - batch consistency is therefore guaranteed.
The PVA – PEG graft copolymer Kollicoat IR is the new instant - release, aqueous
coating polymer from BASF. Due to its low viscosity and excellent mechanical
properties, it permits solid content of up to 30% and leads to fi ne and smooth tablet
fi lm coatings.
In 2005, SEPPIC and BASF Pharma Solutions began collaboration in marketing
tablet fi lm coating systems. From this collaboration, a new range of colored
SEPIFILM coating systems based on Kollicoat (a registered trademark of BASF
Aktiengesellschaft) polymers will be developed to meet individual customer needs.
Kollicoat IR has already been approved in Europe as a fi nished drug in Germany
(a reference member state in a mutual recognition procedure). Common uses in
pharmaceuticals depend on regulations of each country and defi nition of uses.
Actual regulations of a specifi c country and/or application should be checked before
use.
For pharmaceuticals, this combination is based on 88% of Kollicoat ® IR White
and 12% of coloring system SEPISPERSE ™ Dry.
Recommended Equipment
A propeller stirrer is standard equipment even though a defl occulating blade is
very effi cient.
Deionized or distilled water at room temperature.
The blade diameter should be 3 times shorter than the tank ’ s width.
The tank should be 1.5 times higher than wide. The blade should be slightly off
center.
The blade should be positioned close to the bottom of the vessel.
FIGURE 34 Granules of SEPIFILM 1306 green ( . 350). Pigments, lakes, and cellulose fi bers
are thoroughly coprocessed into homogeneous granules.
TABLE 18 SEPIFILM LP (with or without SEPISPERSE Dry)
Manesty XL
Lab01 (A * )
Manesty
Accelacota
(B)
IMA - GS HT/M
(C)
Driacoater
500 (C)
Solid content LP 770 at
12%
LP 014/Dry
at 12%
LP 014/Dry at
11%
LP 014/Dry
at 12%
Batch size, kg 5 100 88.2 3
Spraying rate 15 – 27 mL/min 200 – 300 g/min 180 – 200 mL/min 7 – 15 g/min
Inlet air temperature,
° C
60 70 – 75 62 55 – 60
Outlet air
temperature, ° C
47 — — 42
Bed temperature, ° C 43 40 – 45 36 – 40 —
Atomizing air/pattern
pressure, bars
2 3.5 – 5 2.5 3
Airfl ow, m 3 /h 440 1800 1000 270
Spraying time, min
(% weight gain)
76 (1% w.g.) 60 (2% w.g.) 90 (2.2% w.g.) 70 (3% w.g.)
Mixing Procedure
SEPIFILM Product Range
1. Adjust rotation speed in order to create a vortex.
2. Quickly pour SEPIFILM into the vortex.
3. Reduce mixer speed to avoid drawing air into the liquid (risk of foam
formation).
4. Increase speed again as viscosity builds up.
It is not necessary, though recommended, to keep stirring during the coating
process.
Kollicoat® White + SEPISPERSE Dry
1. Adjust rotation speed to create a vortex.
2. Quickly pour SEPISPERSE Dry into the vortex.
3. Reduce mixer speed to avoid drawing air into the liquid. Stir for 15 min.
4. Pour Kollicoat IR White into the colored dispersion.
5. Stir an additional 5 min.
Some practical conditions for SEPIFILM and equipment machines are shown in
Tables 18 – 20 .
We used SEPIFILM LP in ranitidine core because this is a moisture - sensitive
drug and can be a challenge to formulators because of its tendency to hydrolyze
when exposed to humidity and/or high temperatures.
Cores of 300 mg that contained 50% ranitidine HCl, cellulose microcrystalline
(PH - 102) 27.25%, pregelatinized starch 22%, Aerosil 0.5%, and magnesium stearate
COATING SYSTEMS 1027
1028 TABLET DESIGN
TABLE 19 SEPIFILM Formulations 050 and 752
Driacoater 500 Glatt Coater 1000
Solid content of dispersion SEPIFILM 050 at 15% SEPIFILM 752 at 20%
Batch size, kg 3 80
Spraying rate, g/min/kg 7 (1 nozzle) 3.1 (3 nozzles)
Rotation speed, rpm 10 8
Inlet air temperature, ° C 60 65
Outlet air temperature, ° C 44 —
Bed temperature, ° C 39 – 40 38 – 40
Atomizing air/pattern pressure, bars 3 3.5
Airfl ow, m 3 /h 330 1800
Spraying time, min (3% weight gain) 85 55
TABLE 20 Kollicoat IR White + SEPISPERSE Dry
Manesty Accelacota 24 in.,
300 - mg Propranolol HCl Tablets
Manesty Premier 200,
Placebo Tablets
Solid content of dispersion IR + Dry at 20% IR + Dry at 20%
Batch size, kg 7 180
Spraying rate 5.3 g/min/kg 300 – 375 mL/min
Inlet air temperature, ° C 60 60
Outlet air temperature, ° C 38 – 43 45
Bed temperature, ° C 39 – 41 —
Atomizing air/pattern
pressure, bars
2/1 2.5/2.5
Airfl ow, m 3 /h 220 2200
Spraying time, min
(%weight gain)
37 (3.4% w.g.) 80 (3% w.g.)
0.5% were coated to a 3% weight gain similar to case A * (see Table 18 ). Tablet
weight, diameter, thickness, hardness, and disintegration times were measured after
coating. The fi lm - coated tablets were packaged in bottles with a desiccant. Stability
testing was conducted at 40 ° C/75% RH for 12 months. Application of fi lm coating
(3% wg) resulted in a slight increase in tablet hardness (tablet breaking force 13 –
14 kp). Tablet disintegration time was not signifi cantly affected by the fi lm coating
application (around 13 min) and 100% of the drug was released within 25 min
compared to the USP limit of not less than 80% ( Q ) in 45 min. Figure 35 shows the
dissolution profi les for the uncoated and coated tablets.
No signifi cant changes were recorded for coated tablets after 12 months of
storage for any property measured (Table 21 ). The stability of this formulation is
partly due to the inclusion of pregelatinized starch in the formulation. Starch 1500
acts as a moisture scavenger and retains moisture in its complex structure of glucose
polymer chains. A slight increase in tablet hardness was seen after storage (see
Figure 36 ). No signifi cant changes were seen in the disintegration time after
storage.
FIGURE 35 Dissolution profi le of coated and uncoated ranitidine HCl tablets.
0
20
40
60
80
100
120
0 10 20 30 40 50
Time (min)
Percent released
Uncoated tablet
Coated tablet (3%
WG)
TABLE 21 Stability Data Summary: Test
Test USP Limit Initial 1 month 3 months 6 months 12 months
Breaking force (kp) NMT 1.00% 13.7 14.4 14.2 14.5 14.4
Friability, % 0.0 0.0 0.0 0.0 0.0
Dissolution, T85%,
min
NLT 85% in
45 min
16 16 16 16 16
Assay, % 90 – 110 102 100 100 99 99
Impurities, % NMT 2% 0.5 0.8 1.0 1.2 1.7
Notes: For 40 ° C/75% RH storage conditions. Abbreviations: NMT, no more than; NLT, no less than.
FIGURE 36 Coated tablet hardness on storage, 40 ° C, 75% RH storage conditions.
0
2
4
6
8
10
12
14
16
1 2 3 4 5
Tablet breaking force (kp)
Selected Coating Problems and Practical Solutions
(a) Defective Coatings Caused by poor quality of the core or inadequate coating
formulations.
(b) Chipping Solid content is too high or it is too brittle for want of plasticizer
and the fi lm does not adhere properly to the substrate surface (too lipophilic
surface or surface lacking in porosity).
Solution Revise the formulation for core and coating.
COATING SYSTEMS 1029
1030 TABLET DESIGN
(c) Blistering Drying or spraying is performed at high speed, and solvent may
be retained in the fi lm. They evaporate on postdrying and may then form
blisters in the fi lm.
Solutions Lower the inlet air temperature and reduce the spray rate.
Check if adhesion of the fi lm and core is adequate.
(d) Cracks in Film or Along Edges Caused by too much internal stress, owing
to differences in the thermal expansion of fi lm and core or also caused by
the swelling of the core during the coating operation.
(e) Embedded Particles Particles broken off from the core are embedded in
the fi lm during spraying.
Solutions The core lacks mechanical stability. Check the formulation of
the core.
(f) Picking The fi lm surface contains substances that are not molecularly dispersed
and start to melt at the core bed temperature of the fi lm coating
process. These substances (e.g., PEG, stearic acid) may interfere with the
fi lm - forming polymers and produce holes in the fi lm surface [67] .
Solutions Replace these substances or lower the core bed temperature.
Decrease the speed of praying.
(g) Dull Surfaces The fi lm will be dull and totally devoid of gloss if a coating
process does not produce the requisite smooth surface. This happens if the
spray droplets start to dry before reaching the cores and are too viscous to
form a smooth fi lm.
Solutions (1) Lower the inlet air temperature and reduce the atomizing
air quantity or pressure. (2) Add substances that enhance fi lm formation,
for example, plasticizer or extra solvents.
(h) Twinning The cores permanently stick together.
Solutions Decrease the excessive spraying. Check the tablet shape and
bands. If the tablets are plane or almost plane, this continues until many
of them stick together.
(i) Bridging the fi lm fails to follow the contours of the tablet over break lines
or engravings and settles in these without adhering to the substrate. The
bridging forces in the fi lm exceed the interfacial forces between fi lm and
core.
Solutions change the tablet surface or add plasticizer.
6.3.9 DEVELOPMENT OF PHARMACEUTICAL TABLETS USING
PERCOLATION THEORY
In 1991, Bonny and Leuenberger [40] explained the changes in dissolution kinetics
of a matrix controlled - release system over the whole range of drug loadings on
the basis of percolation theory. For this purpose, the tablet was considered a disordered
system whose particles are distributed at random. These authors derived a
model for the estimation of the drug percolation thresholds from the diffusion
behavior.
Knowledge of the percolation thresholds and the related critical points of the
system allows a rational optimization of the matrix formulation, avoiding the trial -
and - error method usually employed in the pharmaceutical industry. The ideal formulation
of an inert matrix, following percolation theory, must be above the drug
percolation threshold (i.e., the drug plus the initial pores percolate the system). This
fact guarantees the release of the total drug dose. On the other hand, the matrix
must also contain an infi nite cluster of excipient (i.e., the excipient must also be
above its percolation threshold). This percolating cluster of excipient avoids the
disintegration of the matrix during the release process and controls the drug release
[43] .
This kind of system, containing percolating clusters of both drug and excipient,
is called a bicoherent system. Furthermore, in order to decrease the variability in
the biopharmaceutical and mechanical behavior of the matrices, due to little change
in the tablet composition, it is not convenient to formulate the matrices just at the
percolation threshold. In this way, knowledge of the percolation thresholds of drug
and excipient supposes an important decrease in the cost of the optimization process
as well as in the time to market. The percolation thresholds of different pharmaceutical
powders have already been estimated, including drugs such as morphine
hydrochloride [68] , naltrexone hydrochloride [69] , dextromethorphan hydrobromide
[70] , and lobenzarit disodium [71] as well as matrix - forming excipients such
as hydrogenated castor oil, ethylcellulose, and acrylic polymers.
6.3.9.1 Case Study: Optimization of Inert Matrix Tablets for Controlled Release
of Dextromethorphan Hydrobromide
The objective of this work was to estimate the percolation thresholds of dextromethorphan
hydrobromide and Eudragit RS - PM which characterize the release
behavior of these inert matrices in order to rationalize the design of these
controlled - release systems.
Dextromethorphan hydrobromide is an antitussive drug with no analgesic or
addictive action. Its antitussive effect is similar to codeine. The recommended oral
dose for adults is 10 – 30 mg three to six times a day, not to exceed 120 mg daily. It is
absorbed rapidly and completely when taken orally with a lag time of 15 – 30 min
[72] .
In order to estimate the percolation threshold of dextromethorphan hydrobromide,
the matrices were studied from different points of view:
1. Release Profi les and Release Kinetics Figure 37 shows the percentage of drug
released from the studied matrices. As can be appreciated, a very similar behavior
was observed for matrices containing up to 50% w/w of drug. This can be attributed
to the swelling process (approximately 11% v/v) undergone by the matrices during
the release assay. This process makes the infl uence of the percolation threshold on
the release profi les less evident.
Higuchi ’ s kinetic model and Peppas ’ nonlinear regression ( Q = a . + b . t k ) were
employed to study the release data. The results obtained are shown in Table 22 . As
can be seen, the exponent k underwent a change (0.4534 – 0.5472) between matrices
containing 20 and 30% w/w of drug. Even if this is not an important change, it may
be related to some changes in the matrix structure due to the drug percolation
PHARMACEUTICAL TABLETS USING PERCOLATION THEORY 1031
1032 TABLET DESIGN
FIGURE 37 Percentage of drug released vs. time for tablets prepared with different loadings
of dextromethorphan hydrobromide.
100
90
80
70
60
50
40
30
20
10
0
% Dextromethorphan hydrobromide
released
0 120 240 360 480 600 720 840 96010801200
Time (min)
% w/w of
drug
20
30
40
50
60
70
80
90
+ + + + + + + + + + + + + + + + + + + + + + + + +
+
++++++++++++++
+
threshold. The masking effect of the swelling process on the drug percolation threshold
has to be taken into account.
2. Estimation of Drug Percolation Threshold The drug percolation threshold
was calculated using the property . described by Bonny and Leuenberger [40] . This
property is defi ned by the equation
.
.
=
.
b
A C 2 s
(5)
where . is proportional to the square root of the effective diffusion coeffi cient D eff ,
which is expected to obey, in the nearby of the percolation threshold, the scaling
law
D kD eff c = . 0( ) . . .
where D 0 is the diffusion coeffi cient of the drug in pure solvent, k a constant, . the
total porosity of the matrix (sum of initial porosity and porosity due to the dissolution
of the drug), . c the critical porosity or percolation threshold, and . the critical
TABLE 22 Dissolution Data from Dextromethorphan – HBr/Eudragit RS - PM Matrices
Drug Load
(% w/w) n
Q a b t . + Q = a r + b r t k
b ± S. E. r k r
20 248 1.43 . 10 . 4 ± 2.9 . 10 . 7 0.999 0.45335 0.999
30 248 2.47 . 10 . 4 ± 4.7 . 10 . 7 0.999 0.54724 0.999
40 248 3.38 . 10 . 4 ± 1.0 . 1.0 . 6 0.998 0.58981 0.999
50 248 4.43 . 10 . 4 ± 2.0 . 1.0 . 6 0.998 0.59792 0.999
65 241 8.60 . 10 . 4 ± 2.0 . 10 . 6 0.999 0.54536 0.999
70 248 1.15 . 10 . 3 ± 2.0 . 10 . 6 0.999 0.54697 0.999
80 241 1.45 . 10 . 3 ± 5.0 . 10 . 6 0.999 0.57677 0.999
90 85 2.09 . 10 . 3 ± 2.9 . 10 . 5 0.992 0.71910 0.999
TABLE 23 Calculation of Tablet Property b and Related Parameters in Matrices of
Dextromethorphan Hydrobromide
Drug (% w/w) . 0 . n b F b Probability b A . . 10 3
20 0.145 0.300 248 248,833 9.9 . 10 . 16 0.216 0.218
30 0.142 0.378 248 277,630 9.9 . 10 . 16 0.329 0.305
40 0.129 0.453 248 88,245.6 9.9 . 10 . 16 0.450 0.357
50 0.115 0.531 248 79,555.4 9.9 . 10 . 16 0.579 0.413
65 0.091 0.657 241 126,817 9.9 . 10 . 16 0.787 0.687
70 0.093 0.705 248 278,000 9.9 . 10 . 16 0.851 0.887
80 0.083 0.799 241 97,483.4 9.9 . 10 . 16 0.995 1.033
90 0.083 0.898 85 5,324.51 9.9 . 10 . 16 1.133 1.395
exponent for conductivity. This exponent has a value of 2.0 in 3D systems.The values
of . as well as the parameters involved in its calculation are shown in Table 23 .
The percolation threshold of dextromethorphan hydrobromide was estimated as
the intersection with the Y axis from a linear regression of the total porosity, . ,
versus the property . (Figure 38 ). Following the method of Bonny and Leuenberger,
only the . values above p c1 showing a linear dependence on the total porosity
(circles in Figure 38 ) are considered in the regression. The selected . values corresponded
to matrices with 50 – 80% w/w of drug.
The estimated critical porosity is 0.3691 ± 0.0541, considering a 95% confi dence
interval ( P = 0.05). This range corresponds to a dextromethorphan hydrobromide
content of between 23 and 36% w/w.
Estimation of the percolation threshold by visual methods is not very accurate,
mainly due to extrapolation from 2D to 3D systems. Nevertheless, scanning electron
microscopy was employed as an auxiliary technique in order to investigate the distribution
of the particles of dextromethorphan hydrobromide in the matrices.
Figure 39 shows two scanning electron microscopy (SEM) micrographs corresponding
to the tablet side facing the lower punch for matrices containing 20 and
FIGURE 38 Determination of drug percolation threshold. The circles represent the values
selected for the regression, according to its linear behavior. These values correspond to drug
loads between 50 and 80% w/w (three tablets per lot). Each point represents one experimental
datum.
0.8
1
0.6
0.4
0.2
0
0 0.5 1 1.5
., .103
Total porosity
PHARMACEUTICAL TABLETS USING PERCOLATION THEORY 1033
1034 TABLET DESIGN
FIGURE 39 SEM micrographs corresponding to bottom side of matrices using BSE detector.
The light gray particles correspond to dextromethorphan – HBr and the dark gray particles
to the excipient Eudragit RS - PM. ( a ) Matrices containing 20% w/w of drug. ( b ) Matrices
containing 30% w/w of drug.
(a)
(b)
500 .m
500 .m
30% w/w of drug using backscattering electron (BSE) detector at the same magni-
fi cation. In the tablet containing 30% of drug (Figure 39 b ), an infi nite drug cluster
can be observed. The drug particles (light - gray particles) begin to form a connective
network from the left to the right and from the top to the bottom of the micrograph.
In the tablet containing 20% w/w of drug (Figure 39 a ), the particles of the drug
(light - gray particles) seem to form isolated groups in the matrix.
Therefore, considering both micrographs in Figure 39 , a 2D geometric phase
transition can be observed. Figure 39 a shows the drug as gray particles on a black
background, whereas in Figure 39 b there is a black - on - gray array, with black particles
corresponding to Eudragit RS - PM surrounded by a gray background formed
by dextromethorphan hydrobromide particles.
When the cross section of these matrices (20 and 30% w/w drug loading) was
observed (Figure 40 ), the same pattern was found, changing from gray on black
(Figure 40 a , 20% w/w of drug) to black on gray (Figure 40 b , 30% w/w of drug).
Therefore, according to the different methods employed, the drug percolation
threshold in the studied matrices is expected to be between 20 and 30% w/w of
dextromethorphan hydrobromide (total porosity between 30.0 and 37.8% v/v of
drug).
3. Estimation of Excipient Percolation Threshold . In principle, for binary pharmaceutical
systems, two percolation thresholds are expected: the drug percolation
FIGURE 40 SEM micrographs corresponding to cross section of tablets using BSE detector:
( a ) matrices containing 20% w/w dextromethorphan – HBr (light gray particles); ( b ) matrices
containing 30% w/w of drug.
(a)
(b)
500 .m
500 .m
PHARMACEUTICAL TABLETS USING PERCOLATION THEORY 1035
1036 TABLET DESIGN
threshold p c1 and the excipient percolation threshold p c2 . The second is the point
where the excipient ceases to percolate the system.
Nevertheless, in a previous study dealing with inert matrices of naltrexone – HCl
[74] , two different excipient percolation thresholds p c2 were found for the matrix -
forming excipient Eudragit RS - PM: the site percolation threshold related to a
change in the release kinetics and the site - bond percolation threshold derived from
the mechanical properties of the tablet, where the excipient failed to maintain tablet
integrity after the release assay.
An evident change in the release kinetics between tablets containing 80 and 90%
w/w of drug can be observed in Table 23 (from k . 0.57 to k . 0.7 in the Peppas
equation). Therefore, the site percolation threshold of the excipient can be estimated
between the matrices containing 80 and 90% w/w of dextromethorphan
hydrobromide (10 – 20% v/v of excipient). Above this threshold, a percolating cluster
of excipient particles exists. These particles are able to control the drug release
kinetics, but their cohesion forces can be insuffi cient to maintain tablet integrity
after the release assay.
Formulations containing more than 65% w/w of drug were unable to maintain
tablet integrity after the 20 - h release assay. According to this result, the site - bond
percolation threshold of the excipient ranges between 65 and 70% w/w of drug,
corresponding to 29.5 and 34% v/v of excipient. Above this percolation threshold,
that is, for concentrations of excipient > 34% v/v, there is a percolating cluster of
excipient particles bound by suffi cient forces to maintain tablet integrity after drug
release.
In conclusion, according to percolation theory, the studied matrices should be
formulated with drug content between 30 and 65% w/w (37.8 – 66% v/v of total
porosity). These concentrations are optimal to ensure release of the total drug dose,
to have controlled release of the drug, and to avoid disintegration of the matrix. In
order to increase the robustness of the formulation, the limits of this range should
be avoided.
6.3.9.2 Critical Points of Hydrophilic Matrix Tablets
Recently percolation theory is starting to be applied to the study of hydrophilic
matrix systems. Figure 41 shows an example of the changes observed in several
release parameters employed to estimate the critical point and the related percolation
threshold in hydrophilic matrices prepared using KCl as the model drug [73] .
Application of the percolation theory allows explanation of the changes in the
release and hydration kinetics of swellable matrix - type controlled delivery systems.
According to this theory, the critical points observed in dissolution and water uptake
studies can be attributed to the excipient percolation threshold. Knowledge of these
thresholds is important in order to optimize the design of swellable matrix tablets.
Above the excipient percolation threshold an infi nite cluster of this component is
formed which is able to control the hydration and release rate. Below this threshold
the excipient does not percolate the system and drug release is not controlled.
Miranda et al. demonstrated experimentally the infl uence of the particle size of
the components on the percolation threshold in hydrophilic matrices as well as the
importance of the initial porosity in the formation of the gel layer (sample - spanning
cluster of excipient) [74] .
FIGURE 41 ( a ) Higuchi slope; ( b ) normalized Higuchi slope; ( c ) relaxational constant of
Peppas and Sahlin versus percentage of excipient volumetric fraction for batch A (50 – 100 . m
KCl and 150 – 200 . m HPMC K4M).
60
50
40
30
20
10
0
0
5
4
3
2
1
0
0
25
20
15
10
5
0
0 10 20 30 40 50 60 70 80 90 100
10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 100
(a) (b)
(c)
% v/v HPMC % v/v HPMC
% v/v HPMC
y = –2.8783x + 93.63
R2 = 0.8731
y = –0.1124x + 15.01
R2 = 0.9903
y = –0.0172x + 1.7095
R2 = 0.9414
y = –1.4671x + 39.337
R2 = 0.8063
y = –0.2704x + 7.4919
R2 = 0.8973
y = –0.0047x + 0.4418
R2 = 0.9283
6.3.9.3 Case Study: Estimation of Percolation Thresholds in Acyclovir
Hydrophilic Matrix Tablets
The principles of the percolation theory were applied to design controlled - release
matrix tablets containing acyclovir in order to estimate the percolation threshold of
the excipient in acyclovir matrix tablets and to characterize the release behavior of
these hydrophilic matrices in order to rationalize the design of these controlled -
release systems.
Acyclovir is a potent inhibitory of viruses of the herpes group, particularly herpes
simplex virus (HSV I and II) and herpes zoster varicella virus. Unfortunately, acyclovir
has a short half - life (2 – 3 h), and the oral dosage form must be taken fi ve times
daily, which is very inconvenient for patients [75, 76] . Consequently, the aim of this
study was to develop a controlled - release formulation of acyclovir that could be
taken twice daily. The materials used to prepare the tablets were acyclovir (Kern
Pharma, Tarrasa, Barcelona) and hydroxypropyl methylcellulose (Methocel K4M)
(Colorcon) a hydrophilic cellulose derivative as the matrix - forming material.
Binary mixtures were prepared with varying drug contents (60, 70, 80, 90, and
95%) keeping constant the drug and excipient particle size. Table 24 gives the composition
of the studied batches as well as the tablet thicknesses. The mixtures were
compressed on an eccentric machine (Bonals A - 300) without any further excipient.
Cylindrical tablets with a mean dosage of 500 mg and a diameter of 12 mm were
prepared at the maximum compression force accepted by the formulations.
PHARMACEUTICAL TABLETS USING PERCOLATION THEORY 1037
1038 TABLET DESIGN
TABLE 24 Composition of Hydrophilic Matrices Prepared with Acyclovir/ HPMC K4M
(150 – 200 m m) and Percent HPMC plus Initial Porosity
Batch
Percent w/w Percent (v/v) HPMC
+ Initial Porosity
Tablet Thickness
(mm) Acyclovir HPMC K4M
A 95 5 20.76 3.01 ± 0.056
B 90 10 26.41 3.25 ± 0.052
C 80 20 37.60 3.84 ± 0.051
D 70 30 45.11 4.68 ± 0.063
E 60 40 55.82 5.81 ± 0.056
The release profi les were measured with the USP 25 dissolution apparatus 2
(Turu Grau, model D - 6) at 100 rpm in distilled water (900 mL) at 37 ± 0.5 ° C for
12 h. Filtered samples taken at different times were determined for acyclovir content
through ultraviolet absorption at . max (242 nm).
Figure 42 shows the release profi les obtained from hydrophilic matrices formulated
with acyclovir and HPMC K4M 150 – 200 . m.
In order to study the release mechanism of acyclovir from the tablets, the fi tting
of the drug release data to the following kinetic equations has been studied: zero -
order equation, Q = k 0 t ; Higuchi equation [77] , Q = k H t 1/2 ; Korsmeyer – Peppas equation
[78] , Q = kt n ; and Peppas - Sahlin equation [79] , Q k k m m = + d r
2 , where Q is the
amount of drug remaining at time t , k 0 is the zero - order release constant, k H is the
Higuchi rate constant, k is the Korsmeyer – Peppas kinetic constant, n is the exponent
indicative of the release mechanism (for matrix tablets an n value of 0.5 indicates
diffusion control and an n value of 1.0 indicates erosion or relaxation control [80] ,
intermediate values suggest that at least two processes contribute to the overall
release mechanism), k d is the diffusion rate constant, k r is the relaxation rate constant,
and m is the purely Fickian diffusion exponent for a device of any geometric
shape which exhibits controlled release. In our case, the aspect ratios and exponent
values ( m ) are shown in Table 25 [79] . The results obtained are shown in Table 26 .
FIGURE 42 Acyclovir release from matrix tablet with total drug content of 95, 90, 80, 70,
and 60 prepared with acyclovir – HPMC K4M (150 – 200 . m) (mean ± SD, n = 3).
120
100
80
60
40
20
0
0 100 200 300 400
Time (min)
% Acyclovir release
95% acyclovir 90% acyclovir 80% acyclovir
70% acyclovir 60% acyclovir
.
TABLE 25 Aspect Ratios and Exponent Values ( m ) for
Hydrophilic Matrices Studied
Batch Aspect Ratio Exponent (m)
A 3.80 0.45
B 3.59 0.44
C 3.03 0.43
D 2.65 0.42
E 2.16 0.43
TABLE 26 Values of Kinetic constants Derived with Selected Equations in Range
5 – 70% Acyclovir Release for All Batches Studied
Batch A B C D E
Acyclovir,
% w/w 95 90 80 70 60
Zero - order
equation
k 0 1.222 0.122 0.096 0.057 0.042
r 2 0.984 0.974 0.994 0.995 0.987
Sum of squares
total
3545.9 10,352.6 6,531.4 5,149.2 1,268.0
Sum of squares
residual
53.7 278.3 51.8 24.8 16.4
Higuchi
equation
k H 12.440 3.4167 2.8518 1.7683 1.4694
r 2 0.998 0.993 0.956 0.932 0.959
Sum of squares
total
3,545.9 10,352.6 12,367.8 5,149.2 1,268.0
Sum of squares
residual
7.0 66.9 483.6 362.4 54.6
Korsmeyer –
Peppas
equation
k H 3.167 0.290 0.254 0.027 0.041
n 0.782 0.843 0.856 1.114 1.008
r 2 0.999 0.994 0.998 0.998 0.997
Sum of squares
total
13,220.9 26,297.6 33,309.1 13,879.4 5,673.5
Sum of squares
residual
16.7 159.5 65.5 10.1 15.2
Peppas and
Sahlin
equation
k d 2.239 2.056 0.357 . 0.81 . 0.38
k r 1.615 0.161 0.205 0.202 0.127
r 2 0.998 0.999 0.994 0.997 0.997
Sum of squares
total
49,567.0 13,220.9 28,716.1 13,879.4 5,673.5
Sum of squares
residual
42.9 23.3 165.0 43.7 15.2
Notes: k 0 (%/min), zero - order constant; k H (%/min 1/2 ), Higuchi ’ s slope; k (%/min n ), kinetic constant of
Korsmeyer model; n , diffusional exponent; k d (%/min m ), diffusional constant of Peppas and Sahlin model;
k r (%/min 2 m ), relaxational constant of Peppas and Sahlin model; m , diffusional exponent that depends
on geometric shape of releasing device through its aspect ratio (see Table 25 ).
The analysis of the release profi les and the kinetic data indicate two different
behaviors and a sudden change between them. In the fi rst behavior, which corresponds
to the matrices that release the drug at slow rates, the release was controlled
by the fully hydrated gel layer. For these matrices, erosion of the hydrophilic gel
structure has shown an important infl uence on drug release. This is indicated by the
better fi t of the drug release kinetics to the zero - order equation, the n value of
PHARMACEUTICAL TABLETS USING PERCOLATION THEORY 1039
1040 TABLET DESIGN
Korsmeyer – Peppas equation near 1, and the higher value of the relaxation constant
k r in comparison with the diffusion constant k d in the Peppas – Sahlin equation.
Taking into account the drug solubility (2.5 mg/ML), prevalence of the erosion
versus swelling mechanism can be expected. After the transition point, the tablets
allow the free dissolution of the drug when they are exposed to the dissolution
medium due to the fact that the gel layer is not established since the fi rst moment
and, in these conditions, this structure cannot control the drug release. The
Korsmeyer release rate increases from 0.290 to 3.167% min . 1/2 . For these matrices,
according to the Higuchi ( r 2 = 0.998), Korsmeyer ( n = 0.782), and Peppas – Sahlin
(k r < k d ) equations, drug release is governed by the diffusion process.
In hydrophilic matrices the drug threshold is less evident than the excipient
threshold, which is responsible for the release control [73] . In order to estimate the
percolation threshold of HPMC K4M, different kinetic parameters were studied:
Higuchi rate constant, normalized Higuchi rate constant, and relaxation rate constant.
The evolution of these release parameters has been studied as a function of
the sum of the excipient volumetric percentage plus initial porosity. Recent studies
of our research group have found the existence of a sample - spanning cluster of
excipient plus pores in the hydrophilic matrix before the matrix is placed in contact
with the liquid, clearly infl uences the release kinetics of the drug [73] .
Figures 42 – 45 show changes in the different kinetic parameters: the Higuchi rate
constant, normalized Higuchi rate constant, and relaxation rate constant. To estimate
the excipient percolation threshold, these parameters were plotted versus the
excipient volumetric fraction plus initial porosity.
The kinetic parameters studied show a nonlinear behavior as a function of the
volumetric fraction of the excipient plus initial porosity. As an approximation for
estimating the trend of the parameter, one regression line has been performed
below and the other above the percolation threshold. The point of intersection
between both regression lines has been taken as an estimation of the percolation
threshold [73, 74] .
As percolation theory predicts, the studied properties show a critical behavior
as a function of the volumetric fraction of the components. A critical point has been
found between 21 and 26% v/v of excipient plus initial porosity (see Table 24 ). This
critical point can be attributed to the excipient percolation threshold.
FIGURE 43 Higuchi slope (mean ± SD, n = 3) versus percentage of excipient volumetric
fraction plus initial porosity for all batches studied.
% v/v excipient plus initial porosity
KH (%min - 1/2)
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60
FIGURE 44 Normalized Higuchi slope (mean ± SD, n = 3) versus percentage of excipient
volumetric fraction plus initial porosity for all batches studied.
% v/v excipient plus initial porosity
KH (%min - 1/2)/% v/v excipient plus initial porosity
0.7
0.6
0.5
0.4
0.3
0.2
0.1
–0.1
0
0 10 20 30 40 50 60
FIGURE 45 Relaxational constant of Peppas – Sahlin (mean ± SD, n = 3) versus percentage
of excipient volumetric fraction plus initial porosity for all batches studied.
0
0
0.5
1
1.5
2
10 20 30 40 50 60
% v/v of excipiente plus initial porosity
Kr (%/min - 2 m)
The Effective Medium Approximation (EMA), based in some assumptions,
allows us to employ linear regressions as an approximation of the behavior of a
disordered system outside the critical range. Based on EMA theory, two linear
regressions have been performed as an approximation for estimating the percolation
threshold as the point of intersection between both regression lines (see Figures
43 – 45 ). The values of the excipient percolation thresholds estimated for all the
batches studied, based on the behavior of the kinetic parameters, ranged from 25.99
to 26.77%.
Therefore, the results obtained from the kinetics analysis are in agreement with
the release profi les, indicating a clear change in the release rate and mechanism
between matrices containing 90 and 95% w/w of drug (5 – 10% w/w of excipient). The
existence of a critical point can be attributed to the excipient percolation threshold.
From the point of view of percolation theory, this means that above 10% w/w of
HPMC K4M, the existence of a network of HPMC (able to form a hydrated layer
from the fi rst moment) controls the drug release.
PHARMACEUTICAL TABLETS USING PERCOLATION THEORY 1041
1042 TABLET DESIGN
The process of water penetration into hydrophilic matrix tablets was also studied
using a modifi ed Enslin apparatus. This apparatus contains a fritter and a system to
regulate the water level. When the tablet is placed on the fritter, the water is
absorbed from a reservoir which is placed on a precision balance. The amount of
water uptake at each time point was read from the balance as weight loss in the
reservoir. Figure 46 shows the obtained release profi les.
An increase in the rate of water uptake can be observed when the HPMC concentration
decreases. A critical point was found between 90 and 95% w/w of acyclovir.
This range corresponds with the critical point observed in release profi le studies.
The water uptake data were subjected to the Davidson and Peppas model to calculate
the rate of water penetration [81] . The results show a change in the water
uptake constant between the matrices containing 90 – 95% w/w of acyclovir, which
refl ects the presence of the critical point previously observed.
Knowledge of the percolation threshold of the components of the matrix formulations
contributes to improve their design. First, in order to develop robust formulations,
that is, to reduce variability problems when they are prepared at industrial
scale, it is important to know the concentrations corresponding to the percolation
thresholds. The percolation thresholds correspond to formulations showing a high
variability in their properties as a function of the volume fraction of their components.
Therefore, in order to design robust dosage forms, the nearby of the percolation
thresholds should be avoided.
Second, the excipient percolation threshold in hydrophilic matrices represents
the border between a fast release of the drug (below the threshold) and a drug
release controlled by the formation of a coherent gel layer (above the excipient
percolation threshold). Therefore, knowledge of this threshold will allow us to avoid
the preparation of a number of unnecessary lots during the development of a pharmaceutical
formulation, resulting in a reduction of the time to market.
FIGURE 46 Weight gain of systems as function of swelling time for matrix tablet with total
drug content of 95, 90, 80, 70, and 60% prepared with acyclovir – HPMC K4M (150 – 200 . m)
(mean ± SE, n = 3).
95% acyclovir 90% acyclovir 80% acyclovir 70% acyclovir 60% acyclovir
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
800
600
400
200
0
% Water uptake/dry polymer
Time (min)
0 50 100 150 200 250 300 350 400
6.3.10 ULTRASOUND - ASSISTED TABLETING (A NEW PERSPECTIVE)
The compression of a powder is a complex process that is usually affected by different
kinds of problems. These problems have been widely investigated and mainly
concern the volume reduction and the development of a strength between the particles
of the powder suffi cient to ensure tablet integrity [82] . The application of
ultrasonic energy shows a great ability to reduce and even avoid these problems
[83] . Ultrasound refers to mechanical waves with a frequency above 18 kHz (the
approximate limit of the human ear). In an ultrasound compression machine, this
vibration is obtained by means of a piezoelectric material (typically ceramics) that
acts as a transducer of alternate electric energy of different frequencies in mechanical
energy. An acoustic coupler, or “ booster, ” in contact with the transducer increases
the amplitude of the vibration before it is transmitted (usually in combination with
mechanical pressure) to the material to be compressed.
Ultrasound - assisted powder compression has been widely employed in metallurgy
as well as in the plastic and ceramic industries [84] . The fi rst references in the
pharmaceutical industry are two patents in 1993 [85] and 1994 [86] . Since then, some
papers have presented experimental data in this fi eld [45, 87 – 92] .
Two main objectives are pursued nowadays by means of the application of
ultrasound - assisted compression:
1. Increase in the drug dissolution rate due to amorphization of the drug
2. Preparation of controlled - release dosage forms with thermoplastic
excipients
As a consequence of the application of ultrasonic energy, the drug can lose its
crystalline structure. This will result in an increase of the dissolution rate of the
active substance, which can be very adequate for slowly dissolving drugs. Nevertheless,
depending on the storage conditions, the drug can recover, at least partially,
its crystallinity [89, 90] .
To overcome this problem, it has been proposed to use an adequate excipient,
preventing the recovery of the crystallinity, leading in some cases to the preparation
of solid solutions into the die of the tableting machine.
Several analytical techniques, such as infrared (IR) spectroscopy, differential
scanning calorimetry, HPLC, and thin - layer chromatography (TLC), have been used
to investigate possible drug degradation due to ultrasonic energy. No important
permanent modifi cation of the drug has been found, with the exception of the loss
of crystallinity [89, 90] .
Concerning the design of controlled - release dosage forms, using a thermoplastic
excipient (e.g., copolymers of acrylic and metacrylic acid), an important decrease in
the release rate has been found for tablets compressed with the assistance of ultrasonic
energy in comparison with traditional tablets.
Although the effects of the ultrasonic energy on the material are not completely
clarifi ed, this slow release rate has been attributed to different phenomena:
Mechanical Pressure This pressure is exerted by the punches of the ultrasound -
assisted tableting machine. This is the main compression mechanism when
low ultrasonic energies are employed (below 25 J in the mixtures studied by
ULTRASOUND-ASSISTED TABLETING (A NEW PERSPECTIVE) 1043
1044 TABLET DESIGN
Rodriguez et al. [87, 88] ) or when the materials used are not thermoplastic.
In these cases the machine acts as a multiple - impact mechanical press.
Thermal Effects Due to the poor conductivity for ultrasounds (low module of
elasticity and high quantity of air trapped inside) usually exhibited by the
materials included in pharmaceutical formulations, a fast decay of ultrasonic
energy to thermal energy is obtained. This process has been studied, monitoring
the temperature inside the compression chamber by means of a thermistor.
In the studied mixtures [87, 88] , a fast rise in temperature was obtained in
tenths of a second followed by a relatively fast decrease (see Figure 47 ).
The peak temperature obtained for low ultrasonic energy (25 J) is below 80 ° C,
whereas for high energies (125 – 150 J) it is above 140 ° C. In mixtures of ketoprofen
with acrylic polymers [90] , the increase in temperature was slightly
lower. In this respect it must be mentioned that a recent modifi cation of the
ultrasound - assisted tableting machine that involves the suppression of Tefl on
isolators in contact with the powder must result in a faster decrease in temperature
inside the compression chamber. Thermal effects can cause the total
or partial fusion of some components of the formulation. Nevertheless, in the
assayed controlled - release formulations, the components are usually below its
melting points.
Plastic Deformation Plastic deformation results from the combination of
thermal and mechanical effects. The thermoplastic excipient was subjected to
a temperature above its glass transition temperature ( T g ) and to a high - frequency
mechanical pressure that can avoid the elastic recovery of the
material.
Sintering The combination of temperature, pressure, and friction effects can
result in the sintering of particles, so that the limits between them are no
longer evident [46, 87] .
Recent studies [91 – 93] have shown that, for one component of the system undergoing
thermoplastic deformation, the continuum percolation model can be used to
predict the changes in the system with respect to a traditional pharmaceutical
FIGURE 47 Temperature profi le inside compression chamber. (Courtesy of Tecnea Srl.)
0 2 4 6 8 16 18 20 14 12 10
0
20
40
60
80
100
120
Time (sec)
Temperature (°C)
dosage form. The continuum percolation model dispenses with the existence of a
regular lattice underlying the system; therefore, the substance is not distributed into
discrete lattice sites. This model deals with the volume ratio of each component
and a continuum distribution function. The volume ratio is expressed as a space –
occupation probability to describe the behavior of the substance [94, 95] .
The continuum percolation model predicts an excipient percolation threshold
around 16% v/v. This can explain the important decrease in the critical point corresponding
to the excipient percolation threshold, a critical point that governs the
mechanical and release properties of the matrix.
Ultrasound compaction lowers the percolation threshold of the thermoplastic
excipient, resulting in a drastic reduction (about 50%) in the amount of matrix -
forming excipient [98] needed to obtain the controlled-release system as well as in
a better control of the drug release. The structure of the excipient inside the US -
tablets does not correspond to a particulate system but to an almost continuous
medium; therefore, there is no an excipient particle size inside these matrices (see
Figure 48 ). Consequently, the percolation threshold of the active agent is higher
than in traditional tablets. The insoluble excipient almost surrounds the active agent
particles, slowing down the contact with the dissolution medium.
These facts can involve important advantages for the pharmaceutical industry,
such as the preparation of controlled - release inert matrices containing high drug
doses, with very little increase in the weight of the system. This fact is especially
interesting when a high drug dose has to be included in the dosage form, as frequently
occurs in controlled - release systems.
On the other hand, application of ultrasonic energy results in an increase in the
temperature of the die during the compaction process. The consequences of this
fact should be taken into account and cannot be neglected in the case of thermolabile
drugs and/or excipients [87, 88] .
Further research is needed in the area of ultrasound - assisted compression of
pharmaceutical powders, including a higher number of drugs and excipients.
FIGURE 48 SEM micrograph of matrix tablet containing potassium chloride as drug model
and commercial acrylic – metacrylic copolymer. The white KCI particles are surrounded by an
almost continuous dark gray mass of excipient. (Courtesy of M. Mill a n.)
ULTRASOUND-ASSISTED TABLETING (A NEW PERSPECTIVE) 1045
1046 TABLET DESIGN
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FURTHER READING
A. BASF Pharma ingredients generic drug formulations, 2004, 2005.
B. Baveja , S. K. , Ranga Roa , K. V. , and Padmalatha Devi , K. ( 1987 ), Zero - order release
hydrophilic matrix tablets of . - adrenergic blockers , Int. J. Pharm. , 40 , 223 – 234 .
C. Bettini , R. , Colombo , P. , Massimo , G. , Catellani , P. L. , and Vitali , T. ( 1994 ), Swelling and
drug release hydrogel matrices: Polymer viscosity and matrix porosity effect , Eur. J.
Pharm. Sci. , 2 , 213 – 219 .
D. Campos - Aldrete , M. E. , and Villafuerte - Robles , L. ( 1997 ), Infl uence of the viscosity grade
and the particle size of HPMC on metronidazole release from matrix tablet , Eur. J. Pharm.
Biopharm. , 43 , 173 – 178 .
E. Ferrero , C. , Mu n oz - Ruiz , A. , and Jim e nez - Castellanos , M. R. ( 2000 ), Fronts movements
as a useful tool for hydrophilic matrix release mechanism elucidation , Int. J. Pharm. , 202 ,
21 – 28 .
F. U.S. Pharmacopoeia 25 ( 2002 ), National Formulary 20, U.S. Pharmacopeial Convention,
Rockville, MD.
G. V a zquez , M. J. , Peres - Marcos , B. , G o mez - Amoza , J. L. , Mart i nez - Pacheco , R. , Souto , C. ,
and Concheiro , A. ( 1992 ), Infl uence of technological variables on release of drugs from
hydrophilic matrices , Drug Dev. Ind. Pharm. , 18 , 1355 – 1375 .
H. Velasco , M. V. , Ford , J. L. , Rowe , P. , and Rajabi - Siahboomi , A. R. ( 1999 ), Infl uence of drug:
hydroxypropylmethylcellulose ratio, drug and polymer particle size and compression
force on the release of diclofenac sodium from HPMC tablets , J. Controlled Release , 57 ,
75 – 85 .
I. Tu , J. , Wang , L. , Yang , J. , Fei , H. , and Li , X. ( 2001 ), Formulation and pharmacokinetic
studies of acyclovir controlled - release capsules , Drug Dev. Ind. Pharm. , 27 ( 7 ), 687 – 692 .
FURTHER READING 1051
1053
6.4
TABLET PRODUCTION SYSTEMS
Katharina M. Picker - Freyer
Martin - Luther - University Halle - Wittenberg, Institute of Pharmacy, Division of
Pharmaceutics and Biopharmaceutics, Halle/Saale, Germany
Contents
6.4.1 Introduction
6.4.2 Physics of Tablet Formation
6.4.2.1 Tableting Process
6.4.2.2 Final Formation of Tablet
6.4.3 Requirements for Tablet Production Systems
6.4.4 Tablet Manufacturing Process
6.4.4.1 Filling
6.4.4.2 Compression
6.4.4.3 Ejection
6.4.5 Tableting Machines
6.4.5.1 Single - Punch Tableting Machines
6.4.5.2 Rotary Tableting Machines
6.4.5.3 Application of Tableting Machines
6.4.6 Tableting Machine Simulators (Compaction Simulators)
6.4.6.1 Hydraulic Compaction Simulators
6.4.6.2 Mechanic Compaction Simulators
6.4.6.3 Application of Tableting Machine Simulators
6.4.7 Instrumentation of Tableting Machines
6.4.7.1 Force Measurement
6.4.7.2 Displacement Measurement
6.4.7.3 Temperature Measurement
6.4.7.4 Measurement of Time
6.4.8 Analysis of Tableting Process
6.4.8.1 Force – Time Analysis
6.4.8.2 Displacement – Time Analysis
6.4.8.3 Force – Displacement Analysis
6.4.8.4 Force – Displacement – Time Analysis
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1054 TABLET PRODUCTION SYSTEMS
6.4.9 Analysis of Final Tablet Formation
6.4.10 Complete Description of Process of Tablet Formation
6.4.11 Special Accessories of Tableting Machines
6.4.11.1 Optimization of Die Filling
6.4.11.2 Tablet Weight Control
6.4.11.3 Control of Mixing Homogeneity
6.4.11.4 Cleaning
6.4.12 Important Factors during Manufacturing Process
6.4.12.1 Climatization
6.4.12.2 Lubrication
6.4.12.3 Occurring Problems during Manufacturing
6.4.13 Future of Tablet Production Systems
References
6.4.1 INTRODUCTION
Tablet production systems can be defi ned as all machines which are able to produce
tablets. They include tableting machines for production and research as well as
tableting machine simulators, which are able to mimic the production processes of
tableting machines of different size and velocity in order to facilitate scale - up.
Tablets have been produced for more than 150 years. The fi rst tableting machine,
developed by Brockedon in 1972 [1] , was a manually operated single - punch machine.
Currently high - speed tableting machines can produce more than a million tablets
per hour.
However, the amount of drug in the early steps of development still makes the
use of small tableting machines necessary. Thus, before fi nal production a scale - up
from small machines useful for the production of single tablets to high - speed
machines is necessary. Since this scale - up is still based on “ trial and error ” [2] ,
tableting machine simulators can be used to simulate different steps in the
scale - up.
In research tableting machine simulators are often used since they allow precise
and well - adjustable measurements during tableting with only a small amount of
material.
The aim of this chapter is to give an overview of the different techniques used
for production and research and further to show the possibilities, that instrumentation
of tablet production systems gives in order to analyze the tableting process. The
knowledge derived can be used for formulation development as well as to facilitate
tablet production and scale - up. Applied techniques which are necessary for production
in a good manufacturing practice (GMP) environment will also be discussed.
6.4.2 PHYSICS OF TABLET FORMATION
Tablets can be defi ned as two - phase systems which consist of a solid phase (the
compressed powder) and the gaseous phase (the air). The solid phase forms a coher
PHYSICS OF TABLET FORMATION 1055
ent network inside the tablet and a defi ned form with precise outer dimensions
results. Here we defi ne the process of tablet formation as the transformation of the
powder, a noncoherent solid phase, into the compact tablet, and this process lasts
until no further changes are induced by tableting. “ Tableting ” is only part of the
process of tablet formation. It is defi ned as the process by which the powder is
transformed into a coherent form, the tablet. In conclusion tablet formation is the
result of tableting and all the changes induced by tableting [3, 4] .
6.4.2.1 Tableting Process
During the tableting, the powder in the die of the tableting machine is transformed
by the infl uence of lower and upper punches into a coherent form — “ the tablet ”
(Figure 1 ). During tableting different processes occur which are responsible for the
cohesion of the tablet [5, 6] . First the particles are pushed together and reoriented
in the die until they have arranged in the closest packing. This process is followed
by elastic and/or plastic deformation of the particles, as can be observed by confocal
laser microscopy [7] . Some materials show brittle fracture. During brittle fracture
new particles are produced which can again deform or fracture. Under the infl uence
of the applied forces the particles approach each other up to bonding [8] . The
bonded particle collectives continue to deform. The mechanism of deformation and
bonding is dependent on material properties and process conditions. Important
bonding mechanisms are van der Waals forces, surface fi lms, liquid or solid bridges,
and mechanical interlocking [9] .
The nature of the resulting bonds [9, 10] and the extent of particle approach [11]
determine the cohesion of the fi nal tablet and are responsible for the compactibility
of the materials [8] . Further, the formation of bonds occurs not only during the
FIGURE 1 Densifi cation of powder bed: ( a ) particles in die; ( b ) reorientation of particles;
( c , d ) particle deformation and fracture; ( e ) tablet in die.
(a) (b) (c)
(d) (e)
1056 TABLET PRODUCTION SYSTEMS
compression phase but also during decompression of the tablet [12, 13] . Hence
Leuenberger and Ineichen [14] describe tableting as a percolation phenomenon.
Looking closer at the tableting process it becomes clear that the applied forces
during tableting are not only the result of machine movement. When punches are
moving in a machine without material, no force can be measured. The force only
develops when the punches come into contact with the powder bed. The materials
resist pressure deformation, and while the punches are moving, a counterforce
builds up which is the measured force at the punches. In conclusion punch forces
are determined by the material and as a result materials can be characterized by
the measured punch forces.
6.4.2.2 Final Formation of Tablet
After ejection of the tablet from the die of the tableting machine, the tableting
process is fi nished. However, decompression continues and thus the tablet formation
process is not yet fi nished. The remaining stress inside the tablet can resolve and
produce changes in the structure of the tablet. The tablet partially releases the
energy gained during tableting. The tablet often shows the occurrence of relaxation,
which is called elastic recovery or relaxation of the tablet [15, 16] . This relaxation
is primarily axial relaxation and to a small part radial relaxation, as Newton and
Rowley [17] fi gured out. Hiestand [15] called this event “ compression born
repulsion. ”
Van der Voort Maarshalk [18] showed that the axial relaxation of the tablet, that
is, the change in tablet height, is dependent on the stored energy and hinders the
formation of bonds. The elastic recovery [19 – 21] can be measured by measuring
tablet height at different times after tableting. Simultaneously changes in the inner
structure of the tablet can occur, for example, shifts of the crystal planes [22, 23] .
The fusion of amorphous regions and the recrystallization of amorphous parts are
assumed [24 – 26] . These structural changes are induced by the tableting process and
also become visible when transformation of drugs into other polymorphic forms
occurs [27 – 29] . However, even during fi nal formation of the tablets, bonds are
formed [15, 30, 31] , and thus fi nal tablet formation can be defi ned as a valuable part
of the tablet formation process.
6.4.3 REQUIREMENTS FOR TABLET PRODUCTION SYSTEMS
Tablet production systems should be able to produce tablets in a reasonable time
and without loss of material. Usually they consist of two punches and a die, as
schematically shown in Figure 1 . The material is placed into the die cavity, which
is closed on the lower side by the lower punch. A tablet is formed when the powder
is compressed by the punches as described above. The forces evolving to produce
a tablet can be up to 80 kN for pharmaceutical purposes [32] ; for the production of
bigger tablets they are even higher. Usually they range between 10 and 30 kN. Thus
punches and dies are produced from hardened steel.
To withstand the evolving forces, the die has to be fi xed tightly in the machine,
and for that purpose it is accurately fi t in a die table consisting of steel. The die
table is further friction locked to the machine frame.
Tablet production systems can be operated manually; however most of the
systems nowadays operate automatically in order to produce a suffi cient number of
tablets. Further requirements depend on the type of machine, the production
process, the operation mode of the machine, and the production rate of the
machine.
6.4.4 TABLET MANUFACTURING PROCESS
The manufacturing process of tablets principally consists of three stages: fi lling the
die with the powder, compressing the powder, and ejecting the tablet from the
die.
6.4.4.1 Filling
Die fi lling a tableting machine is a volumetric process. In any case the die is fi lled
to the upper edge of the die and the powder surface fl ushes with the surface of the
die and the die table. The fi lling depth is determined by the height of the die cavity,
the fi lling volume is determined by the diameter of the die hole and the fi lling depth.
Both the diameter of the die hole and the fi lling depth are used to calculate the fi nal
fi lling volume.
Three different techniques exist for fi lling the die. The simplest is to use a fi lling
shoe which moves back and forth over the die and fi lls the die to its upper edge.
The surface is leveled off by the fi lling shoe.
Another possibility is a fi lling shoe which does not move. In this case the dies
and the accessory lower punches move below the fi lling shoe and the accessory
upper punches move above the fi lling shoe. In this case the fi lling volume ends at
the upper edge of the die; however, to level off the surface, an additional scraper
is necessary. In addition, many machines use the technique of lowering the lower
punch and with it the powder bed before tableting [33] . This is most helpful to avoid
dusting when the upper punch moves into the die.
The newest fi lling technology is to fi ll the die by centrifugal forces [34] . The
material moves through specially shaped radial channels which approach the die
from the side. In this case the powder volume is determined by the positions of the
lower and upper punches in the die. However, for centrifugal fi lling an absolute
must is a free - fl owing powder. This is a great disadvantage for all non - free - fl owing
powders.
6.4.4.2 Compression
The compression event is the central stage of tablet production. Compression not
only depends on the machine but to a great deal on the material properties of a
tablet formulation. The principal stages of compression have been described above.
Principally, from the machine manufacturing side two different possibilities of compression
exist. Either the lower punch is closing the die from the bottom side and
the upper punch moves downward for compression or both upper and lower punches
move simultaneously toward each other and the powder is compressed from both
sides. In the fi rst case the surface hardness of the tablet is not the same on the upper
TABLET MANUFACTURING PROCESS 1057
1058 TABLET PRODUCTION SYSTEMS
and lower sides; in the second case the surface hardness is the same on the upper
and lower sides.
As already described, the tablet is not completely formed during compression
[35, 36] . When the punches leave each other, the tablet relaxes during decompression,
further relaxing when one punch leaves the tablet and continuing relaxation
after ejection from the die. The process is called decompression as long as a force
is measurable. Afterward the process is called elastic recovery or relaxation of the
tablet.
6.4.4.3 Ejection
During ejection usually the lower punch moves upward to eject the tablet from
the die and the upper punch has already left the die when the process of ejection
starts. Only one machine is presently on the market which ejects the tablets downward
at the bottom of the die [34] . After ejection from the die the tablets are
collected.
6.4.5 TABLETING MACHINES
There exist tableting machines which operate in a different manner and can produce
in between one single tablet and a million tablets per hour . The orientation of the
particles in the machine and their rearrangement, densifi cation, and deformation
depend not only on the material but also on the tableting machine used.
The fi rst machines for the production of tablets were simple hydraulic or manually
operated presses. Later, eccentric and rotary tableting machines were developed.
Today, eccentric tableting machines are only used for research, in early
development, or for special applications.
The newest development for production is a machine which fi lls the dies by centrifugal
force [34] . It is a special rotary tableting machine. Another innovation is a
special machine which operates by ultrasound [37] .
In the following the most important machine types and their working principles
will be described.
6.4.5.1 Single - Punch Tableting Machines
Single - punch machines were the fi rst tableting machines used at the end of the
nineteenth century. The upper punch is lowered by a lever arm on the powder bed
in the die and by reciprocating this procedure single tablets can be produced.
Another possibility is to lower the upper punch by means of a screw. These manually
operated tableting machines are no longer used.
Two other types are still in usage for special purposes. Hydraulically operated
machines are used to produce tablets, for example, for Fourier transform infrared
(FTIR) spectroscopy. In this case the upper punch moves hydraulically onto the
powder bed and the tablet is formed in the die. The other possibility is an eccentric
tableting machine, which will be described in the following.
Eccentric Tableting Machines Eccentric tableting machines are still used in research,
in early development, and for material characterization. They are the machines of
choice when you want to use a single - punch tableting machine. As other single - punch
machines, they work with one pair of punches. In principle, they have a mobile upper
punch and a lower punch which is fi xed during compression. The lower punch only
moves for ejection of the tablet. The densifi cation process is unilateral.
An eccentric valve is driven by a motor and this eccentric valve is responsible
for the movement of the upper punch (Figure 2 ). The eccentric movement determines
the operation and speed of the upper punch. The upper punch is driven by
the eccentric valve into the die, which is closed on the bottom side by the lower
punch, and the tablet is compressed in the die.
The punch forces for compression evolve due to contact with the powder. Thus
the measured punch forces at the upper and lower punches result from the movement
of the upper punch and the resistance of the powder bed toward deformation.
From that it becomes clear that the measured upper punch force is usually higher
than the lower punch force, and thus the upper punch force is the main force used
for material characterization. The tablets show different hardness on upper and
lower surfaces, as do all tablets produced by single - punch machines [38] .
When the upper punch is lifted from the tablet surface, the lower punch moves
the tablet upward for ejection out of the die . However, as is known nowadays, the
lower punch also moves slightly before due to evolving forces.
In eccentric machines, die fi lling is performed by a fi lling shoe which moves back
and forth above the die. When the upper punch moves upward above the die, the
die is fi lled, and when the upper punch starts downward movement, the powder bed
is leveled off. After ejection of the tablet the fi lling shoe pushes the tablet down
from the die table. During this fi lling process with a moving fi lling shoe, demixing
of the product mixture is more easily possible than with a fi xed fi lling shoe. This is
one major disadvantage of eccentric tableting machines.
The production rate varies usually between 10 and 60 tablets/min and is determined
by the number of eccentric movements. An example of an eccentric tableting
machine is given in Figure 3 . Other machines are described by Ritschel and Bauer -
Brandl [32] .
FIGURE 2 Operation of eccentric tableting machine: ( a ) fi lling; ( b ) compression; ( c ) ejection;
( d ) pushing from die table.
(a) (b) (c) (d)
TABLETING MACHINES 1059
1060 TABLET PRODUCTION SYSTEMS
6.4.5.2 Rotary Tableting Machines
Rotary tableting machines are commonly used for tablet production. The principle
of all rotary machines is the same, with one exception, which will be discussed separately.
According to Konkel and Mielck [39] , the information gained with eccentric
and rotary machines complement each other.
Rotary tableting machines work with a number of punch and die sets which move
in a circle. The dies are fi xed in a round die table and the die table circulates.
Together with the dies the lower and upper punches circulate on tracks. The lower
punches close the dies. The densifi cation process is bilateral since both punches pass
the compression wheels and the force is evolving on the upper as well as on the
lower side of the powder bed. The produced tablets show the same hardness on the
upper and lower surfaces.
There are different stages of tablet production which happen simultaneously for
several tablets. The central stage of tableting occurs when the punches pass the
upper and lower compression wheels and the compression wheels determine the
downward movement of the upper punch and the upward movement of the lower
punch: The tablet is formed by the resulting punch forces. The compression wheels
can be positioned either by a fl exible swing or more seldom by an eccentric valve
[40] .
FIGURE 3 Example eccentric tableting machine. (Courtesy of Korsch XP1.)
In Figure 4 this principal stage and the other stages of tablet production are
visible. Except for the compression stage the upper punch is always in an upward
position. The upper punch is above the die and the lower punch closes the die,
determining the fi lling depth; then the dies pass below the fi lling shoe and the
powder fl ows into the die up to its upper edge where the powder bed is leveled off
by a scraper. Whereas the upper punch is still in an upward position, the lower
punch is lowered slightly on the track to keep the whole powder volume during
compression and to prevent dusting [33] . Now the punches start to pass the compression
wheels and the main compression event occurs. Forced by the wheels, both
the upper and lower punches move toward each other, compress the tablet, and
leave each other again. The upper punch lifts into an upward position and the lower
punch moves upward to eject the tablet. After ejection the tablet passes a scraper
and it is pushed down from the die table. An example of a rotary machine is given
in Figure 5 .
Additionally often rotary machines are equipped with precompression wheels
(Figure 4 ). In this case the punches pass the precompression wheels before the main
compression events starts. Precompression has the same stages of compression as
the main compression, but the applied forces are lower. Precompression is deemed
to be helpful to avoid, for example, dusting, capping, or lamination. After precompression
only a lower main compression force is necessary.
The number of dies and complementary punches of a rotary machine can vary
between one and up to hundred. The number of punches and the rotation speed
FIGURE 4 Operation of rotary tableting machine with precompression. (Courtesy of
Fette.)
TABLETING MACHINES 1061
1062 TABLET PRODUCTION SYSTEMS
of the die table determine tablet production. Fast rotary machines have up to
120 rpm of the die table [32] . However, the number of tablets produced per hour
not only is determined by the number of punches and the rotation speed but also
is limited by the deformation properties of the tableted material. Materials need
some time for deformation, and if the time during one compaction cycle is not suf-
fi cient for compression and compaction of the material, no tablets result from the
process.
In order to increase the production rate of rotary tableting machines, double -
sided rotary machines were build which possess two pairs of compression wheels
and two fi lling shoes. Thus during one rotation double the number of tablets are
produced compared with a one - sided rotary machine. If these machines are equipped
with precompression wheels one machine contains four pairs of wheels.
Special Rotary Machines As already mentioned one special rotary machine works
slightly different — called IMA Comprima (Figure 6 ) [34] . In this machine the material
is fi lled by centrifugal force from the side directly into the die. The upper punch
closes the die at the top and the lower punch closes the die at the bottom. However,
when the given volume in the die is fi lled by the powder, both punches move downward
until the die is completely closed. Then the compression process starts and the
dies pass the compression wheels. After compression the tablet is ejected by the
upper punch at the bottom of the die, contrary to all other rotary tableting machines
which eject the tablet at the top of the die.
High-Speed Rotary Tableting Machines High - speed rotary machines work with
the same principles as all other rotary machines. They possess a huge number of
punch and die sets and often two fi lling stations. Another possibility is to use punch
and die sets which are able to produce several tablets simultaneously. Special tooling
can be used for this purpose; however this is not the subject of this chapter. As
FIGURE 5 Example rotary tableting machine: left, machine view; right, detail view into
compression chamber. (Courtesy of Kilian Synthesis 500.)
already mentioned production speed depends to a great extent on product properties.
Excellent powder fl ow is essential since the dies have to be fi lled completely.
This is also most essential for the IMA Comprima, which works with centrifugal
force for die fi lling. Often this machine cannot be used since powder fl ow properties
are not suffi cient. To improve powder fl ow on conventional rotary machines special
fi lling devices have been developed (Section 6.4.11.1 ) .
6.4.5.3 Application of Tableting Machines
In summary, the single - punch tableting machines still being used are mainly eccentric
tableting machines (mostly for research), whereas rotary machines with different
production output are predominantly used for production; and for rotary
machines in most cases it is not machine speed that determines the production rate
but material fl ow and compression properties.
6.4.6 TABLETING MACHINE SIMULATORS
(COMPACTION SIMULATORS)
Tableting machine simulators [41 – 48] have been developed in order to mimic tablet
production systems with a very small amount of powder. Similar to eccentric tableting
machines, tableting machine simulators use one pair of punches. Working only
with a single pair of punches reduces the consumption of tableting materials and
facilitates instrumentation for displacement measurement.
6.4.6.1 Hydraulic Compaction Simulators
The fi rst compaction simulators developed were hydraulic [41 – 45] . The hydraulic
system is electronically controlled. An example is given in Figure 7 . Either compression
force cycles or movement of the punches was freely adjustable. This allowed
FIGURE 6 Working principle of Comprima tableting machine with centrifugal fi lling: left,
operation mode; right, centrifugal fi lling. (Courtesy of IMA Comprima.)
TABLETING MACHINE SIMULATORS (COMPACTION SIMULATORS) 1063
1064 TABLET PRODUCTION SYSTEMS
much variation and the primary aim was to mimic the densifi cation process of a
rotary machine and the mechanical factors infl uencing it.
For example, theoretically, from machine geometries, the force – time profi le of a
rotary tableting machine [49 – 52] can be deduced and calculated and the data are
programmed into the compaction simulator. However, the force – time profi le of a
tableting machine could not be calculated. Too many factors infl uence the measured
force, for example, the tableted material, the geometries of the machine, the machine
wear time, tableting speed, and tableting tools. Similarly, the displacement – time
profi le of a tableting machine, especially a rotary tableting machine, is very diffi cult
to calculate. It has been shown that calculation from machine geometries is only
possible to a certain extent. Mainly the mechanics of a tableting machine cannot be
completely simulated [53, 54] . Thus either an approximated displacement – time
profi le can be used for programming the compaction simulator or approximation
of real punch movement is only possible using recorded data from real tableting
machines.
Thus the simulation of tableting machines needs much effort and a real simulation
is almost impossible because of the hydraulic control. Further the fi lling process
of rotary tableting machines cannot be simulated since die fi lling is usually processed
by a fi lling shoe moving forth and back.
However, hydraulical compaction simulators are still used in research for basic
material characterization. They show the advantage of controlling speed exactly and
of using low and high punch travel speeds, between 10 and 300 mm/s. Mostly a simple
displacement profi le is used for characterization (e.g., a saw tooth or a sine wave
profi le), and the evolving forces at the lower and upper punches are measured.
Further the speed of the punches can be controlled separately and both punches
move freely and independently from each other. Time intervals in which the punches
stand still can be freely set. Thus lots of freedom for material characterization is
possible and these compaction simulators are important tools.
Another advantage of compaction simulators is that only small amounts of material
are necessary to produce a tablet. One single tablet can be produced at low as
well as at high speed of the punches. This is important in order to evaluate defor-
FIGURE 7 Example hydraulically working compaction simulator: left, machine view; right,
detail view into compression chamber. (ESH compaction simulator, Courtesy of Huxley
Bertram.)
mation properties of a formulation already in early dosage form development when
only small quantities of the drug substance are available. Since nowadays the timelines
for production of a new medicine are tight, this advantage of compaction
simulators becomes more and more important.
6.4.6.2 Mechanical Compaction Simulators
More recently mechanical compaction simulators have been developed. The fi rst
was the linear mechanical rotary tableting machine simulator Presster (Figure 8 ),
which was introduced in 1998 [55, 56] . It can mimic the mechanics of different rotary
tableting machines and is called a linear rotary tableting machine replicator. The
name Presster was combined from press and tester.
A single pair of punches moves linearly forth and back on a lower and an upper
punch track. For tableting the punches pass the compression wheels which are
equivalent in dimensions to those of rotary tableting machines used in practice.
These compression wheels are exchangeable. Different machines are simulated by
exchanging them.
The machine speed can be varied and different tableting machines are simulated
by using similar dwell times between 5 and 80 ms. Special tests exhibited that rotary
tableting machines can be simulated with a precision of 1 – 5% [57, 58] . One major
disadvantage of the Presster is that it works with a moving fi lling shoe, and thus the
fi lling process of rotary machine cannot be simulated.
In addition, the present model of the Presster possesses precompression wheels
and thus, besides compression, precompression can be simulated. This is import
FIGURE 8 Detail of Presster. (Courtesy of MCC Corp.)
TABLETING MACHINE SIMULATORS (COMPACTION SIMULATORS) 1065
1066 TABLET PRODUCTION SYSTEMS
when studying the effect of precompression on the fi nal tablet properties. However,
the time between precompression and main compression is determined by the
Presster geometries since the positions of the precompression and main compression
wheels are fi xed.
The newest development for compaction simulation is a mechanical tableting
machine simulator which operates with a cam. Thus it is called Stylcam (Figure 9 )
[59] . The cam is positioned on the lower compression wheel and allows the simulation
of different tableting machines and their dwell times due to different acceleration
of the punches. It was introduced in 2005.
With the Stylcam different dwell times are obtained by adjusting the speed of
the compression wheels. Precompression is simulated by compressing a tablet twice.
Thus the time interval between the precompression and main compression is freely
adjustable. One further advantage of the Stylcam is that it works with a fi xed fi lling
shoe, as on a conventional rotary tableting machine. However, data on the precision
of this instrument are not yet available.
6.4.6.3 Application of Tableting Machine Simulators
Using mechanical compaction simulators allows us to simulate the tableting process
of rotary tableting machines to a greater extent than when using hydraulical compaction
simulators. Thus they will be mainly used in formulation development and
scale - up.
FIGURE 9 Detail of Stylcam. (Courtesy of MedelPharm.)
However, for mechanical compaction simulators the movement of the punches
is mechanically determined and, compared to hydraulic compaction simulators, not
freely programmable. Thus, for basic material characterization and early formulation
development, hydraulical compaction simulators can be advantageous.
6.4.7 INSTRUMENTATION OF TABLETING MACHINES
To describe the tableting process more precisely, tableting machines have been
instrumented since the middle of the last century. Measured values are force, displacement,
and temperature and they are always measured with dependence on
time. Thus time is another variable.
6.4.7.1 Force Measurement
The fi rst instrumentation of a tableting machine for measurement of upper punch
force was performed by Brake [60] . Thus it was for the fi rst time possible to visualize
the compression process with regard to force development which results from
the material stresses during tablet formation. Only shortly after that, similar measurements
were published by another research group [61, 62] .
Besides upper punch force, lower punch force, die wall force [63 – 65] , ejection
force [66] , and tablet scraper force can be measured. Die wall force measurement
will be discussed separately.
For measurement strain gages are mostly used. These strain gages consist of
constantan. They are applied in eccentric tableting machines at the upper or lower
punch holder and in rotary tableting machines at the machine frame or the compression
roll pin. Alternatively piezoelectric crystals can be used which have to be placed
inside the punches [40] .
The sensitivity of force measurement is dependent on the distance between the
force transducer and where the force occurs. Thus for force measurement, instrumentation
of the punches is more advantageous than instrumentation of the machine
frame. However, since punch and die sets have to be exchanged between different
runs of the machines, instrumentation of the punch holder, the machine frame, or
the roller pin is most widely spread.
The most often measured force is the upper punch force. For the eccentric
machine it is the force which controls densifi cation; for rotary tableting machines
upper and lower punch forces have ideally the same values. Schmidt et al. [67]
measured force with a single punch of a rotary tableting machine. Ejection force is
visible as a small lower punch signal which occurs shortly after the end of one compaction
cycle. It is measured by lower punch instrumentation but needs more resolution.
A review of force measurement is given by Bauer - Brandl [68] .
Die Wall Force Measurement During compression of the powder the forces are
evolving not only at the punches but also at the die wall [63 – 65] . Therefore die wall
force measurement complements upper and lower force measurement. Since the
compression process occurs axially, these radially evolving forces are smaller than
the forces at the punches. Measurement of die wall force allows, for example, for
indication on die wall friction, tablet capping, and lamination. Instrumentation for
INSTRUMENTATION OF TABLETING MACHINES 1067
1068 TABLET PRODUCTION SYSTEMS
die wall force measurement is diffi cult and different techniques have been developed
[64, 69, 70] . The die can be instrumented axially or radially and strain gages
or piezoelectric crystals can be used for measurement. Two main effects infl uence
the measured signal: tablet height and tablet position. Related to this, the output
signal can be nonlinear. Piezoelectric foils have been applied which possess the
advantage of independence on tablet position [70] .
One example most recently developed is a split die consisting of three sections
(Figure 10 ) [64] . Integrating the sensing web in a thin middle layer isolates stress
measurement to a narrow band around the tablet and gives much closer approximation
to the true stress. Further die wall force measurement is linear and independent
of tablet height and position as it is uncoupled from all other die wall stresses and
strains. Further it is designed in the shape of a conventional die and can be mounted
without modifi cation into a die table.
6.4.7.2 Displacement Measurement
The fi rst measurement of upper punch displacement was performed in the mid -
1950s by Higuchi and co - workers [62, 71] with the aid of inductive displacement
transducers. By the same instrumentation the movement of the lower punch can be
visualized. Inductive transducers are mounted parallel to the punch and thus give
information on punch position. Alternatively touchless measurement of displacement
is possible. It is important that the transducers be positioned most closely to
the punch in order to minimize the infl uence of machine deformation.
A measurement of displacement on a rotary tableting machine was presented in
1987 by Schmidt and Tenter [72] . Another possibility was presented by Matz and
co - workes [73, 74] . Meanwhile touchless measurement systems for recording displacement
were developed [73, 74] . For all measurements of displacement, correc-
FIGURE 10 Construction details of split die. (Reproduced with permission from ref. 64.)
dp dp
ds dt
wh
wt
Strain gauge
dt
do
di: inner diameter
do: outer diameter
ds: screw hole diameter
dp: alignment pin hole diameter
wa: web height
wt: web thickness
= in.
= in.
= in.
= in.
= in.
= in., in.
38
785
32
18181
16
18
tions which take elastic punch and machine deformation into account are necessary.
M u ller and Caspar [75] showed problems which occur when machine and punch
deformation are not taken into account. Krumme and co - workers gave an extensive
description on this issue for eccentric tableting machines [76, 77] . From punch displacement
measurements tablet height can be calculated, and this height can further
be related to tablet density and porosity.
Again and again it was tried to derive displacement theoretically from machine
geometry, punch geometry, and measured force [49 – 52] . However, until today this
theoretical derivation has not been satisfying and thus experimental testing cannot
be given up.
6.4.7.3 Temperature Measurement
Due to the forces evolving during tableting the compressed material can warm up.
The evolving temperatures can be measured with different methods. In each case
only approximated measurements are possible since either the measurement was
not performed directly inside the tablet or additives were necessary, which can alter
measurement.
Several methods have been applied to determine the temperature increase with
thermal sensors which are installed in the punches, inside the die, or in the powder
bed (Table 1 ) [78 – 87] .
Most recently an analysis technique was developed which allows measurement
of tablet temperature directly after ejection of the tablet on the machine (Figure
11 ) by an infrared sensor [85] . The temperature signal can be directly related to
force and displacement measurement.
6.4.7.4 Measurement of Time
All measured variables can be determined with dependence on time. Thus the
tableting process can be characterized for several variables with dependence on
time.
Two time defi nitions are important: contact time and dwell time. Contact time
can be defi ned as the time during which a contact of powder and punches is measurable,
for example, when the force exceeds a certain limit of 100 N. Dwell time can
be defi ned predominantly for rotary machines as the time during which the punch
heads are completely under the compression wheels and thus the applied force is
constant.
TABLE 1 Methods to Determine Temperature During and Shortly After Compaction
Measurement in punches [78 – 80]
Epoxide punches [81]
Calorimetric measurement [82, 83]
Infrared measurement [84, 85]
Measuring conductivity during tableting with conductive materials [86]
Energy calculations [78, 83, 84]
Determination of melting of materials with certain melting point [87]
INSTRUMENTATION OF TABLETING MACHINES 1069
1070 TABLET PRODUCTION SYSTEMS
Further for description of the tableting process, the entering of the punch into
the die, the compression start at the begin of contact time, and the lifting of the
upper punch from the tablet are important.
6.4.8 ANALYSIS OF TABLETING PROCESS
All measured variables, namely force, time, displacement, and temperature, can be
combined differently and can be analyzed afterward. From the functional relations,
conclusions can be drawn about the compression and compaction behavior of the
materials.
The most basic analysis is the presentation of force versus time or displacement
versus time. These curves are different for eccentric and rotary tableting machines.
The data given in Figures 12 and 13 are valid for the contact time of the compaction
cycle of one single tablet. Due to the eccentric - driven movement of the punches,
the force – time curve can be described by a sharp peak at the maximum force evolving
at the punches and the displacement – time curve can be described with a sharp
peak at the minimum height of the powder bed. For curves of eccentric tableting
FIGURE 11 Infrared sensor unit for measuring temperature directly after tableting [85]
(Martin - Luther - University Halle - Wittenberg.)
Upper
punch
Die
holder
IR sensor
Tablet
FIGURE 12 Force – time and displacement – time profi le for eccentric tableting machine.
Force (kN)
Time (ms) Time (ms)
Displacement (mm)
(a) (b)
machines hardly any dwell time is measurable (Figure 12 ). In contrast, for rotary
tableting machines, the force – time curve and the displacement – time curve are
fl atter at the maximum peak. This is the case due to the dwell time when the punch
heads move completely between the compression wheels. The dwell time is indicated
in Figure 13 . For rotary tableting machines with precompression wheels
additionally force – time curves and displacement – time curves for precompression
can be recorded. They look similar to the curves from the main compression wheels
with the exception that lower forces are applied. Precompression data will not be
discussed in the following since the data can be treated similar to the data of the
main compression event.
Besides this presentation of force and displacement versus time, which are data
directly derived from the tableting machine, other more advanced methods are
possible. Extensive reviews on the methods used can be found in the literature
[54, 88, 89] . In the following only the most important aspects will be discussed.
6.4.8.1 Force – Time Analysis
One method to analyze tableting data is the use of force – time or pressure – time
diagrams. They are easily recordable since displacement measurement is not
necessary.
Some basic parameters can be directly read from the curves. For the force values
upper and lower punch forces and ejection forces should be mentioned, and for the
time values contact time should be mentioned. Deduced parameters such as pressure
and normalized contact time can be calculated and further statistical data are
often used for characterization (Table 2 ). Due to the different shapes of force – time
curves from eccentric tableting machines compared with those from rotary tableting
machines, some parameters can only be calculated from eccentric machine data and
some can only be calculated from rotary machine data.
FIGURE 13 Force – time and displacement – time profi le for rotary tableting machine.
Force (kN)
Displacement (mm)
Time (ms) Time (ms)
(a) (b)
ANALYSIS OF TABLETING PROCESS 1071
1072 TABLET PRODUCTION SYSTEMS
Only eccentric machine data allow us to calculate the R value (maximum upper
punch force/maximum lower punch force), which is an indication of friction. They
also allow us to calculate the time difference between the maximum upper punch
force and the maximum lower punch force. Only dwell time and the minimum force
during the dwell time can be calculated for rotary tableting machine data. The rise
time of rotary machines is defi ned as the time during the compression phase, and
peak offset time is defi ned as the time difference between maximum pressure and
vertical alignment of the punches. Further the infl ection points during the compression
and decompression phases are mostly only calculated for rotary machine
data.
In addition, for force – time diagrams different methods to characterize the tableting
process were developed. These methods can be divided in those applicable to
force – time curves from eccentric and rotary tableting machines [90] and those
applicable only to data from eccentric or rotary tableting machines (Tables 3 – 5 ).
One possibility to analyze the tableting process is to describe the areas under the
curve during compression and decompression and to draw conclusions on plastic
and elastic parts of deformation. Emschermann and M u ller [91] applied this method
to data from eccentric machines (Figure 14 ). Similar area comparisons were performed
by the research group of Schmidt [92 – 94] for rotary machines (Figure 15 ).
They tried to gain information on elasticity by calculating differences between the
area under the plot in the compression phase and the area under the plot in the
decompression phase. A sophisticated technique to interpret area data under one
TABLE 2 Parameters Directly Deduced from Force – Time Profi les
Maximum upper punch force (pressure)
Maximum lower punch force (pressure)
Maximum ejection force (pressure)
Contact time
Normalized contact time
Time at maximum upper compression force
Time at maximum lower compression force
Maximum upper precompression force (pressure)
Maximum lower precompression force (pressure)
Precompression contact time
Normalized precompression contact time
Time at maximum upper precompression force
Time at maximum lower precompression force
TABLE 3 Parameters Calculated for Eccentric and Rotary Tableting Machines
Source Parameters
Chilamkurti [90] Area under curve (AUC)
Height of curve at maximum upper punch force
Width of curve at half - maximum upper punch force
Slope during compression
Slope during decompression
TABLE 4 Parameters Calculated for Eccentric Tableting Machines
Source Parameters
Emschermann [91]
(Figure 14 )
Area under compression curve (compression area)
Area under decompression curve (decompression area)
Compression area/decompression area
Pressure – time function
(modifi ed Weibull
function) [39, 96 – 98]
(Figure 16 )
p p
t t =
.
. .
max, exp upper punch
1
1
1
. .
. .
where . = time parameter
. = asymmetry parameter
Modifi ed Fraser – Suzuki
function [99]
f t H
A
A t t
S
( ) exp
.
ln
( )
. = . ...
...
. + . . ( ). ...
...
...
...
0 693 1
1 177 2
2
r
where A = asymmetry parameter
t r = time parameter
S = deviation of maximum
TABLE 5 Parameters Calculated for Rotary Tableting Machines
Source Parameters
Tenter [72] Area center of gravity
Vogel [92 – 94] (Figure 15 ) t 1 = compression time
t 2 = time at the start of dwell time
t 3 = time at the half of dwell time
t 4 = time at the end of dwell time
t 5 = time at the end of one compression cycle
A 1 = area of densifi cation
A 4 = area of decompression
A 2 = partial area
A 3 = partial area
A 3 / A 2 = area quotient
A 5 = partial area
A 6 = partial area
A 6 / A 5 = area quotient
FIGURE 14 Pressure – time curve analysis [91] .
Pressure
Time
1073
1074 TABLET PRODUCTION SYSTEMS
compaction cycle was developed. The parameters of interest are given in Table 5 .
Further advances were performed by Yliruusi and co - workers [95] .
Another possibility of analysis is to fi t different functions to the force – time data.
The research group of Mielck [39, 96 – 98] described the densifi cation behavior by
the pressure – time function (Figure 16 ). The lower the values of the parameters .
and . , the more plastically the material deforms; the higher the values, the more
elastically the material deforms. The parameter . describes the asymmetry of the
curve and . the time at maximum densifi cation. A similar function, the Fraser –
Suzuki function, which originates from chemical analytics was applied to tableting
data [99] . It can also be used to derive parameters that describe the deformation
behavior of materials. Information on the reversible and irreversible deformation
of the material can be deduced.
FIGURE 15 Pressure – time curve analysis [92, 93] .
Pressure
A5 A6
A1 A2 A3 A4
t2 t1 t3 t4 t5
Time
FIGURE 16 Pressure – time curve analysis with pressure – time function [39, 96 – 98] .
Time
Pressure
6.4.8.2 Displacement – Time Analysis
From displacement – time data a few parameters can be read. The most important
data are given in Table 6 . In addition the data allow us to calculate fast elastic
recovery. The increase in tablet height from the minimum tablet height in the die
up to the lifting of the upper punch from the tablet is called fast elastic recovery.
Since the travel of the punches is analyzed, the data also allow us to determine
the speed of the punches at each point of the compaction cycle. Punch speed is an
important parameter to compare different tableting events [100] . Maximum punch
speed can be determined and used for characterization.
Only a few authors have tried to relate displacement with time. Ho and Jones
[101] determined the slope of porosity over time (rise time). This slope was also
used by Tsardaka [102] for analysis.
6.4.8.3 Force – Displacement Analysis
The most extensively used method to characterize the tableting process is the use
of force and displacement measurements. Usually upper punch force and upper
punch displacement are used. Models which relate force and displacement directly
can be distinguished from those which analyze pressure and volume.
Further, some parameters can be directly read from the curves. The peak offset
time of eccentric tableting machines is defi ned as the time difference between
maximum displacement and maximum compression force [101, 103] .
Relation between Force and Displacement The fi rst information on force –
displacement analysis can be found in F u hrer [104] and Moldenhauer et al. [105] .
Force – displacement diagrams (Figure 17 ) are used to calculate from the areas
enclosed the work or energy necessary for tableting. The force – displacement profi le
includes compression and decompression of the powder to the tablet. The area
between compression and decompression is the area of the compaction energy,
often called the energy of plastic deformation ( EP ) [106] . The area EE is the energy
of elastic deformation. And the last area ( EF ) to complete the triangle start of compression
( D0 ) – maximum force ( Fmax ) – displacement at maximum pressure ( DPM ) can
be interpreted as the energy of friction ( EF ) [107, 108] . The sum of all three energies
is the total energy ET of the tableting machine. The energies can be displaced as an
absolute value or relative to the total energy. Based on these values, Stamm and
Mathis [109] developed the determination of plasticity P as
TABLE 6 Parameters Directly Deduced from
Displacement – Time Profi les
Maximum displacement
Minimum height of tablet
Minimum volume of tablet
Maximum density of tablet
Minimum porosity of tablet
Maximum relative density of tablet
Time at maximum displacement
Time at minimum height of tablet
ANALYSIS OF TABLETING PROCESS 1075
1076 TABLET PRODUCTION SYSTEMS
P
E
E E
=
+
P
P E
(1)
Other authors tried similar attempts [110, 111] and the developed methods were
regarded to be very useful [27, 112 – 115] . Antikainen and Yliruusi [116] more
recently tried to derive further parameters from the diagrams to enable a more
complete characterization. An overview on the possibilities for force – displacement
analysis is given by Ragnarson [117] .
Relation between Volume and Pressure The oldest method of this type of analysis
is to establish a relation between the volume of the tablet and the force necessary
to produce this volume [62, 71] . For exact description the height of the tablet is
determined by displacement measurement and the accuracy of this measurement is
extremely important. Further displacement measurement has to be corrected precisely
for elastic deformation of the punches and the machine in order to use correct
tablet heights.
From tablet height, volume, porosity, and relative density at different stages of
densifi cation can be deduced. These variables are plotted as a function of pressure.
For analysis, for example, the equations of Heckel [118 – 120] , Kawakita, [121, 122] ,
Cooper and Eaton [123] , Walker [124] , Bal ’ shin [125] , and S o nnergaard [126, 127]
can be used. The equations are given in Table 7 . A further overview of these and
other equations used can be found in Celik [88] .
The Heckel equation describes the densifi cation process with fi rst - order kinetics.
A linear equation is obtained with a slope which is inversely proportional to the
yield strength. The slope of the Heckel equation provides information on the plastic
deformation of the powder. It has also been published that the slope of the Heckel
equation can be correlated with the elastic modulus (Young ’ s modulus).
FIGURE 17 Force – displacement diagram for energy analysis [104 – 108] .
Force (kN)
Displacement (mm)
D0 DPM
EP
EF
Fmax
EE
TABLE 7 Parameters Calculated from Force – Time Profi les
Source Parameters
Heckel [118, 119] (Figure 18 )
. =
. ( )= + ln ln . 1
1 D
Kp A
where K = deformation parameter
A = powder bed densifi cation
Yield pressure [120]
Yield pressure
Heckel slope
= 1
Yield strength [118]
Yield strength
Heckel slope
=
.
1
3
Kawakita [121, 122]
p
C ab a p
= + 1 1
where a = porosity of powder bed
b = compression parameter
Walker/Balshin [124, 125]
100 100 V
V
V
W p C rel= . =. +
.
log
where W = compressibility coeffi cient
S o nnergard [126]
V V W p Vee
p pm
rel
/ = . + .
1 log
where W = compressibility coeffi cient
Cooper – Eaton [123]
V aV
V V
V V
a e a e i i
i
n
k p k p * * / / = = .
.
= +
= .
. . .1
0
0
1 2
1 2
where k 1 = deformation pressure for fraction part 1
k 2 = deformation pressure for fraction part 2
a 1 = fraction part 1 of deformation
a 2 = fraction part 2 of deformation
Cooper – Eaton (linearized) [123]
ln ln ln V
V V
V V
Q
p
R * = .
.
= . +
.
0
0
where Q = extent of compressibility
R = sum of fraction parts
The equation of Kawakita describes volume reduction with pressure in the form
of a hyperbolic equation. Walker and Bal ’ shin [125] postulated a logarithmic relation
between applied pressure and volume reduction, which was further modifi ed
by S o nnergard [126] . Cooper and Eaton [123] use an exponential function, which
can also be linearized. Pressure thresholds for deformation mechanisms are determined.
It should be noted that all of these equations and tableting models determine
descriptive parameters.
The equation of Heckel is the most extensively used model and the underlying
porosity – pressure plot is called a Heckel plot (Figure 18 ). The equation for
the linear compression process follows fi st - order kinetics (Table 7 ). Heckel
ANALYSIS OF TABLETING PROCESS 1077
1078 TABLET PRODUCTION SYSTEMS
distinguished measurements which determine the volume of the tablet without pressure
(zero pressure) [119] from those measurements which determine the volume
with pressure [118] . The fi rst method allows determination of the volume after
release of the elastic energy; the second method allows a higher precision repeatability
since it is often diffi cult to determine tablet height at a defi ned time after
ejection. Some milliseconds can cause differences [35, 36] . Years later Sun and
Grant [128] tried to determine the elastic part at pressure measurements. The
experiments showed that deviations in Heckel plots at high pressures are dependent
on the elasticity of the material.
The equation of Heckel has been discussed again and again. One main issue of
critique is that pharmaceutical powders are not purely plastically deforming materials
and thus particle size and deformation mechanisms infl uence the derived parameters
[129, 130] . Already very small errors in displacement determination or the
measurement of true density can induce huge errors in the derived parameters
[75 – 77, 129, 131, 132] . S o nnergaard [126] referred the equation of Walker and
Bal ’ shin for his characterization of materials. He criticized further that the yield
strength derived from the Heckel equation is directly dependent on the true density
of the powders [127] .
Despite this critique of the Heckel equation, the analysis of Heckel plots has
been intensively used for the description of powder compression [128, 133 – 136] .
Gabaude et al. [136] stated that the analysis is quite useful when defi ning preconditions
exactly and apply correct displacement measurement.
Since the development of the equation, it has been tried to derive further information
from it. Rees and Rue [129] determined the area under the Heckel plot.
Duberg and Nystr o m [137] used the nonlinear part for characterization of particle
fracture. Paronen [138] deduced elastic deformation from the appearance of the
Heckel plot during decompression. Morris and Schwartz [139] analyzed different
phases of the Heckel plot. Imbert et al. [134] used, in analogy to Leuenberger and
Ineichen [14] , percolation theory for the compression process as described by
the Heckel equation. Based on the Heckel equation, Kuentz and Leuenberger
[135, 140] developed a new derived equation for the pressure sensitivity of tablets.
FIGURE 18 Heckel plot [118, 119] .
Pressure (MPa)
In(1/1 - Drel)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 50 100
Tsardaka and co - workers [102, 141, 142] presented the Heckel plot with dependence
on time and analyzed deformation in combination with elastic recovery. Additional
areas to describe plasticity were determined from two - dimensional (2D) plots [129] .
Finally, the three - dimensional (3D) model [143, 144] was developed by fi tting a
plane to a 3D data plot on the basis of normalized time, pressure, and porosity
according to Heckel.
6.4.8.4 Force – Displacement – Time Analysis
Force, displacement, and time are the three most important parameters to characterize
the compaction cycles of tableting materials. Even when Hoblitzell
and Rhodes postulated a linear relationship between force – time and force –
displacement data [145] , this could not been exactly proved until today. Thus it is
im portant to analyze these three measured data together.
From force – displacement – time curves some parameters can be directly deduced;
for example, the power during tableting and the maximum power can be determined.
Another important parameter which serves as a measure for viscoelasticity
is the peak offset time [101, 103] . For single - punch tableting machines it is defi ned
as the time difference between maximum displacement and maximum force.
In addition, advanced models as those calculating viscoelasticity and the 3D
model have been developed. They will be described in the following.
Viscoelasticity Models For characterization with viscoelasticity models, simulation
models have been developed on the basis of Kelvin, Maxwell, and Voigt elements.
These elements come from continuum mechanics and can be used to describe
compression.
David and Augsburger [146] were the fi rst to try this method of analysis. Further
tests, for example, determination of complex functions based on methods of numerical
mathematics, were performed by the research group of M u ller [147, 148] .
Although the results were helpful, for exact description the models became rather
complicated and the derived parameters were complex. According to Bauer [149] ,
these models have to be three dimensional for a reasonable description. Another
similar approach was used by the research group of Rippie [12, 13] . They described
the structure evolution in the tablet during decompression by the aid of vectorial
3D models and concluded that fracture and stress contribute to the fi nal structure
of the tablet.
Other research groups derived viscoelastic properties from creep experiments of
the fi nal tablet [150 – 154] . As Tsardaka and Rees [142] determined, stress relaxation
follows a hyperbolic equation.
3D Model Most recently another technique which uses force, displacement, and
time has been developed. 3D modeling is a very useful method to characterize the
tableting process [3, 46, 47, 155 – 159] . Force is expressed as pressure, time has to be
normalized, and from displacement data the porosity according to Heckel [119] is
calculated. It is the only possibility to combine these variables during analysis.
To describe the tableting process the three variables were presented in a 3D plot
(3D data plot) and a plane was fi tted to the data twisted at t = tmax (Figure 19 a ).
From the fi tting process the parameters d (time plasticity), e (pressure plasticity),
ANALYSIS OF TABLETING PROCESS 1079
1080 TABLET PRODUCTION SYSTEMS
FIGURE 19 ( a ) 3D data plot with fi tted plane twisted at t = t max and ( b ) 3D parameter plot
of ( ) DCPD: dicalcium phosphate dihydrate, ( ) spray - dried lactose, ( ) MCC: microcrystalline
cellulose, ( ) theophylline monohydrate, and ( ) HPMC: hydroxypropyl methylcellulose
for data gained with an eccentric tableting machine [47] .
Time (normalized)
Pressure (MPa)
In[1/(1-Drel)]
2,5
2
1,5
1
0,5
0
–0,5
–1
0
0,2
0,4
0,6
0,8
1 0
50
100
150
0.04
0.02
0.00
0
1
2
0.000
0.005
0.010
0.015
0.72
0.74
0.90
0.84 0.72
0.89
0.88
0.72
Increasing .rein, max
d
e (MPa–1
)
.
(b)
(a)
and . (twisting angle, which indicates fast elastic decompression) can be derived
(Table 8 ).
The parameters of the fi tted plane (time plasticity d , pressure plasticity e , and
twisting angle . ) were also exhibited in a 3D plot and this plot is called the 3D
parameter plot. This plot exhibits the compression behavior of the powder. It gives
a simple yet characteristic description of the tableting properties. An example is
given in Figure 19 b .
TABLE 8 Parameters Calculated from Force – Displacement – Time Profi les
Source Parameters
3D model [143,
144, 155]
(Figure 19 )
z
D
t t d p p ep f dt =
. ( )= . + . + + + ln [( )( )] ( ) ( ) max max max
1
1 rel
.
d
D
t
e
D
p
f
D
= . = . =
. ( ) .
.
.
.
ln[ ( )] ln[ ( )]
ln
1 1 1 1 1
1
/ / rel rel
rel
where D rel = relative density
t = time
p = pressure
t max = time at maximum pressure
p max = maximum pressure
. = twisting angle at t max , indicates fast elastic decompession
d = time plasticity
e = pressure plasticity
Time plasticity d describes the plastic deformation of the excipient according to
time [160] . It is infl uenced by tableting speed [157] . With increasing time plasticity
d the powder deforms faster during tableting. Therefore, with increasing densifi cation
time plasticity increases. Pressure plasticity e describes the pressure - dependent
increase of density. The pressure plasticity correlates with the slope of the
Heckel equation [144] . With increasing pressure plasticity e the slope of the Heckel
equation increases in the same direction and the necessary pressure for deformation
(yield pressure [118] or yield strength [120] ) decreases. The twisting angle .
is a measure of the material ’ s elasticity and the ratio between compression and
decompression. Thus, it indirectly describes fast elastic decompression during the
tableting process. When . increases, elasticity decreases. The twisting angle . correlates
with the elastic modulus [144] . In conclusion, materials which deform fast
show high d values, materials which deform easily and with low pressure show
high e values, and those which relax a lot show a lot elasticity and thus low .
values.
Thus the 3D model allows us to characterize the tableting process completely
and to distinguish time - dependent information from pressure - dependent deformation
and elasticity in one step of the analysis.
Temperature Analysis The results gained by determination of temperature during
and shortly after tableting vary strongly and depend on the method used.
A temperature increase of 5 K [79, 80, 161] could be determined with conventional
punches; however, with epoxide punches the measured temperature increase
was as high as 30 K [81] . By calorimetric measurement an increase of 10 up to 30 K
[82] was determined and by infrared measurement the increase was 10 – 15 K
[84] .
Beissenhirtz [86] measured a temperature increase of 30 K indirectly by measuring
conductivity which arose during tableting with conductive materials. Energy
calculations indicate a temperature increase of more than 30 K caused by tableting
ANALYSIS OF TABLETING PROCESS 1081
1082 TABLET PRODUCTION SYSTEMS
[78, 84, 83] . Most recently, partial melting of drugs could be analyzed for materials
whose melting temperature is as high as 94 ° C [87] , and the reversible transgression
of a glass transition temperature of 80 ° C was determined [162] .
All results indicated that temperature increase depends on the material. Further
temperature increase during tableting can contribute to slight changes in material
structure [85] .
6.4.9 ANALYSIS OF FINAL TABLET FORMATION
This characterization of the process of tablet formation has to be completed by
analyzing the changes induced by tableting.
Most important is the elastic recovery of the tablets which starts during decompression
and is fi nished dependent on the material after several days. Elastic recovery
can be defi ned as [163] .
ER % t ( ) min
min
= . .
100
H H
H
(2)
where H min is the minimum height of the tablet under load and H t the height of the
tablet at different times t after tableting.
Elastic recovery gives information on the remaining elasticity of the materials
which is only slowly released. Further it can indicate structural changes inside the
materials and tablets. Structural changes induced by tableting have to be analyzed
by physicochemical techniques, such as spectroscopic and thermoanalytical methods,
X - ray diffraction, scanning electron microscopy, and transmission electron microscopy
[35, 36, 85, 164 – 166] . The analyzed changes will help to better understand the
process of tablet formation and identify the reasons for compactibility of materials.
However, these changes are not the subject of this chapter.
6.4.10 COMPLETE DESCRIPTION OF PROCESS
OF TABLET FORMATION
On the whole, the process of tablet formation can fully be described by combining
the analysis of the tableting process with the fi nal formation of the tablets. The
methods which gives most detailed information of the whole process and simultaneously
is a fast method is the 3D modeling technique in combination loith calculating
the elastic recovery of the tablets. In addition, by combining both these methods
and calculating general plasticity P from time plasticity d , pressure plasticity e , twisting
angle . , and elastic recovery ER, a more general tool for analysis of the process
of tablet formation is available [3, 4] .
Finally the crushing force of the tablets after relaxation gives information on
the formed bonds inside the material and the compactibility. Compactibility has
been described by Leuenberger [167] . For the future it can be expected that a
prediction of compactibility as a result of the process of tablet formation is
possible.
6.4.11 SPECIAL ACCESSORIES OF TABLETING MACHINES
In tablet production it is essential to control tablet weight and tablet homogeneity
in order to ensure a uniform dosage form. For patient safety pharmacopeias demand
that tablet weight is between certain limits. Tablet producers often set their more
narrow specifi cations to ensure that they meet pharmacopeial specifi cations. To
control tablet weight and tablet homogeneity an optimal product mixture, complete
fi lling of the die, exact tooling, and tightly controlled machine conditions are
necessary.
Another demand for patient safety and due to GMP regulations is to ensure the
absence of impurities in the fi nal dosage form, for example, residues from the previous
product or residues from detergents. Thus the process of cleaning of machines
has to be standardized and controlled. In addition optimized short cleaning times
of the machine increase operating time for production. During the last years one
innovation for tableting machines was the development of special accessories for
cleaning in order to reduce standing times.
For special products (e.g., cytostatics or sterile products), it is necessary to
produce tablets in a hermetically closed machine. For these products special containment
solutions have been developed which allow the production in a hermetically
closed machine or behind a wall. Most important is to separate the tablet
production zone strongly from the mechanics of the machine.
In the following tablet fi lling devices, possibilities to control tablet weight
and mixing homogeneity as well as advances in cleaning technology will be
discussed.
6.4.11.1 Optimization of Die Filling
The basics of fi lling have been explained above: The fi lling shoe is moving back and
forth, the fi lling shoe is fi xed, or fi lling is centrifugally controlled. Two problems arise
generally: Either the product demixes and tablet weight and content uniformity are
no longer controlled or the die is not completely fi lled and thus tablet weight also
varies.
Optimal fi lling of the die is determined to a great extent by the material, but the
speed of the machine is also important. At low machine speeds the die is usually
completely fi lled; at high machine speeds this becomes more diffi cult. Thus special
fi lling devices using one or more paddles have been developed to improve fi lling.
One example for a paddle feeder is given in Figure 20 . Alternatively fi lling devices
can be vibrated to improve feeding for materials with bad fl ow characteristics. An
overview is given by Ritschel and Bauer - Brandl [32, 168] .
Besides improved feeding, paddle feeders allow improved mixing uniformity
since the formulation is mixed again shortly before feeding the die. This mechanical
remixing is the only possibility to improve the homogeneity of the mixture. Demixing
is a bigger problem for machines with a moving fi lling shoe than for those with
a fi xed fi lling shoe. Thus moving fi lling shoes are equipped with paddles as a standard.
Fixed fi lling shoes need paddles usually only for materials with bad fl owability
or at high machine speeds.
SPECIAL ACCESSORIES OF TABLETING MACHINES 1083
1084 TABLET PRODUCTION SYSTEMS
6.4.11.2 Tablet Weight Control
To control tablet weight different possibilities exist. The simplest method is to weigh
at preset intervals (in - process control) a number of tablets manually and to adjust
machine settings according to the results when necessary. For high - speed rotary
machines automatically working weighing systems have been developed which
determine tablet weight shortly after ejection [169, 170] . These systems can also
determine tablet height and diameter, which are indirect measures for tablet
uniformity. Simultaneously with tablet weight compression force drifts. A direct
relation between compression force and tablet weight exists. Thus it is possible
to monitor tablet uniformity also by control of compression force. For this purpose
the machines are instrumented with strain gauges or piezoelectric force transducers.
By control of compression, force changes in tablet weight can be directly
detected.
Automatically working control systems are able to eject those tablets separately
which fail the requirements (rejection mechanism), and they adjust the machine
for die fi lling or compression force and collect only those tablets which meet the
requirements. Two alternative principles for automatic tablet weight control and
adjustment are possible, depending on the application and selected machine type.
The principle of control of compression force is based on measurement of the fi nal
compression force under constant tablet height. This principle is used for all applications
where tablet weight accuracy and constant tablet density are less critical.
The principle of control of displacement is based on measurement of tablet
height variations under constant force. This principle is more accurate than the force
control system. It is used for all applications where constant density of the produced
tablets is critical.
Modern systems combine one of these control systems with automatic weighing
of tablets. Weight control will automatically adjust the fi lling depth in order to keep
tablet weight within specifi ed tolerance limits.
6.4.11.3 Control of Mixing Homogeneity
The systems to control weight uniformity are not able to control uniformity of the
mixture. When during fi lling of the die the tableted material demixes, tablet weight
FIGURE 20 Example paddle feeder. (Courtesy of Kilian.)
usually tends to vary. However, these variations can be small and not easy to detect.
To monitor mixing uniformity in the fi nal tablet, most recently spectroscopic techniques
such as Raman spectroscopy and near - infrared (NIR) spectroscopy have
been used [171 – 173] . Special online sensors have been build into the machine and
they measure the spectrum for each tablet. When the mixture is homogeneous, the
appearance of the spectrum will always be the same or between certain limits. If
not, the production can be stopped and adjusted as far as possible. Thus a further
step in quality assurance of tablets has been made. This was partially caused by the
process analytical technique (PAT) initiative of the U.S. Food and Drug Administration
(FDA) [174] .
6.4.11.4 Cleaning
To ensure product quality, cleaning is of utmost importance. Therefore different
standardized cleaning technologies have been developed [175, 176] . Detailed information
may be provided by manufacturers.
Usually, the most effective way to implement cleaning is to design it into a
process which has to be performed after tableting. It involves the addition of spray
systems, tank cleaners, nozzles, and seals into the tableting machine in order to
automate the cleaning process. The automation converts the batch processes to a
continuous operation of tableting cycles and cleaning cycles. Cleaning or washing
in place means an advanced wash liquid preparation system which handles all fi ltering,
preheating, mixing, and pumping of water, detergents, and demineralized water
and provides continuous monitoring and control of cleaning parameters. Another
possibility for standardized cleaning is the wash - off - line procedure. In this case
exchangeable compression modules are especially designed for a fast product
changeover. Different techniques have been developed to exchange the modules.
Either carries or arms or lifting systems are applied or additionally used. The wash -
off - line procedure increases production time by cleaning the exchangeable compression
modules after it is removed from the tablet press. Thus production time is
increased; however, two compression modules are necessary. In this case a special
separate washing system is necessary.
6.4.12 IMPORTANT FACTORS DURING MANUFACTURING PROCESS
To run a tablet production process effectively, robustly and smoothly several factors
have to be kept in mind. Of utmost importance for the process are environmental
humidity during tablet production and adhesion forces between machine punches
and dies. These and other factors can contribute to problems during manufacturing.
In the following the relevance of climatization during tableting, the necessity and
methods of lubrication, and frequently occurring problems during manufacturing
will be discussed.
6.4.12.1 Climatization
Relative humidity (RH) in the production room infl uences the water content of the
materials. This has to be kept in mind during tablet production.
IMPORTANT FACTORS DURING MANUFACTURING PROCESS 1085
1086 TABLET PRODUCTION SYSTEMS
When the materials sorb water, they deform differently compared to the status
before sorption, and compressibility changes [98, 177, 178] . Furthermore the compactibility
of the materials changes and tablets with a different crushing force and
friability result [179] . Even the release from the tablets can be infl uenced [180] .
However, the infl uence of water content on tableting and tablet properties depends
on the material; for example, hydrophilic polymers are mostly infl uenced by RH.
Further, the infl uence of RH during production is most decisive when production
conditions change extremely. Smaller differences up to 10% RH do not infl uence
a robust formulation [3] ; however for critical formulations even these changes are
of importance. Usually the conditions at one production site do not change from
day to day, but great differences have been observed between different seasons of
the year. Since tablet production and the fi nal tablet quality should be the same
throughout the year, often a humidity interval between 40 and 60% RH is used for
tablet production. This is a fi rst step. However, for characterization of material
properties humidity control between 40 and 60% RH is not suffi cient. Material
properties cannot be compared when obtained at different conditions. In this
case humidity control at a certain humidity with a precision of 2 – 5% is absolutely
necessary [3] .
6.4.12.2 Lubrication
Adhesion forces between the material and punches and dies result in sticking of the
tablets at the punches and dies. These adhesion forces are further infl uenced by RH
and this has also to be kept in mind. When the adhesion forces at the punches and
dies are greater than the cohesion forces between the particles inside the tablet, the
tablets stick at the punches and can cap.
To overcome this problem, lubrication is the method of choice [181, 182] . Two
alternatives for lubrication exist: internal lubrication and external lubrication. Internal
lubrication is performed by mixing the tableted product shortly before the
tableting process with a solid lubricant. Thus the lubricant is not only at the surfaces
of the fi nal tablet but also inside the tablet. As a result, internal lubrication lowers
bonding, and this is especially the case for plastically deforming materials [183 – 186] .
The most frequently used and most effective material for internal lubrication is
magnesium stearate [187 – 189] ; however, other hydrophobic or amphiphilic lubricants
are also possible [190 – 196] . Magnesium stearate has one major disadvantage:
It shows a low solubility and remains as a solid after dissolution. Thus the search
for other lubricants is ongoing.
When the lubricant should not be part of the tablet formulation, for example,
when bonding properties of the drug are low, external lubrication is necessary [197] .
For single - tablet production the punches and dies can be manually lubricated with
a fl uid. In production several methods have been developed to place the fl uid on
the surface of punches and dies [198] . Filaments applied at the punches to lubricate
the die or special caps with fl uid lubricant are possible solutions. However, external
lubrication also has disadvantages [199] .
6.4.12.3 Occurring Problems during Manufacturing
The most frequent problems occurring during the manufacture of tablets are high
tablet weight variation, capping and lamination [200] , and further picking and stick
ing at punches and dies. Low product yield, low crushing force, and further tablet
yams and chipping are other problems which have to be solved [33, 201] .
High tablet weight variation can be reduced by using weight control systems.
Further demixing of the tableted material has to be avoided, since demixing results
in higher tablet weight variation and content uniformity can no longer be achieved.
As already discussed, paddle feeders can be used to achieve mixing homogeneity
for problematic products and further spectroscopic techniques can be used for
control.
The problem of capping and lamination can be solved by increasing RH or
adding wetting agents. Further either external lubrication may help. Picking and
sticking of the tablets at punches and dies can be avoided by using lubricants as
discussed above.
A low product yield is caused by loss of material during fast production processes.
On rotary tableting machines this problem is solved by slightly lowering the
lower punches before the compression event starts.
A low crushing force is often caused by the composition of the special formulation.
If the formulation itself is not the reason for a low crushing force, compression
force can easily be increased. Another possible explanation can be low humidity of
the tableted product.
Finally tablets yam and chipping can occur before the tablets leave the die table
of a rotary machine. Usually the lower punches or the tablet scraper of the tableting
machine are not properly adjusted. Another reason can be low crushing force.
In conclusion, for smooth and perfect machine runs, product properties and
machine conditions have to be tightly controlled.
6.4.13 FUTURE OF TABLET PRODUCTION SYSTEMS
In principle tablet production systems have remained the same throughout the last
century. However, major improvements in instrumentation, data acquisition, and
analysis techniques have been made. Nowadays more sophisticated data acquisition
and analysis techniques are available which facilitate and improve interpretation of
tableting data.
In order to facilitate scale - up, more sophisticated simulation systems can be
thought of. They will be a real help for scale - up with small amounts of material as
available in early development of formulations.
Further quality control of the tablets during tableting or shortly after has become
more important. Recent trends show improvements for production in a GMP environment
by isolating the production unit from the machinery. For the near future
the implementation of the PAT initiative of the FDA is conceivable.
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1099
6.5
CONTROLLED RELEASE OF DRUGS
FROM TABLET COATINGS
Sacide Alsoy Altinkaya
Izmir Institute of Technology, Urla - Izmir, Turkey
Contents
6.5.1 Introduction
6.5.2 Tablet Coating Methods
6.5.3 Characterization of Tablet Coatings
6.5.4 Preparation of Asymmetric Membranes
6.5.5 Methods for Optimization of Tablet Coating Formulations
6.5.6 Applications
6.5.6.1 Materials and Methods
6.5.6.2 Results and Discussion
6.5.7 Conclusion
References
Appendix
6.5.1 INTRODUCTION
Controlled - release technology for drug delivery applications is designed to target
the drug to particular places or cells in the body, to overcome certain tissue and
cellular barriers, and to control the duration and level of the drug in the body within
a spefi cied therapeutic window. This usually implies achieving a prolonged, zero -
order release rate over the desired duration of drug delivery. Controlled - release
dosage forms provide sustained drug release and require less frequent drug loading
than conventional forms. Thus, the toxic side effects of the drug are minimized and
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1100 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
patient ’ s convenience and compliance are improved. Controlled - release systems are
usually classifi ed into four categories based on the rate - limiting step of the release
process [1] : (1) diffusion - controlled systems, (2) chemically controlled systems,
(3) swelling - controlled systems, and (4) magnetically controlled systems. Diffusion -
controlled systems are formulated in two main geometries: reservoirs and matrices.
In matrix systems, the drug is generally uniformly distributed or dissolved throughout
a polymer. The release kinetics from these types of systems depend on the
quantity and the diffusivity of drug in the matrix and the geometry of the system.
The release rates from matrix systems usually decrease with time due to an increase
in the path length for diffusion of drug and, thus, their release characteristics are
not generally zero order. In the case of chemically controlled systems, the drug is
either distributed through a biodegradable polymer or chemically bound to a
polymer backbone chain. The drug release from the biodegradable polymer is controlled
by degradation of the polymer through penetration of water or a chemical
reaction [1] . The drug attached to the polymer is released by hydrolytic or enzymatic
cleavage. In swelling - controlled systems, the drug is dissolved or distributed in a
glassy polymer matrix. As water penetrates the dry matrix, the polymer swells and
its glass transition temperature decreases below the temperature in the matrix. As
a result, the glassy – rubbery transition occurs and it allows the release of the drug.
The release rate of the drug out of these systems is mainly controlled by the rate
and degree of swelling. Alternatively, the drug release can also be controlled magnetically
by dispersing the drug and small magnetic beads within a polymer matrix
and exposing them to an oscillating external magnetic fi eld. (The drug is released
as the matrix is exposed to an oscillating external magnetic fi eld.)
Reservoir systems consist of a drug - containing core surrounded by a polymer
membrane. The release rate of drug is controlled by its diffusion through the membrane
[1, 2] . In addition to diffusional release, osmotic pumping mechanisms
contribute to the total drug release rate if either the drug is highly soluble or an
osmotic agent is added to the active core [1 – 4] . Reservoir systems are prepared in
different geometries, such as coated tablets, beads, particles, membrane - based
pouches, and microcapsules. The main advantage of these systems is their ability
to maintain zero - order release rates [1, 2] . This is usually achieved by loading the
powdered form of drug at a level far above the solubility of drug. Then, the concentration
of drug at the internal wall of the reservoir becomes its saturation
concentration and zero - order release occurs until the drug is completely dissolved.
In addition, drug loading can be higher for these systems compared to other controlled
- release systems; thus, the cost of formulation is minimized and the drug is
released at a higher rate for a longer period of time. The major disadvantage of
the reservoir system is rupture of the rate - controlling membrane if it is subject to
dose dumping.
Numerous studies exist in the literature on the drug release from tablet coatings.
In the majority of these studies, diffusional drug release takes place from the coatings
[5 – 17] . Various factors such as the type [8, 10, 15] , and concentration of the
coating material [7, 8, 11] , the type and amount of the plasticizer in the coating
solution [10, 12, 15] , the thickness of the coating [5] , the composition of the tablet
core [6, 14] , the particle size of the coating material [10, 18] , and the weight gain of
the coating [7, 8, 11, 13, 16] were considered during the formulation of tablet coat
ings, and their effects on the drug release rates were investigated. The use of osmotic
tablet coating systems are also described in the literature [3, 4, 17, 19 – 23] . Osmotic
systems utilize osmotic pressure difference as a driving force, and in the simplest
design they consist of an osmotic core containing drug with and without osmotic
agents. Different studies have shown that the release rate of drugs from oral osmotic
pump tablet coating systems is governed by various formulation variables such as
solubility and osmotic pressure of core components [3, 4, 19 – 23] , number [17] and
size of the delivery orifi ce [3, 4, 20, 21, 23] , drug loading [3, 4, 17] thickness of the
coating [3, 4, 19, 21, 23] , composition of the coating solution [3, 4, 17, 20, 21, 23] , and
weight of the coating [17, 20, 22] . The coatings prepared in most of these studies
have dense structures with or without a hole drilled through the coating through
which the drug is delivered. In some cases, drug delivery ports are formed by adding
leachable materials to the coating [24, 25] . The main problem with these systems, in
the absence of a hole, is an excessively prolonged drug release due to the low permeability
of the coating. To increase permeability of the coatings, plasticizers and
water - soluble additives were incorporated in the coating solution, and multilayer
composite coatings [14, 15, 24, 26, 27] or multiple - compartment osmotic tablets were
prepared [17, 22, 28, 29] . The permeability of the tablet coating systems were further
improved by changing the structure of the coatings from dense to porous asymmetric
ones [30 – 42] . Asymmetric membrane tablet coatings are characterized by
a relatively thin, dense skin layer supported on a highly permeable, thicker, and
porous sublayer. The permeabilities and the release rates of the drugs through the
asymmetric - membrane capsule/tablet coatings were determined to be higher compared
to conventional dense tablet coatings [30, 33, 34, 39] . The composition of the
coating solution was found to have a signifi cant effect on the structure of these type
of coatings and thus on the release rate of the drug [30, 33, 35, 36, 39, 40 – 42] . In
addition, it was reported that the drying condition had a signifi cant impact on the
structure of the asymmetric - membrane tablet coating and the release rate of the
drug [41] . A review of the literature studies clearly indicates that asymmetric - type
tablet coating is a new solution for developing controlled drug delivery systems,
since the structure of these types of coatings can be varied easily by changing the
preparation conditions without altering the coating material or signifi cantly varying
the coating thickness.
This work contains six sections in addition to the introduction. Sections 6.5.2 –
6.5.5 present a brief review of manufacturing methods for the application of coating
materials on tablets, characterization methods used to evaluate the uniformity and
defects of the tablet coatings, the techniques commonly used for manufacturing
asymmetric - type membranes, and modeling approaches employed for optimization
of the tablet coating formulations. The aim of the last two sections is to demonstrate
the advantages of asymmetric membrane tablet coatings with respect to their drug
release properties and the factors that affect the morphology of these types of tablet
coatings. To achieve this goal, the in vitro release of a model compound, theophylline,
from asymmetric membrane tablet coatings is determined and the morphology
of the coatings is examined. In addition, the dynamics of the phase inversion is
quantifi ed in terms of ternary - phase diagrams coupled with composition paths
determined from a mathematical model developed previously by our group [43] .
To draw meaningful and objective conclusions from experimental data and derive
INTRODUCTION 1101
1102 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
an empirical expression for the release rate of drug, compositions of the coating
solution are chosen using a statistical experimental design.
6.5.2 TABLET COATING METHODS
Four basic methods are commonly used for the application of coating materials on
drug tablets: (1) pan coating, (2) fl uidized - bed coating, (3) compression (press)
coating, and (4) melt/dry coating.
Pan Coating The pan coating process is the oldest form of pharmaceutical coating
for manufacturing small, coated particles or tablets. In this process, the particles
are tumbled in a pan which is rotated at an angle of usually 45 ° to the horizontal
surface at a speed between 20 and 50 rpm [44] . Coating fl uid is sprayed onto the
particles from a above by means of an air jet. The hot air blown through the coater
evaporates the fl uid and dries the fi lm coating. Pan coating is generally preferred
to coat large tablets since they are exposed to mechanical damage in other coating
operations. The uniformity of the coating applied to the tablets and defects on the
coating are important issues from a practical point of view. Operating conditions
such as drum speed, drum solids loading, the presence/absence of baffl es, air velocity,
and temperature infl uence the movement of tablets within the moving bed, the
circulation and surface exposure times spent by a tablet in the bulk of the moving
bed and on the surface of the bed, respectively, and the rate of drying, which all in
turn determine the uniformity of the coating in a pan coating process [45] . Different
experimental tools such as light emission from a single luminous tablet, photographic
and manual counting, positron emission particle tracking, magnetic resonance
imaging, and near real - time video imaging techniques [45 – 51] were used to
study the motion of tablets in the drum. Simulation of particle movement using
discrete - element modeling has also been used to study the movement of tablets
[52 – 55] . In addition, statistical experimental design was utilized to identify the critical
processing variables that affect coating uniformity and loading of active agent
coated on tablets [56] . Inlet airfl ow, pan speed, inlet air temperature, coating time,
atomization pressure, and fan pressure were investigated as the process variables.
Among these variables, atomization pressure, pan speed, and duration of coating
were found to be critical process variables with respect to uniformity of the
coating.
Fluidized - Bed Coating The basic principle of fl uidized - bed coating is to suspend
tablets in a moving hot - air stream in the upward direction during the coating
process. The coating material is sprayed through a nozzle from the top, the side, or
the bottom into the fl uidized bed. The solvent in the solution is removed from
the coating by the hot - air stream, which also carries the coated tablets/particles.
Fluidized - bed coating provides better coating uniformity due to good solid – fl uid
mixing and minimizes formation of agglomerates. There are various types of
fl uidized - bed coating equipment, the most commonly used confi guration being the
Wurster column coater [44] , in which a draft tube insert is placed coaxially in the
bed to aid the circulation of particles. This column is not suitable for coating of large
particles and tablets due to high erosion of solids associated with the higher velocity
needed to circulate them [57] . Typical operating variables that affect the performance
of the fl uid bed coating in a Wurster column are airfl ow rate, air and bed
temperature, spray rate, gap height between the draft tube and the air distributor
plate, atomizing air pressure, humidity, and solids charge. The product coating uniformity
in the Wurster column coating process is primarily determined by the variation
in coating per pass and the circulation time distribution. Radioactive particle
tracing, magnetic particle tracing, and controlled single - or multiple - pass coating
evaluation techniques were commonly used for detecting particle circulation time
and distribution [57, 58] . In addition, several models have been developed to predict
the amount of material coated on the particles [59 – 65] .
Compaction Coating In this process, the coating is compressed around a preformed
core by using a special tablet press. Compaction coating is especially useful
when the drug itself is unstable in the solution and precipitates from the solution
in a less stable morphology. On the other hand, the large amount of coating material
required limits the applicability of this technology. Another disadvantage of the
process is poor adhesion at the coating – core interface which causes physical instability
(i.e., friability) [66] . The Press coating process is not useful for coating relatively
hard cores which provide essentially no compressibility.
Melt/Dry Coating Film coating processes require water or organic solvent(s). The
use of organic solvents causes environmental pollution and excessive cost of recovery
while a long time is required to remove the aqueous solvent. Both hot - melt and
dry coating techniques eliminate the use of solvents; as a result the processing times
become much shorter and the cost of the process is reduced. Melt coating is possible
for coating materials that have a low melting temperature and acceptable thermostability.
The principal stages in the fi lm formation during dry coating are softening,
melting, and curing [67 – 70] . The process requires larger amount of plasticizer to
partially soften and dissolve the polymer.
6.5.3 CHARACTERIZATION OF TABLET COATINGS
Tablet coatings are applied to improve tablet swallowability, mask unpleasant
tastes and odors, protect the tablet core against water and oxygen, which can
degrade the drug in the core, and control the release rate of the drug. The rate of
dissolution and bioavailability of a drug are primarily infl uenced by the quantity
and quality of the coating applied on the tablet. Thus, the characterization of the
coatings becomes essential in terms of the uniformity and integrity of the coating.
A number of instrumental methods ranging from liquid chromatography [71] to
various noninvasive spectroscopic probes have been introduced and evaluated as
a means of monitoring the coating process. Among these methods, laser - induced
breakdown spectroscopy (LIBS) has been used as a rapid technique for assessing
the uniformity of the coating thickness [72] ; however, the destructive nature of the
method has also been reported [73] . Near - infrared spectroscopy has been employed
for determining the amount of coating applied [74 – 76] . The main disadvantage of
this method has been identifi ed as imprecision in the calibration and validation
models due to uneven distribution of coating from tablet to tablet [77] . Microscopy
CHARACTERIZATION OF TABLET COATINGS 1103
1104 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
techniques provide direct and accurate measurement of the coating thickness but
require laborious sample preparation and are therefore impractical for real - time
process monitoring. Recently, Raman spectroscopy combined with multivariate
data analysis has been reported as a feasible noninvasive technique to quantify
tablet coating thickness and uniformity in the presence of a fl uorescent ingredient
in the coating formulation [77, 78] . Confocal laser scanning microscopy has been
introduced as a novel technique for imaging the fi lm – core interface and surface
defects of fi lm - coated tablets [79] . Surface roughness was also determined as an
important factor in characterizing the tablet coatings using different imaging and
roughness analytical techniques, including optical microscopy, scanning electron
microscopy (SEM), laser profi lometry, and atomic force microscopy (AFM)
[80 – 84] .
6.5.4 PREPARATION OF ASYMMETRIC MEMBRANES
Asymmetric membranes are usually produced by phase inversion techniques. In
these techniques, an initially homogeneous polymer solution becomes thermodynamically
unstable due to different external effects and the phase separates into
polymer - lean and polymer - rich phases. The polymer - rich phase forms the matrix of
the membrane, while the polymer - lean phase, rich in solvents and nonsolvents, fi lls
the pores. Four main techniques exist to induce phase inversion and thus to prepare
asymmetric porous membranes [85] : (a) thermally induced phase separation (TIPS),
(b) immersion precipitation (wet casting), (c) vapor - induced phase separation
(VIPS), and (d) dry (air) casting.
Thermally Induced Phase Separation In the TIPS process, an initially homogeneous
solution consisting of a polymer and solvent(s) phase separates due to a
decrease in the solvent quality when the temperature of the casting solution is
decreased. After demixing is induced, the solvent is removed by extraction, evaporation,
or freeze drying.
Immersion Precipitation (Wet Casting) A homogeneous polymer solution consisting
of a polymer and solvent(s) is cast on a support and is immersed in a nonsolvent
bath. During the immersion, casting solvent diffuses into the nonsolvent
bath and, countercurrently, nonsolvent in the bath penetrates into the solution. The
nonsolvent has a limited solubility in the polymer, and when it reaches its critical
concentration in the solution, precipitation takes place. Then, the solvent and nonsolvent
in the solution are extracted and fi lm is annealed.
Vapor - Induced Phase Separation During the VIPS process, phase separation
is induced by penetration of nonsolvent vapor, into the homogeneous polymer
solution consisting of polymer and solvent(s). Mass transfer is usually much
slower than that in the wet casting process; thus, the VIPS process has been
used to obtain membranes with symmetric, cellular, and interconnected pores
[86, 87] .
Dry (Air) Casting In this process, the polymer dissolved in a mixture of a volatile
solvent and a less volatile nonsolvent is cast on a support and exposed to an air
APPLICATIONS 1105
stream. During drying of the solution, fast solvent evaporation leads to a decrease
in solubility of the polymer, then phase separation takes place.
6.5.5 METHODS FOR OPTIMIZATION OF TABLET
COATING FORMULATIONS
Tablet coating formulation is composed of various formulation factors and process
parameters. The formulation is usually divided into modeling and optimization
phases. The modeling phase consists of preparing series of experimental formulations
by varying the ingredients and process conditions systematically and measuring
their properties. A detailed review of the literature indicates that the response
surface method (RSM) has been widely used for modeling and choosing acceptable
tablet coating and other pharmaceutical formulations. The RSM includes statistical
experimental designs, multiple regression analysis, and optimization algorithms to
search the best formulation for a given set of constraints. Full factorial, orthogonal,
Box – Behnken, central composite, pseudorandom, and Plackett – Burman designs
were used to investigate the effects of tablet core formulation, coating thickness,
and process parameters such as mixing time and speed in the pan coating process,
inlet airfl ow, pan speed, inlet air temperature, coating time, atomization pressure,
and fan pressure in the fl uidized - bed coating process [88 – 92] . Artifi cal neural networks
(ANNs) have also been investigated as an alternative method for modeling
the pharmaceutical formulations [93 – 105] . In the RSM, the pharmaceutical responses
are predicted based on the second - order polynomial equation, which is usually
limited to low levels. When a nonlinear relationship between formulation factors
and response variables is observed, the ANN approach was shown to give better
estimations of optimal formulations [105] .
6.5.6 APPLICATIONS *
Previous sections have reviewed numerous studies in the literature investigating the
release mechanisms from the tablet coating systems, their advantages/disadvantages
as a control release system, and the methods used to characterize and optimize their
formulation. In the following sections, the in vitro performance characteristics of
the asymmetric membrane tablet coatings will be illustrated using release studies
of a model compound theophylline. For this purpose, fi rst the method used for
preparing asymmetric membrane tablet coatings will be explained, then the results
of the dissolution studies will be discussed.
6.5.6.1 Materials and Methods
Preparation and Characterization of Tablet Coatings Tablet cores were prepared
by compressing the drug without any excipient using a hydraulic press operated
at 110 MPa. A stainless steel die with a diameter of 1.2 cm was used to produce
400 - mg drug tablet cores. Asymmetric - membrane tablet coatings were applied
* This article was published in Biochemical Engineering Journal, 28, Sacide Alsoy Altinkaya,
Hacer Yenal, In vitro drug release rates from asymmetric - membrane tablet coatings: Prediction
of phase - inversion dynamics, 131 – 139, Copyright Elsevier (2006).
1106 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
using a dip coating process (Dip Coater Nima, type D1L, serial no. 327). The tablets
were dip coated in polymer solutions prepared by dissolving cellulose acetate
(Aldrich) in a solution of acetone (Merck) and water. The rate of withdrawal of
the tablets from the solution was adjusted to obtain similar fi nal coating thicknesses
for each coating formulation. Immediately after coating, tablets were rotated for
even distribution of the viscous membrane solution, transferred into an environmental
chamber (Angelantoni Industrie, Italy, Challenge series, model number
CH250), and kept there for 2 h to allow for evaporation of both the solvent (acetone)
and nonsolvent (water). The temperature and relative humidity of air in the environmental
chamber were adjusted to 25 ° C and 50%, respectively. Tablets were
allowed to dry further for a minimum of 24 h at room temperature prior to dissolution
experiments. Faster evaporation of acetone and resulting increase in the concentration
of water in the coating leads to a decrease in solubility of the cellulose
acetate (CA); then phase separation takes place. Consequently, asymmetric - membrane
structure forms on the tablet core. Morphology of the coatings was examined
using a scanning electron microscope (Philips, XL - 30SFEG). Samples were
coated with gold palladium using a magnetron sputter coating instrument. The
thickness of the dense skin layer, the overall porosity, and the average pore size
were determined from image analysis of micrographs showing cross sections of the
membranes.
Dissolution Studies The release rate of theophylline from the tablets was determined
by the U.S. Pharmacopeia (USP) XXIII dissolution methodology using a
dissolution tester (Caleva 10ST). According to this standard, 900 ml of dissolution
medium was placed in the vessel and the temperature was maintained constant at
37 ° C using a constant - temperature bath. Then, the tablets were immersed in the
vessel and the solution was stirred at a speed of 50 rpm. To simulate the actual
dissolution environment in the body, the pH of the dissolution medium was
kept at 3 during the fi rst 3.5 h by adding 8.5 vol % phosphoric acid to 900 mL distilled
water and then increased to 7.4 by adding 5.3 M NaOH to the dissolution
medium and kept at this value until the end of the experiment. To determine the
quantity of drug released from the tablets, samples were taken periodically and
assayed by ultraviolet (UV) spectrophotometry (Shimadzu UV - 1601) at a wavelength
of 272 nm. Dissolution experiments were performed on three tablets and
the release profi les were reported as the arithmetic average of the three experimental
runs.
Statistical Experimental Design To determine the infl uence of the composition
of the coating solution on the release rate of drug, experiments were statistically
designed using a commercial software package called Design - Expert [106] . The
system studied in this chapter consists of three components with the following compositional
restrictions:
5 15 70 90 5 15 1 2 3 . . . . . . . . . (1)
where . i is the weight percent of component i and 1, 2, 3 represent CA, acetone,
and water, respectively. Any composition outside these limits will probably fail to
APPLICATIONS 1107
produce a successful asymmetric - membrane coating. In mixture experiments, the
factors are the compositions of the mixture components, and the sum of the fractions
of all components is equal to 1. Therefore the factor levels are mutually dependent.
Thus, factorial experimental designs are not suitable for response surface modeling
of mixtures since such designs require that the experimental treatment combinations
be determined by independent adjustments of each component level. In addition,
a standard response surface design cannot be used either due to the same
constraints. Consequently using the constraint levels shown in Equation (1) , a D -
optimal design was generated by the Design - Expert software package. The 14 experimental
formulations determined are shown in Table 1 . The lower and upper limits
on the weight fraction of each component are required to (a) obtain appropriate
viscosity of the solution and coat the tablets uniformly and (b) induce phase separation,
thus forming a porous membrane structure. These constraints were established
based on preliminary dissolution experiments, available literature data, and the
simulation results reported by Altinkaya and Ozbas [43] .
Of the 14 formulations listed in Table 1 , six experimental runs were required to
fi t the quadratic mixture model, four additional distinct runs were used to check for
the lack of fi t, and fi nally four runs were replicated to provide an estimate of pure
error. Design - Expert used the vertices, the edge centers, the overall centroid, and
one point located halfway between the overall centroid and one of the edge centers
as candidate points. Additionally, four vertices of the design region were used as
check points [106] .
Determination of Phase Diagrams and Composition Paths The dynamics of the
membrane formation process is predicted by combining the kinetics and thermody-
TABLE 1 Theophylline Release Rates from Asymmetric - Membrane Tablet Coatings,
Results of Fitting Release Profi les to Zero - Order Kinetics, and Precipitation Times
Determined from Model Predictions
Compositon (wt %)
Release Rate
(mg/min) R 2
Precipitation
Time a (s)
Cellulose
Acetate Acetone Water
15 80 5 0.036 0.9871 2671/2671
15 80 5 0.036 0.9864 2671/2671
5 90 5 0.45 0.9757 660/660
5 90 5 0.36 0.9864 660/660
15 70 15 0.036 0.9908 3374/3374
15 70 15 0.027 0.9925 3374/3374
5 80 15 0.36 0.9928 1000/1000
5 80 15 0.36 0.9876 1000/1000
5 85 10 0.27 0.9918 675/675
10 80 10 0.054 0.9958 1751/1751
15 75 10 0.054 0.9975 2314/2314
10 85 5 0.036 0.9887 2554/2554
12.5 77.5 10 0.036 0.9902 —
10 75 15 0.063 0.9889 1484/1484
a The fi rst number corresponds to the precipitation time at the tablet – coating interface.
1108 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
namics of the system simultaneously. An appropriate thermodynamic model is
necessary to construct the ternary - phase diagram and to formulate the boundary
conditions of the kinetic model. Phase separation is considered to occur when a
mass transfer path touches the binodal curve. In this study, a robust algorithm developed
previously by our group was used to construct the phase diagram [43] . The
algorithm utilizes Flory Huggins thermodynamic theory with constant interaction
parameters. The composition paths were determined from the kinetic model equations
which consist of coupled unsteady - state heat and mass transfer equations, fi lm
shrinkage, and complex boundary conditions. The details of both the thermodynamics
and kinetic equations can be found in our previous study [43] . We have assumed
that the kinetic model derived for a plane geometry can be used to predict the
membrane formation process on a tablet surface. This assumption is fairly reasonable
since the thickness of the coating is very small and, thus, the cylindrical geometry
can be approximated as the plane geometry.
6.5.6.2 Results and Discussion
Effect of Composition of Coating Solution The effect of changing the composition
of the casting solution is well documented for asymmetric membranes prepared
for separation applications [107 – 114] . However, there are relatively few quantitative
studies illustrating the relationship between the composition of the casting
solution and the drug release rate from the asymmetric - membrane coated tablets/
capsules [30, 33] . To investigate such a relationship, the in vitro release profi les of
the model drug theophylline were measured for the 14 formulations listed in Table
1 and they are shown in the Appendix in Figures A1 – A10 . To fi nd out whether the
drug release from the tablet coatings provides a zero - order release kinetics, each
data set was fi tted to a linear equation. The quality of the fi tted model is determined
by the coeffi cient of determination R 2 and it is defi ned as
R y y
y
2 =
. . .
.
. (2)
where
.y i
i
n
n
Y Y = .
= .
1 2
1
( )
(3)
. ( . ) .y i i
i
n
n
Y Y = .
= .
1 2
1
(4)
denote the sample variance and the prediction error power, respectively. Additionally,
n is the number of experimental data points, Y i is the experimental observation,
Y is the average of the experimental data points, and .Y
i denotes the predicted value
by the fi tted model. The quantity R 2 lies between 0 and 1, and if the value is 1, it
can be said that the fi t of the model is perfect. High R 2 values listed in Table 1 for
each data set indicate that there is an excellent linear relationship between the
concentration of the drug and the release time; thus, all tablet coatings prepared
APPLICATIONS 1109
can provide zero - order or near - zero - order drug release. The release rates for each
coating formulation were estimated from the slope of the average release profi les
and they are also listed in Table 1 .
The drastic change in the release rates with the composition of the coating solution
is shown in Figure 1 . Within the experimental composition range covered, the
highest release rate was observed in the case of the lowest CA (P: polymer) and the
highest acetone (S: solvent) concentrations in the casting solution [polymer (P) –
solvent (S) weight fraction ratio 5/90]. This is caused by the fi nal coating structure
consisting of a very thin and dense top skin layer, and highly porous lower sublayer,
as illustrated in Figure 2 . At the lowest level of polymer concentration (5%), the
FIGURE 1 Release rate of theophylline as function of cellulose acetate(P) – acetone(S)
weight fraction ratios.
FIGURE 2 SEM of cross section of asymmetric - membrane made with 5% water and
CA – acetone weight ratio of 5/90, magnifi cation 2500 . .
1110 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
thicknesses of the dense skin layers of the coatings are very small and similar to
each other; thus, the structure of the lower sublayer becomes an important factor
in determining the release rate of the drug. The porosity of the coating structure in
the case of 90% acetone is so high that an increase in water concentration from 5
to 10% is not suffi cient to produce a more porous fi nal structure and, thus, the
release rate of the drug decreases. A further increase of water concentration to 15%
results in an opposite effect, which makes the release rate increase to a level of
0.36 mg/min due to the dominant effect of water concentration in increasing the
porosity of the lower sublayer.
An increment in the CA concentration from 5 to 10% resulted in signifi cantly
lower release rates at all water concentrations because the structure of the coatings
changed from porous to dense. Keeping the polymer concentration at 10% while
changing the water concentration from 5 to 15% makes the release rates increase.
This behavior is explained by the formation of more porous structures by adding
more nonsolvent into the casting solution, which is in agreement with the observations
of other groups [30, 33] .
When the polymer concentration increases from 10 to 15%, no signifi cant
changes in release rates were observed. As a matter of fact, in the cases of 5%
and 10% water concentrations, the release rates did not change at all. This is due
to the unusual transport characteristics of asymmetric membranes which are
complex functions of the properties of the different regions of the membrane. In
addition to the thickness of the dense skin layer and the porosity of the lower
sublayer, structural factors such as tortuosity, pore size, shape, and connectivity
of the pores also strongly affect the rate of transport through the coating. The
SEM pictures taken at high magnifi cation which are shown in Figures 3 a and 3 b
indicate that the tablet coating prepared with a P/S ratio of 10/85 has a uniform
and narrow pore size distribution with regular elliptic pore shapes, while the
other one (P/S: 15/80) has cylindrical pores with a wide pore size distribution,
forming a connected pore network. As a result, even though the resistance of the
dense skin layer of the coating made with P/S ratio of 15/80 is larger, its lower
sublayer resistance is smaller due to the connected pore network. Consequently,
the release rate of the theophylline becomes the same through both tablet coatings.
Comparison of the scanning electron micrographs in Figures 4 a and 4 b indicates
that the tablet coating made with the P/S ratio of 10/80 has a uniform pore
size distribution, cylindrical pore shapes and high tortuosity while the tablet
coating prepared with the P/S ratio of 15/75 has elliptic, irregular pore shapes
and pores are isolated. Thus, the lower sublayer resistance of the coating made
with the P/S ratio of 10/80 is larger due to the relatively higher tortuosity resulting
in the same release rate with the coating prepared with the P/S ratio of
15/75.
At the 15% polymer concentration level, the release rate increases with the
change in water concentration from 5 to 10%, which is mainly caused by the
increased porosity. However, a further 5% increment in the water concentration
(to 15%) made the release rate decrease back to the same level as in the case of
5% water concentration. Even though higher water concentration favors forming
a more porous structure and a concomitant higher release rate, the acetone level
which decreased below a critical value destroyed this mechanism.
APPLICATIONS 1111
Results of dissolution studies along with morphological observations clearly
indicate that the drug release rate is strongly infl uenced by the morphology of
the membrane. Thus, if one wishes to control the drug release characteristics of the
delivery system, a quantitative understanding of the dynamics and morphology of
the phase inversion process is required. The dynamics of the phase inversion process
can be quantifi ed in terms of the ternary - phase diagram coupled with the heat and
mass transfer model equations. We have obtained information about the structure
of the tablet coating by plotting the composition paths on the ternary - phase diagram
and the polymer concentration profi le at the moment of precipitation. As an illustra-
FIGURE 3 SEM of cross section of asymmetric - membrane made with 5% water and
CA/acetone weight ratios of: ( a ) 10/85, magnifi cation 5000 . ( b ) 15/80, magnifi cation
12,000 . .
(a)
(b)
1112 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
tion, in Figure 5 , concentration paths in time for the tablet coating prepared with
5% CA, 90% acetone, and 5% water are shown. According to this plot, the phase
separation takes place since the concentration paths in time for the drug tablet –
coating and the coating – air interface cross the binodal curve at the same time. In
addition, the coating – air interface enters the phase envelope at a polymer volume
fraction of 0.76, while the tablet – coating interface enters with a volume fraction of
0.023. These two observations imply that the coating will be porous and asymmetric
in which the top layer is more dense than the lower sublayer, which was confi rmed
by the SEM picture in Figure 2 . The predictions, have shown that phase separation
was achieved for all coating formulations, supporting the morphological observa-
FIGURE 4 SEM of the cross section of the asymmetric - membrane made with 10% water
and CA – acetone weight ratios of ( a ) 10/80, magnifi cation 10,000 . , and ( b ) 15/75, magnifi cation
10,000 . .
(a)
(b)
APPLICATIONS 1113
FIGURE 5 Ternary - phase diagram and concentration paths for coating made with 5% CA
dissolved in 90% acetone and 5% water.
FIGURE 6 Concentration profi le of CA in membrane at moment of precipitation for different
CA – acetone weight fraction ratios.
tions and the precipitation times calculated for each case (listed in Table 1 ). Model
predictions can also be used to determine a rough thickness of the high - polymer -
concentration region near the coating – air interface and the pore distribution of the
sublayer structure when the polymer concentration profi les at the moment of phase
separation are plotted. As an illustration, such a plot is shown in Figure 6 for coatings
prepared with 10% CA in the casting solution. Examination of these profi les
1114 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
leads to the following conclusions regarding the effect of increased water concentration:
(1) The polymer concentration at the coating – air interface slightly decreases.
(2) More uniform porosity distribution throughout the lower sublayer is favored.
Also, porosity increases, which is in complete agreement with our release studies
and the observations of other groups [30, 33] .
Effect of Evaporation Condition Previous studies on more traditional applications
have investigated the effect of increased air velocity, that is, forced - convection
conditions for a combination of dry/wet phase inversion techniques to produce
defect - free, ultrahigh fl ux asymmetric membranes with ultrathin skin layers [115 –
117] . To investigate the effect of evaporation condition on the release rate of drug,
tablets were dip coated with CA solution containing 10% CA, 80% acetone, and
10% water and allowed to dry by blowing air across the surface with a blower
(forced convection). As a comparison, tablets coated with the same solution were
air dried under natural free - convection conditions.
As illustrated in Figure 7 , the release profi les of both tablet coatings show a linear
behavior only at small times, and then exponential increases in concentrations were
observed. Based on this behavior, the release profi les were fi tted to an empirical
equation as
C kt eb Ct = + . . 0 1 0 (5)
where k 0 and b 0 are fi tting parameters. The accuracy of Equation (5) for correlating
the release rate data in Figure 7 was confi rmed by high R 2 values, very close to 1 in
both cases. Due to the presence of the second term in Equation (5) , the release rate
FIGURE 7 Release of theophylline from tablet coatings made with 10% CA dissolved in
80% acetone and 10% water. Coated tablets were dried under free - and forced - convection
conditions. The lines correspond to prediction from Equation (5) using k 0 = 1.92 .
10 . 5 mg/(mL·min); b 0 = 0.1179 mL/(mg·min) for forced convection and k 0 = 2.41 .
10 . 5 mg/(mL·min); b 0 = 0.1174 mL/(mg·min) for free convection.
APPLICATIONS 1115
of the drug, dC/dt , is no longer constant and its dependency on the concentration
of drug in the dissolution medium, C , can be expressed as
dC
dt
k e bCt
k b t e
b Ct
b Ct = + + . .
.
0 0
0 0
2
1 1
1
0
0
( )
(6)
Using Equation (6) , the release rate of theophylline from the tablets dried under
forced - and free - convection conditions were determined as 0.047 and 0.078 mg/min,
respectively. It shoud be noted that both of these values correspond to the arithmetic
average of the release rates calculated at each average concentration level shown
in Figure 7 . The difference in release rates can be explained by comparing the scanning
electron micrographs shown in Figures 8 a and 8 b . It can be seen that the cross -
sectional morphology of the tablet coating dried under the forced - convection
condition is dense and nonporous while a porous and asymmetric structure is
observed for the tablet coating dried under free - convection conditions. In the dense
coating, diffusional resistance to transport of the drug occurs through the overall
thickness and is larger than that in the asymmetric porous coating; hence, a lower
drug release rate is observed. To understand the effect of air velocity on the formation
of the coating structure, we have utilized our model predictions. The composition
paths plotted in Figure 9 indicate that, when the speed of air in the drying
atmosphere is signifi cantly increased, the rate of evaporation of solvent (acetone)
increases dramatically and within a short time its concentration at the surface drops
to zero. This situation leads to very strong diffusional resistance within the membrane
solution and, thus, slow evaporation of the nonsolvent (water). Consequently,
phase separation is never achieved and the resulting membrane structure becomes
dense as supported by the SEM picture in Figure 8 a .
Effect of Nonsolvent Type A few studies in the literature have shown that various
membrane morphologies can be obtained by changing the type of nonsolvent in
the casting solution [118 – 120] . To investigate the effect of nonsolvent type, tablet
coatings were prepared from a casting solution of CA in acetone as a solvent and
octanol, formamide, glycerol, and hexanol as nonsolvents. The drastic change in the
release rates of theophylline and percentage of dense skin layer at the surface of
the tablet coating with nonsolvent type is shown in Figure 10 . The decrease in the
release rates is associated with the increase in the thickness of dense skin layer of
the coating. The results illustrated in Figure 10 indicate that the membrane structure
formation during the phase inversion process is strongly infl uenced by the type of
nonsolvent, since each nonsolvent has different volatility, thermodynamic, and
diffusion characteristics. The difference in the rates of evaporation and diffusion
of nonsolvents and change in the miscibility gaps in the case of each nonsolvent
lead to different mass transfer paths; consequently, the morphology of the resulting
membrane structures signifi cantly varies from porous symmetric to dense asymmetric
ones.
Statistical Analysis of Experimental Design The effect of the composition of the
coating solution on the release rate of drug was investigated in detail with the 14
formulations listed in Table 1 . The drug release rate was chosen as an appropriate
1116 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
response variable since zero - order release was easily achieved for all tablet coatings
prepared. The data in Table 1 were best fi t by the special cubic equation
Release rate= + + .
. .
70 94 1 698 37 55 94 66
354 3 49
1 2 3 1 2
1 3
. . . .
.
. . . ..
. . . . 3 357 66 2 3 1 1 3 . . . . . +
(7)
Results of the regression analysis are given in Table 2 . An excellent fi t of the
experimental release rate data to Equation (7) was confi rmed by the high R 2 value
of 0.9801. In addition to the R 2 values, the signifi cance of Equation (7) and each
FIGURE 8 SEM of cross section of asymmetric - membrane made with 10% CA dissolved
in 80% acetone and 10% water: ( a ) coating solution was dried under forced - convection
condition, magnifi cation 6500 . ; ( b ) coating solution was dried under free - convection condition,
magnifi cation 5000 . .
(a)
(b)
APPLICATIONS 1117
term in it to the prediction of the release rate of theophylline was evaluated by the
F statistic or F value. The F statistic is viewed as a ratio that expresses variance
explained by the model divided by variance due to model error or experimental
error and is defi ned as
F
R k
R n k
=
. . .
2
2 1 1
/
/ ( )( )
(8)
FIGURE 9 Ternary - phase diagram and concentration paths for coating made with 10%
CA dissolved in 80% acetone and 10% water. Coating solution was dried under forced -
convection condition.
FIGURE 10 Release rates of theophylline and percentage of dense skin layer of membranes
as function of nonsolvent type. Polymer: CA solvent – acetone.
1118 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
where k is the number of variables in the model. Usually the computed value of F
is compared with the critical F value, F k , n . k , 1 . . , where . is a preselected signifi cance
level. If the value of F is substantially greater than the critical F value ( F critic ), that
is, if . F , the difference between F and F critic , is large, then the regression equation is
considered useful in predicting the response. We have assessed the contribution of
each interaction term by comparing the change in . F and R 2 values between the
full model given in Equation (7) and reduced models. The reduced models were
obtained by deleting a specifi c interaction term in the full model; for example, model
2 includes all terms in Equation (7) except the term involving . 1 . 2 . 3 . The results
in Table 2 indicate that all binary and ternary interaction terms in the full model
are needed for accurate prediction of the release rate since the largest . F values
are calculated for the full model for both signifi cance levels, . = 0.05 and . = 0.01.
According to the criterion mentioned above, among all interactions, CA – acetone
( . 1 . 2 ) was identifi ed as the most infl uential factor on the response since the largest
decrease in both . F and R 2 values compared to those of the full model were
observed when the term . 1 . 2 was deleted from the full model. This simply implies
that changing the CA – acetone ratio in the coating formulation has the most signifi -
cant effect on the release rate. Specifi cally, increasing the ratio of CA to acetone
from 5/90 to 15/80 resulted in a decrease of the release rate from 0.45 to 0.036 mg/
min since the porosity of the membrane decreases and the thickness of the dense
skin layer increases. The ratio of the composition of CA to water ( . 1 . 3 ) was also
found to be an important parameter on the release rate of drug as indicated by the
second largest decrease in R 2 and . F values compared to those of the full model.
Decreasing this ratio from 15/5 to 5/15 caused an increase in the release rate by a
factor of 10 since the thickness of the dense skin layer signifi cantly decreases. Based
on the decrease in . F and R 2 values from those of the full model given in Table 2 ,
the relative importance of each interaction term can be ranked as follows: . 1 . 2 >
. 1 . 3 > . 2 . 3 > . 1 . 2 . 3 .
To further illustrate the simultaneous effects of the factors on the release rate of
drug, a three - dimensional response surface plot based on Equation (7) was generated,
as shown in Figure 11 . As can be clearly seen from this fi gure, the release rate
can be signifi cantly varied just by tailoring the CA – acetone and CA – water ratios
without changing the coating material. In addition, Figure 11 shows that a slight
maximum in release rate is observed as the ratio of composition of acetone to water
increases.
In order to validate the predictive capability of the empirical expression, two
formulations with compositions given in Table 3 were selected randomly from the
TABLE 2 Statistical Analysis of Release Rate Data
Model Number R 2 F values
. = 0.05 . = 0.01
F critic a . F F critic a . F
1 (full model) 0.9801 42.21 4.21 38 8.26 33.95
2 ( . 1 . 2 . 3 ) 0.955 25.13 3.87 21.26 7.19 17.94
3 ( . 2 . 3 ) 0.953 23.57 3.87 19.7 7.19 16.38
4 ( . 1 . 3 ) 0.946 20.64 3.87 16.77 7.19 13.45
5 ( . 1 . 2 ) 0.881 8.65 3.87 4.78 7.19 1.46
a Determined from statistical tables [121] .
FIGURE 11 Three - dimensional response surface plot of release rate as function of composition
of coating solution.
0.40
0.30
0.20
0.09
–0.01
.1 (0.25)
.2 (0.70)
.2 (0.90)
.3 (0.25)
.1 (0.05)
.3 (0.25)
Weight fraction
Release rate (mg/min)
experimental design region. Experimental release rates estimated from the slope of
the release profi les and corresponding rates predicted from Equation (7) are also
listed in Table 3 . According to the results, the maximum absolute percentage difference
between the experimental and predicted release rates is 3.5%. This value lies
within the residuals obtained in deriving Equation (7) . Based on this comparison,
it is fair to conclude that the empirical expression derived in this study can be used
as a tool to predict the release rate of theophylline for any composition within the
experimental design region.
6.5.7 CONCLUSION
This chapter has considered the controlled release of drugs from tablet coating
systems. These systems are still the preferred route of drug administration due to
TABLE 3 Composition of Coating Solution Randomly Selected for Testing Predictive
Capability of Empirical Expression
Compositon (wt %)
Experimental
Release Rate
(mg/min)
Predicted
Release Rate
(mg/min) R 2
Cellulose
Acetate Acetone Water
7.5 82.5 10 0.09 0.125 0.9816
10 82.5 7.5 0.027 0.029 0.9858
CONCLUSION 1119
1120 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
their advantages of ease of administration, maintaining zero - order release rates, and
better patient compliance; thus, they hold the major market share in the pharmaceutical
industry along with the other formulations of oral drug delivery. In tablet
coated systems, diffusion of drug through the coating is usually the rate - limiting step
and the desired drug release rates are achieved by properly selecting the coating
material and adjusting the morphology of the coating using suitable manufacturing
methods. Coating characteristics such as glass transition temperature, crystalline
content, and degree of cross - linking signifi cantly determine the release rate of the
drug. For a selected coating material, the structure of the coating plays a critical role
in achieving useful release rates. The morpholology of the tablet coatings can be
varied by incorporating plasticizer or water - soluble additives in the coating solution,
by blending the polymers, or by applying multilayer composite coatings. Another
approach is to apply asymmetric - and porous - type coatings. The morphological
characteristics of these types of coatings, such as fraction of dense top layer and
porous sublayer and size and shape of the pores, can be varied by optimizing the
composition of the coating solution, evaporation conditions (temperature, relative
humidity, and velocity of air), and the type of solvent or nonsolvent used in the
coating solution. Asymmetric - type coatings can be used to facilitate osmotic delivery
of drugs with low solubilities since high water fl uxes can be achieved. These
coatings allow us to control the release kinetics without altering the coating material
or signifi cantly varying the coating thickness.
Currently, considerable research efforts have been directed toward developing
protein drug delivery systems due to discovery of numerous protein and peptide
therapeutics. The delivery of protein drugs is usually limited to parenteral administration
and frequent injections are required due to their short half - lives in the blood.
In this regard, development of oral delivery systems is necessary for patient compliance
and convenience. The challenge in the design of the oral delivery systems is
that they should protect the incorporated drugs from chemical and enzymatic degradation
until the drug reaches the delivery site. The protein drug should not be
infl uenced by pH or bacteria and enzymes along the gastrointestinal (GI) tract and
should be delivered at the desired site with a desired effi ciency. To achieve site -
specifi c delivery for protein - and peptide - based drugs, one of the strategies that has
often been investigated is to coat the drug core with polymers that can respond to
the stimuli of local environments such as pH and enzymes. For water - soluble protein
drugs, such as insulin, an additional protective coating is usually required to isolate
the drug from the surrounding water. Composite tablet coating materials which
combine the enzymatic susceptibility and protective properties of polymers can be
another solution for this problem. A systematic comparison study among various
polymer combinations is required to select the appropriate coating materials for
specifi c drugs. Not only the selection of the coating material but also the manufacturing
technique for the preparation of tablet coatings plays a critical role and still
remains one of the most challenging subjects in the controlled drug delivery area.
Proteins are very sensitive to conditions that can occur during the coating process.
Mechanical stresses during the preparation, exposure to a hydrophobic organic
solvent, intermediate moisture level during hydration, and interaction between
protein and polymer can easily inactivate the protein - based drug. Therefore, more
research focused on both optimization of coating materials and manufacturing
methods for encapsulating the protein and peptide drugs is necessary.
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REFERENCES 1127
1128 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
FIGURE A2 Release of theohylline from tablet coatings made with 10% CA dissolved in
85% acetone and 5% water.
FIGURE A1 Release of theohylline from tablet coatings made with 5% CA dissolved in
90% acetone and 5% water.
APPENDIX
The in vitro release profi les of the model drug theophylline are shown below in
Figures A1 through A10 .
FIGURE A4 Release of theohylline from tablet coatings made with 5% CA dissolved in
85% acetone and 10% water.
FIGURE A3 Release of theohylline from tablet coatings made with 15% CA dissolved in
80% acetone and 5% water.
APPENDIX 1129
1130 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
FIGURE A6 Release of theohylline from tablet coatings made with 15% CA dissolved in
75% acetone and 10% water.
FIGURE A5 Release of theohylline from tablet coatings made with 10% CA dissolved in
80% acetone and 10% water.
FIGURE A8 Release of theohylline from tablet coatings made with 10% CA dissolved in
75% acetone and 15% water.
FIGURE A7 Release of theohylline from tablet coatings made with 5% CA dissolved in
80% acetone and 15% water.
APPENDIX 1131
1132 CONTROLLED RELEASE OF DRUGS FROM TABLET COATINGS
FIGURE A10 Release of theohylline from tablet coatings made with 12.5% CA dissolved
in 77.5% acetone and 10% water.
FIGURE A9 Release of theohylline from tablet coatings made with 15% CA dissolved in
70% acetone and 15% water.
1133
6.6
TABLET COMPRESSION
Helton M. M. Santos and Jo a o J. M. S. Sousa
University of Coimbra, Coimbra, Portugal
Contents
6.6.1 Introduction
6.6.2 Theory of Particle Compaction
6.6.3 Compactibility
6.6.4 Tablet Compression
6.6.5 Equipment for Tablet Compression
6.6.6 Tablet Press Tooling
6.6.7 Tablet Engraving
6.6.8 Tablet Shape and Profi le
6.6.9 Tablet Bisect
6.6.10 Problems during Tablet Manufacturing
6.6.10.1 Capping and Lamination
6.6.10.2 Picking and Sticking
6.6.10.3 Mottling
6.6.10.4 Weight and Hardness Variation
References
6.6.1 INTRODUCTION
Tablets are the most important pharmaceutical dosage from and their design has
always been of great interest to pharmaceutical engineering. Since tablets are made
by a process of die compaction, although commonly called tablet compression, many
investigations have been involved in the task to describe the mechanisms involved
in this process. Nevertheless, some considerations should be taken regarding the
defi nitions of stages involved in tablet compression. Compression is one of two
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1134 TABLET COMPRESSION
stages involved in the compaction of a two - phase system due to the application of
an external force. It is defi ned as the reduction in the bulk volume of a material as
a result of gaseous phase [1, 2] . The second stage is consolidation, which is described
as an increase in the mechanical strength of the material resulting in particle -
to - particle interaction [1, 2] .
It is suggested that four mechanisms are basically involved in the process of
compression of particles: deformation, densifi cation, fragmentation, and attrition.
The process of compression is briefl y described as follows: small solid particles are
fi lled in a die cavity and a compression force is applied to it by means of punches
and then the formed monolithic dosage form is ejected. The shape of the tablet is
dictated by the shape of the die while the distance between the punch tips at the
point of maximum compression governs the tablet thickness, and the punch tip
confi guration determines the profi le of the tablet. The compression cycle in a conventional
rotary tablet press will be described in detail in this chapter and is illustrated
in Figure 1 .
The physical and mechanical properties of tablets, such as density and mechanical
strength, are signifi cantly affected by the process. Since tablet compression relies on
the ability of particulates to be compacted, the need to control the critical properties
of the materials with respect to readiness or ability to compact is an important issue
to the formulator.
In order to compress a powder or granulation product into a tablet of specifi c
hardness, a defi ned compression force must be applied. As pointed out by Shlieout
et al. [3] , by compressing a constant mass of powder, any variation in the applied
force causes a change in the measured force. In addition, the substance itself plays
an important role, that is, if it is of good compressibility, then the force needed for
compression would be low. It is well known that this compressibility will depend on
powder characteristics such as crystal habit and thermodynamic behavior.
The structure and strength of tablets are often discussed in terms of the relationship
between the properties of the particulate material and the properties of the
formed tablet. The properties of a powder that control its evolution into a tablet
FIGURE 1 Compression cycle. (Courtesy of Thomas Engineering.)
(a) Creating thecavity
Fill cam
(b) Filling the cavity
Suction
gravity
feeder
(c) Metering thecavity
Weight adjustment ramp
and head
scraper
(d) Containing the fill
Tail over die
Pull down after
weightcontrol
Punch-holding device
(e) Precompression
Solid formation
(f) Compressing
Solid formation
(g) Removing solid
Ejection cam
during compression, which will also relate to the fracture toughness and the tensile
strength of the tablets, are the compression mechanics of the particles and their
dimensions. Generally, all materials have the ability to store some elastic strain;
however, its extent will greatly vary for different materials and will depend upon
the intrinsic nature of the material. There are many instances where a brittle material,
or its surface, reduces signifi cantly its cohesion or adhesion compared to that
of a ductile material [4] .
6.6.2 THEORY OF PARTICLE COMPACTION
Basically, the process of tablet compression starts with the rearrangement of particles
within the die cavity and initial elimination of voids. As tablet formulation is a
multicomponent system, its ability to form a good compact is dictated by the compressibility
and compactibility characteristics of each component. Compressibility
of a powder is defi ned as its ability to decrease in volume under pressure, and compactibility
is the ability of the powdered material to be compressed into a tablet of
specifi c tensile strength [1, 2] . One emerging approach to understand the mechanism
of powder consolidation and compression is known as percolation theory. In a
simple way, the process of compaction can be considered a combination of site and
bond percolation phenomena [5] . Percolation theory is based on the formation of
clusters and the existence of a site or bond percolation phenomenon. It is possible
to apply percolation theory if a system can be suffi ciently well described by a lattice
in which the spaces are occupied at random or all sites are already occupied and
bonds between neighboring sites are formed at random.
The transitional repacking stage is driven by the particle size distribution and
shape. This will determine the bulk density as the powder or granulation product is
delivered into the die cavity. In this phase, the punch and particle movements occur
at low pressure. The particles fl ow with respect to each other, with the fi ner particles
entering the void between the larger particles, and thus the bulk density of the
granulation is increased. Various techniques have been utilized to determine the
degree of the two consolidation mechanisms in pharmaceutical solids (initial packing
of the particles and elimination of void spaces), namely the rate dependency technique.
By applying this technique, stress relaxation data based on the Maxwell
model of viscoelastic behavior indicate virtually no rate dependency for elastic or
brittle materials. There is also an increase in the calculated yield pressure with an
increase in punch velocity for viscoplastic materials such as maize starch and polymeric
materials. This is attributed to the reduction of time necessary for the plastic
deformation process to occur [6] . For brittle materials such as magnesium and
calcium carbonates there is no observed change in the yield pressure with increasing
punch velocity [6] .
When a force is applied to a material, deformation occurs. When this deformation
completely disappears after cessation of the external force, further deformation
occurs. Deformations that do not completely recover after release of the stress are
known as plastic deformations. The force required to initiate a plastic deformation
is known as the yield stress. When the particles are so closely packed that no further
fi lling of the voids can occur, a further increase of the compressional force causes
deformation at the points of contact. Both plastic and elastic deformation may occur,
THEORY OF PARTICLE COMPACTION 1135
1136 TABLET COMPRESSION
although one type will predominate for a given material. The ability of materials to
be compressed relies on their deformation behavior. The known extreme cases are
as follows. For elastic bodies, the force applied to consolidation will be fully given
back (action equals reaction). This is expressed as a completely elastic deformation.
For plastic bodies, the force applied will be saved as energy in the body and will
express no elastic deformation at all. During tablet building, these two processes
never occur alone but occur only in combination, as mentioned before.
As the external force is increased, the stresses within the particles become great
enough and cracks may occur. Fragmentation furthers densifi cation with the infi ltration
of the smaller fragments into the void spaces being responsible for increasing
the number of particles and formation of new and clean surfaces that are potential
bonding areas. The mechanisms of fragmentation and plastic deformation are not
independent since both phenomena modify particle size distribution, and larger
particles do not act as small particles with respect to plastic deformation [7] .
The bonding of particles is governed by different mechanisms. The three most
considered theories are mechanical theory, intermolecular theory, and liquid surface
fi lm theory. The fi rst theory assumes that under pressure the individual particles
experience elastic, plastic, or brittle deformation and that the edges of the particles
intermesh, forming a mechanical bonding. According to Parrot [2] , intermolecular
theory states that under pressure the molecules at the points of true contact are
close enough so that van der Waals forces interact to consolidate the particles.
Liquid surface fi lm theory relies on the presence of a thin liquid fi lm, which may be
the consequence of fusion or solution, at the surface of the particle, induced by the
energy of compression. Even tough the applied force is not high, it is locally transmitted
to small areas of true contact so that a very high pressure will exist at the
contact surfaces. This high pressure plays an important role in the melting point and
solubility of the material and proves to be essential to bonding. It follows that after
releasing the pressure, solidifi cation of the fused material would form solid bridges
between the particles. An important consideration has been proposed by Zuurman
et al. [8] to explain the action of some excipients during this phase. One of these
excipients is magnesium stearate, which is widely used as a lubricant in order to
prevent tablets from sticking to the die and punches and minimize wear of tooling.
It has been proven that magnesium stearate can have an adverse effect on bonding
between particles. The decrease of tablet strength is always considered to be the
result of reduction of interparticle bonding due to the addition of a lubricant.
The production of tablets with the desired characteristics depends on the stresses
induced by elastic rebound and the associated deformation processes during decompression
and ejection. Ideally, if only elastic deformation occurred, with the sudden
removal of axial pressure the particles would return to their original form, breaking
any bonds that may have been under pressure.
Finally, as the lower punch rises and pushes the tablet upward, there is a continued
residual die wall pressure and considerable energy may be expanded due to the
die wall friction. As the tablet is removed form the die, the lateral pressure is
relieved, and the tablet undergoes elastic recovery with an increase in the volume
of the portion removed from the die.
The compression cycle profi les may be used to characterize the consolidation
mechanisms of powders as they help to characterize the extent of pressure distribution
within the powder bed as well as the formed tablet. The compression behavior
of powder mixtures is usually characterized using the well - known Heckel equation
[9, 10] , which describes the relationship between the porosity of a compact and the
applied pressure and is based on the assumption that the densifi cation of the bulk
powder in the die follows fi rst - order kinetics:
ln
r
1
1.
= +
.
kP A
(1)
where . r is the relative density of the compact at pressure . , P is the applied pressure,
and K and A are constants. The constants A and k are determined from the
intercept and slope, respectively, of the extrapolated linear region of a plot of ln(1/1
. . r ) versus . (compaction pressure). The Heckel constant k is related to the reciprocal
of the mean yield pressure, which is the minimum pressure required to cause
deformation of the material undergoing compression. The intercept obtained from
the slope of the upper portion of the curve is a refl ection of the densifi cation after
consolidation. A large value of k indicates the onset of plastic deformation at relatively
low pressure. Thus, K appears to be a material constant. The correlation
between k and the mean yield pressure P y gives Equation (2) . The constant A is
related to the densifi cation during die fi lling and particle rearrangement prior to
bonding [11] :
k
P
= 1
y
(2)
A high . r value indicates that there will be a high volume reduction of the product
due to particle rearrangement. The constant A has been shown to be equal to the
reciprocal of the mean yield pressure required to induce plastic deformation. A
larger value for A (low yield pressure) indicates the onset of plastic deformation at
relatively low pressure, a sign that the material is more compressible.
The Heckel plot allows an interpretation of the mechanism of bonding. A nonlinear
plot with small value for its slope (a small Heckel constant) indicates that the
material undergoes fragmentation during compression. When the plot is linear, it
indicates that the material undergoes plastic deformation during compression.
In addition to the Heckel approach, other techniques may be applied to the
characterization of powder compression. One of these approaches was proposed by
Cooper and Eaton [12] :
V V
V V
a
k
P
a
k
P
0
0
1
1
2
2 .
.
= . ( )+ . ( ) s
exp exp
(3)
where V is the volume of the compact at pressure P (m 3 ), V 0 is the volume of
compact at zero pressure (m 3 ), V s is the void - free solid material volume (m 3 ), a 1 , a 2 ,
k 1 , and k 2 are the Cooper – Eaton constants.
The Kawakita equation [13] describes the relationship between volume reduction
and applied pressure according to Equation (4) , where P is the applied pressure, V 0
is the initial bulk volume, V is the volume at pressure P, a and b are the constants
characteristic of the powder under compression, and C is the degree of volume
reduction [Equation (5) ]:
THEORY OF PARTICLE COMPACTION 1137
1138 TABLET COMPRESSION
P
C
P
a ab
= + 1 (4)
C
V V
V
= . 0
0
(5)
In the Kawakita equation the particle density is not introduced in the calculations
since the model operates on the relative change in volume, which gives the same
result whether the relative or the absolute volume is used. The problem in the calculation
of this equation is to fi nd the correct initial volume V 0 .
6.6.3 COMPACTIBILITY
Compactibility of a powdered mixture is defi ned as the ability of the material to be
compressed into a tablet of a specifi ed strength without changing its composition.
Investigations have demonstrated that binary mixes of identical composition could
have different organizations, depending on the surface energy and particle size of
the fraction used. Actually, it has been demonstrated that it is possible to control
the organization of binary mixes by modifying the particle sizes of the fractions
blended if they have the appropriate surface energies [6] .
Generally, only powders that form hard compacts under an applied pressure
without exhibiting any tendency to cap or chip can be considered as readily
compactible. The compactibility of pharmaceutical powders can be characterized
by its tensile strength and indentation hardness, which can be used to determine
three dimensionless parameters: strain index, bonding index, and brittle fracture
index.
To calculate the work of compaction during tableting, it is necessary to have
accurate values of force and punch displacement. Differences in the dynamics of
powder densifi cation between eccentric and rotary machines were pointed out by
Palmieri et al. [14] after compression of microcrystalline cellulose, lactose monohydrate,
and dicalcium phosphate dehydrate at different compression pressures. The
effect of the longer dwell time of the rotary machine press on the porosity reduction
after the maximum pressure is reached is more noticeable in a ductile material such
as microcrystalline cellulose. It has been shown that Heckel parameters obtained in
the rotary press are in some cases different from those recovered in the eccentric
machine because of the longer dwell time, machine defl ection, and punch tilting
occurring in the rotary press, although theoretically they could better describe the
material densifi cation in a high - speed production rotary machine.
Williams and McGinity [15] studied and compared the compaction properties of
microcrystalline cellulose from six different sources using tableting indices. It was
demonstrated that storage of compacts at elevated humidity conditions prior to
determining the tableting indices decreased the magnitude of the tensile strength,
dynamic indentation hardness, and bonding index. Based on the differences in
physicomechanical properties observed for the tableting indices, the authors stated
that microcrystalline cellulose products from different sources are not directly interchangeable
and showed that the tensile strength, indentation hardness, bonding
index, and brittle fracture index for compacts composed of microcrystalline cellu
lose in combination with either talc or magnesium stearate generally decreased as
the amount of the lubricant was increased over the concentration range of 0 – 9%.
Similar results were observed for admixtures of sodium sulfathiazole in combination
with either talc or magnesium stearate. It was also demonstrated that the tensile
strength, indentation hardness, and bonding index increased, and the brittle fracture
index decreased as the percent of microcrystalline cellulose was increased in a
mixture with sodium sulfathiazole.
The results of a study conducted by Muller and Augsburger [16] suggest that
the pressure – volume relationship determined during powder bed compression is
affected by the instantaneous punch speed profi le of the displacement – time waveform
for all materials studied, even though they deform by different mechanisms.
It appears that the instantaneous punch speed profi le of the particular displacement
– time waveform is a confounding factor of Heckel analysis.
Moisute acts as a plasticizer and infl uences the mechanical properties of powdered
materials for tablet compression. In the case of microcrystalline cellulose, at
moisture levels above 5% the material exhibits signifi cant changes consistent with
a transition from the glassy state to the rubbery state [17] . The possible infl uence of
moisture on the compaction behavior of powders was also analyzed by Gupta et al.
[18] . This work evaluates the effect of variation in the ambient moisture on the
compaction behavior of microcrystalline cellulose powder.
The work conducted by Gustafsson et al. [19] evaluated the particle
properties and solid - state characteristics of two different brands of microcrystalline
cellulose (Avicel PH101 and a brand obtained from the alga Cladophora sp.)
and related the compaction behavior to the properties of the tablets. The difference
in fi bril dimension and, thereby, the fi bril surface area of the two celluloses
were shown to be the primary factor in determining their properties and
behavior.
The compaction properties of pharmaceutical formulations can be studied experimentally
using a variety of techniques, ranging from instrumented production
presses to compaction simulators, and methods of analysis. The results are usually
plotted as porosity – axial stress functions, which is of interest to compare different
materials. However, there are some drawbacks on this type of evaluation. As mentioned
by Cunningham et al. [20] , this approach is defi cient once it considers only
the average stress along the direction of compaction, ignoring radial stress transmission
and friction.
There have been some attempts to overcome the analysis of compaction
problems, mostly by introducing numerical modeling approaches. The modeling
approaches often used in compaction analysis are (a) phenomenological continuum
models, (b) micromechanically based continuum models, and (c) discrete - element
models. The parameters that should be analyzed when tableting is under development
are as follows:
1. Understanding the formulation and compositional effects on the compaction
process, including axial loading and unloading along with ejection
2. Determination of the stress distributions within the powder compact, including
residual stresses
3. Optimization of the tablet tooling design
COMPACTIBILITY 1139
1140 TABLET COMPRESSION
4. Estimation of the density distributions within a tablet that may infl uence dissolution
or mechanical properties
5. Estimation of the compaction force necessary to obtain tablets having given
properties
6. Taking into account the effect of the tablet material on the stress distribution
on tooling to aid tool design
7. Assessment of the origin of defect or crack formation
8. Optimization of more complex compaction operations such as bilayer and
trilayer tablets or compression - coated tablets
The demonstration of the validity of the continuum - based modelling approach
to tablet compaction requires familiarity with fundamental concepts of applied
mechanics. Under the theory of such a mechanism, powder compaction can be
viewed as a forming event during which large irrecoverable deformation takes place
as the state of the material changes from loose packing to near full density. Moreover,
it is important to defi ne the three components of the elastoplastic constitutive
models which arose from the growing theory of plasticity, that is the deformation
of materials such as powder within a die:
1. Yield criterion , which defi nes the transition of elastic to plastic deformation
2. Plastic fl ow potential , which dictates the relative amounts of each component
of plastic fl ow
3. Evolution of microstructure , which in turn defi nes the resistance to further
deformation
It is also known that the compression process can be described using static and
dynamic models. In the case of static models, time is not considered, although it is
a very important factor in the deformation process. The viscoelastic reactions are
time dependent, especially for the plastic fl ow.
Recently Picker [21] proposed a three - dimensional (3D) model to help explaining
the densifi cation and deformation mechanism experienced by differently deforming
materials during compression. According to the author, a single description of
the processes during tableting is possible, and thus densifi cation and deformation
properties can be clearly distinguished with a single model. This issue has been
investigated over the last years, and a comprehensive approach has been developed
for the analysis of compaction using continuum mechanics principles. This approach
is based on the following components:
1. Equilibrium equations (balance of forces transmitted through the material)
2. Continuity equation (conservation of mass)
3. Geometry of problem
4. Constitutive behavior of powder (stress – strain behavior)
5. Boundary conditions, including loading (e.g., displacement and velocity) and
friction between tooling and powder
6. Initial conditions (e.g., initial relative density of powder)
Due to the signifi cant nonlinearity in material properties and contact stresses, a
typical powder compaction problem cannot be solved analytically without major
simplifi cations, and thus a numerical approach is required.
The tableting properties of materials also depend on their deformation behavior.
It is apparent that the tablet tensile strength is a strong function of the plastic work
required for its formation but not a function of the elastic work recovered. Consequently,
it is likely that strong and ductile interparticle functions, whose formation
dissipated a signifi cant plastic work, result in strong and tough compacts [4] .
It should be mentioned that the material parameters do change with compaction
and the use of constant material values, which is often applied, is not necessarily
appropriate given the evolving microstructure of the deforming powder. The experimental
characterization and accompanying analysis allow these material properties
to be evaluated within the comprehensive framework of continuum mechanics,
which can be useful in analyzing and predicting the effects of constitutive behavior,
friction, geometry, loading schedule, and initial condition, for example, initial relative
density and powder fi ll confi guration.
Ruegger and Celik [22] investigated the effect of punch speed on the compaction
properties of pharmaceutical powders with one particular objective: to separate out
differences between the effect of the compression and decompression events. Tablets
were prepared using an integrated compaction research system. The loading and
unloading speeds were varied independently of one another. In general, when the
compression speed was equal to the decompression speed, the tablet crushing
strength was observed to decrease as the punch velocity increased. When the
compression speed was greater than or less than the decompression speed, the
results varied, depending on the material undergoing compaction. The authors also
stated that the reduction of the unloading speed had a similar effect on the direct -
compression ibuprofen; however, even greater improvement in the crushing
strength was observed when the loading speed was reduced. As a major conclusion,
it was demonstrated that the strength of tablets can be improved and some tableting
problems such as capping can be minimized or prevented by modifying the rates of
loading/unloading.
It is important to notice that, in the case of interacting materials, the compatibility
of a binary mix will depend mostly on the compatibility of the percolating material
[23] . Accordingly, several industrial applications can be made over these fi ndings. In
the development phase, it is possible to modify the formulation of interacting
systems to increase the drug content without losing the compatibility of the mix,
whereas in the production phase, it is possible to increase the compatibility of a
poorly compatible active ingredient by sieving or preferably by milling an excipient
with good compression qualities without changing the composition of the mixture.
6.6.4 TABLET COMPRESSION
The process of tablet compression is divided into three stages: fi lling, compression,
and ejection of the tablet (see Figure 1 ).
During the fi rst stage of a compression cycle the lower punch falls within the die,
creating a cavity which will contain the powder or granulation product that fl ows
TABLET COMPRESSION 1141
1142 TABLET COMPRESSION
from a hopper. The fi ll volume is determined by the depth to which the lower punch
descends in the die. At this moment the particles of the powder or granulation
product fl ow with respect to each other, thus resulting in a close packing arrangement
and the physical characteristics of the material (particle size, particle size distribution,
density, shape, and individual particle surface properties) associated with
process parameters such as fl ow rate and compression rate, and the relationship
between the die cavity and the particle diameter will defi ne the number of potential
bonding points between the particles. The packing characteristic of the product to
be compressed is greatly affected by the shape of the particles. Since the product to
be compressed comprises components of different nature, the voidage of a closely
packed system is considerably changed.
When the upper punch goes down, its tip penetrates the die, confi ning the powder
or granulation product, letting the particle rearrangement stage to continue and
initiating the compression stage as the compression force is applied. As a result,
forces resulting from the compression force are transmitted through the interparticulate
points of contact created in the previous stage. The porosity of the powder
bed is gradually decreased, the particles are forced into intimate proximity to each
other, and stress is developed at the interparticulate points of contact. Once the
particles have formed contacts, they will deform plastically under the applied load.
Deformation of the particles will be characterized by elastic, plastic, fragmentation,
or a combination of these phenomena, which will depend on the rate and magnitude
of the external applied load, the duration of locally induced stress, and the physical
properties of the product under compression. When the particles are in suffi ciently
close proximity, they are bonded. Particles bond as a result of mechanical interlocking,
which is described as entanglement of the particles, phase transition at the points
of contact, and intermolecular forces, namely the van der Waals force, hydrogen
bonding, and ionic bonding.
After formation of the tablet by application of a compression force follows the
decompression stage, where the compression force is removed and the upper punch
leaves the die. Then, the formed tablet undergoes a sudden elastic expansion
followed by a viscoelastic recovery during ejection when the lower punch moves
upward.
6.6.5 EQUIPMENT FOR TABLET COMPRESSION
The equipment employed for tablet compression is generally categorized according
to the number of compression stations and dislocation mode. Therefore, eccentric
model presses have only one compression station (one die and one pair of punches,
upper and lower) while rotary models have multiple compression stations (each
station with one die and one pair of punches, upper and lower). The basic difference
between the two types of compression equipment is that for eccentric models the
compression force applied during compression is due to the upper punch whereas
for rotary models it is mainly applied by the lower punch.
A rotary tablet press machine (Figure 2 ) comprises a housing in which the compression
set and subsets (upper and lower roller assemblies) are mounted, the turret
head, the upper cams, the weight control assembly and the lower cams, the hopper,
the feeder assembly, the take - off chute, the aspiration assembly, the gear box and
the electrical unit, and the lubrication system.
The compression zone is located on the back side of the equipment and employs
a maximum load force limited by the type of tooling being used. It is of paramount
importance to note that, if a load force is applied over the indicated limit, the press
unit will not function properly, resulting in premature wear or possible damage to
the tooling. The compression set comprises the hopper and feeder system, the die
table, the upper and lower compression rollers, the upper and lower turrets, the
excess - material scraper, the tablet stripper, the recirculation channel, and the aspiration
system.
The hopper is usually made of stainless steel and has the shape of a funnel to
contain and deliver the product to be compressed. It may be provided with a window
for the observation of the product level and may also be provided with low - level
sensors that signal an alarm, shut off the engine, or activate the feeding mechanism
to deliver the product when it falls below this level. The feeder system usually consists
of three sections (in the case of force feeders) and is ideal for press performance
at high speed. The fi rst section of a force feeder system is where the hopper is connected
and is responsible for the fl ow of the product from the hopper to the next
sections. The second section is where the die cavities are fi lled to their maximum
capacities, and the third section is where the weight control adjustment takes place.
These sections contain paddle systems which prevent packing of the product. The
FIGURE 2 Rotary tablet press machine: ( a ) left-side view; ( b ) black - side view. (1) Cabinet,
(2) compression, (3) turret, (4) gear, (5) weight control assembly and lower cams, (6) plate
cams, (7) guarding, (8) hopper system, (9) feed frame assembly, (10) take - off chute,
(11) aspiration assembly, (12) electrical system.
(a) (b)
EQUIPMENT FOR TABLET COMPRESSION 1143
1144 TABLET COMPRESSION
speed of the paddles is adjustable and should be synchronized with the die table in
order to prevent tablet weight variation. Better adjustment of the paddle speed
could be achieved when keeping the lowest standard deviation of the compression
force. The feeder system height above the die table is usually kept between 0.05 and
0.10 mm. When the product to be compressed is of very fi ne particles, this height
should be kept at 0.025 mm.
Presses are commonly equipped with a powder aspiration system which is connected
to a vacuum source in order to remove excess powder from the die table.
This assembly is essential for a high - speed press working for extended periods of
time. Special attention must be taken when the powder product comprises an active
ingredient of fi ne particle size. In this case, aspiration should be minimal in order
to prevent loss of the active ingredient.
The compression subsets comprise the upper roller assembly and the lower roller
assembly. The upper roller assembly is located on the roof plate of the press and
utilizes an adjustment system for the regulation of the insertion depth. The lower
roller assembly is located on the underside of the die table and utilizes a device for
the regulation of the tablet edge thickness.
The turret head is fi xed to the main shaft of the gear box. It is manufactured in
two pieces (upper and lower) which guarantee the alignment between punches and
dies. The gear box is mounted on the lower section under the die table and is responsible
for transmitting the draft movement of the motor toward the turret head.
The upper cams are responsible for guiding the upper punches around the circumference
of the turret head. It comprises the fi lling stage track, which guides the
upper punches in an up position during its passage over the feeder system; the upper
lowering cam, which guides the upper punches down in order to keep their tips
covering the cavities (precompression position) and directs the upper punches to
their compression stage; the upper compression roller, which guides the upper
punches to their compression position; and the upper fi lling cam, which guides
the upper punches back to the fi lling track.
The weight control assembly, which comprises the weight adjustment cam, is
located in the lower section of the press and is regulated by an adjustment system.
The lower cams are also located in the lower section of the press and comprise the
preweight control (or fi ll cam), the weight adjustment cam, the lower lowering cam,
and the ejection cam. The preweight control guides the lower punches to the full - fi ll
position. The weight adjustment cam guides the lower punches up to the desired
fi ll position. The lower lowering cam guides the lower punches to the precompression
position. The ejection cam guides the lower punches and the formed tablets to
the discharge position. It is recommended to operate the weight adjustment cam in
the approximate center of the fi ll cam and because of this the fi ll cam is removable
and available in different sizes having a range of approximately 10 mm with an
increment range of 4 mm. The choice of the adequate fi ll cam for the operation of
a tablet press with a particular product should be based on the density of the
product. According to Figure 3 , the fi ll cam can be adequately chosen when taking
into account the density of the material to be compressed and thus the material
column height in the die cavity.
Rotary tablet presses could be designed to be single, double, or triple sided. A
single - sided press comprises one hopper, one set of compression rolls, and one take -
off chute unit whereas double - and triple - sided presses comprise two and three each
of these units, respectively. Irrespective of the design of the rotary tablet press, the
compression cycle is described as follows (Figures 1 and 4 ).
The powder or granulation product contained in the hopper fl ows to the feeder
which spread the product through a large area over the die table in order to provide
enough time to fi ll the die cavity. The die cavity is created when the fi ll cam guides
the lower punches to the full - fi ll position and enters the feeder area. Note that the
die cavity is fi lled with an excess of the product at this stage of the cycle. Right after,
the weight adjustment ramp and head guide the lower punches to the desired fi ll
position. The excess of the product is removed by the scraper and is pushed back
by the excess product stripper when entering the recirculation channel. At this stage
the lower punches are guided to the fi rst and second lower compression rols (precompression
and main compression rolls, respectively) while the upper punches are
guided by the upper lowering compression roll to the precompression position and
to the compression position by the main compression roll. As the upper punch
penetrates the die cavity until a predefi ned height, the main compression roll applies
the compression load over the lower punch, compressing the product in the die.
Soon after compression, the upper lifting cam allows the upper punch to leave the
die cavity. Simultaneously, due to the ejection cam, the lower punch is pulled,
FIGURE 3 Fill cam depth as function of product column height.
Fill cam (mm)
6 10 14 18 22
Product
column
height
(mm) 8 12 16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
EQUIPMENT FOR TABLET COMPRESSION 1145
1146 TABLET COMPRESSION
ejecting the formed tablet to the die table. The ejected tablets are then stopped by
a scraper and allowed to escape through a chute and collected. At this time the fi ll
cam geometry makes the lower punch go down and a new compression cycle
begins.
6.6.6 TABLET PRESS TOOLING
Punches and dies are essential tools in the tableting process and therefore are critical
to the quality of the tablets produced. Both tools are designed for long life under
normal conditions of working, but, in spite of this, they are not proof against careless
handing.
It is important for those working with a tablet press to be familiar with the terminology
used in the industry concerning the punches and dies. Table 1 describes
the commonly used terms related to press tooling. Some of the press tooling parts
can be identifi ed in Figure 5 .
When considering a tableting operation, it is important not only to select the
appropriate press tooling in terms of dimensional data but also to consider the
material of which tools are made. Performance of the press tooling will in part be
a function of the material selected for its manufacture. Usually the material and
hardness of the compression tooling are left to the manufacturer ’ s discretion. There
are various types of steel available for the manufacture of press tooling. It is important
to recognize the individual characteristics of the steel regarding its composition
and the percentage of each constituent element. Usually only a small amount of
alloying element is added to steels (usually less than 5%) for the purpose of improving
hardness and strength corrosion resistance, stability at high or low temperatures,
and control of grain size. Some of these elements are as follows:
1. Carbon Principal hardening element. As the carbon content increases, its hardness
increases. Increases the tensile strength of the steel.
FIGURE 4 Compression cycle on rotary tablet press. (Courtesy of Thomas Engineering.)
Upper lifting cam
Upper lowering cam
Feeder Scraper
Ejection cam
Tail over die
Pull down after
weight control cam
Weight adjustment
ramp and head
Fill cam
Main compression rolls
Precompressionon
rolls
Main compression rolls
Precompressionon
rolls
TABLET PRESS TOOLING 1147
TABLE 1 Tooling Terminology
Band Area between opposing cup profi les formed by die wall
Bakelite tip
relief
Undercut groove between lower punch tip straight and relief; assures
sharp corner to assist in scraping product adhering to die wall
Barrel/shank Surface controlled by turret punch guides to ensure alignment with die
Barrel - to - stem
radius
Provides smooth transition from tip length to barrel
Barrel chamfer Chamfers at ends of punch barrel, eliminates outside corners
Barrel fl utes Vertical slots machined into punch barrel to reduce bearing surface
and assist in removing product in punch guides
Cup depth Depth of cup from highest point of tip edge to lowest point of cavity
Die Component used in conjunction with upper and lower punches; accepts
product for ocmpaction and is responsible for tablet ’ s perimeter size
and confi guration
Die bore Cavity where tablet is made, shape and size determine the tablet
Die chamfer Entry of die bore
Die groove Groove around periphery of die to allow die to be fi xed in press
Die height/
overall length
Overall height of die
Die lock Mechanism used to lock die in position after it is installed in die table
Die outside
diameter
Outside diameter of die, compatible with die pockets in press
Die taper Gradual increase in die bore from point of compaction to mouth of
bore, assists ejection
Head End of punch which guides it through press cam track
Head/dwell fl at Flat area of head that receives full force of compression rolls at time
that tablet is being formed
Inside head
angle
Area of contact with lower cam and upper cam
Key Prevents rotational movement of punches ensuring alignment to
shaped and multihole dies
Keying angle Relationship of punch key to tablet shape; position will be infl uenced
by tablet shape, take - off angle, and turret rotation
Land Area between edge of punch cup and outside diameter of punch tip
Neck Relieved area between head barrel which provides clearance for die
Outside head fl at
angle/radius
Contact area with press cams and initial contact with pressure rolls
Overall length Total punch length as measured from head fl at to end of tip
Tip face/cup Portion of punch tip that determines contour of tablet face including
tablet embossing
Tip length Straight portion of stem effective inside die bore
Tip relief Portion of punch stem which is undercut or made smaller than punch
tip straight; most common for lower punches in order to reduce
friction from punch tip and die wall
Tooling set Complete set of punches and dies to accommodate all stations in tablet
press
Tooling station Upper punch, lower punch, and die which accommodate one station in
tablet press
Relief/undercut Mechanical clearance between stem and die bore, sharp edge between
tip straight and undercut areas acts to clean die
Stem Area of punch opposite head which begins at end of barrel and
extends to tip
Working length Length of punch from bottom of cup to head fl at; together, upper and
lower working lengths control tablet thickness and weight; also
known as overall length, bottom of cup (OLBC)
1148 TABLET COMPRESSION
FIGURE 5 Identifi cation of common parts of press tooling (upper and lower punches and
die) according to ISO 18084, 2005: (1) upper punch, (2) lower punch, (3) die, (4) key, (5) land,
(6) stem, (7) barrel - to - stem chafer, (8) cup depth, (9) tip face, (10) blended land, (11) face,
(12) bore, (13) die grove, (14) protection radius or shoulder, (15) chamfer or radius, (16) outer
diameter, (17) tip straight, (18) relief, (19) barrel - to - stem radius, (20) working length of tip,
(21) overall length, (22) barrel, (23) working length, (24) barrel - to - neck radius, (25) neck - to -
head radius, (26) inside head angle, (27) neck, (28) head, (29) head outer diameter,
(30) outside head angle, (31) head fl at, (32) key orientation angle, (33) upper punch face
key position, (34) barrel diameter.
2. Manganese Increases ductility and hardenability of the steel. Also increases
the rate of carbon penetration during carbonizing and imparts excellent wear
resistance.
3. Nickel Improves the toughness and impact resistance of the steel and mildly
increases its hardness.
4. Chromium Increases the hardness of the steel and improves its wear or abrasion
resistance. It helps to limit grain size. If added in amounts greater than
5%, it can impart corrosion and wear resistances.
5. Molybdenum Improves hardenability and increases tensile strength of the
steel.
6. Vanadium Produces a fi ne grain size and improves fatigue strength of the steel,
just like molybdenum.
TABLET PRESS TOOLING 1149
7. Tungsten Is used in tool steels to maintain hardness at elevated
temperatures.
8. Copper Increases corrosion resistance; nevertheless its content has to be
controlled, otherwise the surface quality and hot - working behavior are
compromised.
The carbon steels comprise alloying elements not exceeding the defi ned limits of
1% carbon, 0.6% cooper, 1.65% manganese, 0.4% phosphorus, 0.6% silicon, and
0.05% sulfur. For the alloy steels, the limits exceed those for the carbon steels and
may also include elements not found in carbon steels. The alloy steels have a specifi c
designation according to the American Iron and Steel Institute (AISI). Such designation
is a four - digit number where the fi rst digit stands for the class of the alloy
(e.g., 1, carbon; 2, nickel - chromium; 3, molybdenum; 4, chromium), the second digit
designates the subclass of the alloy, and the last two digits designate the amount of
carbon in 0.01%. The stainless steels comprise at least 10% chromium with or
without the addition of any other alloying element. The tool steels are carbon steel
alloys with an excess fo carbides which impart hardness and wear resistance. According
to the AISI, tool steels are grouped as water hardening (W), shock resisting (S),
cold - work oil hardening (O), cold - work medium - alloy air hardening (A), cold - work
high - alloy high chromium (D), low alloy (L), carbon tungsten (F), low - carbon mold
steels (P1 – P19), other mold steels (P20 – P39), chromium - based hot work (H1 – H19),
tungsten - based hot work (H20 – H29), molybdenum - based hot work (H40 – H59),
high - speed tungsten based (T), and high - speed molybdenum based (M).
The appropriate steel for press tooling should be selected based upon the toughness
and wear resistance required by the application, and therefore it is mandatory
to have satisfactory knowledge regarding the abrasiveness, corrosiveness, and lubricity
of the product intended for compression as well as the desired dimensions of the
punch tip. The toughness of the steel regards its ability to resist shock and its wear
resistance regards the ability to resist physical damage or erosion due to product
contact.
For a clear understanding, the Thomas Engineering Press Tooling Manual [24]
states the following:
Punches manufactured from high carbon/high chromium steel may exhibit improved
wear resistance characteristics, however under extreme compression force, the cup may
crack due to the brittle nature of the steel. Steels with lower carbon and chromium
levels will act conversely. While these steels may be useful in some applications, the
majority will require a more moderate balance of toughness and wear resistance.
Steel selection for dies is not as critical. In most cases, high wear resistance steel is
preferred.
The bulk of pharmaceutical tablet press punches are manufactured from S1, S5, S7, or
408 (11% chromium, 8% nickel) tool steel. The S series steels provide a good combination
of shock and wear resistance and have a proven record of performance in tableting
operations. At one time, 408 or 3% nickel steel was the industry standard because of
its superior shock resistance toughness . The S grades however, which have only a slight
loss in ductibility by comparison, offer much improved wear characteristics and have
all but replaced 408 as the preferred general purpose punch steel. A2, D2 and D3 are
high carbon/high chromium steel used for their excellent wear resistance. Among all
the steels commonly used for press tooling, D3 has the highest wear resistance. However,
1150 TABLET COMPRESSION
its low toughness rating typically limits its use to dies only. D2 rates slightly lower in
abrasion resistance than D3 but its increased toughness makes it suitable for punch
use, provided the cup design is not too fragile. A2 is a compromise between the general
purpose S grades and D2 in both toughness and wear. It can be used for punches as
well as dies.
Tungsten carbide, while not actually a steel, is extremely wear resistant and is commonly
used to line dies. Punch tips can be manufactured from tungsten carbide;
however, the cost of tooling is quite high and restricted to applications where tip fracture
due to high compression forces is not likely.
Ceramic materials such as partially stabilized zirconia can also be used as die liners.
Ceramics offer high wear and corrosion resistance and lower tablet ejection forces than
either steel or carbide due to their low coeffi cient of friction.
S1, S7 and 408 provide some protection against mildly corrosive materials. More severe
corrosion problems however, demand the use of stainless steel (440C) tooling. From
the standpoint of wear, 440C falls between the S and D grades of tool steel. Its low
toughness rating (comparable to D3) requires a strong cup design if tip fracture problems
are to be avoided.
One measure of tool steel quality is the rate of inclusions. Inclusion are unwanted
impurities or voids and are present to some degree in all steels. After heat treatment,
inclusions give rise to localized areas of stress concentration where microscopic cracks
can later develop. Remelting of the steel at the foundry will further reduce a tool steel ’ s
level of impurities; therefore improving the quality of the steel and subsequently its
performance in the tooling environment. In cases where punch tip fracture is a problem,
tooling suppliers may recommend a remelted or premium grade of particular steel as
a means of eliminating the problem.
Concerning the confi guration of compression tooling, the most commonly used
are the so - called B (19 mm, or 3/4 in.) and D (21 mm, or 1 in.) tooling types. Additionally,
these two types are classifi ed into three specifi cations: the North American
TSM ( Tableting Specifi cation Manual ) [25] , the European Union (EU) standard,
and the Japan Norm (JN). The North American TSM is used in the United States
and is the only standard offi cially supported by the governing body and published
by the American Pharmacists Association. The EU standard and the JN are generally
used in Europe and the Far East, respectively. In spite of the existence of these
standard specifi cations, there are tablet press manufacturers that use their own
confi gurations for tooling which have the disadvantage of being restricted to a specifi
c tablet press. Figures 6 and 7 depict the three standard confi gurations of compression
tooling. In addition, the International Organization for Standardization
(ISO) standard 18084:2005 [26] comprises specifi cations of the main dimensions,
including tolerances and characteristics of punches and dies.
Regarding the importance of compression tooling to the performance of the
tablet press and the quality of the tablets, it is of paramount importance that
punches and dies are handled with care. The fi rst criterion is the identifi cation
of the tooling; that is, punches and dies should be identifi ed according to the standard
and be designated by “ upper punch without key, ” “ upper punch with key, ”
“ lower punch with key, ” “ lower punch without key, ” or “ die, ” the reference of the
standard (e.g., TSM, EU, JN, ISO), and the punch or die diameter. Punches and
dies should also have a marking that includes at least the manufacturer ’ s identifi cation,
the number of the punch in the series, and/or the identifi cation number. Upon
TABLET PRESS TOOLING 1151
FIGURE 6 Tooling standards confi gurations.
receipt, after manufacturing, and prior to inspection, the punches and dies should
be carefully and thoroughly cleaned and dried. Then, tooling should be lightly oiled,
packed, and stored in a dry, cool place.
Damage to the punches and dies should be avoided. Therefore, they should not
be transported from place to place without protective package. During transportation,
installation, and removal of tooling from the tablet press, cleaning, inspection,
and storage, care must be taken to avoid hitting the tips of the punches.
The visual and dimensional inspection of punches and dies should be carried out
periodically. Visual inspection should be performed each time punches and dies are
installed in and removed from the tablet press. Under normal conditions, slight wear
is to be expected. When abnormal or excess wear is detected, the cause should be
immediately investigated, inspecting the cams or components which touch the
affected area of the tool. The importance of visual inspection resides on the fact
that it may ensure the optimum life of the punches and dies, performance of the
tablet press, and consistency and appearance of the tablets. In addition to the visual
inspection, it is also recommended that dimensional inspection be performed at
specifi c intervals throughout the life span of the punches and dies. The dimensional
inspection not only ensures the consistency of hardness, weight, and thickness of the
tablets, but also proves to be critical in diagnosing potential and real problem areas
with regard to the tableting process and press. A typical schedule for the dimensional
inspection may be as follows: 50%, 75%, 85%, 90%, and 95% of the historical
or projected life cycle of the punches and dies. Therefore, the history or data base
should be maintained for each set of tooling. Nevertheless, there is no general
agreement on what dimensions of the punches and dies should be included in a
1152 TABLET COMPRESSION
FIGURE 7 Tooling head confi gurations.
Standard TSM (B-type punches) TSM domed (B-type punches)
Standard TSM (D-type punches) TSM domed (D- type punches)
EU standard Japan norm
dimensional inspection. Some believe that a 100% inspection should be carried out
while others defend that only critical dimensions (e.g., working length, cup depth,
and overall length) should be inspected, believing that measuring any other dimension
is either unnecessary, since it rarely if ever changes and therefore is not worthy
of the time and expense of measuring, or cannot be properly measured with current
equipment and is better served by a visual inspection. What is important when
inspecting compression tooling is that the dimensional values are consistent within
the set and tolerances and within specifi cations. Before proceeding with inspection,
the measuring instruments should be calibrated to be certain that the dimensional
values obtained are accurate and true.
6.6.7 TABLE ENGRAVING
Engraving is the most common method for tablet surface marking identifi cation.
The engraving method could be embossed (letters or symbols are raised on the
tablet surface and cut into the punch tip face) or debossed (letters or symbols are
cut into the tablet surface and raised on the punch tip face). For engraving on the
tablet surface some specifi cations should be considered: stroke width, angle of
engraving, radius, depth, spacing, and engraving area.
Generally, for uncoated tablet application, a stroke width between 15 and 20%
of the letter height having an engraving angle of 30 ° is recommended. The radius
should be between 50.8 . m (0.002 in.) and a value derived from dividing the stroke
width by 2 times the cosine of the engraving angle. It is important to note that radii
smaller than 50.8 . m or exceeding the maximum value are diffi cult to machine since
it may decrease depth and defi nition of engraving. The depth is a function of the
engraving angles, stroke, and radius for a given tablet size and, as a general rule, the
depth should not exceed 50% of the stroke width, or no less than 88.9 . m (0.0035 in.).
Spacing between letters or symbols should be a minimum of one stroke width. The
available engraving area is based upon letter distortion due to the curvature or
radius of the cup and thus, as a general rule, letter distortion is defi ned by the ratio
of the outside depth of the engraving to the specifi ed depth. Generally, distortion
is present when this ratio exceeds 1 : 3.
When engraving is considered for fi llm - coated tablets, the recommended stroke
width should be the same as recommended for uncoated tablets. The recommended
engraving angle should be 35 ° . However, engraving angles up to 40 ° can be used
in extreme applications to allow coating solution fl ow. For stroke widths of 203.2 . m
(0.008 in.) or less, a 30 ° angle is recommended to maintain minimum engraving
depth. The recommended radius should be between 76.2 and 152.4 . m and a value
derived by dividing the stroke width by 2 times the cosine of the engraving angle.
It is important to note that radii will be determined by the fl owability of the coating
solution and coating process. As a general rule, stroke depth should be at least
177.8 . m. However, shallower depths can be used, provided that the fi lm coating
process is properly developed taking this factor into account. The spacing between
the letters and symbols and the available engraving area considerations are the same
as for uncoated tablets. The following equations can be used to determine stroke
width and engraving radii:
S H H = - 0 15 0 20 . . (6)
R R
S
min max m
cos
= = 50 8
2
. .
.
(7)
6.6.8 TABLET SHAPE AND PROFILE
The more popular standard geometric shapes of tablets are the round and the caplet
shapes. Other tablet shapes include the oval, elliptical, square, diamond, rectangular,
and polygonal. The shape of tablets plays an important role in terms of aesthetics,
process (printing, fi lm coating, packaging, and shipping), and acceptability by the
consumer (identifi cation, help with swallowing).
TABLET SHAPE AND PROFILE 1153
1154 TABLET COMPRESSION
In terms of design, the profi le of a tablet also plays an important role in the aesthetics,
packaging, orientation for printing processes, and handling. The profi le of a
tablet is important in the fi lm coating process and even in helping with the swallowing.
Applying a bisect score onto the tablet surface enables the tablet to be easily
divided into smaller dosage amounts.
Nowadays it is common for tooling suppliers to use software to provide 2D and
3D technical drawings of tablets and tooling. Such software may provide accurate
details of tablets and tooling using only tablet dimensions as input and therefore
enables fast evaluation by the manufacturing department prior to ordering prototypes.
Figure 8 illustrates some tablet shapes and profi les.
Flat - face, bevelled - edge tablets have many advantages due to their fl atness, which
provides the most compact tablet weight per volume weight, uniform hardness since
the compression force is exerted evenly on the cup face, and engraving with no distortion.
This tablet profi le proves to be ideal for small tablets, especially when
engraved, although the engraving area may be limited by the 381 . m (0.015 in.)
radius on the bevel. On the other hand, compression tooling displays an inherent
weakness in the punch cup design at the point where the bevel edge meets the cup
fl at. Attention must be paid since these types of tablets cannot be coated as they
will stick together, or twin.
FIGURE 8 Common tablet shapes and profi les: (A) standard convex, (B) compound cup,
(C) convex beveled, (D) fl at faced plain, (E) fl at faced bevel edged, (F) fl at faced radius
edged, (G) lozenge, (H) modifi ed ball, (I) core rod with hole in center, (J) capsule, (K) modi-
fi ed capsule, (L) oval, (M) bullet, (N) arrow head, (O) triangle, (P) arc triangle, (Q) square,
(R) pillow or arc square, (S) rectangle, (T) modifi ed rectangle, (U) diamond, (V) pentagon,
(W) hexagon, (X) octagon, (Y) almond.
A B C D E
F G H I J
K L M N O
P Q R S T
U V W X Y
Shallow and standard concave tablets have the great advantage of displaying a
maximum allowable area available for engraving without distortion as a result of
the moderate curvature of the cup profi les and the absence of a bevel. The shallow
and standard cup confi gurations are the strongest profi les per punch tip diameter.
In addition, such profi les allow consistent distribution of the compression force over
the cup face due to the slight curves involved in the cup, thus contributing to the
production of tablets of uniform hardness. Nevertheless, caution must be taken
concerning the cup depth since when it approaches the cup edge it may be less than
the depth of the engraving. The major disadvantage of these profi les may be due to
the angle of the cup profi le to the tablet sidewall, which may lead to chipping at the
tablet edge during fi lm coating or handling.
Caplet - shaped tablets are easier to swallow, aesthetically pleasing, and chipping
at the tablet edge generally does not occur during fi lm coating or handling due to
the angle of the cup profi le to the tablet sidewall. Nevertheless, the increased curvature
of the cup reduces compression force by approximately 50% compared to
the shallow and standard concave profi les. Distortion of engraving may also be a
problem because of the more extreme curvature. During fi lm coating caplet tablets
have the potential of sticking together, or twinning, as the tablet sidewalls are parallel.
This problem could be alleviated by applying a 76.2 mm (0.003 in.) drop (15.24 –
20.32 cm, or 6 – 8 in. radius) to the sidewalls.
The concave oval profi le displays a maximum allowable area for engraving and
a uniform distribution of the compression force over the cup face. Such an advantage
is a consequence of the mild curvature of the cup profi le and absence of a bevel.
Structurally, concave oval tablets are the strongest of the non - round - shaped tablets.
However, due to the angle of the cup profi le to the tablet sidewall, chipping at the
tablet edge may occur during fi lm coating or handling.
The compound cup profi le could be used to provide round or oval tablets. This
profi le provides a good tablet weight per volume but simultaneously presents a
weak cup edge, thus being the weakest of all cup confi gurations. Because of this, the
maximum compression force is limited to the minor cup radius on the round shapes
and the minor cup radius on the minor side for the oval shapes. In addition, the
available engraving area is limited to the blending point of the two radii.
6.6.9 TABLET BISECT
Usually known as score or break line, the tablet bisect has the purpose of easily
breaking the tablet in predetermined small dosages. According to the TSM, the
bisect types range from the most functional (the pressure sensitive, or type G) to
the least functional (partial, or type H). Each bisect type has its own characteristic,
as can be seen in Figure 9 . Generally, the bisect is placed on the upper punch, especially
when its depth exceed 40% of the cup depth, in order to avoid problems
during ejection of tablets. Nevertheless, the bisect can be placed on the lower punch
either when the upper punch is supposed to contain embossed characters or printing
that makes diffi cult the existence of the bisect or when its depth does not exceed
40% of the cup depth. When it is desired to apply a bisect to the upper tablet ’ s
surface but there is interference of engraving or printing, then a modifi ed bisect
design should be considered.
TABLET BISECT 1155
1156 TABLET COMPRESSION
When considering applying a bisect to a tablet ’ s surface, careful attention should
be taken with respect to the tablet ’ s cup depth, band thickness, and hardness. Considering
these aspects, the specifi cations for the bisect size are determined taking
into account the tablet ’ s size, engraving or printing, and desired bisect design.
The TSM acknowledges two different confi gurations of bisect for concave tablets:
protruding and cut fl ush. The protruding confi guration follows the curvature of a
radiused cup and extends past the tip edge of the punch. The cut fl ush confi guration
FIGURE 9 Tablet bisect for concave tooling (according to TSM): (A, B, C) pressure -
sensitive (type G), (D, E, F) cut through (type D) or European style, (G, H, I) decreasing
(type C), (J, K, L) standard protruding (type A), (M, N, O) standard (type E), (P, Q, R) short
(type B), (S, T, U) partial (type H).
Top view Profile (end) view Profile (side) view
A B C
D E F
G H I
J K L
M N O
P Q R
S T U
50% max of
band thickness
95% of
cup depth
75% of
cup depth
Cup radius
Bisect
radius
Break
radius
Break
radius
is the most popular bisect confi guration since one may experience problems with
the protruding confi guration. This is explained by the fact that the protruding bisect
may run into the tip edge of the lower punch if they become too close during the
compression cycle of the press.
Among the bisect styles acknowledged by the TSM, the cut - through, also known
as the European style, can only be applied on radiused cup designed tablets. Other
styles are the standard, the short, and the partial bisects. Compared to the standard
style, the cut - through style is said to have the advantage of letting patients better
break the tablet into smaller subunits. On the other hand, because the cut - through
is wider at the center, it decreases the available tablet surface area for engraving or
printing.
6.6.10 PROBLEMS DURING TABLET MANUFACTURING
Due to either formulation or equipment, some problems can arise during the tablet
compression process, such as capping and lamination, picking and sticking, mottling,
double printing, weigh variation, and hardness variation. It is the early detection
and accurate diagnosis of any of these fl aws that can avoid tablet compression
process failure and consequently improve its reliability, safety, reduce process downtime
and the overall operating cost.
Often, some of the above - mentioned problems are not detected during the development
of a particular tablet formulation, only appearing during scale - up as the
processing speed is increased. Some of the problems experienced during tableting
can be solved by shifting the formulation or alleviated by altering the tableting
conditions.
6.6.10.1 Capping and Lamination
Capping and lamination are common problems that can be experienced during
tableting. Capping is defi ned as the splitting of one or both lids of a tablet from its
body. Lamination is a precursor to capping since it involves the occurrence of layers
in a compact parallel to the punch face. Sometimes capping is noticed not during
the process but during physical testing, such as friability and hardness.
An incipient theory proposed by Train [27] related lamination to radial elastic
recovery of the compacted material during ejection. A once - accepted theory formulated
that capping and lamination are the result of air entrapped in the tablet
under pressure which tries to escape during ejection [28] . This theory is no longer
widely accepted. Disagreement arises from the fact that some formulations cap or
laminate even at low press speeds. Today, it is believed that the entrapped air may
be related to capping but does not affect lamination.
A widely accepted theory for lamination presented by Long [29] and reformulated
by Ritter and Sucker [30] attributes capping to the residual die wall pressure.
This pressure is said to cause internal shear stresses in the tablet causing the propagation
of cracks, which results in lamination or capping. The propagation of cracks
can be prevented by plastic relaxation of shear stresses. Therefore, materials having
suffi cient plasticity may not be susceptible to lamination. Some properties of the
powder mixture, such as moisture content, type and amount of the binder, and
PROBLEMS DURING TABLET MANUFACTURING 1157
1158 TABLET COMPRESSION
particle size, are important formulation variables that could be assessed in order to
impart plasticity, thus diminishing capping and lamination tendencies.
Normally, drugs such as paracetamol, mannitol, ibuprofen, phenazone, and mefenamic
acid have poor compression properties and produce tablets that are weak
and frequently exhibit capping. Materials that deform elastically or exhibit time
dependence are more susceptible to capping and lamination and/or strength reduction,
especially as tableting rate is increased. The effect of punch velocity is most
marked when transferring a material from an eccentric to a rotary press or when
scaling up to larger production size tablet presses.
In addition to the possible causes of capping and lamination discussed previously,
one should also consider the possibility that shape of the tooling and tooling defects
are sources of capping. In such cases the problem can simply be alleviated by repairing
or altering press tooling.
Usually the process of capping can be evidenced as an increase in tablet height
within a few seconds after tablets are ejected from the die.
A technique generally applied to characterize and prevent the capping and lamination
of a material intended to be compacted is using the brittle fracture index
(BFI). The BFI was designed by Hiestand et al. [31] and measures the ability of a
material to relieve stress by plastic deformation around a defect. It is obtained by
applying Equation (8) and compares the tensile strength of a tablet with a hole in
its center ( T 0 ), which acts as a built - in stress concentrator defect, with the tensile
strength of a similar tablet without a hole ( T ), both at the same relative density:
BFI = ( ). ...
...
0 5 1 0 .
T
T
(8)
It is said that a material showing a moderate to high BFI value ( > 0.5) is prone to
laminate and cap during the process. A low value of BFI is desirable to minimize
lamination and capping during tablet production.
Indentation hardness is another measure which fi nds wide application in the
pharmaceutical industry for the assessment of capping and lamination tendency. The
indentation hardness measurement employs an indentation hardness tester and is
defi ned as the hardness of a material determined by either the size of an indentation
made by an indenting tool under a fi xed load or the load necessary to produce
penetration of the indenter to a predefi ned depth. An instrumented indentation
hardness tester can be employed for that purpose since it has the ability to measure
the intender penetration ( H ) under the applied force ( F ) throughout the testing
cycle and is therefore capable of measuring both plastic and elastic deformation of
the material under test.
Another technique for the assessment of capping and lamination tendency which
has been increasingly employed in the research - and - development phase of tablet
manufacturing is acoustic emission. This technique relies on the fact that an abrupt
change in stress within a material to be compacted generates the release of a transient
strain energy designated as acoustic emission which results in a mechanical wave that
propagates within and on the surface of a structure [5, 32] . Thus, this technique can
discriminate between capped and noncapped tablets based on comparing the measured
level of acoustic emission energy against a decision threshold.
If it is desirable to overcome capping and lamination during the tableting process,
the use of ultrasound - assisted presses could be a reliable solution. However, use
this technique is still very recent since reports in the scientifi c literature extend only
over the last decade [33] .
In general, when capping and lamination are possible problems during tablet
manufacture, an option could be the slower removal of force during decompression.
This could be useful since capping tendency increases with increasing rates of
decompression. However, better improvements could be achieved if the compression
and decompression events are treated separately. By determining the effect of
reducing either the loading or unloading speeds on the individual materials, it could
be possible to increase crushing strength and eliminate or minimize the incidence
of capping and lamination to greater extents. Thus, there is the need for a machine
that is capable of customizing compaction profi les so that each formulation can be
manufactured under an optimum set of conditions.
6.6.10.2 Picking and Sticking
Picking refers to adherence of powder to the punch surface. It is more problematic
when the punch surfaces are engraved with logos or letters such as B, A, or O in
order to produce debossed tablets. Sticking occurs when powder tends to adhere to
the die leading to the development of an additional pressure to surpass friction
between the formed compact and the die wall. As a result, the produced tablets
show a rough surface at their edges. Furthermore, sticking can cause picking or
damage the press punches by blocking the free movement of the lower punches
leading to an increase of compaction pressure.
Various approaches can be used to solve picking and sticking problems during
tablet manufacturing, namely optimization of press tooling, process parameters, and
formulation. Generally, it is important to fi nd the optimal combination of formulation
and process parameters, particularly when market image tablets are to be
produced.
In relation to formulation adjustment, an antisticking agent (talc is commonly
used for this purpose) can be added to the powdered formulation in order to
eliminate picking and sticking during manufacturing. Colloidal silicon dioxide may
be the right choice when picking is evident since this excipient can impart smoothness
to the punch surfaces. However, when adding colloidal silicon dioxide to the
powder formulation, it would be necessary to add an extra lubricant in order to
avoid sticking and facilitate ejection of tablets from the dies. In addition to the
need for an extra excipient in the powder formulation, press tooling may need to
be adjusted to improve tableting. For the production of market image tablets,
logo or letters on the punches should be as big as possible. Additionally, punch
tips may be plated with chrome in order to give a smooth and nonadherent
surface.
When a lubricant such as stearic acid or propylene glycol or any other raw material
of low melting point is present in the powder formulation, the heat generated
during tableting may cause softening of these ingredients, thus leading to sticking.
To overcome this problem, it may be needed to refrigerate the powder load to be
tableted or to equip the press machine with a cooling unit.
PROBLEMS DURING TABLET MANUFACTURING 1159
1160 TABLET COMPRESSION
6.6.10.3 Mottling
Mottling is defi ned as an uneven coloration of tablets or nonuniformity of color
over the tablet surface. One of the possible causes of mottling may be the difference
in color between the active principle and excipients, but sometimes it may be the
result of degradation of the active ingredient which imparts spot zones over the
surface of the tablets.
Nonetheless, when colored compressed tablets are needed for aesthetic reasons,
the foremost cause of mottling is dye migration to the periphery of granules
during the drying process [34] . To overcome this problem, one should consider
changing the solvent used for wet granulation or the binder agent, using a low drying
temperature, or decreasing the particle size of the excipient. Another way to overcome
mottling was demonstrated by Zagrafi and Mattocks [35] and suggests the
inclusion of an adsorbent agent such as wheat or potato starch to the formulation.
The adsorbent agent is said to adsorb the dye, retarding its migration then decreasing
mottling.
6.6.10.4 Weight and Hardness Variation
Weight and hardness variation are common problems experienced when tableting.
Tablet weight is mainly affected by factors such as powder variation, tablet press
condition and tooling, and fl ow of powder on the tablet press.
Inconsistent powder or granulate density and particle size distribution are
common sources of weight variation during tablet compression. Problems related
to the density of the powder or granulate are often associated with overfi lling of
the die and recirculation of the product on the tablet press. A variation of particle
size distribution of the powder or granulate can be the result of segregation due to
transfer or static electricity. It might also vary because the product cannot withstand
the handling and mechanical stress it undergoes before reaching the tablet press.
Weight variation can arise as a result of a poorly prepared or operated tablet
press. To solve this problem, one should inspect the press performance. Attention
must be taken when dealing with a new die table on a load tablet press. In such a
case, operation of the tablet press must regard the up - and - down motion of the
punches within 76.2 . m of the setting without neglecting the conditions of the pressure
rolls and cams.
Inspection of the critical dimensions of tablet press tools is recommended. At
least three dimensions of the upper and lower punches should be inspected: the
working length, the cup depth, and the overall length. The working length is the key
factor affecting tablet weight. Therefore, the length of each punch must be correct
and identical. The cup depth and the overall length are not critical with regard to
controlling tablet weight. Therefore comprehensive inspection and evaluation of the
press tooling are essential to minimize deviation of tablet thickness, weight, and
hardness.
During the course of a compression operation it is also important to not neglect
the level of the product in the hopper. Head pressure is a critical factor related to
the amount of product in the hopper. The more product present in the hopper, the
greater the head pressure, and vice versa. Therefore, when the head pressure varies,
so does the weight of the tablets. So, in order to maintain a constant head pressure,
thus reducing a potential variation of weight, compression should be conducted
within a narrow range of the powder or granulate product in the hopper.
The fi ll cam is another factor that can have a profound effect on tablet weight.
The choice of an adequate fi ll cam regarding some characteristics of the powder or
granulate product allows the die cavity to be properly overfi lled. Usually, in order
to maintain consistent tablet weight during compression, it is recommended to
overfi ll the cam by 10 – 30% of its volume. Basically, any tablet press part that is
ultimately related to the powder product fl ow can have a mild or profound impact
on weight control. It is important to remember that the scraper blade tends to
become worn by die table rotation and powder product abrasion. Therefore, periodic
inspection of its condition and replacement are recommended. Nevertheless,
the scraper blade proper condition is important but also its adjustment since if it is
not set up correctly, powder product may accumulate on the die table, leading to
problems with weight control.
Tablet hardness variation is intimately related to weight variation and, accordingly,
to the infl uence of compression variables such as dwell time, tablet thickness,
and working length of the punches. Thus, to solve a hardness variation, consistency
of the tablet weight must be checked fi rst. If the predefi ned weight is achieved but
hardness is out of limits, then precompression and compression forces should be
adjusted while keeping tablet thickness within target limits. Although dwell time
might be a source of hardness variation, adjustment of this parameter may be detrimental
to the whole process since the compression rate is slowed. Occasionally,
when the tablet weight target is kept within limits but hardness varies, the problem
may be due to the formulation. As mentioned previously, the correct use of punches
and dies is of paramount importance and periodic inspection is mandatory in order
to ensure the compression process has not been compromised. So, when it becomes
hard to achieve tablet hardness, it is recommended to fi rst verify tablet weight and
thickness consistency and then try to adjust the precompression. The choice to
increase the tablet weight even if it is within limits or to reduce the tablet press
speed is not convenient and should be used only when there are no more options.
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I. Characterization of mechanical behavior of powder and powder/tooling friction ,
J. Pharm. Sci. , 93 ( 8 ), 2022 – 2039 .
21. Picker , K. M. ( 2004 ), The 3D model: Explaining densifi cation and deformation mechanisms
by using 3D parameter plots , Drug. Dev. Ind. Pharm. , 30 ( 4 ), 413 – 425 .
22. Ruegger , C. E. , and Celik , M. ( 2000 ), The effect of compression and decompression speed
on the mechanical strength of compacts , Pharm. Dev. Technol. , 5 ( 4 ), 485 – 494 .
23. Barra , J. , Falson - Rieg , F. , and Doelker , E. ( 1999 ), Infl uence of the organization of binary
mixes on their compactibility , Pharm. Res. , 16 ( 9 ), 1449 – 1455 .
24. Press Tooling Manual ( 2003 ), Thomas Engineering Inc. Hoffman Estates, IL , USA . pp.
1 – 38 .
25. Tableting Specifi cation Manual , 7th ed., American Pharmacists Association, p. 130.
26. International Organization for Standardization (ISO) , 18084: 2005 , 1st ed, ISO , Geneva ,
pp. 1 – 13 .
27. Train , D. ( 1956 ), An investigation into the compaction of powders , J. Pharm. Pharmacol. ,
8 ( 10 ), 745 – 761 .
28. Burlinson , H. ( 1968 ), Tablets and Tabletting , Heinemann , London .
29. Long , W. M. ( 1960 ), Radial pressures in powder compaction, Powder Metall. , 6 , 73 – 86 .
30. Ritter , A. , and Sucker , H. B. ( 1980 ), Studies of variables that effect tablet capping , Pharm.
Tech. , ( 3 ), 57 – 65 , 128.
31. Hiestand , E. N. , Bane , J. M. , Jr ., and Strzelinski , E. P. ( 1971 ), Impact test for hardness of
compressed powder compacts , J. Pharm. Sci. , 60 ( 5 ), 758 – 763 .
32. Joe Au , Y. H. , Eissa , S. , and Jones , B. E. ( 2004 ), Receiver operating characteristic analysis
for the selection of threshold values for detection of capping in powder compression ,
Ultrasonics , 42 ( 1 – 9 ), 149 – 153 .
33. Rodriguez , L. , Cini , M. , Cavallari , N. , Passerini , N. , Saettone , M. F. , Monti , D. , and Caputo ,
O. ( 1995 ), Ultrasound - assisted compaction of pharmaceutical materials , Farm Vestn. , ( 46 ),
241 – 242 .
34. Armstrong , N. A. , and Palfrey , L. P. ( 1989 ), The effect of machine speed on the consolidation
of four directly compressible tablet diluents , J. Pharm. Pharmacol. , 41 ( 3 ), 149 – 151 .
35. Zografi , G. , and Mattocks , A. M. ( 1963 ), Adsorption of certifi ed dyes by starch , J. Pharm.
Sci. , 52 (Nov.), 1103 – 1105 .
REFERENCES 1163
1165
6.7
EFFECTS OF GRINDING IN
PHARMACEUTICAL TABLET
PRODUCTION
Gavin Andrews , David Jones , Hui Zhai , Osama Abu Diak , and
Gavin Walker
Queen ’ s University Belfast, Belfast, Northern Ireland
Contents
6.7.1 Introduction
6.7.2 Milling Equipment
6.7.2.1 Ball Mill
6.7.2.2 Fluid Energy Mill
6.7.2.3 Hammer Mill
6.7.2.4 Cutting Mill
6.7.3 Powder Characterization Techniques
6.7.3.1 Powder Sampling
6.7.3.2 Particle Density and Voidage
6.7.3.3 Particle Surface Area
6.7.3.4 Particle Shape
6.7.4 Effect of Particle Size Reduction on Tableting Processes
6.7.4.1 Wet Granulation Processes
6.7.4.2 Mixing Processes
6.7.4.3 Flowability of Pharmaceutical Powders
6.7.4.4 Compression Processes
References
6.7.1 INTRODUCTION
The importance of size reduction in relation to pharmaceutical active agents and
excipients is well known, and the aim of this chapter is to identify methods for particle
size reduction, discuss how particle size and shape are characterized, and
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1166 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
recognize the importance of controlling particle characteristics to ensure the success
of pharmaceutical powder processing and the manufacture of elegant pharmaceutical
products. An initial overview of the implications of size reduction within
pharmaceutics and the importance of comminution in relation to variability of
active pharmaceutical ingredient (API) surface area, effi cacy, and ultimately dosing
regimen required to maintain optimum therapeutic effects will be addressed. This
will encompass examples from a diverse range of dosage forms, including oral,
parenteral, and topical systems. The effects of particle size on the essential characteristics
of powders intended for compression (tablets, capsules) such as fl uidity and
compressibility will be addressed. The need for uniformity of size and the effects
of particle size distribution on the homogeneity of mixing/blending and in essence
on the uniformity of APIs within the fi nal manufactured dosage form will be
highlighted.
6.7.2 MILLING EQUIPMENT
There are many factors that must be taken into consideration in choosing milling
equipment. Some of these factors are related to required product specifi cations such
as particle size distribution, but additionally, physical and chemical properties of
the material such as particle shape and moisture content must also be taken into
consideration. Furthermore, other factors that are related to production requirements
(mill capacity and the required production rate) must be carefully balanced
to ensure the correct choice of milling equipment.
6.7.2.1 Ball Mill
A ball mill consists of a hollow cylinder mounted such that it can be rotated on its
horizontal longitudinal axis (Figure 1 ). The length of the ball mill is slightly greater
than its diameter. A ball mill reduces particle size by subjecting particles to impact
and attrition forces generated by moving steel balls or pebbles (grinding medium)
that typically occupy 30 – 50% of the total volume of the mill. It is common for a
ball mill to contain balls of different diameters that aid size reduction. Generally,
larger diameter balls have a higher tendency to act upon coarse feed materials
FIGURE 1 Ball mill in operation showing correct cascade action.
(a) (b) (c)
MILLING EQUIPMENT 1167
whereas smaller diameter balls facilitate the formation of fi ne product by reducing
void spaces between the balls.
The most important factors governing the performance of the mill and the
achievement of the desired particle size are as follows:
1. Amount of material required for subsequent testing (sample volume)
2. Speed of rotation of ball mill
A high volume of powder feed produces a cushioning effect whereas small
sample volumes cause a loss of effi ciency and abrasive wear of the mill parts. The
amount of material to be milled in a ball mill may be expressed as a material - to - void
ratio (ratio of the volume of material to that of the void in the ball charge). As the
amount of material is increased, the effi ciency of a ball mill is increased until the
void space in the bulk volume of ball charge is fi lled; then, the effi ciency of milling
is decreased by further addition of material.
Rotational speed is the most signifi cant factor controlling the particle size speci-
fi cation. The optimum speed of rotation is dependent on mill diameter. At low
angular velocities the balls move with the drum until the force due to gravity
exceeds the frictional force of the bed on the drum, and the balls then slide back
to the base of the drum. This sequence is repeated, producing very little relative
movement of balls so that size reduction is minimal. At high angular velocities the
balls are thrown out onto the mill wall by centrifugal force and no size reduction
occurs. At about two - thirds of the critical angular velocity where centrifuging occurs,
a cascading action is produced. Balls are lifted on the rising side of the drum until
their dynamic angle of repose is exceeded. At this point they fall or roll back to the
base of the drum in a cascade across the diameter of the mill. By this means, the
maximum size reduction occurs by impact of the particles with the balls and by
attrition.
The critical speed of a ball mill is the speed at which the balls just begin to centrifuge
with the mill. Thus, at the critical speed, the centrifugal force is equal to the
weight of the ball. At and above the critical speed, no signifi cant size reduction
occurs. The critical speed n c is given by the equation
n
D c = 76 6 .
where D is the diameter of the mill.
A larger mill reaches its critical speed at a slower revolution rate than a smaller
mill. Ball mills are operated at from 60 to 85% of the critical speed. Over this range,
the output increases with the speed; however, the lower speeds are for fi ner grinding.
An empiric rule for the optimum speed of a ball mill is
n D = . 57 40log
where n is the speed in revolutions per minute and D is the inside diameter of the
mill in feet.
1168 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
In practice, the calculated speed should be used initially in the process and modi-
fi ed as required.
The use of a ball mill is advantageous in that it may be used for both wet and
dry milling and additionally can be successfully employed in batch and continuous
operation. Also, the installation, operation, and labor costs involved in ball milling
are extremely low in comparison to other techniques, which makes this technique
economically favorable.
6.7.2.2 Fluid Energy Mill
Fluid energy milling acts by particle impaction and attrition that are generated by
a fl uid, usually air (Figure 2 ). Fluid energy mills can reduce the particle size to
approximately 1 – 20 . m. A fl uid energy mill consists of a hollow toroid that has a
diameter of 20 – 200 . m, depending on the height of the loop, which may be up to
2 m. Fluid is injected as a high - pressure jet through nozzles at the bottom of the
loop with the high - velocity air, giving rise to zones of turbulence into which solid
particles are fed. The high kinetic energy of the air causes the particles to impact
with other particles with suffi cient momentum for fracture to occur. Turbulence
ensures that the high levels of particle – particle collision produce substantial size
reduction by impact and attrition.
The design of fl uid energy mills provides an internal classifi cation system according
to their particle size in which the fi ner and lighter particles are discharged and
the heavier, oversized particles, under the effect of centrifugal force, are retained
until reduced to a signifi cantly smaller size.
FIGURE 2 Fluid energy mill.
Centrifuging action
throws coarser
particles outward
Classifier removes
fine particles
and fluid
Solids inlet
Fluid inlet jets
Zone of
turbulence
MILLING EQUIPMENT 1169
6.7.2.3 Hammer Mill
The main mechanism of size reduction produced by a hammer mill is by impaction
that is generated from a series of four or more hammers hinged on a central shaft
and enclosed within a rigid metal case (Figure 3 ). During milling the hammers swing
out radially from the rotating central shaft. The angular velocity of the hammers
produce strain rates up to 80 s . 1 , which are so high that most particles undergo brittle
fracture. As size reduction continues, the inertia of particles hitting the hammers
reduces markedly and subsequent fracture is less probable, so that hammer mills
tend to produce powders with narrow particle size distributions. Particle retention
within the mill is achieved using a screen, which allows only suffi ciently milled particles
(defi ned particle size) to pass through. Particles passing through a given mesh
can be much fi ner than the mesh apertures, as particles are carried around the mill
by the hammers and approach the mesh tangentially. For this reason, square, rectangular,
or herringbone slots are often used. According to the purpose of the operation,
the hammers may be square faced or tapered to a cutting edge or have a
stepped form.
The particle size achieved may be controlled variation in the speed of the hammers
and additionally by careful selection of the size and type of screen. During the
operation of a hammer mill the speed of rotation is critical such that below a critical
impact speed the rotor turns so slowly that a blending action rather than milling is
obtained. Such operating conditions result in signifi cant rises in temperature. Moreover,
at very high speeds, there is the probability of insuffi cient time between successive
passes of the hammers for a signifi cant mass of material to fall from the
grinding zone.
The hammer mill is particularly useful in achieving particles in the approximate
size range of 20 – 40 . m and additionally in producing a particle size distribution that
is extremely narrow. The equipment offers ease of use and high levels of fl exibilty
(speed and screen may be rapidly changed allowing rapid variation in achievable
particle size), is easy to clean, and can be operated as a closed system, thus avoiding
operator exposure to potent dusts and potential explosion hazards.
FIGURE 3 Hammer mill.
Hammers
Feed
Screen
Product
1170 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
6.7.2.4 Cutting Mill
Particle size reduction using a cutting mill involves successive cutting or shearing a
sample using a series of knives attached to a horizontal rotor (Figure 4 ). This rotary
motion pushes the sample against a series of stationary knives that are attached to
the mill casing. Size reduction occurs by fracture of particles between the two sets
of knives, which have a clearance of approximately a few millimetres. As with a
hammer mill a screen is fi tted at the base of the mill casing and acts to retain material
until a suffi cient degree of size reduction has occurred.
6.7.3 POWDER CHARACTERIZATION TECHNIQUES
6.7.3.1 Powder Sampling
Powdered materials are used in a wide range of industries, no more so than in the
pharmaceutical industry wherein powders are used for the manufacture of a wide
range of dosage forms, the two most common being tablets and hard gelatin capsules.
Orally administered solid dosage forms are the preferred and most patient
convenient, primarily because of the ease of administration and the convenience of
handling. Pharmaceutically, orally administered solid dosage forms are generally
more favorable because of increased stability in comparison to their liquid counterparts
(suspensions, syrups) and the increased control they offer in manipulating
drug dissolution in vivo to suit end - use requirements. Solid dosage forms administered
via the oral route are an intricate blend of pharmaceutical excipients (diluents,
FIGURE 4 Cutter mill.
Screen Product
Feed
Stationary knives
Rotating knives
binders, disintegrants, glidants, lubricants, and fl avors) and APIs. In order to successfully
manufacture acceptable pharmaceutical products, these materials must
be adequately mixed and/or granulated to ensure that the resultant agglomerates
possess the required fl uidity and compressibility and, in addition, avoid demixing
during postgranulation processes. Moreover, the fi nal characteristics of tablets or
capsules such as drug dissolution rate, disintegration time, porosity, friability and
hardness are signifi cantly infl uenced by the properties of the powder blends used
during their manufacture.
During product manufacture large volumes of powder blends are fed through
production equipment/processes, and it is essential to be able to accurately
determine, defi ne, and control powder properties to ensure reproducible manufacture
and product performance. Therefore the characterization of the physicochemical
properties of powder blends is extremely important. It is well accepted
that there are inherent diffi culties in characterizing the entire mass of a bulk
powder blend or process stream, so it is essential to remove and analyze discrete
samples.
Sampling is a useful technique that allows an appropriate aliquot to be withdrawn
from the bulk so as to collect a manageable amount of powder which is representative
of the batch [3] , in other words, every particle should have an equal chance of
being selected [4] . However, there are many circumstances that may result in the
selection of nonrepresentative samples and hence the defi nition of powder characteristics
that are not a true estimation of the entire bulk powder. Typically, powder
masses with an extremely wide particle size distribution or diverse physical properties
are highly likely to be heterogeneous, which may result in high levels of variability
and samples that do not represent bulk mass. Moreover, powder characteristics
may change because of the attrition and segregation during transfer that can make
sampling extremely diffi cult.
It is well accepted that two types of sampling errors are possible when removing
small masses of powder from bulk [5] .
1. Segregation errors, which are due to segregation within the bulk and can be
minimized by suitable mixing and the use of a large number of incremental
samples to form a larger test sample.
2. Statistical errors, which arise because the quantitative distribution in
samples of a given magnitude is not constant but is subject to random fl uctuations.
Consequently, it is an example of a sampling error that cannot
be prevented but can be estimated and indeed reduced by increasing the
sample size.
Therefore, sampling procedures are of the greatest importance in order to reduce
the effect of nonuniform size segregation and nonrandom homogeneity of a system
to achieve statistically meaningful sampling results. Careful attention and faithful
observance must be demonstrated and it is extremely important that sampling
occurs when the powders are in motion [6] and samples are withdrawn from the
whole stream for equal periods of time, rather than part of the stream for all of the
time [3] .
POWDER CHARACTERIZATION TECHNIQUES 1171
1172 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
TABLE 1 Stationary Bulk Sampling
Sampling Devices Procedure of Sampling
Application and
Characteristics
Low volume powder
sampler (Figure
5 a )
In operation the sampler is
inserted into the product to be
sampled. At a specifi c sampling
depth the operator pushes
down on the T bar, which
opens the sampling chamber.
When released the spring -
loaded T bar will close the
sampling chamber.
Used for small quantity of
sample powders. The
sampler has a sampling
chamber volume
approximately equal to
2 mL.
Pneumatic lance
sampler (Figure
5 c )
A gentle fl ow of air out of the
nozzle allows the probe to move
through the powder bed. At the
site, the air is slowly reversed to
draw up a sample, which is
collected against a porous plate
at the end of the probe [7] .
Minimizes powder
disturbance and therefore
is better than a sample
thief, but bias still cannot
be avoided [8] .
Scoop sampler A single swipe of the scoop
completely across the powder
bulk collects the sample. Each
collection should use opposite
directions.
Suitable only for materials
that are homogeneous
within the limits set by
the quantity of material
taken by the scoop. It
may be used for non -
free - fl owing or damp
materials where
instrumental methods
are inappropriate [9] .
Thief/spear probe
sampler
(Figure 5 b )
One or more cavities are stamped
in a hollow cylinder enclosed by
an outer rotating sleeve. The
thief is inserted into sample
with the cavities closed, once
opened the sample fi lls the hole.
The cavities are closed and the
thief is withdrawn. It must be
ensured that samples are
withdrawn from different
locations
Thief samplers belong to
two main classes, side
sampler (has one or more
cavities along the probe)
and end sampler (has a
single cavity at the end of
the probe), which are the
most common used for
stored non - fl owing
material [10] .
There are a number of sampling techniques for particle sampling, which can be
classifi ed in many different ways. Here, particle sampling techniques are divided
into three parts: stationary bulk sampling (Table 1 and Figure 5 ), fl owing stream
sampling (Table 2 and Figure 6 ), and subsampling (Table 3 and Figure 7 ). The
sampling devices, procedures and application overview of the common used techniques
in corresponding fi elds are shown as follow.
FIGURE 5 Stationary bulk sampling: ( a ) low - volume powder sampler; ( b ) thief/spear
probe sampler; ( c ) pneumatic lance sampler [7] .
(a)
(b)
(c)
Press
Side sampler
End sampler
Airflow
Porous plate Fluidizing jets
6.7.3.3 Particle Density and Voidage
Particle density may be defi ned as the total mass of the particle divided by its total
volume; however, depending upon the different defi nitions of the total volume (or
the different ways to measure the particle volume), there are various defi nitions of
particle density in existence (see Table 4 ).
In order to get clear understanding of the subtle differences between the defi nitions
of various particle density types, an illustration can be formed as shown in
Figure 8 .
Particle Density Methods Density is defi ned as the ratio of mass to volume, so
the density determination can be separated into two steps: measurement of mass
and measurement of volume. Determining the mass of an object is rather straightforward;
however, it is much more diffi cult to directly determine the volume of a
solid. The volume of a solid object with a regular geometric shape may be calculated
POWDER CHARACTERIZATION TECHNIQUES 1173
1174 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
TABLE 2 Flowing Stream Sampling
Sampling
Devices Procedure of Sampling Application and Characteristics
Auger sampler
(Figure 6 a )
[Line sampler
for stream]
A pipe with a slot is placed
inside the process stream,
permitting easy capture of
powder through the process
stream cross section when
rotated. Samples are
subsequently then delivered
into a separate container by
gravitational forces. [6]
While this is often used for
stream sampling, it is diffi cult
to collect a representative
sample when stream is
heterogeneous [10] .
Constant - volume
sampler
(Figure 6 b )
[Point sampler
for stream]
Sampling occurs when the stream
falls down through a pipe and a
constant - volume container is
inserted or withdrawn from the
stream system.
Designed to extract a constant
volume of homogeneous
granular material for
subsequent chemical analyses
and is not suitable for
withdrawing samples for
physical analyses [11] .
Diverter sampler
(Figure 6 c )
[Cross -
sectional
sampler for
stream]
The whole stream is diverted by
opening a sliding cover or
pivoting an external fl ap in the
bottom of a gravity - fl ow chutes
or pipes or screw conveyors
[12] . The samples could be
removed to a low - angle laser
light-scattering instrument then
returned to the process stream
[6] .
The process could be automated
and highly suitable method
for online particle size
measurement. [7]
Full stream
sampler
(Figure 6 d )
[Cross -
sectional
sampler for
stream]
Samples are withdrawn from
conveyors, carried out only on
the return stroke.
Extremely useful for dusty
materials provided the trough
extends the whole length of
the stream and does not
overfi ll [6] .
mathematically; however, in most conditions, the shape of a particle is often irregular,
especially in powder technology, which makes it extremely diffi cult to measure
geometrically. Therefore, various methods have been developed to determine the
volume of particles and powders. The two most in use in both laboratory and industrial
settings are liquid and gas displacement methods. The different values of particle
density can also be expressed in a dimensionless form, as “ relative density ” (or
specifi c gravity), which is the ratio of the density of the particle to the density of
water.
The discussion that follows will give an overview of the common methods used
in particle density measurement.
FIGURE 6 Flowing stream sampling: ( a ) auger sampler [6] ; ( b ) constant - volume sampler
[6] ; ( c ) diverter sampler; ( d ) full - steam sampler.
(a) (b)
(c) (d)
Powder flow
Sample
Process stream Process stream
Discharge position Sampling position
To analyzer
Stream
Step 1
Normal position
Step 2
Sampling stroke
Step 3
Discharging sample
Samples
Stream
Measurement of Particle Density
1. Liquid Pycnometry Method There are several British standards that deal
with liquid pycnometry applied to specifi c materials [18 – 23] . A pycnometer bottle
is weighted empty (M1), and then full of liquid (M2). Following these two initial
measurements, two subsequent measurements are made: a sample of powder
approximately one - third of maximum container volume (M3) and the bottle fi lled
to capacity containing the sample and water (M4). Great care is required in the fi nal
step to ensure that the liquid is fully wetted and all the air removed. Variations in
recorded weight also arise depending on how much liquid escapes when the ground
glass stopper is inserted in the liquid - fi lled container. It is extremely important that
the liquid used in this procedure does not solubilize or react with the solid particles.
Moreover, the solid particles must not absorb the selected fl uid.
2. Gas Pycnometry Method Principally this method is similar to liquid pycnometry
in that volume determination is achieved by detecting the pressure or volume
change associated with the displacement of a gas (rather than liquid) by a solid
object. Given that this method is largely dependent upon the diffusivity of the gas,
helium is often used since it has a low molecular weight and a small atomic radius,
allowing high diffusivity into small pores. Sample volumes are often displayed on a
POWDER CHARACTERIZATION TECHNIQUES 1175
1176 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
TABLE 3 Subsampling
Sampling Devices Procedure of Sampling Application and Characteristics
Coning and
quartering
(Figure 7 a )
A cross - shaped cutter is used
to separate the sample heap
(which is fi rst fl attened at
the top) into four equal
parts. The segments are
drawn apart and two
opposite quadrants are
combined together. This
procedure is repeated at
least 4 times until a small
enough sample has been
generated.
The fi rst choice for non - free -
fl owing powders and
nonfl owing powders. Prone to
operator bias as fi ne particles
remain in the center of the
cone and should never be used
with free - fl owing powders [13] .
Oscillating hopper
sample divider
(Figure 7 c )
Hopper (paddle) oscillates and
powder falls into two
collectors placed under the
hopper (paddle).
Used for small quantity of
samples. Sample size can be
controlled by monitoring time
over each collector [7] .
Revolving sample
splitter (Figure
7 f )
The revolving feeder
distributes the sample
material equally (in time)
over a number of radial
chutes, assuming constant
rotational speed [14] .
Very easy to perform and several
versions are available that are
suitable for free - fl owing
powders, dusty powders, and
cohesive powders. Handling
quantities can vary from 40 L to
a few grams.
Riffl e/chute splitter
(Figure 7 e )
The sample is introduced to a
rectangular area, divided by
parallel chutes leading to
two separate receptacles
[14] .
Well - accepted method for sample
reduction that is highly suitable
for free - fl owing powders. Used
to produce samples with a
minimum volume of 5 mL.
Spinning riffl er
(Figure 7 d )
a steady stream of powder is
run into a rotating basket of
containers [8] .
Useful in subsampling large
samples [15] . Suitable for free -
fl owing materials [13] .
Table sampler
(Figure 7 b )
In a sampling table, powder
fl ows down from the top of
an inclined plane, holes and
prisms splitting the powder.
The powder that reaches the
bottom of the plane is the
sample.
Used for sample reduction with
the advantages of low price and
lack of moving parts.
digital counter on the testing equipment [24] ; however, such volumes are easily
calculated using the pressure change and the ideal gas law, PV = nRT . The true
density of the particle can be measured using this method if the particles have no
closed pores, while the apparent particle density can be measured if there are any
closed pores. Additionally, if open pores are fi lled with wax, envelope volumes may
FIGURE 7 Subsampling: ( a ) coning and quartering; ( b ) table sampler [6] ; ( c ) oscillating
hopper (paddle) sample divider [6] ; ( d ) spinning riffl er (BSI); ( e ) riffl e/chute splitter (BSI);
( f ) revolving sample splitter [14] .
Sample
(b) (a)
(c) (d)
(e) (f)
Repeat
Discard
AAA
Container 1
Container 1
Container 2
POWDER CHARACTERIZATION TECHNIQUES 1177
1178 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
TABLE 4 Defi nitions of Density Terms
Density
Types Density Defi nitions
Volumes in Defi nition
Solid
Material
Volume
Closed -
Pore
Volume
Open -
Pore
Volume
Interparticle
Void
Volume
Absolute
powder
density
Mass of powder per unit of
absolute volume, which is
defi ned as the solid matter
after exclusion of all the
spaces (pores and voids)
(BSI)
Apparent
particle
density
Mass of particles divided by its
apparent particle volume,
which is defi ned as the total
volume of the particle,
excluding open pores but
including closed pores (BSI)
Apparent
powder
density
Mass of powder divided by its
apparent powder volume,
which is defi ned as the total
volume of solid matter,
including open pores and
closed pores and interstices
(BSI)
Bulk
density
Mass of the particles divided
by the volume they occupy,
which includes the space
between the particles
(ASTM)
Effective
particle
density
Mass of a particle divided by
its volume, including open
pores and closed pores (BSI)
Envelope
density
Ratio of the mass of a particle
to the sum of the volumes of
the solid in each piece and
voids within each piece,
which is, within close - fi tting
imaginary envelopes,
completely surrounding each
piece (ASTM)
Skeletal
density
Ratio of the mass of discrete
pieces of solid material to
the sum of the volumes of
the solid material in the
pieces and closed pores
within the pieces (ASTM)
Tap
density
Apparent powder density
obtained under stated
conditions of tapping (BSI)
True
density
Mass of a particle divided by
its volume, excluding open
pores and closed pores (BSI)
Note: BSI = British Standards Institute [16] , ASTM = American Society for Testing and Material [17] .
, included; , excluded.
Eliminating
interparticle
void
volume
Bulk volume Envelope/effective
volume
Apparent/skeletal
volume
True/absolute
volume
Porous particles
in container
Bulk density Envelope/effective
density
Apparent/skeletal
density
True/absolute
density
Eliminating
open pores,
cracks
volume
Eliminating
closed pores
volume
FIGURE 8 Various density types. The density value increases from bulk density to true
density while the volume value decreases from bulk volume to true volume.
be determined and the difference between envelope and apparent volume can yield
the open - pore volume, which is sometimes used as a measure of porosity.
3. Hydrostatic Weighing Method The volume of a solid sample is determined
by comparing the mass of the sample in air with the mass of sample immersed in a
liquid with a known density. The volume of sample may be calculated using the
difference between the two measured mass values divided by the density of the
liquid. This method can be used to determine the bulk or apparent volume. It is
extremely important that the suspending liquid does not interact with the powder
under investigation.
4. Float – Sink or Suspension Method This method involves placing a solid
sample into a liquid with known and adjustable density. The density of liquid is
incrementally adjusted until the sample begins to sink – fl oat (ASTM C729 - 75 [25] ),
or is suspended at neutral density in the liquid (ASTM C693 - 93 [26] ). At the point
of equilibrium the density of the sample is equal to the density of the liquid.
5. Bed Pressure Drop Method This technique is based on making measurements
of bed pressure drop as a function of gas velocity at two voidages, when gas
is passed through the bed of powder in the laminar fl ow regime [24] . During measurement
pressure changes for at least four velocities must be measured. The effective
particle density . p can be calculated using the equation
s
s
1
2
3
= .
b1
b2
p b2
p b1
where s is the gradient of pressure drop with gas velocity, . b is the bulk density, . p
is the particle effective density.
6. Sand Displacement Method The sand displacement method is another useful
way of measuring the envelope density of a particle using fi ne sand as the displacement
media. Sand is mixed with a known amount of particles, then the density of
the sample particles can be determined from the difference of the bulk density
between sand alone and that with samples.
7. Mercury Porosimetry Method Mercury is a nonwetting liquid that must be
forced to enter a pore by application of external pressure. Consequently it is an
extremely useful and convenient liquid for measuring the density of powders and/or
particles. This method can measure the apparent and true density of one sample by
POWDER CHARACTERIZATION TECHNIQUES 1179
1180 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
applying different pressures. At atmospheric pressure, mercury will resist entering
pores smaller than about 6 . m in diameter, but at pressures of approximately 60,000
psi (414 MPa) mercury will be forced to enter pores with diameters as small as
0.003 . m [27] .
Measurement of Bulk Density Bulk density is very important in determining the
size of containers used for handling, shipping, and defi ning storage conditions for
pharmaceutical powders and granules. It is a property that is also pertinent in defi ning
the size of hoppers and receivers for milling equipment and for sizing blending
equipment in the scale - up to pilot and to commercial production [28] . The concept
of bulk density is the mass of particles divided by the bulk volume, which includes
not only the envelope volume of particles but also the spaces between particles, so
it should not be confused with particle density [24] .
The most convenient method to measure bulk density is to fi ll the particles into
a known volume container (usually cylindrical), level the surface, and weigh the
particles in the container. The bulk density is calculated by the mass of the particles
divided by the volume that can be read from the scale of the measuring cylinder.
In order to minimize experimental errors, the container should be ideally at least
1 L in volume, and the ratio of length and diameter should be about 2 : 1. Also it is
recommended to leave the sample for approximately 10 min to achieve an equilibrium
volume (density) value before making readings.
Given that the bulk volume associated with the particle mass is a mixture of air
and solid material, the bulk density value is highly dependent on sample history
prior to measurement. Calculation of the tapped density can then be achieved by
tapping the bulk powder a specifi ed number of times (to overcome cohesive forces
and remove entrapped air) to determine the tapped volume of the powder. The
tapped and bulk density values can be used to defi ne the fl owability and compressibility
of a powder using Carr ’ s index and the Hausner ratio.
6.7.3.4 Particle Surface Area
Surface area is one of the most important characteristics in particle technology.
Particles with a different surface area will express different physical properties
that will subsequently affect many applications and ultimately fi nal dosage form
properties.
Similar to particle density, there are various defi nitions relating to particle surface
area [16] :
1. Adsorption surface area : the surface area calculated from an adsorption
method.
2. BET surface area : the surface area calculated from the Brunauer, Emmett,
and Teller theory of multilayer adsorption of a gas on a solid surface.
3. Calculated surface area : the surface area of a powder calculated from its particle
size distribution.
4. Effective permeability mass - specifi c surface : the effective volume - specifi c
surface divided by the effective solid density, determined by permeametry.
5. Effective permeability volume - specifi c surface : the effective surface area
divided by the effective solid volume, determined by permeametry.
6. Permeability surface area : the surface area of a powder calculated from the
permeability of a powder bed under stated conditions.
7. Specifi c surface area ( S w ): the surface area of a unit mass of material determined
under stated conditions, where S w is usually expressed in centimeters
squared per gram or meters squared per gram and can be used for quality
control purposes [28] .
Particle Surface Area Determination Methods From the standard defi nitions of
particle surface area, it can be seen that various determination methods are used
for surface area measurement, such as adsorption (including Langmuir ’ s equation
for monolayer adsorption and the BET equation for multilayer adsorption), particle
size distribution, and permeability methods. The different methods are rarely in
agreement because the value obtained depends upon the procedures used and also
on the assumptions made in the theory relating the surface area to the phenomena
measured. The most common methods used for measuring particle surface area are
described below.
1. Gas Adsorption Method Gas adsorption methods measure the surface area
of particles/powders through measurement of the amount of gas adsorbed onto the
sample surface. The methods can measure both external and internal surfaces
(including open pores in the particles) and can yield physically meaningful average
particle sizes with nonporous materials [24] . The amount of gas adsorbed depends
upon the nature of the solid (adsorbent) and the pressure at which adsorption takes
place. The amount of gas (adsorbate) adsorbed can be found by determining the
increase in weight of the solid (gravimetric method) or the amount of gas removed
from the system due to adsorption by application of the gas laws (volumetric
method [6] ). The adsorption used in this method is physical adsorption, which is a
relatively weak interaction between samples and gases and therefore can be removed
by evacuation.
In this method, a graph of the number of moles of gas adsorbed per gram of
solid, at constant temperature, against the equilibrium gas pressure is called an
adsorption isotherm. A point must be chosen on this isotherm corresponding to the
completion of the adsorbed monolayer in order to calculate S w [29] .
2. Permeametry Method This method is based on the fact that the fl ow rate of
a fl uid through a bed of particles depends on the pore space, the pressure drop
across the bed, the fl uid viscosity, dimensional factors such as the area of the bed,
and specifi c surface area ( S w ). The determination of permeability can be made either
under continuous steady - state fl ow (constant fl ow rate) or under variable - fl ow
(constant - volume) conditions.
All of the permeability methods are based on the Kozeny – Carman equation,
which is used to calculate a surface area of a packed powder bed from its permeability.
The Kozeny – Carman equation is expressed as [16]
S
A p
K Lq k =
.
.. .
.. .
.
. .
3
2 1
.
( )
POWDER CHARACTERIZATION TECHNIQUES 1181
1182 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
where S k = effective permeability volume - specifi c surface of powder assuming only
viscous fl ow occurs in determination (Kozeny – Carman term)
A = cross - sectional area of bed of powder perpendicular to direction of fl ow
of air
. = porosity of bed of powder
. p = pressure difference across bed of powder
K = Kozeny constant
L = linear dimension of bed of powder parallel to direction of fl ow of air
(commonly known as height of powder bed)
. = viscosity of air at its temperature at time of determination
q = rate of fl ow of incompressible fl uid through bed of powder
The specifi c surface area calculated here only involves the walls of the pores of
the bed and excludes the pores within the particles. Therefore, the surface area
measured in this method can be much smaller than the total surface area measured
by gas adsorption methods [24] .
3. Particle Size Distribution Method The surface area of particles can be determined
using particle size and particle shape values. The “ equivalent spherical diameter
” is used in this technique and many attempts to measure the surface area using
this method have led to values that are signifi cantly less than the true value (large
deviations arising from inability to defi ne particle shape due to surface irregularities
and porosity). Surface area values calculated from particle size distribution methods
will in effect establish the lower limit of surface area due to the implicit assumptions
of sphericity or other regular geometric shapes and by ignoring the highly irregular
nature of real surfaces [30] .
Besides the three methods introduced above, there are many other methods of
surface area determination: Any surface - dependent phenomenon can be used for
such measurement [24] . Some available methods (mercury porosimetry, adsorption
from solution, adsorption of dyes, chemisorption, density methods, and secondary
ion mass spectroscopy) are explained in more detail elsewhere [6, 30, 31, 32] .
6.7.3.5 Particle Shape
Particle behavior is a function of particle size, density, surface area, and shape.
These interact in a complex manner to give the total particle behavior pattern [28] .
The shape of a particle is probably the most diffi cult characteristic to be determined
because there is such diversity in relation to particle shape. However, particle shape
is a fundamental factor in powder characterization that will infl uence important
properties such as bulk density, permeability, fl owability, coatablility, particle
packing arrangements, attrition, and cohesion [33 – 36] . Consequently it is pertinent
to the successful manipulation of pharmaceutical powders that an accurate defi nition
of particle shape is obtained prior to powder processing.
A number of methods have been proposed for particle shape analysis, including
shape coeffi cients, shape factors, verbal descriptions, curvature signatures, moment
invariants, solid shape descriptors, and mathematical functions (Fourier series
expansions or fractal dimensions); these are beyond the scope of this chapter but
have been adequately described in other texts [37] .
In the most simplistic means of defi ning particle shape, measurements may be
classifi ed as either macroscopic or microscopic methods. Macroscopic methods
typically determine particle shape using shape coeffi cients or shape factors, which
are often calculated from characteristic properties of the particle such as volume,
surface area, and mean particle diameter. Microscopic methods defi ne particle
texture using fractals or Fourier transforms. Additionally electron microscopy
and X - ray diffraction analysis have proved useful for shape analysis of fi ne
particles.
Particle Shape Measurement
1. Shape Coeffi cients and Shape Factors There are various types of shape
factors, the majority based on statistical considerations. In essence this translates to
the use of shape factors that do refer not to the shape of an individual particle but
rather to the average shape of all the particles in a mass of powder. However, a
method developed by Hausner [38] that uses three factors — elongation factor, bulkiness
factor, and surface factor — may be used to characterize the shape of individual
particles (Table 5 ).
2. Determining Particle Shape by Fourier Analysis Fourier transforms have
been previously used to determine particle shape and the rollability of individual
particles from the coeffi cients of the resulting series [39] . Moreover, fast Fourier
transforms have been successfully used to determine coeffi cients and a particle
“ signature ” by plotting ln An versus ln n , where An is the n th Fourier coeffi cient
and n is the frequency [29, 40, 41] . In brief, Fourier method consists of fi nding the
centre of gravity of a particle and its perimeter, from which a polar coordinate
system is set up. Amplitude spectra of a fi nite Fourier series in closed form are used
as shape descriptors of each particle [42] . Several research papers have focused on
the characterization of individual particle shape using Fourier grain analysis or
morphological analysis [43 – 44] . The method has also been extended to the measurement
of particle shapes in a blend [45] and to relate particle attrition rate in a milling
operation to particle shape [46] .
3. Determining Particle Shape by Electron Microscopy Electron microscopy
has been used for the examination of fi ne powder dispersions and will provide
information on particle shape perpendicular to the viewing direction. Standard
shadowing procedures may be useful in obtaining information on shape in the third
dimension. Scanning electron microscopy can give direct and valuable information
on the shape of large particles [47] .
4. Determining Particle Shape by X - Ray Diffraction Broadening The broadening
of X - ray diffraction lines is primarily a measure of the departure from single -
crystal perfection and regularity in a material and can therefore be used to
characterize particle shape. This is the only method that gives the size of the primary
crystallites, irrespective of how they are aggregated or sintered, and is of great value
for determining the properties of fi ne powders [48, 49] .
5. Other Methods for Particle Shape Determination Gotoh and Finney [50]
proposed a mathematical method for expressing a single, three - dimensional body
POWDER CHARACTERIZATION TECHNIQUES 1183
1184 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
TABLE 5 Shape Coeffi cients and Shape Factors
Coeffi cients and Factors Symbols Defi nitions and Equations
Volume shape coeffi cient . v
.v = V
d3
where V = average particle volume
d = mean particle diameter
Surface shape coeffi cient . s
.s = S
d2
where S = average particle surface
d = mean particle diameter
Volume – surface shape
coeffi cient
. vs
. .
. vs
s
v
=
where . v = volume shape coeffi cient
. s = surface shape coeffi cient
Shape factor . 0
. . o m n = v
where . o = shape factor for equidimensional
particle and thus represents part of . v
which is due to geometric shape only
. v = volume shape coeffi cient
m = fl akiness ratio, or breadth/thickness
n = elongation ratio, or length/breadth
Sphericity shape factor . w Sphericity = (surface area of sphere having same
volume as particle) / (surface area of
actual particle)
Circularity shape factor Circularity = (perimeter of particle outline) 2 /
4 . (cross - sectional or projection
area of particle outline)
Source: From refs. 6 and 42 .
by sectioning it as an equivalent ellipsoid with the same volume, surface area, and
average projected area as the original body. Moreover, wedge - shaped photodetectors
to measure forward light - scattering intensity have also been explored for determination
of crystal shape [51] . More recently a technique referred to as time of
transition (TOT) that was fi rst introduced in 1988 has also been used for the analysis
of particle size and shape [52, 53] .
6.7.4 EFFECT OF PARTICLE SIZE REDUCTION ON
TABLETING PROCESSES
Particle size plays a critical role in the effi cacy of a drug product. It can impact not
only bioavailability but also the effi ciency and success of production process and
ultimately the properties of the fi nal dosage form.
6.7.4.1 Wet Granulation Processes
The particle size of an active pharmaceutical ingredient can have signifi cant effect
on the processing behavior of a formulation, such as granule growth during wet
granulation and hence the resulting granule characteristics. The particle size of the
starting material can affect the strength and deformability of moist granules and
hence their behavior during the wet granulation process.
The effect of particle size on granule growth is a function of several interacting
factors, the balance of which largely depends on the nature of the material and the
experimental conditions. Differences in granule structure and porosity, resulting
from changes in starting material particle size, can also affect other characteristics
(e.g., compressibility) of the granulation.
Badawy et al. [57] studied the effect of DPC 963 (a nonnucleoside reverse transcriptase
inhibitor) particle size on the granule growth, porosity, and compressibility
of granules manufactured by a high - shear wet granulation process. It was found that
DPC 963 granule growth in the high - shear granulator and the resulting granule
compressibility and porosity were sensitive to relatively small changes in drug substance
particle size. Decreasing the particle size resulted in more pronounced
granule growth and enhanced the porosity and compressibility of the granulation.
Higher pore volume for the granulation manufactured using the active ingredient
with a smaller particle size may be the reason for its higher compressibility. The
high granulation porosity resulted in an increased fragmentation propensity and
volume reduction behavior of the granulation that led to increased compressibility.
The more porous granulation has higher tendency to densify upon application of
the compression force, resulting in closer packing of the particles.
6.7.4.2 Mixing Processes
Mixing may be defi ned as a unit operation that aims to treat two or more components,
initially in an unmixed or partially mixed state, so that each unit of the components
lies as nearly as possible in contact with a unit of each of the other
components [2] . Whenever a product contains more than one component, mixing
will be required in the manufacturing process in order to ensure an even distribution
of the active component(s).
It is well accepted that mixing solid ingredients is usually more effi cient and
uniform if the active ingredient and excipients are approximately the same size,
which ultimately provides a greater uniformity of dose [1] . Particle size and particle
size distribution are important in the powder - mixing process since they largely
determine the magnitude of forces, gravitational and inertial, that can cause interparticulate
movement relative to surface forces, which resist such motion. As a
consequence of high interparticulate forces, as compared with the gravitational
forces, powders of less than 100 . m mean particle diameter sizes are not free
fl owing. Powders that have high cohesive forces due to interaction of their surfaces
can be expected to be more resistant to intimate mixing than those whose surfaces
do not interact strongly [2] .
In moving from one location to another, relative to neighboring particles, a particle
must surmount a certain potential energy barrier that arises from forces resisting
movement. This effect is a function of both particle size and shape and is most
EFFECT OF PARTICLE SIZE REDUCTION ON TABLETING PROCESSES 1185
1186 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
pronounced when high packing densities occur. Ideal mixing may be achieved when
all the particles of the powder mix have similar size, shape, and density characteristics
whereas segregation (demixing) may occur when powder blends are not composed
of monosized near - spherical particles but contain particles that differ in size,
shape, and density. Segregation is more likely to occur if the powder bed is subjected
to vibration.
The main reason for segregation in powder blends is the difference in the particle
size of the components of the particles contained within the blend. Due to the high
diffusivity of small particles, such materials move through the voids between larger
particles and so migrate to the lower regions of the powder mix. Moreover, during
mixing operations, extremely fi ne particles have a high tendency to be forced
upward by turbulent air currents as the powder blend tumbles and subsequently
become isolated from the mixing process through continuous suspension above the
blend. When mixing is stopped, these particles will sediment and form a layer on
top of the coarser particles.
It is important to control the particle size distribution of pharmaceutical granules
or powder blends because a wide size distribution can lead to a situation with a high
probability of segregation. If this occurs within the hoppers of tablet machines,
nonuniform products may be manufactured due principally to large weight variations.
Tablet dies are fi lled by volume rather than weight, and consequently, the
establishment of different regions within a hopper containing granules of different
sizes (and hence bulk density) will contain a different mass of granules. This will
lead to an unacceptable distribution of the active pharmaceutical content within the
batch of fi nished product, even though the drug is evenly distributed by weight
throughout the granules.
6.7.4.3 Flowability of Pharmaceutical Powders
Due to the relatively small particle size, irregular shape, and unique surface characteristics,
many pharmaceutical powders have a high tendency to be extremely
cohesive. This high level of cohesion results in “ sticky ” powders that have poor
fl owability, commonly resulting in large mass variability within the fi nal product
owing to unpredictable and variable fi lling of tablet dies.
Powders with different particle sizes have different fl ow and packing properties,
which signifi cantly alter the volume of powder expelled from manufacturing equipment
during, for example, encapsulation or tablet compression. In order to avoid
such problems, the particle sizes of the active pharmaceutical ingredient and other
powder excipients should be defi ned and controlled during formulation so that
problems during production are avoided. Most notably, powder fl owability is of
critical importance in the successful production of acceptable pharmaceutical dosage
forms. High levels of fl owability within pharmaceutical powders is not just important
in the fi nal stages of manufacture but is essential for many industrial processes,
particularly mass transport.
Poor or uneven powder fl ow can result in excess entrapped air within powders,
which may induce capping or lamination in specifi c high - speed tableting equipment.
Moreover, uneven powder fl ow that is a direct result of the presence of excess fi nes
within a powder blend will also promote increased particle – die wall friction, lubrica
tion problems, and very importantly increased dust contamination hazards to operating
personnel.
Although particle size is a signifi cant factor controlling the fl owability of pharmaceutical
powders or granules, other factors must be considered. The presence of
molecular forces between particle/granule surfaces increases the probability for
cohesion and adhesion between solid particles. Cohesion may be defi ned as the
attractive forces between like surfaces, such as component particles of a bulk solid,
whereas adhesion may be defi ned as the attractive force between two unlike surfaces,
for example, between a particle and a tablet punch. It is extremely important
to appreciate that cohesive forces acting between particles in a powder bed are
attributed mainly to short - range nonspecifi c van der Waals forces that are signifi -
cantly altered as particle size and relative humidity change.
Cohesion and adhesion are phenomena that occur at the surface of a solid and
hence particles with an extremely large surface area will have greater attractive
forces than those with a smaller surface area. Consequently particle surface area
will have a dramatic effect on the fl owability of pharmaceutical powders. Typically,
fi ne particles with very high surface - to - mass ratios will be more cohesive than larger
particles, which are infl uenced more by gravitational forces. Particles larger than
250 . m are usually relatively free fl owing, but as the size falls below 100 . m, powders
become cohesive and fl ow problems are likely to occur. Powders having a particle
size less than 10 . m are usually extremely cohesive and resist fl ow under gravity.
Although it has been previously stated that particles with similar particle sizes
are desirable for pharmaceutical processes, a bulk powder mass with a narrow particle
size distribution accompanied with dissimilar particle shapes can produce a
bulk mass with inherently different fl ow properties, owing principally to differences
in interparticle contact area.
6.7.4.4 Compression Processes
In general, the strength of a compressed powder depends on the inherent ability of
the powder to reduce in volume during compression and the amount of interparticulate
attraction in the fi nal compact. The decrease in compact volume with
increasing compression load is attributed normally to particle rearrangement, elastic
deformation, plastic deformation, and particle fragmentation. Pharmaceutical materials
normally consolidate by more than one of these mechanisms [58, 59] . Unmodi-
fi ed paracetamol crystals exhibit poor compressibility during compaction, resulting
in weak and unacceptable tablets with a high tendency to cap [60] . Moreover the
incidence of capping and lamination during production, following ejection of tablets
from the die, depended on the plastic and elastic behaviors of the excipients used
[61] . It has been suggested that materials undergoing plastic deformation, in contrast
to elastic deformation, display enhanced bond formation and produce strong
tablets.
The effect of particle size on the compression properties of paracetamol oral
dosage forms has been previously reported [62] . Heckel analyses plots indicated
that the predominant mechanism of compaction of paracetamol was fragmentation
with larger particle fractions experiencing more fragmentation than the smaller
particles. Furthermore, Heckel analysis also indicated that, for a given applied
EFFECT OF PARTICLE SIZE REDUCTION ON TABLETING PROCESSES 1187
1188 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
pressure, the larger particles of paracetamol produced denser compacts than the
smaller particles. The results of elastic – plastic energy ratios indicated that the
majority of energy involved during compaction of paracetamol was utilized as
elastic energy. This suggested a massive elastic deformation of paracetamol particles
under pressure, resulting in weak and capped tablets. It was found that larger particles
exhibited less elastic recovery and elastic energy compared to smaller particles.
This was attributed to increased fragmentation of larger particles, resulting in
increased bonding between particles due to the formation of more new, fresh, and
clean particle surfaces.
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38. Hausner , H. H. ( 1967 ), Characterization of the Powder Particle Shape in Particle Size
Analysis , Society for Analytical Chemistry , London .
39. Schwarcz , H. P. , and Shane , K. C. ( 1969 ), Measurement of particle shape by Fourier
analysis , Sedimentology , 13 ( 3 – 4 ), 213 – 231 .
40. Meloy , T. P. ( 1969 ), Screening , AIME , Washington, DC .
41. Meloy , T. P. ( 1977 ), Fast Fourier transforms applied to shape analysis of particle silhouettes
to obtain morphological data , Powder Technology , 17 , 27 – 35 .
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1190 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION
42. Ehrlich , R. , and Full , W. E. ( 1984 ), Fourier shape analysis — a multivariate pattern recognition
approach , in Beddow , J. K. , Ed., Particle Characterization in Technology , Vol.
II, Morphological Analysis, CRC Press , Boca Raton, FL .
43. Meloy , T. P. , Clark , N. N. , Durney , T. E. , and Pitchumani , B. ( 1985 ), Measuring the
particle shape mix in a powder with the cascadograph , Chemical Engineering Science ,
40 ( 7 ), pp. 1077 – 1084 .
44. Alderliesten , M. ( 1991 ), Mean particle diameters. part II: standardization of nomenclature
. Part. Part. Syst. Charact. , 8 , 237 – 241 .
45. Fairbridge , C. , Ng , S. H. , and Palmer , A. D. ( 1986 ), Fractal analysis of gas adsorption
on syncrude coke , Fuel , 65 , 1759 – 1762 .
46. Shibata , T. , and Yamaguchi , K. ( 1990 ), paper presented at the Second World Congress
Particle Technology, Sept., Part 1, Kyoto, Japan.
47. Johari , O. , and Bhattacharyya , S. ( 1969 ), The application of scanning electron microscopy
for the characterization of powders , Power Technol ., 2 , 335 .
48. Hillard , J. E. , Cohen , J. B. , and Paulson , W. M. ( 1970 ), Optimum Procedures for determining
ultra fi ne grain sizes , in Burke , J. J. , Reed , N. L. , and Weiss , V. , Eds., Ultrafi ne
Grain Ceramics , Syracuse University Press , Syracuse, New York , pp. 73 .
49. Oel , H. J. ( 1969 ), Crystal growth in ceramic powders , Gray , T. J. , and Frechette , V. D. ,
Eds., Kinetics of Reactions in Ionic Systems , Plenum , New York , p. 249 .
50. Gotoh , K. , and Finney , J. L. ( 1975 ), Representation of the size and shape of a single
particle , Powder Tech ., 12 , 125 – 130 .
51. Heffel , C. , Heitzmann , D. , Kramer , H. , and Scarlett , B. ( 1995 ), paper presented at the
6th European Symp. Particle Size Characterization, Partec 95, Nurenberg, Germany.
52. Karasikov , N. , Krauss , M. , and Barazani , G. ( 1988 ), in Lloyd , P. J. , Ed., Particle Size
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53. Manohar , B. , and Sridhar , B. S. ( 2001 ), Size and shape characterization of conventionally
and cryogenically ground turmeric (Curcuma domestica) particles , Powder Technol .,
120 , 292 – 297 .
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powders, Part I. Homogeneous expansion , Powder Technol ., 26 , 35 – 46 .
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size on the characteristics of granulation manufactured in a high - shear mixer , AAPS
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pressure curves for the characterization of volume reduction — Mechanisms in powder
compression , Powder Technol ., 46 , 67 – 75 .
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Effect of compression force, compression speed, and particle size on the compression
properties of paracetamol , Drug Dev. Ind. Pharm ., 27 ( 9 ), 935 – 942 .
1191
6.8
ORAL EXTENDED - RELEASE
FORMULATIONS
Anette Larsson ,1 Susanna Abrahmsen-Alami, 2 and Anne Juppo 3
1 Chalmers University of Technology, G o teborg, Sweden
2 AstraZeneca R & D Lund, Lund, Sweden
3 University of Helsinki, Helsinki, Finland
Contents
6.8.1 Introduction
6.8.1.1 Background
6.8.1.2 Biopharmaceutical Aspects on Oral ER Formulations
6.8.1.3 Infl uence of Drug Properties
6.8.1.4 Principles for Extended Drug Release
6.8.2 Insoluble Matrix Tablets
6.8.2.1 Principles of Formulation and Release Mechanisms
6.8.2.2 Manufacturing of Insoluble Matrix Tablets
6.8.3 Membrane - Coated Oral Extended Release
6.8.3.1 Principles of Formulation and Release Mechanisms
6.8.3.2 Manufacturing of Oral Membrane - Coated Systems
6.8.4 Hydrophilic Matrix Tablets
6.8.4.1 Principles of Formulation and Release Mechanisms
6.8.4.2 Manufacturing of Hydrophilic Matrix Tablets
6.8.5 Comparison and Summary of Different Technologies
6.8.6 Other Oral ER Formulations
References
6.8.1 INTRODUCTION
6.8.1.1 Background
In order to achieve therapeutic effect, a drug needs to reach the right place in the
body at the right time. For some drugs, this may be achieved by simple solutions or
solid dosage forms with an instant drug release while, for others, one has to modify
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1192 ORAL EXTENDED-RELEASE FORMULATIONS
the drug release. To understand the literature within the area of modifi ed drug
release, it is important to be aware of the standard terms used for dosage forms
within this fi eld. Malinowski and Marroum have summarized these terms in
the book Encyclopedia of Controlled Drug Delivery [1] . The authors state that
modifi ed - release (MR) formulations refer to “ dosage forms for which the drug
release characteristics of time course and/or location are chosen to accomplish
therapeutic or convenience objectives not offered by conventional dosage forms. ”
One group of MR formulations is the delayed - release dosage form, which does not
release the drug immediately after administration. One example of delayed - release
formulation is the enteric - coated formulations. Another subgroup of MR formulations
is the extended - release (ER) dosage forms, which are the focus of the present
chapter. According to a defi nition from the U.S. Pharmacopeia (USP), ER formulations
can be referred to as dosage forms that allow at least a twofold reduction in
the dosing frequency compared to conventional dosage forms [2, 3] .
The interest in oral ER formulations has dramatically increased in recent years.
This can be seen in Figure 1 , where the bars in the diagram correspond to the
number of publications found in a search in the database SciFinder Scholar 2006
[4] that include the words oral extended release . This increase in publications con-
fi rms that there are many ongoing activities in this fi eld. The expression oral extended
release occurs in the database for the fi rst time in 1954, when Yamanaka et al. utilized
the slow dissolution rate of various salts of a drug (pyrimidine penicillin) to
extend the period of time when the drug had a clinical effect [5] . In 1959 Robinson
and Suedres made a formulation of sulfamethylthiadiazole together with hydrogenated
castor oil, which was suspended in an aqueous vehicle, creating a formulation
with extended drug release [6] . Later, in the late 1950s and early 1960s Sj o gren and
Frykl o f compressed active substances (e.g., pentobarbitone sodium and theophyl-
FIGURE 1 Number of publications containing the words “ oral extended release ” identifi ed
in the database SciFinder Scholar 2006 for the fi ve - year period 1960 – 2005 [4] .
0
200
400
Number of publications
600
800
1000
1200
1400
1600
1800
2000
2001–
2005
1996–
2000
1991–
1995
1986–
1990
1981–
1985
Five-year periods
1976–
1980
1971–
1975
1966–
1970
1961–
1965
>1960
INTRODUCTION 1193
line) together with polyvinyl chloride (PVC) and obtained extended drug release
from these insoluble matrix tablets [7] . In an early publication they showed that
increased dose loadings or addition of channeling agents increased the drug release
rate. It is also interesting to note that the most popular ways to prepare oral
ER formulations today were already mentioned in an early review from the early
1970s [8] .
6.8.1.2 Biopharmaceutical Aspects on Oral ER Formulations
The clinical effect of low - molecular - weight substances is often related to the concentration
of the drug in the blood plasma. Classical blood plasma profi les for both
immediate - release (IR) and ER formulations are shown in Figure 2 . It is well known
that a drug only has a clinical effect when the concentration in the blood plasma is
above the minimum effective concentration (MEC). If the concentration of the
active substance is above the maximum safe concentration (MSC), the side effects
will be unacceptable. The interval between the MEC and MSC is called the therapeutic
window or therapeutic range, and the time when the concentration is above
the MEC is called the “ duration ” of the drug. One aim of ER formulations is to
increase the time the substance is above its MEC by continuous release of the drug
from the formulation. Under optimal conditions the rate - limiting step in the drug
absorption process of an ER formulation is its release rate, which then can be
directly related to the concentration of the drug in the blood plasma. When the drug
release rate from an ER formulation is constant, the blood plasma concentration
will be constant under ideal conditions, whereas ER formulations with time -
FIGURE 2 ( a ) Schematic picture of blood plasma concentration profi le after administration
of a drug to an individual, including the MSC, MEC, therapeutic range, and duration.
( b ) Repeated administration of IR formulation (four times daily) of a drug with short pharmacokinetic
half - time and administration once daily of an ideal ER formulation with constant
drug release (broken line) or ER formulation with nonconstant drug release rate
(dotted line).
0
5
10
15
20
25
30
Conventional IR
Ideal ER
ER 0
5
10
15
20
25
30
n
Time (h)
Blood plasma concetration
Time (h)
Blood plasma concentration
MEC
MSC
Duration
Therapeutic range
MEC
MSC
(a) (b)
0 5 10 15 20 25 0 5 10 15 20 25
1194 ORAL EXTENDED-RELEASE FORMULATIONS
dependent drug release rate may give rise to time variations in the concentration
of the drug in the blood plasma (Figure 2 ).
The ER concept might offer several advantages, such as reduction in administration
frequency, reduction of side effects, less irritation in the gastrointestinal tract,
and improved patient compliance. Speers and Bonnano have also mentioned some
economic aspects of ER formulations, such as the possibility to patent line extensions
and to reduce manufacturing costs since fewer units are required to obtain
the same effect [9] . On the other hand, ER formulations may have several drawbacks,
for example, large variations in effect between patients due to varying physiological
factors within the patient group, limited transit time for the ER formulation,
drug stability problems during the gastrointestinal passage, and more severe complications
such as dose dumping.
In order to obtain a clinical effect by an orally administered drug, it is, for
example, required that the drug is (i) dissolved and released from its formulation,
(ii) transported over the mucosal barrier, and (iii) has passed from the lumen to the
systemic blood circulation without being metabolized by, for example, the lumen
or the liver. The drug dissolution rate and the rate of absorption of the dissolved
active substance as well as the relation between these processes are important, in
particular the dissolution process since the absorption of undissolved drug particles
can be disregarded.
The ER formulations can be a single - unit, monolithic system or multiple - unit
systems containing many individual units with extended release. Multiple - unit
systems consist of many small pellets and are normally produced by extrusion and
spheronization or coating on inert spheres [2, 10] . The composition and ER mechanism
can vary for multiple - unit systems, and some examples are membrane - coated
reservoir systems and polymer - or lipid - based matrix systems, where the matrices
can be made of both soluble and insoluble carriers [11 – 16] .
From a biopharmaceutical point of view, the multiple - unit systems have many
advantages, for example, a more consistent gastrointestinal transit compared to
larger monolithic systems [17] . The gastrointestinal transit times for monolithic and
multiple - unit systems were compared in a study by Abrahamsson et al. [17] . It was
found that the gastric emptying time for the small multiple units was considerably
shorter than that of larger monolithic systems (on average 3.6 and 9.6 h, respectively).
The transit times through the small intestine were approximately equal,
whereas the transit time in the colon for the multiple units was longer compared to
the monolithic system, which was explained by different infl uence of the motility
on the different systems. Another advantage with multiple - unit compared to monolithic
systems is that the effects of dose dumping become less severe [2] . A breakage
and instant drug release from one pellet will have considerably lower effect than
breakage of one monolithic system.
There may also be development and manufacturing advantages and disadvantages
with multiple - unit systems compared to monolithic systems. The dose for
multiple - unit systems may be easier to adjust since one can readily increase the
number of pellets in the formulation and thus increase the dose. One can adjust the
drug release profi les for multiple - unit systems by mixing pellets with different
release profi les. The multiple - unit systems offer the possibility to mix pellets containing
different active substances, which can be an advantage for the patients who
then only need to take one formulation at a time (containing more than one active
INTRODUCTION 1195
substance) instead of several formulations. Disadvantages with multiple - unit systems
may also exist; for example, the time to develop the multiple - unit systems may well
be longer than for monolithic systems. There are greater challenges in the scaleup
procedure for the multiple - unit systems since several expensive and specialized
types of equipment may be needed [18] . For fi lm - coated formulations, additional
changes in the drug release rate may be obtained upon storage as a consequence
of aging of the fi lm. However, this change can be suppressed by introducing a curing
step in the production [10] .
6.8.1.3 Infl uence of Drug Properties
Drug properties that are important to consider during development of IR tablets
are metabolism, stability, permeability, and solubility [18, 19] . In the development
of ER formulations, these aspects are also important, but in addition to IR formulations,
they must be considered in relation to the different environments that the ER
formulations meet during their passage through the gastrointestinal tract, and some
of these aspects will be discussed briefl y below. It can be mentioned that, based on
these initial properties for drug candidates, Thrombre has constructed a feasibility
assessment fl ow chart for ER formation development [18] .
The pharmacokinetic half - life for a drug may give an indication of whether a
conventional or ER formulation is to be chosen. For drugs with short biological t 50
(less than 1 – 2 h), devices that continuously release the active substance are required
[2, 18] . For drugs with lifetimes longer than about 10 h, ER formulations may not
add any benefi ts compared to IR formulations. However, for drugs with half - lives
between these limits, ER formulations may be a good alternative to IR
formulations.
The stability of a drug in the solid state or in aqueous solution is a critical parameter
when selecting an appropriate manufacturing process. A drug in an oral ER
formulation reaches aqueous environments with, for example, variations in pH (1 –
8), ionic strength, and bile salt concentration, which requires high chemical stability
of the drug [19] . Furthermore, the substance should be stable not only against
chemical degradation such as hydrolysis but also against enzymatic degradation
(metabolism) during the passage from the lumen to the systemic blood circulation.
The drug is released from oral ER formulations along the whole gastrointestinal
tract. This implies that, in contrast to IR formulations, drug permeability must be
good along the whole gastrointestinal tract for drugs in ER formulations [19] . Furthermore,
the solubility and the dissolution rate of the drug are extremely important
to consider, since these factors will directly infl uence the release rate for the drugs
from ER formulations. The dissolution rate can be described as the fl ux J of dissolved
material from a drug particle and, according to the Noyes – Whitney equation,
it is [20, 21] :
J
D
h
C C = . ( ) s b (1a)
where D is the drug diffusion coeffi cient, h is the thickness of the stagnant diffusion
layer around the particles, and C s and C b are the concentrations of the drug at the
particle surface and in the surrounding bulk media, respectively. For substances in
1196 ORAL EXTENDED-RELEASE FORMULATIONS
their most stable solid - state form, the concentration C s close to the particle surface
is equal to the saturated concentration. However, when a substance is in a different
polymorphic state or in an amorphous state, C s can be larger than the saturation
concentration.
Many drugs are weak acids or bases with one or several p K a values. According
to the Henderson – Hasselbalch equation, the solubility of an acidic drug will depend
on the p K a and pH as
pH p a
i = + K
S
S
log
0
(2)
where S i and S 0 are the concentrations of the drug ’ s un - ionized and ionized forms,
respectively. Since the pH varies along the gastrointestinal tract, the solubility and
the dissolution rate [Equation (1a) ] of the drug depend on the position of the drug
in the gastrointestinal tract. Furthermore, for some drugs, such as indomethacin, the
dissolution of the acid drug may lead to a changed microenvironmental pH within
the stagnant layer and thus also infl uence the dissolution rate [22, 23] .
6.8.1.4 Principles for Extended Drug Release
The main principles related to ER systems are as follows:
(i) Insoluble matrix formulations
(ii) Membrane - coated solid dosage forms including osmotic pump systems
(iii) Soluble hydrophilic matrix formulations
Below we will discuss each of these formulation principles in terms of basic
release mechanisms and the advantages or drawbacks associated with the different
formulations and manufacturing processes. However, drug release from all kinds of
ER formulations starts with hydration of the formulation and water diffusion into
the system. The presence of water in the formulation facilitates the start of the dissolution
process of the drug, whereby the dissolved drug can be released from the
formulations.
The driving forces for transport of water and drug are the differences in chemical
potentials between the formulation and its surrounding. Due to the similarities in
the driving forces for the dissolution of a drug and the release from an ER formulation,
one can modify Equation (1a) to
dM
dt
JA
DA
h
C C t= = . ( ) s b (1b)
where M t is the amount of active drug that is released at the time t , D the drug diffusion
coeffi cient, and J the fl ux of the drug from the formulation. The other parameters
in Equation (1a) have been adjusted to fi t the drug release from the formulation
and therefore A becomes the surface area of the releasing system in Equation (1b)
(e.g., the area of the membrane - coated tablet), h the diffusion pathlength, and C s
and C b correspond to the concentration of the dissolved active drug at the surface
of the drug particles/formulation and in the bulk solution surrounding the ER
device, respectively. Depending on the exact type of ER system, some modifi cations
in Equation (1b) may be needed to fully describe the drug release. One important
factor in the equation is the difference in concentration ( Cs . Cb ), and Cb is often
assumed to be zero due to release under so - called sink conditions. For active substances
in their most stable solid state, the remaining concentration, Cs , is equal to
the saturation concentration in that medium. However, as mentioned above, Cs can
be oversaturated or depend on pH. This means that the drug release from formulations
depends on the solid - state properties and p Ka of the drug as well as the pH
to which the formulation is exposed [24] . Since the pH varies along the gastrointestinal
tract, the drug release will be dependent on the position of the formulation in
this tract. Several attempts to avoid pH - independent drug release has been made,
for example, by including buffers [25 – 27] .
6.8.2 INSOLUBLE MATRIX TABLETS
6.8.2.1 Principles of Formulation and Release Mechanisms
The history of insoluble matrix tablets goes back to the beginning of the 1960s, when
H a ssle and Abbott developed the Duretter and the Gradumet, respectively [2] .
Since then, many ER tablets based on this principle have been developed. Looking
at the homepage of the U.S. Food and Drug Administration (FDA [28] ) and searching
for “ insoluble matrix tablets ” produces more than 140 hits, which indicates that
this research area is still active.
The term insoluble matrix tablet refers to tablets in which the drug is embedded
in an inert carrier that does not dissolve in the gastrointestinal fl uids. The carrier
material in insoluble matrix tablets can be based on insoluble lipids or polymers,
both matrix builders whose function it is to keep the matrix together during the
passage through the gastrointestinal tract and thus prolong the diffusion path of the
drug before it is released from the formulation. The drug can be dispersed or dissolved
or both in a matrix carrier (see Figure 3 ) and, depending on the formulation,
different mechanisms can be regarded to take place:
• Dissolved drug in the matrix diffuses through the matrix.
• Dissolved drug in the matrix diffuses through pores in the matrix.
• Dispersed drug dissolves and diffuses through the matrix.
• Dispersed drug dissolves and diffuses through pores in the matrix.
FIGURE 3 Schematic pictures of insoluble matrix systems. Left: Drug (light gray) molecularly
dissolved in carrier material (black); middle: drug particles dispersed in carrier material;
right: drug particles dispersed in carrier material at higher drug loading, leading to
continuous network of drug.
INSOLUBLE MATRIX TABLETS 1197
1198 ORAL EXTENDED-RELEASE FORMULATIONS
As early as 1963, Higuchi [29] derived an expression for drug release from
insoluble matrix systems. In this historical paper, Higuchi derived two equations for
two different geometries, the simple planar sheet matrix system with infi nite area
and spherical pellets. Furthermore, two special cases were treated, one where the
matrix is a homogeneous matrix without pores and another where the matrix contains
pores. In the system without pores, the drug is assumed to diffuse through the
homogeneous matrix. For matrix systems with pores the effi ciency in transport
through liquid - fi lled pores is greater than through the solid matrix carrier. Therefore,
the main contribution to the drug release is transport in the pores. This is
gained by penetration of the surrounding medium into the pores, where it dissolves
the drug. The dissolved drug can diffuse through the pores and be released at the
surface of the matrix. The simplest theoretical treatment of the drug dissolved in
the matrix carriers assumes the following [30] :
• There is no breakage of the matrix.
• There is no dissolution of the matrix.
• There is no resistance to drug transport in the boundary layer surrounding the
device.
• There is no accumulation (e.g., adsorption) of the drug in the device.
• The saturated concentration C s,m of the drug in the matrix is constant during
the process.
• The drug - loading concentration C 0 is larger than C s,m .
• The drug concentration around the matrix is zero, C b = 0 (sink conditions).
• The diffusion constant D m in the matrix is independent of the drug
concentration.
• The partition coeffi cient K between the matrix material and the surrounding
release medium is independent of the drug concentration.
A handy derivation of the equation describing the release from planar homogeneous
matrix systems can be found in a book by Wu [30] . It is derived for a
sheet with the area A and assumes that the concentration of dissolved drug
inside the matrix is constant and equal to the saturation concentration C s,m .
Under these assumptions, the amount of drug, M t , that is released at time t can be
predicted as
M ADKC t C C t= . ( ) [ ] m s,m s,m 2 0
0 5 . (3)
When the matrix contains a drug - fi lled network (Figure 3 , right image), water can
diffuse and dissolve the drug, and this creates a pore structure. The equation
describing the drug release from matrices with networks is modifi ed to include
information about the created pore structure [Equation (4) ]. This can be described
by the porosity . (the volume of the pores in proportion to the total volume of the
device) and the tortuosity . of the pores (a measure of how much the diffusion
path is lengthened due to lateral excursions). Also the diffusion coeffi cient D m and
C s,m in Equation (3) are replaced with D and C s in Equation (4) , corresponding to
the diffusion coeffi cient and the solubility of the drug in the solution inside the
pores, respectively:
M A
D C t
C C t= . ( ) ...
...
.
.
. s
s 2 0
0 5 .
(4)
The most common types of insoluble matrix tablets are those containing pores.
From the equations above one can see that the drug - release depends on the solubility
of the drug, the drug - loading concentration, and the diffusion coeffi cient, which
is related to the molecular size of the drug. The area of the insoluble matrix tablet
also affects the drug release and can be changed by altering the dimensions or the
geometry of the tablet. The drug release from insoluble matrix tablets also depends
on the porosity and pore structure of the tablet, and the drug release rate increases
with increasing porosity.
A comparison of Equations (3) and (4) shows that, in both equations, the amounts
of released drug are directly dependent on the area of the device, the square root
of the time t , the drug - loading concentration C 0 , the respective saturated drug concentrations,
and the drug diffusion coeffi cients. In addition, the release rate (the
time derivate of the amount of released material) depends on the square root of
time and can be stated as
dM
dt t
t . 1 (5)
As pointed out above, for ideal ER formulations, the rate - limiting step for drug
absorption is the release rate from the ER formulation. Thus, since the release rate
from an insoluble matrix system depends on time, the concentration of drug in the
blood plasma will also be time dependent and not constant (Figure 2 ), which may
be a therapeutic drawback. Another factor infl uencing the concentration of drug in
the blood plasma is the gastrointestinal transit times. When the transit times of the
formulations vary, the reproducibility between different administration occasions
in one patient will be low, and furthermore, great variation in the patient group
may be obtained. However, these conclusions are valid for all ER formulations
based on matrix systems and not limited to insoluble matrix systems only.
The equations above are valid when no depletion of drug occurs inside the
device. The equations for release rate will be much more complex when depletion
of the drug can occur [30] . However, it has been shown that, when the amount of
released material is less than approximately 60%, the release rate will depend on
time as t . 0.5 [29, 30] .
6.8.2.2 Manufacturing of Insoluble Matrix Tablets
Insoluble matrix tablets need a carrier, which can be a lipid - or polymer - based
excipient [7, 31 – 36] . Some suggestions of carrier materials can be found in Table 1 .
The table also presents the number of hits found upon searching the FDA ’ s homepage
[37] for the number of times an excipient is registered as a component in oral
extended, sustained, or controlled formulations. This list gives an indication of how
often these excipients are commercially used in oral ER formulations but does not
automatically tell us the exact formulation or exact mechanistic effect of the excipient.
The choice of carrier material is important, and one should be aware of possible
INSOLUBLE MATRIX TABLETS 1199
1200 ORAL EXTENDED-RELEASE FORMULATIONS
TABLE 1 FDA Registered Oral ER Formulations Containing Commonly Used
Excipients in Insoluble Matrix Formulations
Excipient
Number of Hits on
FDA Homepage Content Interval (mg)
Lipid based
Carnauba wax 9 46 – 300
Stearyl alcohol 4 25 – 244
Glyceryl behenate 3 15 – 51
Castor oil 2 23
Cottonseed oil, hydrogenated 2 58 – 402
Cetyl alcohol 2 44 – 59
Paraffi n 2 50 – 150
Stearic acid 2 26 – 180
Castor oil, hydrogenated 1 295 – 410
Vegetable oil, hydrogenated 1 228
Mineral oil 1
Microcrystalline wax 0
Insoluble polymer
Ethylcellulose 9 15 – 309
Ammonia methacrylate copolymer 5 37 – 138
Polyvinyl acetate 1 46
Polyethylene 0
Inorganic
Calcium phosphate (dibasic) 6 33 – 335
Source : http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm .
exposure of lipid - based formulations to erosion, which can be the result of enzymatic
degradation of the lipids [38] . This will of course also infl uence the drug
release rate.
In the compositions of insoluble matrix systems, excipients other than the carrier
material are needed to obtain products with processability and that meet requirements
from the pharmacopedias. Examples of categories of excipients included in
insoluble matrix tablets are binders, lubricants, glidants, colorant, taste maskers,
and channeling agents. As mentioned above, the drug release rate can be regulated
by the porosity in the insoluble matrix system. The properties and amounts of drugs
and excipients that can create pores will have a large impact on the release rate.
Examples of channeling agents are sugars, salts such as sodium chloride, and polyols
[2] . The pore structure also depends on other factors such as the particle sizes of
the excipients and the drug, the size and porosity of the granules, and the compaction
pressure.
The choice of process steps depends on the properties of the drug and the chosen
excipients. For insoluble matrix tablets one often mixes the active substance with
the excipients. Either this mixture can be directly compressed to matrix tablets or
the powder mixture can be exposed to a granulation technology to enlarge the
particle sizes. One such technique is dry granulation, that is, compaction of the
mixture in, for example, a roller compactor, followed by milling to desirable granule
sizes. Another granulation technique is melt granulation, where the melted granulation
liquid agglomerates the particles to granules. The most common granulation
method for insoluble matrix tablets is probably wet granulation [2, 7] with aqueous -
based or organic granulation liquids. The wet granulated masses are dried in fl uid
bed driers or ovens. In order to increase the drying speed, microwaves can be used.
The powder mixtures or granules are compressed in ordinary tableting machines
[7] . However, it is an advantage if the compaction pressures can be carefully monitored,
since this pressure may infl uence the porosity and thus the drug release. The
fi nal tablet can be coated to, for example, mask the taste.
Alternative production methods to the traditional compaction of powder to
insoluble matrix tablets are available, some of which are based on melting technologies,
but of course these methods rely on the ability of the carrier materials
or additives to melt. The drug is commonly dissolved or dispersed in the melt.
This melt can be fi lled into hard gelatin capsules [31] or it can be spray chilled by
pressing it through a nozzle into a vessel containing solid carbon dioxide [39] . Hot -
melt extrusion of polymer - based systems to form multiple - unit systems has been
investigated. The carrier material in these cases can be, for example, Eudragit
[40 – 42] .
6.8.3 MEMBRANE - COATED ORAL EXTENDED RELEASE
6.8.3.1 Principles of Formulation and Release Mechanisms
One way to protect the drug from being directly released is to coat the system with
an insoluble fi lm. The drug is suspended or dissolved in a reservoir system which
can consist of monolithic or multiple - unit systems. The MR fi lms surrounding the
reservoir will be insoluble and thus give the system extended drug release properties
or they can become soluble by external trigging. The latter case is defi ned as a
delayed - release formulation, which means that the formulation does not release
directly after administration. This can be achieved, for example, by enteric coatings,
where the fi lm - forming materials are insoluble in aqueous solutions at low pH but
soluble at high pH values. This results in delayed release from an enteric - coated
formulation, since the pH is low in the stomach. When the units are transported
into the intestine, the pH increases, the fi lm dissolves, and the drug can be released
immediately. This type of formulation will not be further discussed here, but the
work of Hogan is recommended for further information on these systems [43, 44] .
In ER reservoir systems, a membrane surrounds a reservoir of the drug, also
called the core of the system. The membrane controls the drug release and the
driving force is the difference in chemical potential over the membrane, which can
be correlated with a concentration gradient over the membrane. The transport of
the drug through the ER membranes can be divided into three different mechanisms
[45 – 47] :
• Diffusion through the membrane
• Diffusion through pores and cracks in the membrane
• Osmotic transport through pores, cracks, or drilled holes
The overall drug release process for common membrane - coated systems has been
shown to pass through three different time periods (Figure 4 ) [48] . During the initial
MEMBRANE-COATED ORAL EXTENDED RELEASE 1201
1202 ORAL EXTENDED-RELEASE FORMULATIONS
period, the amount of released drug will be low. The water penetrates the membrane
and at the same time starts to dissolve the material in existing pores, consisting
of water - soluble sugars, salt, or polymers. The water that has penetrated the
membrane and reached the core starts to dissolve the active substance in the reservoir
[11] . The concentration of the active drug increases continuously until its saturation
concentration has been reached and pseudoequilibrium between the solid
material and saturated solution inside the membrane has developed. The osmotic
pressure in the reservoir will depend on the concentrations of all dissolved species
inside the membrane, and an increase of the osmotic pressure may lead to membrane
rupture. After an initial lag period, the number of cracks and pores becomes
constant, and the osmotic pressure and the concentration of dissolved species in the
reservoir and in the membrane reach their steady - state levels. When these parameters,
which are the driving forces for the drug release, are constant, the drug release
rate will also be constant. Therefore, a second period with time - independent drug
release rate will occur.
When the solid drug material inside the membrane is completely dissolved and
the concentration of the dissolved drug decreases, a fi nal period with declining
FIGURE 4 Release from membrane - coated reservoir system, where the three different
stages are depicted (initial lag period, “ steady - state ” period, and fi nal depletion period). A
schematic picture of a formulation with the drug reservoir (dark grey) surrounded by the
membrane (middle grey) is presented at the top. The dissolution medium penetrates the
membrane and dissolves the drug (light gray), and pores/cracks are formed through which
the drug can be released.
0.0
20.0
40.0
60.0
80.0
100.0
0 5 10 15 20 25
Time (h)
Fraction released (%)
release rate will be entered. Ragnarsson et al. [49] have shown that the solid material
disappears earlier as the drug solubility increases and that the third stage with
time - dependent and decreasing release rate appears earlier compared to drugs with
lower solubility.
The contribution of diffusion to the release process can be modeled by using
Fick ’ s fi rst law. For diffusion of a substance through the membrane, it will turn out
as (assuming sink conditions)
dM
dt
JA
D K A
h
C C
D K AC
h
t= = . ( )= m m
s b
m m s (6)
where K m is the partition coeffi cient for the drug between the membrane and solution,
D m is the diffusion coeffi cient in the membrane, A is the area of the membrane,
h is the thickness of the membrane, and C s and C b are the concentrations on the
inside of the membrane surface and in the bulk, respectively. The equation for diffusion
through pores or cracks resembles Equation (6) :
dM
dt
JA
D KA
h
C C
D KAC
h
t= = . ( )=
.
.
.
. s b
s (7)
where . and . are introduced to describe the porosity and tortuosity in the
membrane, respectively. The parameters D m and K m in Equation (6) are replaced
in Equation (7) by D and K , which are the diffusion coeffi cient in the solution
inside the pores and the partition coeffi cient between the solution and materials
surrounding the liquid - fi lled pores, respectively. Equations (6) and (7) depend on
the concentration gradient over the membrane, and both are independent of the
time.
The osmotic contribution to the drug transport is described by the so - called
Kedem – Katchalsky equations (based on nonequilibrium thermodynamics) [50, 51] .
A simplifi ed version is
dM
dt
JA
AC L
h
t= = s p... (8)
where . . is the osmotic pressure difference over the membrane and L p and . are
the hydraulic permeability and the refl ection, respectively.
A comparison of Equations (6) – (8) shows important similarities; they depend on
the solubility of the drug, the area of the device, and the thickness of the membrane.
This means that an increased solubility, larger area of the membrane, and thinner
membranes will facilitate the drug release rate. This can be exemplifi ed by a study
by Ragnarsson and Johansson [11] , who showed that, for different salt forms of
metoprolol, an increased solubility also increased the drug release rate, which was
predicted from the equations. Furthermore, Equations (6) – (8) show constant and
time - independent release rates. This constant amount of released drug will be a
biopharmaceutical benefi t since it theoretically makes it possible to achieve a constant
concentration of the drug in the blood plasma.
MEMBRANE-COATED ORAL EXTENDED RELEASE 1203
1204 ORAL EXTENDED-RELEASE FORMULATIONS
One special type of ER formulation based on coated reservoir systems is the so -
called osmotic - controlled oral drug delivery system or osmotic pump. Pure osmotic
systems have semipermeable membranes; that is, water can permeate the membrane
but not other substances. These semipermeable membranes can be made of,
for example, cellulose acetate [44] , and for such formulations the dominating release
mechanism is the osmotic pressure [Equation (8) ]. The oldest formulation based on
osmotic release was OROS from Alza Corporation [47] . In order to achieve drug
release through the semipermeable membrane, a laser hole was drilled, but today
many osmotic formulations instead use pores fi lled with water - soluble materials.
The aspects of the formulation and different types of commercially available formulations
are summarized in the review by Verma et al. [52] . Some advantages with
osmotic pumps compared to other ER formulations such as hydrophilic matrix
systems are (i) the time - independent drug release (often mentioned as zero - order
release), (ii) the superior in vivo – in vitro correlation which facilitates further formulation
development, and (iii) less variation between fasted and fed states. Potential
drawbacks may be high initial development costs and lack of in - house
competence. Drawbacks from an economic point of view may be the necessity to
pay royalties and the need for special equipment associated with laser drilling
technology.
6.8.3.2 Manufacturing of Oral Membrane - Coated Systems
The fi rst step in the production of membrane - coated systems is to prepare the
drug reservoir, the core, of the system. The process steps for producing the core
depend on the size of the core. In monolithic membrane - coated systems, the core
can be a fi lled capsule or a tablet which is produced in the traditional ways. This
may include mixing of active substance and excipients, possibly granulation and
drying, fi lling into capsules, or compaction into tablets. The production of cores for
multiple - unit systems (often termed pellets) is more sophisticated and may be performed
in different ways. One, and probably the most common way, is to produce
cores for multiple - unit systems by extrusion and spheronizing [53 – 55] . An alternative
methodology is to coat an inert core, e.g. glass or nonpareil beads, with the
active substance and the excipients [11] . When the drug - containing core is manufactured,
the process continues with the coating of the release - controlling
membrane.
The composition of the core depends on the properties of the drug and the
excipients, the chosen production chain, and whether the systems should be a monolithic
or multiple - unit system. The compositions and production steps are reviewed
by Tang et al. [56] . In general, the core will include the active substance together
with the fi ller materials and, if necessary, solubilizers and lubricants/glidants. Classical
fi ller materials are lactose and microcrystalline cellulose, but also other materials
such as dextrose, mannitol, sorbitol, and sucrose can be used. However, it should
be remembered that the dissolution of the fi ller material might infl uence the osmotic
pressure. The effect of fi ller solubility has been investigated by Sousa et al. [54] ,
who found a relation between the solubility of the fi ller materials and drug release
rate. For fi ller materials with large water solubility, there is a great risk that the
membrane will rupture due to the development of an excessive osmotic pressure,
which will infl uence the drug release rate.
The choice of fi lm - forming materials and fi lm - coating techniques is critical for
the drug release rate [54] . The ER membrane should remain intact during the
release, which implies that it should not dissolve or erode. As fi lm - forming material,
water - insoluble substituted cellulose derivatives such as ethylcellulose have been
suggested [53, 57] as well as synthetic polymers such as methylacrylates (e.g.,
Eudragit NE 30D, RS30D or RL30D, where NE stands for nonionic and RS/RL
correspond to cationic polymers) [55] . A commercial technology platform is available
under the name Eudramode, which is a platform for development of multiple -
unit systems with extended drug release based on Eudragit [58, 59] . Other
fi lm - coating materials such as shellac and zein have been used, but a drawback with
these naturally occurring materials is the variation in quality. To obtain a fi lm with
satisfying release properties, channeling agents such as the hydrophilic polymers
hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and polyethylene
glycol (PEG) or other water - soluble materials such as sodium chloride or
lactose may be used [2, 57] . To improve the mechanical properties of the fi lm and
thus avoid ruptures and cracks in the fi lm, plasticizers may be added to the formulation.
Examples of plasticizers are PEGs, diethyl phthalate, triacetin, mineral oils,
glycerol, and chlorobutanol [60] .
The fi lm coating may be performed in different types of equipment. For coating
of larger units, such as tablets or capsules, a rotating drum is often used, for example
an Accela Coater, but other similar equipment may also be used [10, 53, 57, 61] .
This type of equipment contains a perforated pan that rotates and thus mixes the
bed of units. At the same time, a coating liquid is applied to the moving units by
means of a spray gun, where the mixing of the units ensures uniform coverage of
the coating. The fi lm coating is dried by blowing a stream of hot air onto the surface
of the tablets. For all types of coating processes, there are many parameters, such
as the temperature and relative humidity of the inlet air, drum rotation speed,
spraying rate, and droplet size of the coating liquid, that have to be adjusted in order
to produce good coated fi lms.
For coating smaller units, such as pellets, the fl uid bed coating technique is used
[56] . This is an attractive technique in which the starting material is placed in the
coater and heated air is blown through a base plate. This leads to vigorous mixing
of the pellet units. By changing the pressure of the incoming air stream, the material
becomes suspended in the air. This happens when the bed starts to fl uidize and the
bed will then have fl uidized properties similar to the properties of ordinary liquids.
There are different designs of fl uid bed: top spray coating, bottom spray coating (or
Wurster coating), or tangential coating. They differ with regard to placement of the
spray guns. In top spray coating, the liquid is sprayed from the top of the equipment
and the droplets hit the particles moving in opposite direction. In the bottom spray
coating, the sprayed liquid drops and particles fl ow in the same direction, which
avoids the problem of blocking the spray guns that may occur in top spray equipment.
In the more rarely tangential spray coating equipment the base plate rotates
and the spray guns are spraying in a tangential direction to the spiral moving particles
[10, 62] .
A critical parameter for obtaining fi lms with desirable properties is the creation
of coating droplets, a process often referred to as atomization, that is, when a bulk
liquid is dispersed in air to form a spray or a mist [61] . Atomization is done by
letting pressurized air and bulk solution pass simultaneously through a nozzle (spray
MEMBRANE-COATED ORAL EXTENDED RELEASE 1205
1206 ORAL EXTENDED-RELEASE FORMULATIONS
gun). The air divides the droplets into smaller units, but in spite of the name atomization,
the droplets are dispersed not at atomic scale but rather in the nano - to
micrometer - scale range [61] .
The mechanism for fi lm formation depends on whether the polymer is dissolved
or dispersed as small latex particles in the solution. For both technologies the fi lm
formation process starts with wetting and spreading of the coating droplets on the
surface of the reservoir system [10] . For the case with polymer dissolved in solution,
the fi lm formation mechanism continues with evaporation of the solvent. This leads
to an increase of the polymer concentration, and at a certain limit the polymers
precipitate and a coated fi lm is formed. In the case of ER fi lms, the most commonly
dissolved polymer is ethyl cellulose and the most commonly used solvent is ethanol
but other organic solvents can also be used. The use of organic solvent is problematic
from the SHE (safety, health, and environmental) point of view and therefore
these aspects must be considered before starting to use organic - based coating
process technologies. An alternative process methodology is to disperse particles
of the fi lm - forming polymer in an aqueous solution [63] . Film formation of dispersed
particles undergoes the following steps: evaporation of water, close packing of the
particles, deformation of the particles, and annealing of the particles by migration
of individual polymer chains between the particles to form a coherent, smooth fi lm
coating. The fi rst steps of the process occur in the coating equipment but the last,
the annealing step, may continue days after the coated product has left the coating
equipment.
6.8.4 HYDROPHILIC MATRIX TABLETS
6.8.4.1 Principles of Formulation and Release Mechanisms
Several recent informative review articles on hydrophilic matrix systems have been
published [e.g., 24 , 64 ]. Hydrophilic matrix tablets are composed of an active substance,
a hydrophilic polymer, release modifi ers, lubricants, and glidants. The technology
goes back to the mid - 1960s when Lapidus and Lordi [65, 66] and Huber
et al. [67] determined the drug release from hydrophilic matrix systems. The release
mechanism for this type of formulation starts with dissolution of hydrophilic matrix
polymers and the formation of a highly viscous polymer layer around the tablet
(Figure 5 ) [64] . This layer is often referred to as a gel layer even though it normally
contains only physical entanglements and not chemical cross - linkers, which is traditionally
required for gels. However, the gel layer surrounds the inner (more or
less dry) part of the tablet and this part is called the core. In traditional hydrophilic
tablets, the active substance particles are embedded in the matrix carrier. The dissolution
process of the active substance can start when the carrier material has dissolved
in water and formed the aqueous gel layer, since without exposure to water
the active substance cannot dissolve. Therefore, the “ dry ” core will shield the active
drug from dissolution, which is one reason for the extended drug release from this
type of formulation.
The release process for hydrophilic matrix tablets can be schematically described
as in Figure 5 . The left side of the fi gure shows a hydrophilic tablet undergoing dissolution,
swelling, and release. An interface between the solution and gel layer, here
called the erosion front, can be identifi ed, and the polymer chains and drug molecules
are released at this front. In the gel layer, the polymer concentration will
decrease (Figure 5 ) from a highly concentrated solution at the swelling front, the
interface between the gel layer and more or less dry core, to a diluted polymer
solution at the erosion front [68] . Some authors have suggested that the polymer
concentration at the erosion front is related to the overlap concentration [68 – 70] .
At the same time, the water content in the gel layer gradually increases from the
center of the tablet toward the erosion front, and thus the dissolution of the active
substance particles can start already in the gel layer. However, the volume fraction
of dissolved drug depends on the amount of available water, which in turn is a function
of the position in the gel layer [64, 71] . Assuming that the drug saturation
concentration is equal in water solutions and polymer gels, the volume fraction
dissolved drug will correlate with the volume fraction water available, and the
volume fraction drug will gradually increase with increasing distance from the core.
Far from the core, the variations in the concentration of polymer and water are less
pronounced and, as long as solid particles coexcite with the saturated drug solution,
the volume fraction of the drug will theoretically be almost constant. A third front
has also been introduced, the diffusion front, which corresponds to the position in
the gel layer where all of the active substance has dissolved. Between the swelling
and dissolution front, dissolved and undissolved drug particles will coexist, but
between the dissolution and erosion fronts, only dissolved drug molecules occur. In
this region, the diffusion of drug out from the matrix will give rise to a decrease in
the volume fraction of the drug.
Achieving a mechanistic understanding of drug release from hydrophilic matrix
tablets is not a trivial task since the release depends on the properties of both the
polymer and the drug. The swelling process is directly related to the properties of
the polymers, and this is an important factor for drug release, since the polymer
swelling process can, for example, transport individual drug particles through the
FIGURE 5 ( a ) Hydrophilic matrix system shown with core (dark gray) and drug parti cles
(small dark gray particles). The swelling, diffusion, and erosion fronts are depicted.
( b ) Dependencies of volume fraction polymer (solid line) and dissolved drug (broken line)
as function of position in matrix system together with swelling, diffusion, and erosion fronts.
Top shows how the solid drug particles diminish in size. ( c ) Examples of drug release as
function of time for erosion - controlled ( n = 1, solid line) and diffusion - controlled systems
(broken line, n = 0.5).
0
25
50
75
100
Time (h)
Fraction released (%)
Swelling front Diffusion front Erosion front
(a) (b)
0 6 12 18 24
(c)
HYDROPHILIC MATRIX TABLETS 1207
1208 ORAL EXTENDED-RELEASE FORMULATIONS
gel layer, which has been shown by Adler et al. [72] . Macroscopically, polymer
swelling can be observed as an increase in the size of the tablet. On a molecular
level, the swelling depends on the dilution and transport of water into the gel layer.
This transport is driven by changes in the chemical potential, and the main contribution
is the increase of conformational entropy when the polymer chains are diluted
[73] . The kinetics of the swelling process may vary. A faster polymer release rate
compared to the swelling rate results in a movement of the erosion front toward
the center of the tablet and the size of the tablet will diminish. Conversely, the tablet
size increases when the polymer release is slower than the swelling process. When
the swelling rate is on the same order as the polymer release rate, the position of
the erosion front (i.e., tablet size) will remain constant.
The swelling front between the gel layer and amorphous (or semicrystalline) core
material has traditionally been described as corresponding to a transition of the
solid states of the polymers. The polymers in the core, for example in HPMC tablets,
are in a glassy state, and the polymer material is transformed to a rubbery state due
to the fact that water acts as a plasticizer and decreases the glass transition temperature
[74] . This rubbery state can be regarded as a polymer solution, and therefore
the glassy - to - rubbery state transition can be regarded as a dissolution process
of the polymer, where the dissolution rate will determine the position of the swelling
front. A commonly described special case for hydrophilic tablets is the so - called
front synchronization, which is when the movements of the swelling front and the
erosion front occur equally fast. This special case corresponds to a constant gel layer
thickness.
Depending on the drug solubility and the dissolution, swelling, and release processes
of the polymer, either of two different drug mechanisms can be observed:
erosion - or diffusion - controlled drug release (Figure 5 ). One way to characterize
these two mechanisms is to compare the drug and the polymer release. The erosion
mechanism is characterized by equal release of the polymer and the drug, whereas
when the release of the drug is faster than that of the polymer, this is called diffusion
- controlled drug release [64] . The diffusion - controlled mechanism occurs when
the diffusion front, the border between undissolved and dissolved drug, is displaced
in the gel layer and the drug can effi ciently diffuse out from the gel layer. The
erosion mechanism dominates when the diffusion and erosion fronts overlap. This
means that drug particles may be released from the surface of the gel layer. When
this occurs, the drug particles will dissolve faster in the free solution than in the gel
layer due to the fact that stirring is more effi cient in the free solution, which results
in a decreased thickness of the unstirred boundary layer around the particles and
thus an increased dissolution rate [Equation (1a) ]. Whether the release mechanism
will be diffusion or erosion controlled depends on (i) the polymer release rate,
which governs the position of the rate erosion front, and (ii) the drug dissolution
rate, which governs the position of the diffusion front. The position of the diffusion
front and the dissolution rate depend on the solubility of the drug. Lower solubility
of the drug gives slower dissolution rates and hence the diffusion front can overlap
more easily with the erosion front, which yields erosion - controlled drug release. On
the other hand, large solubility of the drug will give diffusion - controlled drug release
[75 – 78] .
Traditionally, the drug release rate from hydrophilic matrix systems has been
modeled as [79, 80]
Q
dM
dt
dt kt t n = = .0
(9)
where Q is the accumulated amount of released drug and k and n are constants.
The values of n have been suggested to describe the drug release mechanisms.
Release from a planar surface with n = 1 has been shown to correspond to erosion -
controlled drug release and n = 0.5 to pure diffusion - controlled drug release. This
is strictly only valid when the polymer release has n = 1, but this is often the case.
In practice, for hydrophilic matrix systems, one often fi nds n to be between 0.5
and 1, indicating that both the diffusion of the drug and the polymer erosion infl uence
drug release. For other geometric shapes, such as tablet shapes, the limits for
n shift to 0.45 and 0.89 for diffusion - and erosion - controlled release, respectively
[81 – 83] .
Another popular way to describe the drug release is to characterize the infl uence
of the relative contributions of erosion and diffusion to drug release as [84]
Q at bt m m = + 2 (10)
where a , b , and m are constants. The fi rst factor in Equation (10) should represent
the Fickian diffusional contribution and the second term to the erosion contribution
to the drug transport. One mechanistic drawback with this approach is that it treats
the diffusion and erosion processes as independent of each other, which they are
probably not in any practical case.
The drug release from hydrophilic matrix tablets has been found to vary with the
polymer parameters, the composition of the formulation, and the process parameters.
Examples of important polymer - related parameters with signifi cant infl uence
on drug release are the viscosity and the hydrophilicity of the polymer, where polymers
of larger viscosity grades give lower release rates and longer durations of the
release [85] and an increased hydrophilicity gives larger swelling and faster drug
release, which was found by comparing different degrees of substitution of HPMC
[86] . The drug release rate also depends on the composition of the formulation.
When components with high water solubility, such as lactose, are included in the
matrix, the drug release increases, which can be seen as a dilution of the gel - forming
material [69, 87] . Similarly, an increased amount of a soluble active substance also
increases the release rate of drugs, probably also due to the corresponding decrease
of the relative amount of hydrophilic polymer [88, 89] . The size and shape of the
tablet also infl uence drug release, and the release rate increases as the area - to -
volume ratio increases [90 – 93] . The infl uence of particle size on the drug release
rate has also been investigated [89, 94, 95] . The size of polymer particles seems to
have low infl uence on drug release when there is enough gelling polymer available
to quickly form a coherent gel layer. In contrast, at low amounts of hydrophilic
matrix polymer, gel formation may be too slow, which makes polymer particle size
important. In this case, the matrix may disintegrate before it develops a coherent
gel layer [95 – 97] . The effect of drug particle size on the drug release rate depends
on the solubility of the substance. Varying the particle size of drugs with high solubility
seems to have little infl uence [89] , whereas the release rate may depend on
the particle sizes of drugs with moderate solubility [24, 89] .
HYDROPHILIC MATRIX TABLETS 1209
1210 ORAL EXTENDED-RELEASE FORMULATIONS
The effects of process parameters on drug release have been discussed in the
literature. Different granulation technologies, such as dry granulation [98 – 100] or
wet granulation [101 – 103] (which includes fl uid bed granulation [104, 105] ), have
been used. Also direct compression has been used for production of hydrophilic
matrix tablets [95] . The effects on the choice of production steps may be critical
when the relative fraction of the polymers is low in the formulations. The effect of
the compaction pressure on drug release has also been studied. Several authors [89,
88, 106, 107] have found that, while the compaction pressure has a signifi cant effect
on the tensile strength of the tablets, it has a minimal infl uence on drug release.
This can be due to the fact that, when a coherent gel layer is formed, only the
parameters governing the performance of this gel layer are important, and since
parameters such as porosity do not affect the gel layer, they are of low importance
for the drug release rate.
6.8.4.2 Manufacturing of Hydrophilic Matrix Tablets
The traditional way of producing hydrophilic matrix tablets resembles the production
of the core for membrane - coated tablets and insoluble matrix tablets. It includes
a mixing step, possibly a granulation step, a compaction step, and sometimes a
coating step. However, one large difference between the production of insoluble
and soluble matrix systems is notable; the latter matrix type has strong interactions
with water, which complicates the production steps when water is present [108] .
Therefore, for hydrophilic matrix systems with large fractions of hydrophilic polymers,
traditional wet granulation with water as granulation liquid may cause problems
with formation of hard lumps [108] . To avoid this problem, a new technique
using foam granulation has been suggested [109, 110] . During granulation, the
foams will fl ow on the top of already foam - wetted particles, which may lead to
superior distribution of the granulation liquid. An alternative wet granulation
method is to use organic solvents such as ethanol as granulation liquid [111] .
However, even if the production of granules with good compaction properties can
be maintained in this way, it may, as already mentioned, be an advantage to consider
the SHE aspects before choosing organic solvents as granulation liquid. Another
alternative to wet granulation is to use dry granulation technologies such as roller
compaction [98, 99, 100] . Although this technique has several advantages compared
to traditional wet granulation with water, it may result in lower tensile strengths of
the tablets [112] . An alternative to granulation technologies is direct compression.
This can be done by purchasing special direct - compression qualities of the hydrophilic
polymers. A drawback that always arises in relation to direct compression is
the diffi culty to achieve content uniformity of the tablets, a problem that increases
with decreasing doses of the active drug. Therefore, special care should be taken
with regard to formulations with low doses of active substance when direct compaction
is used [2] .
Water - soluble hydrophilic matrix systems may also be extruded, both to
monolithic systems and to multiple - unit systems [12, 14 – 16] . Polyethylene oxide
(PEO) and chloropheniramine maleate have, for example, been extruded to a
monolithic unit [113] . This manufacturing method proved more feasible for mixtures
between low - and high - molecular - weight PEO, since systems containing only
high - molecular - weight PEO proved too viscous and diffi cult to extrude. It was also
shown that degradation of PEO to lower molecular weight might occur due to the
high temperature used in the extruder. Extrusion to small pellets based on hydrophilic
polymers hydroxyethyl cellulose (HEC) and HPMC has been performed, and
isopropyl alcohol instead of water was used to avoid lump formation [12] . Also
Carbopol [14 – 16] , pectin [114] , and xanthan [115, 116] have been extruded and used
as matrix carrier materials for extended release.
The choice of hydrophilic polymer is one determining factor for the drug release
rate, as discussed above. The most common type used for hydrophilic matrix systems
is HPMC (hypromellose, hydroxypropyl methyl cellulose) [24, 83] but some alternatives
are HEC [117 – 119] , HPC [120, 121] , methyl cellulose [121] , xanthan [122 – 127] ,
PEO [68, 128] , Carbopol [129 – 131] , pectin [131 – 133] , and alginate [134 – 136] ; see
Table 2 . HPMCs are available in several approved degrees of substitution [60] . To
achieve different release durations, different viscosity grades of the polymers may
be used (the higher the viscosity grade, the slower the release rate). If the desired
viscosity is not available, one can mix two polymer grades with different viscosity
grades [68] .
6.8.5 COMPARISON AND SUMMARY OF DIFFERENT TECHNOLOGIES
The main principles for oral extended drug release reviewed here are the
membrane - coated reservoir systems and hydrophilic or hydrophobic matrix systems.
They all have advantages and drawbacks and Table 3 summarizes some aspects of
the different formulation principles.
There are a number of commercial variants of the above - mentioned principles.
Examples of commercial matrix - based formulations can be found in a review by
Varma et al. [137] . One of the oldest commercial hydrophilic matrix systems is
TABLE 2 FDA Registered Oral ER Formulations Containing Commonly Used
Excipients in Hydrophilic Matrix Tablets
Hydrophilic Polymer a
Fraction of
Excipient (%)
Number of Hits on
FDA Homepage
Other Names
of Excipients
Hydroxypropyl methyl
cellulose [24]
10 – 80 12 Hypromellose,
HPMC,
MHPC
Hydroxypropylcellulose
[120, 121]
15 – 35 4 HPC
Polyethylene oxide [68, 128] 5 – 85 4 PEO
Sodium alginate [134] 5 – 50 3
Xanthan gum [125, 137 – 140] 10 – 99 3
Hydroxyethyl cellulose
[117 – 119]
40 – 97.5 3
Carbomer [129] 20 – 75 1 Carbopol,
polyacrylic
acid
Methyl cellulose [121] 5 – 75 1 MC
Source : http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm .
a Examples of references using them are given.
COMPARISON AND SUMMARY OF DIFFERENT TECHNOLOGIES 1211
1212 ORAL EXTENDED-RELEASE FORMULATIONS
TABLE 3 Comparison between Three Drug Release Principles
Aspects
Insoluble Matrix
Systems
Membrane - Coated
Systems
Hydrophilic Matrix
Systems
Drug solubility Dose dependent
[18]
Dose dependent
[18]
Whole range
Release
mechanism
Diffusion controlled Diffusion and
osmotic
Diffusion to erosion
controlled
Release profi le Q . t0.5 Q . t1 Q . tn , 0.4 < n < 1
Main release
dependence
(except drug
properties)
Channeling
components and
processes
parameters
Properties of
membrane
Properties of
polymer carrier
Composition
alternatives
Many approved and
functional
excipients
available
Pore - channeling
excipients may
determine drug
release
Small changes in
formulation may
change release
rate
Many approved
and functional
excipients
available
Release can be
changed by fi lm
and core
compositions
Many polymers are
based on natural
material which
may give large
batch - to - batch
variation
Many grades of
available polymers
exist
Easy to regulate drug
release by means
of composition and
polymer properties
Many polymers are
based on natural
material, which
may result in large
batch - to - batch
variation
Manufacturing
aspects
Uses traditional
production
technologies and
is inexpensive
Release depends on
process
parameters
Coating process can
be used to
control drug
release
Film coating
process
dominates
release, which
may be sensitive
in relation to
many different
process
parameters
Release is mainly
robust against
process parameters
Granulation with
water is diffi cult
In vivo – in
vitro
correlation
Release depends on
fasted and fed
state
Good Release depends on
fasted and fed
state
TIMERx, which is based on xanthan gum mixed with, for example, locus bean gum
[138] . A mixture of these polymers has a special property; the different polymers
interact strongly with each other and this interaction produces gels with large viscosity.
Variants of osmotically controlled ER formulations, some of which are commercially
available, are reviewed by Verma et al. [52] .
6.8.6 OTHER ORAL ER FORMULATIONS
Here some new oral ER technologies based on principles other than those mentioned
above will be presented. It is beyond the scope of this chapter to cover all
systems and details thereof, and the interested reader is recommended to make use
of the literature included in the references.
One promising technology is to let the active drug interact with the excipients,
for example, by covalent binding between the drug and excipient. The azoaromatic
cross - linkers between the drug and excipient can exemplify this. These special cross -
linkers break due to bacterial degradation in the colon, but not until they are
exposed to this bacterial fl ora [139 – 141] . This means that the drug is hindered from
release before it reaches the colon. This is an example of site - specifi c delivery to
the colon, an area that is reviewed by a special issue of the journal Advanced Drug
Delivery Reviews 2005 (volume 57, number 2).
Another example of modifi cations of ER systems is when electrostatic interaction
between charged drugs and excipients (with opposite charge) is used. This
concept can be valuable for drugs with pH - dependent solubility. If, for example,
the drug is a weak base, it will have a large solubility at low pH, and a major part
of the drug may be rapidly released already in the stomach, which is not desirable.
An introduction of polyions with opposite charge can result in electrostatic interaction
between the drugs and the polyions, which can change the solubility or decrease
the diffusion rate of the drug through the ER formulation. This concept is, for
example, used when propranolol hydrochloride interacts with sodium carboxymethylcellulose
(NaCMC) in HPMC matrices [142] . In addition, buffering of the ER
systems by organic or inorganic buffers has been used to obtain pH - independent
drug release [27, 143, 144] . A recent publication by Riis et al. showed that insoluble
inorganic buffers such as magnesium hydroxide and magnesium oxide provided
stable drug release over longer periods of time than when more soluble buffer
systems were used [26] .
Sophisticated systems based on responsive gels are promising alternatives in
terms of oral extended release. The principle behind many of these systems is that
the formulation should react and undergo some kind of transition due to a trigger
signal [145] . In general, the triggers can be various factors such as temperature, light,
pressure, electrosensitivity, or interactions with specifi c molecules such as glucose
or antigens. For oral drug delivery systems, the triggers can also be a biological
change in the gastrointestinal environment, such as a change in the bacteria fl ora,
as mentioned earlier [139] . It can also be a physiochemical change, such as a pH
change in the gastrointestinal tract. Hydrogels composed of copolymers of
poly(acrylic acid) and covalently attached Pluronic surfactants is one such example
that reacts on pH changes. The cross - linked microgels can be loaded with drug and
tailored to collapse in low pH but swell and release the drug at high pH [146] .
A novel method for producing ER formulations is a technology called three -
dimensional printing (TheriForm technology) [147] , which is similar to the one used
in ordinary printers. The ink is here replaced with an active substance and carrier
material. The layer - by - layer “ printing ” provides controlled placement of the active
drug and thus of the release from the device [148] .
Even if there have been advances in oral drug delivery technologies during the
last 50 years, many highly sophisticated drug delivery systems have failed and have
OTHER ORAL ER FORMULATIONS 1213
1214 ORAL EXTENDED-RELEASE FORMULATIONS
not reached the market [149] . This can be due to several reasons and in many cases
the formulation is probably not the cause. However, on occasion one may be
tempted to agree with Rocca and Park in their review of prospects and challenges
in the oral drug delivery: “ breakthrough technologies are required to generate novel
dosage forms raising drug delivery to higher level ” (p. 52) [149] .
ACKNOWLEDGMENT
Sven Engstr o m, Chalmers University of Technology, is acknowledged for his useful
suggestions and comments.
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ROLE OF NANOTECHNOLOGY
SECTION 7
1225
7.1
CYCLODEXTRIN - BASED
NANOMATERIALS IN
PHARMACEUTICAL FIELD
Erem Bilensoy and A. Atilla Hincal
Hacettepe University Faculty of Pharmacy, Ankara, Turkey
Contents
7.1.1 Introduction
7.1.1.1 Cyclodextrins in Pharmaceutical Field
7.1.2 Application of Cyclodextrins in Nanoparticles
7.1.2.1 Incorporation of Drug – Cyclodextrin Complexes in Nanoparticulate Delivery
Systems
7.1.2.2 Preparation of Nanoparticles from Cyclodextrins
7.1.2.3 Effi cacy and Safety of Amphiphilic Cyclodextrin Nanoparticles
7.1.3 Conclusion
References
7.1.1 INTRODUCTION
Cyclodextrins (CDs) have a wide range of application in the pharmaceutical fi eld
due to their unique structure, which allows them to include hydrophobic molecules
in their apolar cavity and to mask the physicochemical properties of the included
molecule. This results in the enhancement of drug bioavailability by improving
aqueous solubility and the physical and chemical stability of the drug, masking
undesired side effects such as irritation, taste, or odor, and overcoming compatibility
problems or interactions between drugs and excipients.
Parallel to the increasing interest and successful licensing and commercialization
of nanoparticulate pharmaceutical products, CDs have also been incorporated
into nanoparticulate drug delivery systems for several purposes. This can be
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1226 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
achieved by two approaches: (1) complexation of active ingredient with an appropriate
CD and entrapment into polymeric nanoparticles to solve problems arising
from the drug ’ s physicochemical properties or (2) modifi cation of CDs to render
an amphiphilic character to these molecules, which allows CDs to self - align into
nanoparticles in the form of nanospheres, nanocapsules, solid lipid nanoparticles,
nanosize liposomes, and nanosize vesicles with or without the presence of surface -
active agents.
In light of current research, this chapter will deal with the following issues
concerning the use of CDs and derivatives as nanomaterials for drug delivery:
use of CDs (natural and synthetic) derivatives in the pharmaceutical fi eld and
application of CDs in nanoparticulate drug delivery systems. A major part of the
chapter will be focused on new CD derivatives, amphiphilic CDs, and the characterization,
effi cacy, and safety of nanoparticles prepared from amphiphilic
CDs.
7.1.1.1 Cyclodextrins in Pharmaceutical Field
Natural Cyclodextrins Cyclodextrins are cyclic oligosaccharides obtained by the
enzymatic degradation of starch. Major natural CDs are crystalline, homogeneous,
nonhygroscopic substances which have a toruslike macroring shape built up from
glucopyranose units, as seen in Figure 1 [1 – 3] . Cyclodextrins are named depending
on the number of glucopyranose units. Major industrially produced CDs are named
as follows; . - CD, possessing six units, . - CD, possessing seven units; and . - CD, possessing
eight units.
Natural CDs have been demonstrated to have a special structure; that is, glucose
residues in the CD ring possess the thermodynamically favored 4 C 1 chair conformation
because all substituent groups are in equatorial position. Cyclodextrins behave
like rigid compounds with two degrees of freedom: rotation at the glucosidic links
C(4) – O(4) and C(1) – O(4) and rotations at the O(6) primary hydroxyl groups at the
C(5) – C(6) band. As a consequence of this chair conformation, all secondary hydroxyl
groups at C(2) and C(3) are located at the broader side of the CD torus in the
equatorial position. Hydroxyl groups on C(2) point toward the cavity and hydroxyl
groups on C(3) point outward. The primary hydroxyl groups at the C6 position are
located at the narrower side of the torus. These hydroxyl groups ensure good water
solubility for the natural CDs. The cavity of the torus is lined with a ring of C . H
groups (C3), a ring of glucosidic oxygen atoms, and another ring of C . H groups
(C5). Thus, the cavity of CDs exhibits an apolar character. This is accompanied by
a high electron density and Lewis base property. The physicochemical characteristics
and inclusion behavior of CDs are a direct consequence of these special binding
conditions [4, 5] . Certain physicochemical characteristics of natural CDs are given
in Table 1 .
Cyclodextrin Derivatives Natural CDs were reported to form total or partial
inclusion complexes with several drugs to improve the aqueous solubility and stability
under physiological or ambient conditions, reduce or mask completely the side
effects associated with the included drug, and increase compatibility of the drug
with other drugs in the formulation or excipients while improving patient compliance
by masking the taste or odor of the active ingredient [6 – 8] .
FIGURE 1 Schematic representation of natural CD structure and modifi cation sites.
HOCH2
HOCH2
CH2OH
CH2OH
CH2OH
CH2OH CH2OH
O
O
O
O
O
O O
O
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
OH
HO
OH
OH
OH
OH
OH
OH
6
2
3
OH (6)
OH (3) OH (2)
Primary face
Secondary face
Apolar cavity
Modification
sites
INTRODUCTION 1227
TABLE 1 Some Physicochemical Characteristics of Natural Cyclodextrins
Characteristics . - CD . - CD . - CD
Number of glucose units 6 7 8
Molecular weight, g/mol 972 1135 1297
Internal diameter, A 4.7 – 5.2 6.0 – 6.4 7.5 – 8.3
External diameter, A 14.2 – 15.0 15.0 – 15.8 17.1 – 17.9
Depth, A 7.9 – 8.0 7.9 – 8.0 7.9 – 8.0
Solubility in water (25 ° C), g/L 145 18.5 232
Crystal water, w % 10.2 13.2 – 14.5 8.13 – 17.7
Approximative cavity volume in 1 mol
CD, A 3
174 262 472
Melting point, ° C 250 – 260 255 – 265 240 – 245
Half - life in 1 M HCl at 60 ° C, h 6.2 5.4 3.0
Crystal forms (from water) Hexagonal
plate
Monoclinic
parallelogram
Quadratic
prism
Partial molar volumes in solution, mL/mol 611.4 703.8 801.2
1228 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
In contrast to the advantageous nature of CDs for molecular inclusion, their
surface makes it more diffi cult for the highly hydrophilic CD molecule to interact
with lipophilic biological membranes. For this reason, natural CDs have been chemically
modifi ed to alter their water solubility, interaction with biological membranes,
and drug release properties.
Two of the natural CDs are known to be parenterally unsafe due to nephrotoxic
effects [9] . The etiology of the nephrotoxicity of . - and . - CD is unknown but is
believed to be related to either CD uptake by kidney tubule cells resulting in disruption
of intracellular function or the extraction of lipid membrane components
by the CDs. The latter is suggested to be of validity since there seems to be a linear
correlation between the ability of some CDs to disrupt cellular membranes and
kidney nephrotoxicity [2, 6] . The ability of CDs to cause red blood cell hemolysis
and membrane irritation seems also to correlate with their ability to extract lipid
membrane components: cholesterol and phospholipids [10, 11] .
Modifi cation of natural CDs has been the aim of many research groups to
improve safety while maintaining the ability to form inclusion complexes with
various substrates. Some groups have also focused on improving the interaction
between the pharmaceuticals and the CDs while others have attempted to prepare
materials that can be chemically defi ned more precisely.
Methylated CDs are obtained by methylation of CDs on either all C2 secondary
and C6 primary hydroxyl groups [dimethyl cyclodextrins (DIMEB)] or all the
hydroxyl groups C2, C3, and C6 [trimethyl cyclodextrins (TRIMEB)]. Major disadvantage
of methylated CDs is that their solubility decreases with increasing temperature
and they are reported to be hemolytic [12] . This is a result of partial
methylation of the hydroxyls of . - CD which leads to stronger drug binding but also
to stronger hemolysis [12] .
Hydroxypropylated CDs are statistically substituted derivatives because hydroxypropylation
does not result in selective substitution as with methylation. While the
reaction proceeds, the reactivity of the hydroxyl group changes, and this results in
a mixture of products with various degrees of substitution. Their dissolution is
endothermic so there is no decrease in solubility with increasing temperature
[6, 13] . It is necessary to note that degree of substitution in hydroxypropylated CDs
is inversely correlated with their inclusion capability [14, 15] . Hydroxyalkylated CDs
are commercially available as tablets, ocular collyrs [16] , and excipients under the
trademarks Encapsin and Molecusol.
Sulfobutylether - . - cyclodextrins (SBE - . - CDs) are water soluble and parenterally
safe. An advantage over hydroxypropylated - . - CDs (HP - . - CDs) is that higher sulfobutyl
group substitution often results in higher drug complexation ability [17] .
Inability of the SBE - . - CDs, especially the commercially available product (SBE) 7M -
. - CD (Captisol), to form strong complexes with cholesterol and other membrane
lipids, arising from their polyanionic nature causing Coulombic repulsions, results
in a little or no membrane disruption [6, 13] and reduced hemolysis [18] . Captisol
is used in parenteral and ocular systems as well as osmotic tablets and also as a
freeze - drying excipient [19] .
Branched CDs (mono - or di - glucosyl, maltosyl and glucopyranosyl . - and . -
CDs) are more resistant to . - amylase than natural CDs. Natural . - CD and monoglucosyl
-. - CD are stable in rat blood because they have no linear glycosidic bond.
Branched CDs exert a lower hemolytic activity on human erythrocytes and are
APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 1229
weaker than natural CDs. Their inclusion capability is more or less drug dependent.
Steroids were reported to show a slightly higher affi nity to branched CDs than to
natural CDs [19] .
The water solubility of acylated CDs decreases proportionally to their degree of
substitution, whereas their solubility in less polar solvents such as ethanol increases.
Acylated CDs of hydrophobic nature are useful for controlling the release rates of
water - soluble drugs [20] .
Ionizable . - CDs possess interesting solubility properties, too. Solubility in water
for these derivatives depends on the pH of the solution. Carboxymethylethyl - . -
cyclodextrin (CME - . - CD) is prepared as an enteric - type carrier system. The presence
of a carboxymethyl group causes a pH - dependent solubility range in water,
meaning that it is only slightly soluble in the low - pH region, but freely soluble in
neutral and alkaline regions due to the ionization of the carboxyl group (p Ka about
3 – 4) [21] . Inclusion - forming capability of CME - . - CD is dependent on drug properties,
including size, shape, and charge of the molecule.
Sulfated CDs are of anionic nature and are interesting from chemical and biological
points of view because of their angiogenic and antiviral properties [22, 23] . Sulfated
derivatives are also reported to have a heparin - like activity, resulting in
increase in blood - clotting times, which limits their injectable dose [7] .
Low - molecular - weight CD polymers (MW 3000 – 6000 Da) are soluble in water
whereas high - molecular - weight CD polymers (MW > 10,000 Da) can only swell in
water and form insoluble gels [24, 25] . Insoluble cross - linked bead polymers seem
to be applicable as wound - healing agents for the treatment of wounds like burns or
ulcers. Iodine has been complexed with such a CD polymer as an antiseptic wound
healing agent [24] .
7.1.2 APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES
7.1.2.1 Incorporation of Drug – Cyclodextrin Complexes in
Nanoparticulate Delivery Systems
Nanoparticles are of pharmaceutical interest due to their active and passive targeting
properties and their ability to deliver poorly solube drugs and drugs with stability
problems. Nanoparticles are considered more stable than liposomal delivery
systems. However, a major drawback is associated with the drug - loading capacity
of polymeric nanoparticles. Classical emulsion polymerization procedures result in
considerably low drug - loading capacities. This results in the administration of excessive
quantities of polymeric material which may impair the safety of the drug delivery
system [26, 27] .
For this reason, different techniques are used to improve the drug - loading
properties of polymeric nanoparticles. Cyclodextrins are used for this reason to
improve water solubility and sometimes the hydrolytic or photolytic stability of
drugs for better loading properties [8] . Drug – CD complexes act to solubilize or stabilize
active ingredients within the nanoparticles, resulting in increased drug concentration
in the polymerization medium and increased hydrophobic sites in the
nanosphere structure when large amounts of CDs are associated to the nanoparticles
[27, 28] .
1230 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
The antiviral agent saguinavir was complexed to HP - . - CD to increase saquinavir
loading into polyalkylcyanoacrylate nanoparticles by providing a soluble drug reservoir
in the polymerization medium that is the basis of nanoparticle formation [29] .
A dynamic equilibrium was observed between the complex, dissociated species of
the complex, and the forming polymeric nanoparticle. During nanoparticle formation,
free drug was believed to be progressively incorporated into a polymer network,
driven by the drug partition coeffi cient between the polymer and polymerization
medium. Simultaneous direct entrapment of some CD – drug complex was also suspected
[28 – 30] .
Incorporation of the steroidal drugs hydrocortisone and progesterone in complex
with . - CD and HP - . - CD reduced the particle size for solid lipid nanoparticles
(SLNs) below 100 nm. Steroids were demonstrated to be dispersed in the amorphous
state. Compexation to CDs resulted in higher drug - loading properties for the
more hydrophobic drug hydrocortisone and lower in vitro release for both drugs
when they are complexed to CDs rather than their free form [31] .
The in vivo behavior of nanoparticles obtained from drug – CD complexes was
also evaluated. HP - . - CD addition in the polymerization medium of polyethylcyanoacrylate
(PECA) nanospheres improved the subcutaneous absorption of metoclopramide
in rats. PECA nanospheres with HP - . - CD provided the highest drug
concentration and enhanced drug absorption compared with those with dextran or
with drug solution. However, in addition to drug absorption from subcutaneous
sites, HP - . - CD also enhanced the drug elimination by enhancing the drug absorption
to reticuloendothelial tissues [32] .
Progesterone complexed to HP - . - CD or DM - . - CD was loaded into bovine
serum albumin (BSA) nanospheres. Dissolution rates of progesterone were signifi -
cantly enhanced by complexation to CDs with respect to free drug. Nanospheres of
100 nm loaded with drug – CD complexes provided a pH - dependent release profi le
and good stability in an aqueous neutral environment [33] .
In another approach, CD properties of complexation were combined with those
of chitosan. Complexation with CD was believed to permit solubilization as well as
protection for labile drugs while entrapment in the chitosan network was expected
to facilitate absorption. Chitosan nanoparticles, including complexes of HP - . - CD
with the hydrophobic model drugs triclosan and furosemide, were prepared by ionic
cross - linking of chitosan with sodium tripolyphosphate (TPP) in the presence of
CDs. Nanoparticles were then prepared by ionotropic gelation using the obtained
drug – HP -. - CD inclusion complexes and chitosan. Cyclodextrin and TPP concentration
largely affected particle size but the zeta potential remained unchanged with
different parameters. On the other hand, drug entrapment increased up to 4 and 10
times by triclosan and furosemide, respectively. The release profi le of nanoparticles
indicated an initial burst release followed by a delayed release profi le lasting up to
4 h [34] .
Recently a CD – insulin complex was encapsulated in polymethacrylic acid – chitosan
– polyether[polyethylene glycol (PEG) – propylene glycol] copolymer PMCP
nanoparticles from the free - radical polymerization of methacrylic acid in the presence
of chitosan and polyether in a medium free of solvents or surfactants. Particles
had a size distribution of 500 – 800 nm. The HP - . - CD inclusion complex with insulin
was encapsulated into the nanoparticles, resulting in a pH - dependent release profi le
as seen in Figure 2 . The biological activity of insulin was demonstrated with enzyme -
APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 1231
linked immunosorbent assay (ELISA). Cyclodextrin complexed to insulin encapsulated
into mucoadhesive nanoparticles was believed to be a promising candidate for
oral insulin delivery [35] .
7.1.2.2 Preparation of Nanoparticles from Cyclodextrins
Cyclodextrins are used as excipients in the preparation of nanoparticles by three
approaches:
1. Preparing nanoparticles from polymers under the presence of CDs in the
medium
2. Preparing nanoparticles from polymers incorporating or conjugated to CDs
3. Preparing nanoparticles directly from amphiphilic CDs
Nanoparticles consisting copolymers of aminoethylcarbamoyl - . - cyclodextrin
(AEC - . - CD) and ethylene glycol diglycidyl ether (EGDGE) were prepared by an
interfacial polyaddition reaction in a mini – emulsion system. By combining these
two technologies, namely, cross - linking and modifi cation of hydroxyl groups, a novel
functional nanoparticle based on . - CD was introduced as a novel material of nanobiotechnology
[36] .
Nanoparticles prepared fromn CDs are promising targeted delivery systems.
Transferrin, an iron - binding glycoprotein ligand for tumor targeting, was conjugated
to . - CD polymers and adamantane – PEG5000 through carbohydrate groups self -
assembled into sub - 100 - nm particles in a recent study [37] . A CD - containing polyca-
FIGURE 2 pH - dependent release profi le for insulin complexed to HP - . - CD and encapsulated
in nanoparticles. ( Reprinted from S. Sajeesh and C. P. Sharma, International Journal of
Pharmaceutics , 325, 147 – 154, 2006, Copyright 2006, with permission from Elsevier. )
100
90
80
70
60
50
40
30
20
10
0
0 50 100 150 200 250 300 350
Time (min)
Percentage of release
HPCDI pH7.4
HPCDI pH1.2
I pH7.4
I pH1.2
1232 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
tion was used for nucleic acid condensation into nanoparticles [38] and the second
component, adamantane - terminated modifi er for stabilizing nanoparticles to minimize
interactions with plasma and to target cell surface receptors, was incorporated
in this system. The particles were demonstrated to mediate transferrin - mediated
delivery of nucleic acids to cultured cells [37] .
Transferrin - containing CD polymer - based nanoparticles were studied as nucleic
acid delivery system that can be modifi ed for targeted delivery of small interfering
ribonucleic acid (siRNA) to cancer cells. Molecular studies showed that the siRNA
CD nanoparticles reduced levels of Ewing ’ s transcript by 80% and inhibited growth
of cultured Ewing ’ s tumor cell line. It was also reported that this delivery system
indicated a lack of toxicity [39] .
A new tadpole - shaped polymer was synthesized via a coupling reaction of PLA
onto mono[6 - (2 - aminoethyl)amino - 6 - deoxy] - . - cyclodextrin (CDenPLA). A hydrophilic
head consisting of the CD group was believed to bind proteins and the PLA
tail gave the amphiphilic property [40] . BSA was incorporated into nanoparticles
of CDenPLA using both nanoprecipitation and double - emulsion techniques, as can
be seen in Figure 3 [40] . A similar process was used to couple PLGA onto amino -
. - CD and ethylenediamino - bridged bis( . - CD) to afford amphiphilic conjugates
forming nanoparticles with the nanoprecipitation technique. This approach was
believed to be promising for protein delivery since BSA structure was unchanged
FIGURE 3 TEM photographs of BSA - loaded . CDen47PLA nanoparticles prepared
with different techniques and their magnifi ed images. ( Reprinted from H. Gao, Y. W. Yang,
Y. G. Fan, and J. B., Ma, Journal of Controlled Release , 112, 301 – 311, 2006. Copyright 2006,
with permission from Elsevier. )
0.5 mm 100 nm
0.5 mm 100 nm
DE1 DE2
NP1 NP2
APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 1233
after encapsulation into nanoparticles and during its release. Nanoparticles were
reported to be stable after freeze drying [40, 41] .
Nanoassemblies were formed by mixing solutions of . - CD polymer and dextran
hydrophopbically modifi ed with alkyl chains (C12) and loaded with the model
hydrophobic drug benzophenone. Nanoassemblies were characterized as 110 – 190 nm
with relatively low drug - loading values ranging between 0.3 to 1.01% w/w. Authors
suggested that hydrophobic model drug and hydrophobically modifi ed dextran
compete for the apolar CD cavity; however, benzophenone does not impede the
hydrophobic dextran to interact with . - CD polymer to form supramolecular assemblies
at the nanoscale [42] .
Another group has worked on the oligo(ethylenediamino) - . - cyclodextrin modi-
fi ed gold nanoparticles (OEA - CD - NP) as a vector for DNA binding. Possessing
many hydrophobic cavities at the outer space, OEACD - NP was believed to have a
capability of carrying biological and/or medicinal substrates into cells. Presence of
the CD moieties was suggested to be the key parameter in the high affi nity to DNA
for the gold nanoparticles. In addition, CD moieties were demonstrated to reduce
the cytotoxicity of gold nanoparticles arising from the gold clusters that impair
plasma membrane functions and lead to cell death [43] .
Nucleic acid delivery was also studies by Park et al. using CD - based nanoparticles
prepared from . - CD - modifi ed poly(ethylenimine) (CD - PEI). The inclusion - forming
capability of . - CD was used in order to immobilize the nanoparticles on solid surfaces
(adamantine - modifi ed self - assembled monolayers). CD - PEI nanoparticles
were proposed as delivery systems onto solid surfaces to attain specifi c and high
affi nity loading. The interaction is schematized in Figure 4 [44] .
FIGURE 4 Schematic representation of . - CD – adamantane inclusion complex formation
and immobilization of CD nanoparticles on adamantine - modifi ed surface. ( Reprinted with
permission from ref. 44 . Copyright 2006 by the American Chemical Society. )
+
=
O O O
O
O
O
O O
O
O
O
O
O
O
OH
OH
OH
OH
OH
HO
OH
HO
HO
HO
HO
HO
HO
HO
HO HO
OH
OH
OH OH
OH
Scheme 1A
Adamantane .-cyclodextrin Inclusion complex
CD-PEI/DNA
complex CD-PEI
nanoparticle
Specific
binding with
high affinity AD-modified
surface
50-.m gold layer
Glass slide
1234 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
Amphiphilic Cyclodextrins Nanoparticles have been obtained spontaneously
from modifi ed CDs of amphiphilic structure since the last decade. This approach
differs from the previously discussed approaches in that amphiphilic CDs do not
require the presence of another polymer or macromolecule or even surfactants.
Amphiphilic CDs have been synthesized to solve problems of natural CDs that
limit their pharmaceutical applications. The main reasons for the synthesis of amphiphilic
CDs are as follows:
1. Enhancement of interaction of CDs with biological membranes through a
relative external hydrophobicity
2. Modifi cation or enhancement of interaction of CDs with hydrophobic drugs
arising from the high number of long aliphatic chains and by increasing
the number of hydrophobic sites for possible interactions with hydrophobic
molecules
3. Allowing self - assembly of CDs resulting in the spontaneous formation of
nanosize carriers in the form of nanospheres and nanocapsules
The unique advantage of amphiphilic CDs is that they possess self - assembling properties
that are suffi cient to form nanoparticles spontaneously without the presence
of a surfactant as well as the capability of including hydrophobic molecules in their
cavity and within the long aliphatic chains [45 – 47] . Amphiphilic CDs can be classi-
fi ed according to their surface charge.
Nonionic Amphiphilic Cyclodextrins Nonionic amphiphilic CDs are obtained by
grafting aliphatic chains of different length on the primary and/or secondary face
of the CD glucopyranose unit. Different derivatives depicted in Figure 5 are named
after their structure:
1. Lollipop CDs [48] are obtained by grafting only one aliphatic chain to
6 - amino - . - CD.
2. Cup - and - ball CDs were synthesized by the introduction of a voluminous group
such as the tert - butyl group which is linked to the end of the aliphatic chain
in order to prevent self - inclusion of the pendant group [49, 50] .
FIGURE 5 Schematic representation of some nonionic amphiphilic CDs.
Lollipop
CD
Cup-and-ball
CD
Medusa-like CD
Skirt-shaped CD
Bouquet-shaped
CD
APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 1235
3. Medusa - like CDs are obtained by grafting aliphatic chains with length between
C10 and C16 to all the primary hydroxyls of the CD molecule [51 – 53] .
4. Skirt - shaped CDs consist of . - and . - CDs per - modifi ed with aliphatic esters
(C2 – C14) on the secondary face [54 – 57] .
5. Bouquet - shaped CDs result from the grafting of 14 polymethylene chains to
3 - monomethylated . - cyclodextrin, meaning seven chains on each side of the
CD ring molecule [58] . Per(2,6 - di - O - alkyl) CDs where the alkyl chain may be
propyl, butyl, pentyl, 3 - methylbutyl, or dodecyl also take part in the bouquet
family [4] .
6. Cholesteryl CDs were recently introduced as more complicated derivatives
[59] . They have been designed assuming that CD is the hydrophilic head group
and cholesterol is the hydrophobic part.
Interfacial properties of nonionic amphiphilic CDs have been demonstrated by
different groups [60 – 62] . It was found that length, structure, and bond type of the
aliphatic chain play important roles upon the surface - active characteristics of amphiphilic
CDs. Alignment of the amphiphilic CD molecule at the air – water interface
was demonstrated to be aliphatic chains perpendicular to and a CD ring parallel
to the fi lm [62] . Inclusion - forming capability of nonionic amphiphilic CDs also has
been reported with various model molecules of a different nature. It was suggested
that leaving the wider side of the cavity, that is, the secondary face, unsubstituted
may facilitate entrance of the drug in the cavity of the amphiphilic CD [63 – 65] .
Cationic Amphiphilic Cyclodextrins Recently cationic amphiphilic CDs were
obtained and characterized carrying an amino group as an ionic group. Heptakis(2 -
. - amino - O - oligo(ethyleneoxide) - 6 - hexylthio) - . - CD, a “ stealth ” cationic amphiphilic
CD because of the oligoethylene glycol group it carries, was synthesized [66] . The
structural properties of cationic amphiphilic CDs were believed to be due to the
balance between hydrophobic tails such as thioalkyl chains and hydrophilic components
such as ethylene glycol oligomers. The presence of ethylene glycol chains in
particular was believed to increase the colloidal stability of the supramolecular
aggregates formed by cationic amphiphilic CDs. Amphiphilic alkylamino - . - and
. - CDs were also reported regarding their synthesis and characterization [67] .
A series of polyamino - . - CDs have been synthesized by Cryan et al. [68] and
complete substitution by amine groups at the 6 - position. Neutral CDs have been
shown to interact with nucleic acids and nucleotides and to enhance their transfection
effi ciency in vivo. Cationic CDs have shown even greater ability to bind nucleotides
and enhance delivery by viral vectors. The major advantage of polycationic
CDs and their nanoparticles is their enhanced ability to interact with nucleic acids
combined with their self - organizational properties [68] .
Anionic Amphiphilic Cyclodextrins Anionic amphiphilic CDs possess a sulfate
group in their structure to render an anionic nature to the molecule. An effi cient
regiospecifi c synthetic route to obtain acyl - sulfated . - CDs was introduced in which
the upper rim is functionalized with sulfates and the lower rim with fatty acid esters
[69] . These derivatives were able to form aggregates in aqueous medium.
Sulfated amphiphilic . - , . - , and . - CDs were demonstrated to form 1 : 1 inclusion
complexes with the antiviral drug acyclovir. Noncovalent interactions between
1236 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
acyclovir and nonsulfated amphiphilic CDs (nonionic amphiphilic CDs) appeared
to take place both in the cavity of the CD and inside the hydrophobic zone generated
by alkanoyl chains. However, in the case of sulfated anionic amphiphilic CDs,
the interactions appear to take place only in the hydrophobic region of the alkanoyl
chains [70] .
Fluorine containing anionic . - CDs were fi rst introduced by Granger et al. [71]
functionalized at the 6 - position by trifl uoromethylthio groups. They exhibit an
amphiphilic behavior at the air – water interface and are good candidates for a new
class of amphiphilic carriers. P e roche et al. [72] described the synthesis of new
amphiphilic perfl uorohexyl - and perfl uorooctyl - thio - . - CDs and their alkyl analogue,
nonanethio - . - CD. The ability of these products to form nanoparticles was
also investigated by photon correlation spectroscopy and imaging techniques such
as scanning electron microscopy (SEM) and cryo – transmission electron microscopy
(TEM).
Fluorophilic CD derivatives have been obtained as a result of combinations
of CDs and a linear perfl uorocarbon [73] . 2,3 - Di - O - decafl uorooctanoyl - . - CD was
obtained with a protection – deprotection synthetic method and characterized further
by thin - layer chromatography (TLC), Fourier transform infrared (FTIR) spectroscopy,
differential scanning calorimetry (DSC), elemental analysis, and time - of - fl ight
mass spectrometry (OF - MS).
7.1.2.3 Safety and Effi cacy of Amphiphilic Cyclodextrin Nanoparticles
Amphiphilic CDs yield nanoparticles spontaneously in the form of nanospheres
or nanocapsules depending on the preparation technique. Nanoparticles have been
manufactured using three different techniques. However, the nanoprecipitation
technique is generally preferred since it is a simple technique resulting in unimodal
distribution. The general preparation techniques for amphiphilic CD nanoparticles
are as follows:
1. Nanoprecipitation [74 – 76]
2. Emulsion/solvent evaporation [77]
3. Detergent removal [78]
Nanocapsules are also prepared according to the same techniques. Amphiphilic CD
and the oil Miglyol or benzyl benzoate are dissolved in suitable organic solvent
(acetone, ethanol). The solution is poured into aqueous phase under constant stirring
and the nanocapsules form spontaneously. Organic solvent is then evaporated.
Resulting nanocapsules vary in size between 100 and 900 nm according to the preparation
process and technological parameters [79] .
Particle sizes of nanocapsules are mostly affected by the size of the oil droplet
formed during the preparation along with the molar concentration and nature of
amphiphilic CD. Nanospheres, on the other hand, are not signifi cantly affected
by amphiphilic CD concentration and can be formed with very high concentrations
of amphiphilic CDs. The modifi cation site of the CD (primary or secondary face) is
infl uential for nanosphere size since modifi cations on the secondary face result in a
larger surface area. The presence and concentration of a surfactant such as Pluronic
F68 do not affect the particle size of nanospheres and nanocapsules [80] . Nano
APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 1237
spheres and nanocapsules of amphiphilic CDs were imaged with different microscopic
techniques such as cryo - TEM, atomic force microscopy (AFM), and scanning
transmission microscopy (STM). SEM imaging, on the other hand, results in shrinkage
or disruption of the nanoparticles due to electron bombardment. Figures 6 a
and 6 b present TEM photomicrographs after freeze fracture for 6 - O - CAPRO - . - CD
nanospheres and nanocapsules, respectively [62, 66] .
Drug loading into amphiphilic nanospheres and nanocapsules is governed by the
loading technique used. Amphiphilic CD nanoparticles can be loaded with the following
techniques:
1. Conventional Loading Drug solution is added to the organic phase during
preparation.
2. Preloading Nanoparticles are prepared directly from preformed drug –
amphiphilic CD complexes.
3. High Loading Nanoparticles are prepared directly from preformed drug –
amphiphilic CD complexes and further loaded by the addition of drug solution
in the organic phase.
A high - loading technique results in two - to threefold increase in drug entrapment.
Other factors infl uencing drug loading to amphiphilic CD nanospheres are
related to drug physicochemical properties such as drug – CD association constant
k 1:1 , representing the affi nity of the drug to the CD cavity, oil/water partition coeffi -
cient, and aqueous solubility. The affi nity of the drug to the CD cavity is correlated
with drug - loading capacity. Lipophilic drugs interact both with the CD cavity and
the long aliphatic chains situated on either the primary or the secondary face.
Drug release properties of amphiphilic CD nanospheres are affected by various
parameters, including drug lipophilicity, drug – CD association constant, and loading
technique with release profi les varying from 2 to 96 h depending on the above
parameters. Nanocapsules, on the other hand, exert somewhat different drug release
profi les that are mostly dependent on lipophilicity and aqueous solubility of the
drug. Lipophilicity of the drug is inversely correlated with the rate of release, as
seen in Figure 7 [81] . Nevertheless, preparing nanoparticles directly from preformed
FIGURE 6 Cryo - TEM images of 6 - O - CAPRO - . - CD nanospheres ( a ) and nanocapsules
( b ). [ ( a ) Reprinted from E. Memisoglu, A. Bochot, M. en, D. Duchene, and A. A. Hincal,
International Journal of Pharmaceutics , 252, 143 – 153, 2003. Copyright 2003 with permission
from Elsevier .)
(a) (b)
S
1238 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
inclusion complexes helped reduce the initial burst effect observed in general for
nanospheres due to their very large surface area.
Cancer Therapy Nanoparticles were fi rst prepared with the concept of targeting
colloidal carriers of nanosize to tumor tissues via the leaky vasculature in tumor
regions. Since then nanoparticulate drug carriers have been associated with cancer
therapy through passive and active targeting to cancer cells. Thus, amphiphilic CD
nanoparticles were mainly focused on cancer therapy and its different aspects.
Tamoxifen, an antiestrogen drug used for the fi rst - line and adjuvant therapy for
metastatic breast cancer as long - term chemotherapy, has been incorporated into
amphiphilic CD nanoparticles prepared using the amphiphilic CD, . - CDC6 seen in
Figure 8 in order to reduce the severe side effects associated with the nonselective
cytotoxicity of this drug. Tamoxifen citrate – loaded nanospheres and nanocapsules
with approximately 65% entrapment effi ciency liberated the drug with a controlled -
release profi le up to 6 h when the high - loading technique is used [82] . Anticancer
effi cacy of tamoxifen citrate – loaded nanospheres and nanocapsules was demonstrated
to be equivalent to tamoxifen citrate solution in ethanol against MCF - 7
human breast cancer cells. Transcription effi ciency of the tamoxifen citrate nanocapsules
and nanospheres was evaluated against MELN cells in the presence of 17 -
. - estradiol (E2) for the inhibition of E2 - mediated luciferase gene expression. It was
found that transcription effi ciency of tamoxifen citrate – loaded nanospheres and
nanocapsules were concentration dependent [83] .
Paclitaxel, an anticancer drug with bioavailability problems arising from its very
low aqueous solubility, its tendency to recrystallize when diluted in aqueous media,
FIGURE 7 In vitro release profi les of steroids with different physicochemical properties
from . - CDC6 nanocapsules (HCR HL, hydrocortisone high loaded, HCR CL, hydrocortisone
conventionally loaded; TST HL, testosterone high loaded; TST CL, testosterone conventionally
loaded; PRO HL, progesterone high loaded; PRO CL, progesterone conventionally
loaded).
Cumulative drug (% released from
Time (h)
0
20
40
60
80
100
120
0 2 4 6 8 12 14 16 18 20 22 24 10
nanocapsules)
HCR CL HCR HL TST CL TST HL PRO CL PRO HL
APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 1239
and solubilizers used in its commercially available injectable formulations, has been
loaded into nanoparticles prepared from amphiphilic . - CD modifi ed on the primary
face with 6C aliphatic esters, 6 - O - CAPRO - . - CD, seen in Figure 8 . Paclitaxel - loaded
6 - O - CAPRO - . - CD nanospheres and nanocapsules were characterized with a diameter
of 150 nm for nanospheres and 500 nm for nanocapsules with high entrapment
effi ciencies. Blank nanoparticles were proven to be physically stable in aqueous
dispersion for 12 months. The in vitro release of paclitaxel from nanoparticles was
completed in 24 h [84] . Amphiphilic . - CD nanoparticles were compared to the commercial
vehicle Cremophor EL in terms of hemolysis and cytotoxicity. 6 - O - CAPRO -
. - CD nanospheres in particular were found to be signifi cantly less hemolytic than
paclitaxel solution in the Cremophor vehicle on human erythrocytes. Cytotoxic
effects of blank nanoparticles were assessed against L929 mouse fi broblast cells and
a vast difference in cytotoxicity of up to 100 - fold reduction was observed for amphiphilic
CD nanoparticles.
Drug - loaded nanoparticles were also evaluated for their safety and effi cacy.
Paclitaxel - encapsulated 6 - O - CAPRO - . - CD nanospheres and nanocapsules were
evaluated for their physical stability in a one - month period in aqueous dispersion
form with repeated particle size and zeta potential measurements and AFM imaging
to evaluate recrystallization in aqueous medium. Paclitaxel - loaded amphiphilic CD
nanoparticles were found to be physically stable for a period of one month whereas
recrystallization occurs within minutes when diluted for intravenous (IV) infusion
[85] . Finally, paclitaxel - loaded amphiphilic nanoparticles were demonstrated to
show similar anticancer effi cacy against MCF - 7 cells when compared to paclitaxel
solution in a cremophor vehicle [85] .
Our group is currently working on the formulation of another potent anticancer
drug, camptothecin, that is clinically inactive due to its very low water solubility and
poor stability under physiological pH, which causes the drug to be converted from
its active lactone form to its inactive carboxylate form. Two different amphiphilic
FIGURE 8 Amphiphilic . - CD derivatives modifi ed with 6C aliphatic esters on ( a ) secondary
face, . - CDC6 and ( b ) primary face, 6 - O - CAPRO - . -CD.
O
6
2
3
OH
O
O
OH
O
C
C O
O
7
O
6
2
3
OH
OH
OH
O
O
C O
7
(a) (b)
1240 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
. - CD nanospheres, . - CDC6 and 6 - O - CAPRO - . - CD, have succeeded in maintaining
camptothecin in its active lactone form with considerable loading values and
release profi les prolonged up to 96 h [86, 87] .
Cationic amphiphilic CDs, heptakis[2 - . - amino - O - oligo(ethylene oxide] hexylthio
-. - CD nanoparticles, have encapsulated anionic porphyrins (TPPS) by entangling
these molecules within the aliphatic chains aligning both faces of the cationic
amphiphilic CD. These nanoparticles were demonstrated to preserve the photodynamic
properties of the entrapped photoactive agent. The photodynamic effi cacy
of the carrier/sensitizer nanoparticles was proven by in vitro studies on tumor HeLa
cells showing signifi cant cell death upon illumination with visible light [88] .
Oxygen Delivery Amphiphilic and fl uorophilic . - CD derivatives perfl uoro - . - CDs
were used to prepare nanocapsules with a single - step nanoprecipitation technique.
Highly fl uorinated materials have multiple properties, such as repellance to water
and oil, unique dielectric, rheological, and optical properties, as well as exceptional
chemical and biological inertness. The fl uorinated chains, due to their strong hydrophobic
and fl urorophilic character, impart unique properties to the vesicles,
including enhanced particle size stability, prolonged intravascular persistence, and
increased drug encapsulation capability. Thus, 2,3 - di - O - decafl uorooctanoyl - . - CD
nanoparticles were believed to be a suitable carrier for oxygen solubilization and
delivery. Oxygen delivery of perfl uorinated amphiphilic CD nanocapsules was compared
to water and showed a prolonged delivery of oxygen. Fluorophilic nanocapsules
were believed to overcome fl uorocarbon emulsions as oxygen carriers due to
their higher number of particles in the colloidal solution which will permit a greater
rate of dissolved oxygen [73] .
Oral Delivery Amphiphilic . - CD nanocapsules loaded with indomethacin have
been evaluated in vivo. The nanocapsules have been applied to the rat model. It was
reported that the gastrointestinal mucosa of the rat was signifi cantly protected from
the ulcerogenic effects of the active ingredient indomethacin in free form. Drug
encapsulation yield in the nanocapsules were > 98% and the drug content per CD
unit was 7.5% w/w [89] .
Cytotoxicity The cytotoxicity of nanocapsules was investigated against L929
mouse fi broblast cells and human polymorphonuclear PMNC cells with MTT assay
[90] . Cell viability values of different nanocapsule and nanosphere formulations on
L929 and PMNC cells indicated that nonsurfactant . - CDC6 nanocapsules were less
cytotoxic than nanocapsules containing surfactants. The cytotoxicity of the nanoparticles
mostly arises from surfactant presence and was concentration dependent
[90] .
Nanospheres of . - CDC6 prepared without surfactant and with Pluronic F68 of
varying concentrations between 0.1 and 1% were found to be slightly less cytotoxic
than nanocapsules to both L929 and human PMNC cells. It was concluded that
cytotoxicity increased with increasing concentration of surfactant and the most suitable
percentage for surfactant if required was found to be 0.1% [80] .
Sterilizability Three different sterilization techniques — autoclaving, fi ltration,
and gamma sterilization — were evaluated for amphiphilic CD nanoparticles of
. - CDC6 loaded with the model drug tamoxifen [90] . It was found that fi ltration was
not suitable for injectable amphiphilic CD nanoparticles since nanoparticle sizes
were too close to fi lter pore sizes of 0.22 . m. Autoclaving did not affect the nanoparticle
yield but caused a signifi cant increase in particle size and aggregates. Gamma
irradiation realized with a dose of 25 kGy was demonstrated to be a suitable sterilization
technique since no signifi cant change was observed for mean diameter, zeta
potential, drug entrapment effi ciency, and in vitro release profi les for nimodipine -
loaded . - CDC6 nanospheres and nanocapsules . The in vitro release profi le of sterile
and nonsterile nanospheres and nanocapsules of . - CDC6 loaded with nimodipine
is seen in Figure 9 [90] .
7.1.3 CONCLUSION
Cyclodextrins have been involved in nanoparticulate drug delivery systems by
increasing the solubility of the drug via complex formation, forming nanoparticles
in the presence of another polymer/macromolecule, forming nanoparticles by conjugation
to polymers, or modifi cation of natural CDs to render this molecule an
amphiphilic character. This chapter mainly focused on the potential of amphiphilic
CDs as promising carriers for anticancer drugs with bioavailability problems, oxygen
delivery for the treatment of ischemia, or the safe oral administration of drugs with
gastrointestinal side effects.
FIGURE 9 In vitro release profi le of tamoxifen from . - CDC6 nanospheres and nanocapsules
before and after gamma sterilization. ( Reprinted from E. Memisoglu - Bilensoy and
A. A. Hincal, International Journal of Pharmaceutics , 311, 203 – 208, 2006. Copyright 2006 with
permission from Elsevier. )
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6
Time (h)
Cumulative TMX citrate (% released)
Nonsterile NC Sterile NC Nonsterile NS Sterile NS
CONCLUSION 1241
1242 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD
Many new studies are available to modify amphiphilic CDs further by giving
them “ stealth ” properties or targeting moieties such as transferring to enable the
active targeting of CD - based nanoparticles to tumor tissues. Amphiphilic CD nanocarriers
now emerge as promising delivery systems for poorly soluble anticancer
drugs, DNA and oligonucleotide delivery, and photodynamic and targeted tumor
therapy. These systems are proven to be nonhemolytic and noncytotoxic and are
capable of prolonging the release of drugs with different properties.
ACKNOWLEDGMENTS
The authors wish to thank the TUBITAK Turkish Council of Scientifi c and Technical
Research, projects SBAG - CNRS - 3 and SBAG - CD - 66, and the Hacettepe University
Research Fund, project 0202301005, for fi nancial support of the amphiphilic
cyclodextrin research carried out by our group at Hacettepe University, Faculty of
Pharmacy, Department of Pharmaceutical Technology.
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7.2
NANOTECHNOLOGY IN
PHARMACEUTICAL
MANUFACTURING
Yiguang Jin
Beijing Institute of Radiation Medicine, Beijing, China
Contents
7.2.1 Introduction
7.2.2 Nanomaterials
7.2.2.1 Types of Nanomaterials
7.2.2.2 Manufacturing and Processing of Nanomaterials
7.2.3 Nanotechnology for Drug Delivery
7.2.3.1 Nanocarriers
7.2.3.2 Nanosuspensions
7.2.3.3 Self - Assembled Drug Nanostructures
7.2.4 Nanomedicine
7.2.5 Perspective
References
7.2.1 INTRODUCTION
Nanotechnology is the ability to produce and process nanosized materials or manipulate
objects within the nanoscale. The nanoscale commonly indicates the range
from 1 to 100 nm [1] . However, some scientists regard the nanoscale range from 1
to 200 nm [2] , even to 1000 nm [3] . Making a comparison with a human hair, it is
about 80,000 nm wide. Nanotechnology is a broad, highly interdisciplinary, and still
evolving fi eld which involves the production and application of physical, chemical,
and biological systems. Nanotechnology is likely to have a profound impact on our
economy and society in the early twenty - fi rst century, perhaps comparable to that
of information technology or advances in cellular and molecular biology. Science
and engineering research in nanotechnology promises breakthroughs in areas such
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1250 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
as materials, manufacturing, electronics, medicine, health care, energy, environment,
biotechnology, information technology, and national security. It is widely felt
that nanotechnology will lead to the next industrial revolution [4] .
The idea of nanotechnology was fi rst presented by physicist Richard Feynman.
His lecture entitled “ Room at the Bottom ” in 1959 unveiled the possibilities available
in the molecular world. Because bulk matter is built of so many atoms, there
is a remarkable amount of space within which to build. Feynman ’ s vision spawned
the discipline of nanotechnology, and his dream is now coming true [5] . Along with
continually increasing multidisciplinary applications of nanotechnology, many new
terms with nanotechnology characteristics appear, for example, nanomechanics [6] ,
nanooptics [7] , nanoelectronics [8] , nanochemistry [9] , nanomedicine [10] , nanobiotechnology
[5, 11] , nanolithography [12] , nanoengineering [13] , nanofabrication [14] ,
and nanomanufacturing [15] . A very broad sense term, nanoscience is often used.
More and more new words with nano as a prefi x will be created to fi t for the nowadays
nanoworld. In fact, applications of nanotechnology in medicine and biotechnology
have made great progresses in the recent two decades.
All developed countries including the United States, Japan, and Europe invest
a great deal of money in nanotechnology. The National Science Foundation (NSF)
of the United States is a leading agency in the national nanotechnology initiative,
funding nanotechnology investments at $ 373 million in 2007, an increase of 8.6%
from 2006 and of nearly 150% since 2001 [16] . Developing countries such as China
and India also invest a lot in this increasing fi eld so as not to stay far behind developed
countries. Cancer therapy and research are hottest applied fi elds of bionanotechnology.
In 2004, the U.S. National Cancer Institute (NCI) launched a $ 144
million cancer nanotechnology initiative, and the investment increased largely in
the following two years [17] . At the same time, investment from public resources
or companies is much higher than that from governments.
The application of nanotechnology in pharmacy has a long history, before the
prevalence of the nanoconcept. It was well known 50 years ago that very small drug
particles have a high solubility in solvents, resulting from the too large surface area
when particle size decreased to a very small level, that is, the nanoscale, although
this scale had not been mentioned yet. In 1965, Banham created liposomes (lipid
vesicles) consisting of phospholipids which had a small size, typically ranging from
10 nm to several micrometers. It was soon found that liposomes were excellent drug
carriers, and more importantly they had site - specifi c distribution capability in vivo
depending on their size. It is well known that nanosized liposomes are inclined to
deposit in the mononuclear phagocyte system (MPS), including liver, spleen, lung,
and marrow. Therefore, nanotechnology was introduced in drug delivery very long
ago. Now various nanomaterials are used to deliver drugs, and some nanosystems
delivering active agents are available on the market. Undoubtedly, nanotechnology
plays a key role in future pharmaceutical development and pharmacotherapy.
7.2.2 NANOMATERIALS
7.2.2.1 Types of Nanomaterials
Nanomaterial is a general term. Although nanomaterials are defi ned as solid or
liquid materials at the nanoscale, the nanoscale range remains unclear. Many scien
NANOMATERIALS 1251
tists regard materials that are one dimensional and 1 nm to less than 100 nm as
nanomaterials. However, some scientists treat larger materials (e.g., less than
200 nm) as nanomaterials [2] . In spite of the different views, nanomaterials show
unique characteristics that are different from those of bulk materials. Rapid development
of nanotechnology in varied disciplines helps to create various kinds of
nanomaterials. In terms of shape differences, nanomaterials can be classifi ed as
nanospheres, nanovesicles, nanoshells, nanotubes, nanohorns, nanofi bers, nanowires,
nanoribbons, nanorods, nanosticks, nanohelices, and so on, and they can
appear in any shape imagined. In terms of state differences, nanomaterials can also
be classifi ed as nanoparticles with solid cores, nanoemulsions with liquid cores, and
nanobubbles with air cores. Images of some nanomaterials of various shapes are
shown in Figure 1 .
Nanoparticle is the most usually used term, having a broad meaning. From a
narrow sense, nanoparticles are always used to indicate all ball - like nanomaterials,
and therein the term nanosphere is also used. Nanocapsules are core – shell nanoparticles,
wherein trapped drugs are gathered in a core coated with a hard shell, though
generally nanoparticles have uniformly dispersed drugs within the whole particle.
Nanovesicle is not a familiar term, for example, liposomes have an inner phase and
an outer phase (dispersing medium) that exist together in nanovesicles [18] . In light
of drug nature, especially solubility, drugs are entrapped in an inner phase or bilayers
(shells). In addition, nanosuspension often appears in the pharmaceutical fi eld,
meaning drug nanocrystal dispersion in liquid media [19] . Needle - shaped nanocrystals
are more common than globe - shaped ones. Nanogels are newly developed
based on hydrogels, being similar to nanoparticles after lyophilization [20] . Recently,
a special kind of nanomaterial consisting of drugs was created for drug delivery,
called self - assembled drug nanostructure (SADN), which is formed by the self -
assembly of amphiphilic prodrugs in aqueous media [21, 22] .
Some special nanomaterials are of great interest due to their unique properties.
Dendrimers are versatile, well - defi ned, nanosized monodispersing macromolecules
which are hyperbranched synthesized polymers constructed by repetitive monomer
units. They are perfect nanoarchitectures with size from 1 nm to more than 10 nm
depending on the synthesis generation. Drugs can be entrapped into the branches
FIGURE 1 Some typical nanomaterials.
Nanospheres Nanorods Nanovesicles
Nanotubes Nanofibers Nanohelices
1252 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
of dendrimers or conjugated with them on the high reactive surfaces [23] . The fi rst
fullerene discovered was the buckyball, also known as buckminsterfullerene. It was
discovered by Smalley, Curl, and Kroto in 1985 [24] , who shared a Nobel Prize in
1996 for the discovery. Buckyball is roughly spherical cages of 60 carbon atoms
(C60 ) arranged in interlocking hexagons and pentagons, like the patches on a soccer
ball. Fullerenes have attracted considerable research interest, partly because of
their unique structures and further because, once suitably dissolved, they display a
diverse range of biological activity [25] . Quantum dots (QDs) are semiconductor
nanocrystals commonly consisting of CdSe or ZnS. Besides their utilization as
electronic materials, QDs have recently been applied to biomedical areas after
modifi cation. The new generations of QDs have far - reaching potential for the study
of intracellular processes at the single - molecule level, high - resolution cellular
imaging, long - term in vivo observation of cell traffi cking, tumor targeting, and
diagnostics [26] .
Although many types of nanomaterials are created continually, the most important
and basic issues are nanoscale effects and the subsequent particular functions.
Nanomaterials with varied shapes and components provide different platforms to
achieve more functions. In the area of pharmaceutical manufacturing, people focus
on the drug delivery function of nanomaterials. Furthermore, the rapid development
of modern medicine has led to the belief that traditional drug dosage forms
such as tablets, capsules, and injections may not treat some vital diseases well,
perhaps not at all. Some advanced techniques developed in other disciplines should
be considered to apply to medicine. Nanomaterials can load and deliver drugs in
vivo as well as display special properties such as high dispersion, adhesive property,
and site - specifi c distribution in vivo. Modifi ed nanomaterials further possess new
functions, for example, they may be thermally sensitive, pH sensitive, magnetically
sensitive, and ultrasound sensitive.
Nanotechnology has a great effect on pharmaceutical manufacturing. The unique
functions of nanomaterials promise considerable benefi t to pharmacotherapy over
traditional drug preparations. When drug - loaded nanomaterials go through the
gastrointestinal tract, high dispersion and adhesion can lead to tight contact of
nanomaterials with mucous membranes, enhancing drug absorption. Nanomaterials
have been applied in all routes of administration, including oral, injection (intravenous,
subcutaneous, intramuscular, intra - articular cavity, and other possible injection
sites), intranasal, pulmonary inhalation, conjunctiva, topical, and transdermal,
possibly showing various required effects. Some of the characteristics and pharmaceutical
applications of nanomaterials are given in Table 1 . More applications will
continue to be developed.
7.2.2.2 Manufacturing and Processing of Nanomaterials
When material dimensions reach the nanoscale, quantum mechanical and thermodynamic
properties that are insignifi cant in bulk materials dominate, causing these
nanomaterials to display new and interesting properties. The manufacturing and
processing of nanomaterials may become diffi cult due to the unique properties. The
very small size of nanomaterials produces a very large surface - to - volume ratio, that
is, a great number of molecules/atoms locate on surfaces. High surface energy leads
to nanomaterials easily agglomerating to diminish energy unless enough hindrance
NANOMATERIALS 1253
TABLE 1 Characteristics and Applications of Some Nanomaterials in Pharmacy
Types of
Nanomaterials Characteristics
Applications in
Pharmacy References
Nanoparticles Solid nanosized particles
consisting of polymers,
lipids, or inorganic
materials spherically
shaped most of the
time, entrapped
compounds dispersing
in the whole particle
Loading all kinds
of active agents,
including drugs,
vaccines, diagnostic
agents, and imaging
agents for good
bioavailability,
targeted delivery, and
controlled release
27, 28
Nanocapsules Core – shell nanoparticles
with entrapped
compounds gathering
in the core
Loading all kinds of
active agents for
same aims as
nanoparticles,
possibly protecting
entrapped agents
29
Liposomes Lipid vesicles with
entrapped compounds
in inner phase or
bilayers depending on
physicochemical
property
Loading all kinds of
active agents for
good bioavailability,
targeted delivery, and
controlled release
30, 31
Niosomes Nonionic surfactant
vesicles with similar
property as liposomes
Loading all kinds of
active agents for
same aims as for
liposomes
32
Nanoemulsions Nanoscale emulsions Loading drugs, as a
method to prepare
nanoparticles
33, 34
Polymeric
micelles
Micelles consisting of
amphiphilic polymers
Loading hydrophobic
drugs in the core for
solubilization,
targeted delivery, and
controlled release
35, 36
Nanogels Nanosized hydrogels
consisting of cross -
linked hydrophilic
polymers
Loading various
compounds for
controlled release
or targeting
20
Dendrimers Well - defi ned, nanosized,
monodispersing
macromolecules with
hyperbranched
structures
Loading all kinds of
active agents for
good bioavailability,
targeted delivery, and
controlled release
23
1254 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
Types of
Nanomaterials Characteristics
Applications in
Pharmacy References
Carbon
nanotubes
(CNTs)
Nanosized tubes as if
rolling up a single
layer of graphite sheet
(single - walled CNTs;
SWNTs) or by rolling
up many layers to
form concentric
cylinders (multiwalled
CNTs; MWNTs) with
diameters of . 1 nm
and large length –
diameter ratio
Linking a wide variety
of active molecules
with functionalized
CNTs
37
Fullerenes Very tiny balls consisting
of 60 carbon atoms
with diameter of
. 0.7 nm
Water - soluble
carboxylic acid C 60
derivatives acting as
antimicrobials, being
linked to a variety of
active molecules
25, 38, 39
Quantum dots Tiny nanocrystals
commonly consisting
of semiconductor
materials in the range
of 2 – 10 nm, glowing
upon ultraviolet (UV)
light
Mainly as probes to
track antibodies,
viruses, proteins, or
deoxyribonucleic acid
(DNA) in vivo
26, 40
Nanosuspensions Drug nanocrystals
dispersing in aqueous
media commonly
stabilized by
surfactants
Suitable for insoluble
drugs to obtain good
bioavailability and
targeting
19
Self - assembled
drug
nanostructures
Nanostructures
consisting of
amphiphilic prodrugs
Suitable for hydrophilic
drugs to obtain good
bioavailability,
targeting, and
controlled release
21, 22
prevents them from agglomeration. As a result, manufacturing and processing of
nanomaterials become hard issues. Anyway, many successful methods have been
found to manufacture stable nanomaterials.
“ Top down ” and “ bottom up ” are two basic ways to manufacture nanomaterials.
From its apparent meaning, the top - down method starts with a bulk material and
then breaks it into smaller pieces using mechanical, chemical, or other forms of
energy. Microchip manufacturing is the most common example of the top - down
approach to produce nanomaterials. While this is an effi cient approach for some
industries, the process is generally labor and cost intensive. In contrast, the bottom -
up method produces nanomaterials from atomic or molecular species via chemical
reactions or physicochemical interactions such as self - assembly, allowing the precur-
TABLE 1 Continued
NANOMATERIALS 1255
sor molecules/particles to grow in size. Self - assembly leads to gaining the lowest
energy state of molecules and makes molecules reorient naturally to obtain ordered
aggregates. Carbon nanotubes, liposomes, and the SADNs are examples of nanomaterials
that are manufactured using the bottom - up approach. A deep understanding
of chemical and physical properties of precursor molecules/particles is needed
to design and manufacture nanomaterials using the bottom - up approach. Both top -
down and bottom - up approaches can be performed in gas, liquid, supercritical fl uid,
solid state, or vacuum. Anyway, when bulk materials corrupt, energy is required,
and certainly the obtained nanoscale materials stay at a higher energy state than
their parents. Whereas in the bottom - up approach molecules self - assemble into
ordered aggregates with controlled behavior. Considering the higher energy of self -
assembling monomolecules dispersing in media, their aggregation should be an
energy - diminishing procedure and proceed spontaneously (Figure 2 ).
One of the largest hurdles of nanomanufacturing is how to scale up production.
In the laboratory, manufacturing nanomaterials is diffi cult enough as highly
advanced tools and carefully clean environments are required. Therefore, scale - up
manufacturing in factories becomes a great challenge, hard to achieve. The most
successful mass nanomanufacturing to date has occurred with computer microprocessors
where companies have been able to etch circuit boards at 65 nm or smaller.
Most manufacturers are interested in the ability to control (a) particle size, (b)
particle shape, (c) size distribution, (d) particle composition, and (e) degree of particle
agglomeration. Neither the top - down nor bottom - up approach is superior at
the moment. Each has its advantages and disadvantages. However, the bottom - up
approach may have the potential to be more cost - effective in the future.
Clinical applications require that biomedical nanomaterials have good biocompatibility
or biodegradability. Therefore, biodegradable polymers (synthetic or
natural), small molecules such as lipids, and some bioabsorptive inorganic salts such
as calcium phosphate are preferred. Other materials such as poly(ethylene glycol)
(PEG) is eventually excreted from body so they can also be selected. Materials that
are nonbiodegradable or not easily removed from the body, such as carbon nanotubes
and quantum dots, should be carefully considered as drug carriers, although
they have already been used to deliver drugs or genes. More importantly, before
any nanomaterial can be used in a clinic, the acute and long - term toxicity and side
effects must be estimated in detail. So a novel discipline, nanotoxicology, is of great
interest [41] . In addition, problems of large - scale production of nanomaterials, for
example, the uniformity and stability of products, cannot be ignored. Some nanomaterials,
including liposomes, polymeric or lipid nanoparticles, nanosuspensions,
and SADNs, are described in detail in the following sections. The common manufacturing
methods of pharmaceutical nanomaterials are listed in Table 2 , though
some are only used in the laboratory.
FIGURE 2 Two basic methods to manufacture nanomaterials.
Top down Bottom up
Nanomaterials Atoms or molecules Bulk materials
1256 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
TABLE 2 Manufacturing Methods of Some Nanomaterials in Pharmacy
Types of
Nanomaterials Materials Involved a Manufacturing Methods References
Nanoparticles
Polymeric
nanoparticles
Various natural polymers,
e.g., albumin, gelatin,
alginate, collagen,
chitosan; biodegradable
synthetic polymers, e.g.,
poly(lactic acid) (PLA),
poly(lactide - co -
glycotide) (PLGA),
poly( . - caprolactone)
(PCL), poly(methyl
methacrylate),
and poly(alkyl
cyanoacrylate);
derivatives of
cyclodextrin and starch;
some modifi ed
polymers (e.g.,
PEGylated polymers)
also used
Monomer
polymerization,
precipitation, solvent
evaporation, salting
out
42 – 44
Solid lipid
nanoparticles
(SLNs)
Mainly glycerides and
fatty acids, surfactants
also used
High - pressure
homogenization,
microemulsion
technique, solvent
evaporation
27
Inorganic
nanoparticles
Calcium salts (e.g.,
calcium carbonate and
calcium phosphate),
gold
Precipitation 45 – 47
Nanocapsules Various polymers, e.g.,
poly( iso -
butylcyanoacrylate)
(PIBCA), PLA, PLGA,
PCL
Interfacial
polymerization of
monomers or
interfacial
nanodeposition of
polymers
29, 48, 49
Liposomes Phospholipids and
cholesterol,
phospholipid
derivatives, e.g., PEG –
polyethylene (PE), also
added
Many methods used,
mainly fi lm
hydration, reverse -
phase evaporation,
injection, freeze
drying
50
Niosomes Noionic surfactants, e.g.,
sorbitan monostearate
(Span 60)
As for liposomes 32
Nanoemulsions Oil and surfactants High - pressure
homogenization,
ultrasonic
emulsifi cation, phase
inversion
34
NANOMATERIALS 1257
Types of
Nanomaterials Materials Involved a Manufacturing Methods References
Polymeric
micelles
Poloxamer - like block
copolymers; PEG and
lipophilic polymer
copolymers; PEGylated
lipids
Dialysis, emulsifi cation,
or fi lm method
35, 36
Nanogels Cross - linked hydrophilic
copolymers, e.g.,
Pluronic –
poly(ethylenimine)
(PEI) and polyethylene
oxide (PEO) – PEI
Covalent conjugation of
polymers
20, 51, 52
Dendrimers Dendritic macromolecules
with repetitive moieties
Divergent or
convergent synthesis
23, 53
Carbon
nanotubes
Carbon, but only the
water - soluble
derivatives of CNTs
used in pharmacy
CNTs formed by
chemical vapor
deposition (CVD)
in presence of Fe
catalyst, water -
soluble CNT
derivatives obtained
by acid processing
followed by
conjugation with
drugs
54, 55
Fullerenes (C 60 ) Carbon, but only the
water - soluble
derivatives of C 60 used
in pharmacy
C 60 obtained by arc
discharge method
using graphite
electrodes or in a
benzene fl ame,
water - soluble C 60
derivatives obtained
by acid processing
followed by
conjugation with
drugs
38, 56
Quantum dots Water - soluble derivatives
of semiconductor
materials (e.g., ZnS,
PbS, CdSe, InP) used in
pharmacy
QDs obtained via
pyrolysis of
organometallic
precursors, water -
soluble QD
derivatives obtained
by chemical reaction
57 – 59
Nanosuspensions Pure drugs and stabilizers
(including surfactants
or polymers)
Precipitation, wet
milling,
homogenization
19, 60, 61
Self - assembled
drug
nanostructures
Polar drugs with proper
conformation and lipids
with long chains (e.g.,
glycerides, fatty acids,
cholesterol)
Amphiphilic prodrugs
obtained by synthesis,
subsequently SADNs
obtained by injection
method
21, 22
a Organic solvents may be involved and subsequently removed.
TABLE 2 Continued
1258 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
7.2.3 NANOTECHNOLOGY FOR DRUG DELIVERY
7.2.3.1 Nanocarriers
High - throughput screening technologies in drug discovery present an effi cient way
to fi nd new potential active agents. But in recent years it has become evident that
the development of new drugs alone is not suffi cient to ensure progress in pharmacotherapy.
Poor water solubility of potential active molecules, insuffi cient bioavailability,
fl uctuating plasma levels, and high food dependency are the major and
common problems. Major efforts have been spent on the development of customized
drug carriers to overcome the disappointing in vivo fate of those potential
drugs. For drug carriers the followings are considered: nontoxicity (acute and
chronic), suffi cient drug - loading capacity, possibility of drug targeting, controlled -
release characteristic, chemical and physical storage stability (for both drugs and
carriers), and feasibility of scaling up production with reasonable overall costs.
Nanocarriers have attracted great interest because they are desirable systems to
fulfi ll the requirements mentioned above.
Over the past decade nanocarriers as nanoparticulate pharmaceutical carriers
have been shown to enhance the in vivo effi ciency of many drugs both in pharmaceutical
research and the clinical setting, including liposomes, micelles, nanocapsules,
polymeric nanoparticles and lipid nanoparticles. They perform various
therapeutically or diagnostically important functions. More importantly, many
useful modifi cations have been made, including the increased stability and half - life
of nanocarriers in the circulation, required biodistribution, passive or active targeting
into the required pathological zone, responsiveness to local physiological stimuli
such as pathology - associated changes in local pH and/or temperature, and ability
to serve as imaging/contrast agents for various imaging modalities (gamma scintigraphy,
magnetic resonance imaging, computed tomography, ultrasonography). In
addition, multifunctional pharmaceutical nanocarriers have already made a promising
progress [62] . Some of those pharmaceutical carriers have already found their
way into clinics, while others are still under preclinical investigation. This section
presents two of the most promising nanocarriers, that is, liposomes and nanoparticles,
especially their manufacturing, characteristics, and applications.
Liposomes Liposomes (lipid vesicles) have a relative long history, fi rst discovered
by Banham in 1965 [63] . In the following decades, liposomes rapidly became a useful
drug carrier. During the 1990s, many liposome - based drugs reached the market in
the United States and Europe. The history of liposomes is the procedure of nanotechnology
application to biomedicine. Phospholipids have particular structural
conformation, leading to their self - assembly into bilayers with lipid chains inside
and polar head groups outside during hydration. Importantly, phospholipids are the
primary components of cell membranes so that liposomes have good biocompatibility
without toxicity. The formation of liposomes is almost spontaneous, wherein a
bottom - up procedure is involved [64] . When relatively free phospholipid molecules
meet water, their polar head groups have affi nity with water while lipid chains
repulse water, which subsequently leads to their aggregation due to hydrophobic
interaction, and then bilayers consisting of phospholipids are formed spontaneously.
Closed vesicles are further formed by bilayer bending (Figure 3 ). Before phospho
FIGURE 3 Structures of phospholipids and formation of liposomes.
H3C
CH3
N+
CH3
O
O O
O–
P
O
O
O
O
CH3
H3C
Dipalmitoyl phosphatidylcholine
(DPPC)
3D optimized structure of DPPC
Polar head group
Lipid chains
Self-assembly
Bilayer
Vesicle
(liposome)
lipids become “ free, ” bulk phospholipids must be dispersed throughout, forming a
thin fi lm, dissolution or emulsifi cation, wherein additional energy is sometimes
needed. Liposomes may have a size ranging from 10 nm to more than 10 . m mainly
depending on composition and manufacturing approaches. A number of reports
about the preparation of liposomes can be found in the literature and a detailed
description of liposomes is in Chapter 7.1 of this handbook. In this section the
NANOTECHNOLOGY FOR DRUG DELIVERY 1259
1260 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
preparation, characteristics, and applications of nanosized liposomes are presented
as well as some modifi ed liposomes and recent progress.
Although various methods of manufacturing liposomes are reported, three types
are usually involved: hydration of lipid fi lm, interface aggregation of lipid molecules
by emulsion - like process, and lipid solutions dispersing into nonsolvents by an
injection - like process or controlled mixture. Practical methods are thin - fi lm hydration
[65] , reverse - phase evaporation [66] , ethanol injection [67] , polyol dilution [68] ,
double emulsions [69] , proliposome method [70] , and high - pressure homogenization
[71] . Liposomes may have various morphologies related to manufacturing methods,
mainly multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs), and small
unilamellar vesicles (SUVs). Liposomes can be further processed by sonication,
detergent depletion, membrane fi ltration [72] , and lyophilization [73] to make them
fi ner and more uniform or stable. For example, MLVs are sonicated to SUVs.
The composition of liposomes is a key factor in their manufacturing. Phospholipids
are major components of liposomes. In terms of resources, phospholipids are
classifi ed as natural, semisynthetic, and wholly synthetic phospholipids. Natural
phospholipids also have different resources (e.g., soybean, egg yolk). In terms of
polar head groups, phospholipids are classifi ed as phosphatidylcholine (PC), phosphatidylethanolamine
(PE), phosphatidylserine (PS), phosphatidylinositol (PI),
phosphatidylglycerol (PG), and phosphatidic acid (PA), where PC and PE are the
most used. Different polar head groups result in varied surface charged liposomes
that then infl uence the stability and in vivo distribution. Because PS, PI, PG, and PA
have negative charges, the liposomes containing them are negatively charged. Sometimes,
other lipids such as N,N. - dioleoyl - N,N. - dimethylammonium chloride
(DODAC) and stearylamine are mixed with phospholipids to prepare positively
charged liposomes. Cholesterol is commonly used with phospholipids because cholesterol
can make liposomal membranes stronger [50] . The mole percentage of
cholesterol in the liposomal composition is commonly not more than 50%. Lecithin
(an often used term in the lipid fi eld) as a phospholipid from natural resources (e.g.,
soybean lecithin and egg lecithin) is often used to manufacture liposomes, which is
actually a mixture composed of various kinds of phospholipids though PC dominates.
The long - chain fatty acids constituting phospholipids also have many types,
such as lauric (C12), myristic (C14), palmitic (C16), and stearic (C18). In general,
unsaturated fatty acids occur in natural phospholipids. Dimyristoyl phosphatidylcholine
(DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine
(DSPC), and dipalmitoyl phosphatidylethanolamine (DPPE) are the most
common synthetic phospholipids. The length of the lipid chain infl uences the gel –
liquid crystalline phase transition temperature ( Tc ) of phospholipids, wherein longer
chained lipids lead to higher Tc . For example, DPPC has a Tc of 41 ° C while DSPC
has a Tc of 54 ° C [50] .
Drug entrapment is an important parameter in manufacturing liposomes which
is infl uenced by many factors: the types, molecular weights, and physicochemical
properties of drugs; the types, sizes, and compositions of liposomes; and the manufacturing
methods. In addition, entrapped drugs may leak during storage. Drugs may
be entrapped in one of two parts of liposomes, the inner phase or bilayers, depending
on the physicochemical property of the drugs. Water - soluble drugs prefer the
aqueous inner phase while lipophilic drugs prefer the hydrophobic environment of
bilayers. Macromolecules such as peptides and proteins can adsorb onto bilayers,
wherein electrostatic interaction can infl uence entrapment. Those drugs insoluble
in both water and oil are hard to entrap. Rather than common manufacturing
approaches, more promising methods are adopted to improve drug entrapment.
Ionic gradient methods can increase the entrapment effi cacy of some ionic drugs,
including the pH gradient method [74] , the ammonium sulfate gradient method [75,
76] , the acetate gradient method [77] , and the manganese ion gradient method [78] .
Lyophilization of liposomes is a good preservation method which can prevent
entrapped drugs from leaking, liposome precipitation and agglomeration due to
gravity and thermal movement, and possible hydrolysis of phospholipids (resulting
in production of toxic lyso - phospholipids). Generally, lipophilic drugs have a high
entrapment effi cacy, though drug loading is limited, because they insert into bilayers
tightly. Therefore, an effi cient method increasing entrapment effi cacy is to prepare
the lipophilic derivatives of hydrophilic drugs [79] .
The manufacturing of nanosized liposomes can be performed using the methods
mentioned above. However, the small size of nanoliposomes is diffi cult to achieved
by methods such as fi lm hydration. Molecular self - assembly occurs in the injection
method, and then the size and morphology of obtained liposomes can be well controlled.
In fact, liposomes that result from the injection method are uniform and
small enough, to the nanoscale, and usually SUVs are obtained. Because of the very
low toxicity of ethanol, the ethanol injection method is usually used and is described
as follows to show the process of manufacturing liposomes [50] . A scale - up manufacturing
process of the ethanol injection method has been established [80 – 82] . The
obtained liposome size is mostly less than 300 nm:
(a) Handling and storage of lipids is important. Store organic solutions of phospholipids
in a sealed glass container layered with argon or nitrogen below
. 20 ° C, preferably at . 78 ° C. When transferring a portion of the material, allow
it to reach room temperature before opening the bottle. Saturated phospholipids,
that is, lipids composed of completely saturated fatty acids, such as
DPPC, are stable as powders. However, storage of these lipids as described
above is highly recommended. Unsaturated phospholipids are extremely
hygroscopic as powders, which will quickly absorb moisture and become
gummy upon opening the storage container. Always dissolve such lipids in a
suitable solvent (preferably chloroform) and store it in a glass container at
. 78 ° C.
(b) Prepare materials such as phospholipids, cholesterol, other additives, ethanol,
injector, beaker, agitation machine, and evaporation device before manufacturing.
Calculate the amount of these agents according to the request of the
last products. A fi ne - gauge needle to a 1 - mL glass syringe is preferred. Dissolve
lipid components (including lipophilic drugs) in ethanol. Dissolve water
- soluble drugs in water or aqueous media as dispersing media.
(c) Rapidly inject the ethanol solutions into agitated aqueous media with the
tip under the surface. A homogeneous and almost transparent liquid will be
obtained. Repeat this process and notice that the percentage of ethanol in the
last product is not more than 7.5%. Collect all liquids and remove ethanol by
evaporation, dialysis, or gel fi ltration. The last liposomal suspensions can be
further concentrated through evaporating water. Sterilize them by autoclave.
They may be lyophilized when needed.
NANOTECHNOLOGY FOR DRUG DELIVERY 1261
1262 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
Separation of nonentrapped drugs from liposomes (or purifi cation of liposomes) is
an important process after manufacturing. The size difference between liposomes
and unincorporated materials is the basis of separation. Gel chromatography, dialysis,
and centrifugation are usual approaches. The drug entrapment percentage of
liposomes can be obtained after separation. The whole drugs in liposomal suspensions
or the entrapped drugs can be determined by dissolving liposomes with organic
solvents or solubilizing liposomes with detergents to release drugs. The morphology
of liposomes can be investigated by negatively stained transmission electron microscopy,
cryo - electron microscopy, or freeze - fracture electron microscopy. The size
distribution of liposomes is usually analysized by photon correlation spectroscopy
(laser light scattering) [50] .
Beyond conventional liposomes, functional liposomes are designed to achieve
various therapeutic effects. Conventional liposomes manufactured by natural phospholipids
or commonly used synthetic phospholipids such as PC and PE are negatively
charged. However, cationic liposomes can form complexes with peptides or
nucleic acids through electrostatic interaction and prefer to adsorb onto the surfaces
of cell membranes, subsequently improving interaction with cells and penetrating
into cytosol or phagocytosis. Therefore, cationic liposomes have become a standard
transfection agent in cell manipulation [83] . Furthermore, they become primary
nonviral gene delivery carriers [84] .
Liposomes show site - specifi c distribution in the MPSs after intravenous (IV)
administration due to opsonization by the complement system [85] . The diseases in
MPSs can benefi t from the drug targeting. But this is a bad result for diseases in
other tissues. Long - circulating liposomes are then developed for targeting to non -
MPS tissues. The long - circulating effect results from hydrophilic polymers coated
on liposomes. For example, the half - life of the long - circulating liposomes can be
extended to 20 h in rat. They are also called sterically stable liposomes or Stealth
liposomes. The lipid conjugate of PEG, PEG – DSPE, is commonly used and inserts
into bilayers and hinders plasma protein adsorption. The enhanced permeability and
retention (EPR) effect of solid tumors makes long - circulating liposomes a very
useful tool for anticancer therapy [86] . However, in recent years it was reported that
in most cases PEGylated liposomes were cleared very rapidly from circulation with
repeated injection. But doxorubicin PEGylated liposome is an exception. The production
of anti - PEG immunoglobulin (Ig) M following injection is the major reason,
and the spleen also plays a key role [87] . However, a more recent case has appeared.
A modifi ed phospholipid – methoxy(polyethylene glycol) conjugate was recently
synthesized through the methylation of phosphate oxygen moiety which could
prevent PEGylated liposomes from being activated by a complement system in vivo
followed by achieving a true long - circulating effect [88] .
Other functional liposomes are mainly stimuli - responsive liposomes. The
pH - sensitive liposomes contain pH - sensitive lipids such as 1,2 - dioleoyl - sn - 3 -
phosphatidylethanolamine (DOPE) showing an inverted hexagonal confi guration
in a low - pH environment and release entrapped drugs in the low - pH environment
of tumor tissues due to liposomal membrane destabilization [89] . Temperature - sensitive
liposomes are prepared from special lipids such as DPPC whose phase transition
temperature ( Tc = 41 ° C) is proper to perform clinical anticancer therapy. When
up to Tc , the fl uidity of liposomal membranes increases sharply, followed by
entrapped drugs releasing [90] . Some thermosensitive polymers can also be used to
manufacture temperature - sensitive liposomes [91] . Magnetoliposomes load ultra-
fi ne magnetite, preferring to accumulate in the local tissue within the magnetic fi eld
[92] . Immunoliposomes load attached monoclonal antibodies to treat some severe
diseases such as cancer [93] .
Liposomes have been successfully applied to many drugs, diagnostic agents,
imaging agents, transfection agents, vaccines, and so on. Liposomes have been tried
in almost all routes of administration: oral, injection (intravenous, subcutaneous,
intramuscular, intra - articular cavity, and other possible injection sites), intranasal,
pulmonary inhalation, conjunctiva, topical, and transdermal. The most signifi cant
application fi eld of liposomes is still anticancer therapy. After a long - time research
for 30 years, some liposomal products have reached the market (Table 3 ). The major
problems in manufacturing liposomes are scale - up production, effi cient sterilization,
and stable storage.
TABLE 3 Liposomal Drugs Approved for Clinical Application
Drug Product Name
Composition of
Liposomes and
Other Major
Excipients Indication Company
Daunorubicin DaunoXome DSPC , cholesterol Kaposi ’ s
sarcoma
Gilead Sciences
Doxorubicin Mycet Egg PC,
cholesterol
Combinational
therapy of
recurrent
breast cancer
Zeneus
Doxorubicin Doxil/Caelyx MPEG – DSPE,
HSPC,
cholesterol,
ammonium
sulfate, sucrose,
histidine
Refractory
Kaposi ’ s
sarcoma;
ovarian
cancer;
recurrent
breast cancer
Alza/SP Europe
Amphotericin
B
AmBisome
(lyophilized
product)
HSPC, cholesterol,
DSPG, . -
tocopherol,
sucrose,
disodium
succinate
Fungal
infections
Gilead Sciences
Cytarabine DepoCyt DOPC, DPPG,
cholesterol,
triolein
Lymphomatous
meningitis
SkyePharma
Morphine DepoDur DOPC, DPPG,
cholesterol,
tricaprylin,
triolein
Pain following
major
surgery
SkyePharma
MPEG = methyl PEG; HSPC = hydrogenated soy phosphatidylcholine; DSPG = disteroylphosphatidylglycerol;
DOPC = dioleoylphosphatidylcholine; DPPG = dipalmitoylphosphatidylglycerol.
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1264 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
Nanoparticles Nanoparticles attract much attention of pharmaceutical scientists
because of their controllable manufacturing, uniform preparations, and low cost.
The major difference between nanoparticles and liposomes is that the former has a
solid core while the latter only has inner aqueous phase and thin bilayers. In addition,
in the case of liposomes, the entrapped water - soluble drugs exist only in solutions
of the inner phase, while lipophilic drugs are only limited in the small space
of bilayers. Therefore, the drug - loading effi ciency (drug – lipid ratio) of liposomes is
always limited. In the case of nanoparticles, drugs exist in the solid state, and high
drug loading is possibly achieved. Unlike liposomes, nanoparticles may be composed
of various materials, and biodegradable materials are preferably used. Furthermore,
modifi ed materials based on traditional natural and synthetic materials
are also frequently used to manufacture nanoparticles to achieve more functions,
which then highly enlarges the lists of used materials. In addition, more manufacturing
methods of nanoparticles are optional than liposomes. Therefore, nanoparticles
are relatively ideal nanocarriers for most drugs.
Nanoparticles can be classifi ed as three types, polymeric nanoparticles, lipid
nanoparticles, and inorganic nanoparticles, depending on the major components.
Polymeric nanoparticles are exploited earlier, while lipid nanoparticles are of great
interest in recent years due to very good biocompatibility. The development of
polymeric nanoparticles is highly related to polymer science. Besides a great deal
of natural polymers, more and more biodegradable polymers are synthesized, which
allows pharmaceutical scientists to have enough optional subjects. Solid lipid
nanoparticles (SLNs) composed of solid lipids have a profound advantage of no
biotoxicity [94] . Inorganic nanoparticles are currently exploited only a little [45 – 47] ,
and the major problems may be their poor biodegradability and relatively low drug -
loading effi ciency.
Polymeric Nanoparticles Polymeric materials for manufacturing nanoparticles
include synthetic poly(lactic acids) (PLA), poly(lactide - co - glycolide) (PLGA),
poly(. - caprolactone) (PCL), poly(methyl methacrylates), and poly(alkyl cyanoacrylates);
natural polymers (albumin, gelatin, alginate, collagen), and modifi ed
natural polymers (chitosan, starch). Polyesters, alone and in combination with other
polymers, are most commonly used for the formulation of nanoparticles. PLGA
and PLA are highly biocompatible and biodegradable. They have been used since
the 1980s for numerous in vivo applications (biodegradable implants, controlled
drug release). The U.S. Food and Drug Administration (FDA) has approved PLGA
for human therapy [95] . More recently, formulations based on natural polymers
have been developed and are on the market. For example, a wonderful natural
polymer, chitosan, has permeability enhancer abilities, allowing the preparation of
organic solvent free mucoadhesive particles [42] .
Nanoparticles of synthetic polymers are usually manufactured by dispersion of
preformed polymers. Although many methods can be used, they may be classifi ed
as monomer polymerization, nanoprecipitation, emulsion diffusion/solvent evaporation,
and salting out. An appropriate method is selected mainly depending on
polymer and drug natures. Polymerization of polymer monomers has been developed
usually using poly(alkyl cyanoacrylate) [96, 97] . Organic solvents are usually
used in polymerization. A detailed description of this method is not provided
here.
The nanoprecipitation method is commonly adopted to entrap lipophilic drugs,
and low polydispersity is probably achieved [42] . In general, the organic solution
containing drugs and polymers is added a nonsolvent to lead to polymers precipitating
together with drugs. The size of formed nanoparticles can be adjusted by the
polymer and nonsolvent amounts in the organic phase. Nanoparticles can be separated
from solvents and unincorporated drugs with centrifugation followed by spray
drying or freeze drying when needed. The stability and drug recovery yield of
nanoparticles depend on the ratio of drugs to polymers [98] . Recently, this technique
has also been used to entrap hydrophilic compounds into PLGA and PLA
nanoparticles [99, 100] , especially peptides and proteins [101] .
Another common method to manufacture polymeric nanoparticles is the emulsion
diffusion or solvent evaporation technique, which is used to entrap hydrophobic
or hydrophilic drugs. Generally, the polymer and hydrophobic drugs are dissolved
in a partially water miscible organic phase (e.g., benzyl alcohol, propylene carbonate,
and ethyl acetate). The organic solution is emulsifi ed in aqueous media containing
a suitable surfactant [i.e., anionic sodium dodecyl sulfate (SDS), nonionic
poly(vinyl alcohol) (PVA) or cationic didodecyl dimethyl ammonium bromide
(DMAB)] under stirring. The diffusion of the organic solvent and the counterdiffusion
of water into the emulsion droplets induce polymeric nanoparticle formation.
The organic solvent is evaporated. Also hydrophilic drugs could be entrapped into
a water - in - oil (W/O) emulsion containing polymers and then undergo the above
process. Then a water - in - oil - in - water (W/O/W) emulsion is obtained. After evaporation
of total organic solvent, the drug - loaded nanoparticles can be separated.
Polymer nature, polymer concentration, solvent nature, surfactant molecular mass,
viscosity, phase ratio, stirring rate, temperature, and fl ow of water all affect nanoparticle
size [102] .
The salting - out method is also used. Polymers are dissolved in water - miscible
organic solvents such as acetone or tetrahydrofuran (THF). The organic phase is
emulsifi ed in an aqueous phase that contains the emulsifi er and salts of high concentration.
Typically, the salt solution used contains 60% (w/w) magnesium chloride
hexahydrate or magnesium acetate tetrahydrate with a polymer - to - salt ratio of 1 : 3.
In contrast to the emulsion diffusion method, no diffusion of solvents occurs due
to the presence of high concentrated salts. The fast addition of pure water to the
O/W emulsion under mild stirring reduces the ionic strength and leads to the migration
of the organic solvent to the aqueous phase, inducing nanoparticle formation.
The fi nal step is purifi cation by cross - fl ow fi ltration or centrifugation to remove the
salting - out agent. Common salting - out agents are electrolytes (sodium chloride,
magnesium acetate, or magnesium chloride) or nonelectrolytes, such as sucrose.
Polymer concentration and molecular weight, stirring rate and time, and the nature
and concentration of surfactant and solvent are all important parameters. This
method would allow avoiding the use of organic chlorinated solvents and large
amounts of surfactants [102] . Furthermore, formulation of nanoparticles with
natural polymers is performed by ionic gelation (chitosan), coacervation (chitosan,
gelatin), and desolvation (gelatin) [102, 103] . These mild methods have the advantage
of producing organic solvent - free formulations.
Additional advantages can be obtained by changing nanoparticle surface properties,
for example, good stability, mucoadhesion, and long circulation time.
For example, the in vivo long - circulating effect is achieved either by coating
NANOTECHNOLOGY FOR DRUG DELIVERY 1265
1266 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
nanoparticle surfaces with hydrophilic polymers/surfactants or by incorporating biodegradable
copolymers containing a hydrophilic moiety. Like long - circulating liposomes,
PEG - containing polymers are frequently used to manufacture long - circulating
nanoparticles. PEGylated copolymers (PLA – PEG, PLGA – PEG and PCL – PEG) are
used [42, 104] , and the long - circulating effect also results from the adsorption or covalent
conjugation of some hydrophilic polymers with the hydrophobic surface of
nanoparticles [105, 106] . Moreover, the active targeting of nanoparticles can be
achieved by incorporating the conjugate of the polymer and target - directed molecule,
such as (Arg - Gly - Asp) RGD , (trans - activator transcription) TAT peptides [107] , and
monoclonal antibody [108] . Recently, self - assembled nanoparticles have aroused
great interest, consisting of amphiphilic macromolecules, such as hydrophobically
modifi ed glycol chitosan, which can also entrap drugs or peptides [109] .
Before nanocarriers go into clinical applications, some issues must be considered,
including drug - loading capacity, possibility of drug targeting, in vivo fate of the
carrier (interaction with the biological surrounding, degradation rate, accumulation
in organs), acute and chronic toxicity, scaling up of production, physical and chemical
storage stability, and overall costs. A certain advantage of polymer systems
is the wealth of possible chemical modifi cations. Possible problems of polymeric
nanoparticles derive from residues of organic solvents used in the production
process, polymer cytotoxicity, and the scaling up of production. Polymer hydrolysis
during storage has to be taken into account and lyophilization is often required to
prevent polymer degradation [94] .
Solid Lipid Nanoparticles The outstanding advantage of lipid nanoparticles is
perfect biocompatibility because their raw materials are the components of our
body, preferring to be used or degraded by the body. Solid lipids are usually used
as the major component of lipid nanoparticles — hence the name solid lipid nanoparticles.
However, the used solid lipids generally become liquid at a high temperature
to adapt to the preparation of SLNs. Compared with polymeric nanoparticles, the
materials used for SLNs are simpler. The frequently used lipids are glycerides
of various fatty acids, which also exist in the emulsions for parenteral nutrition.
Large - scale production of SLNs can be achieved in a cost - effective and relatively
simple way using high - pressure homogenization and microemulsion. Another useful
method is solvent emulsifi cation/evaporation. The SLN introduced in 1991 represents
an alternative carrier system to traditional colloidal carriers, such as liposomes
and polymeric nanoparticles. SLNs combine advantages of the traditional systems
but avoid some of their major disadvantages [27] . The proposed advantages of SLNs
include [94] :
• Possibility of controlled drug release and drug targeting
• Increased drug stability
• High drug payload
• Incorporation of lipophilic and hydrophilic drugs feasible
• No biotoxicity of the carrier
• Avoidance of organic solvents
• No problems with respect to large - scale production and sterilization
Solid lipids, emulsifi ers, and water are generally the ingredients involved for
manufacturing SLNs. The term lipids is used in a broader sense and includes triglycerides
(e.g., stearin), partial glycerides (e.g., Imwitor), fatty acids (e.g., stearic
acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). All categories of
emulsifi ers may be used to stabilize the lipid dispersion, and the combination
of emulsifi ers prevents particle agglomeration more effi ciently. The choice of the
emulsifi er depends on the administration route and is more limited for parenteral
administration.
High - pressure homogenization (HPH) has emerged as a reliable and powerful
technique for the preparation of SLNs. Homogenizers of different sizes are commercially
available from several manufacturers at reasonable prices. In contrast
to other techniques, scaling up of HPH is out of the question in most cases. High -
pressure homogenizers push a liquid with high pressure (100 – 2000 bars) through a
narrow gap (in the range of a few micrometers). The fl uid accelerates over a very
short distance to very high velocity (over 1000 km/h). Very high shear stress and
cavitation forces disrupt the particles down to the submicrometer range. Typical
lipid contents are 5 – 10%, though higher lipid concentrations (up to 40%) may be
used. Two general approaches of HPH, hot and cold homogenization, can be used
for manufacturing SLNs (Figure 4 ) [27] .
Microemulsions (transparently appearing with droplet size less than 100 nm) are
thermodynamically stable systems, and the choice of optimal formulation containing
oil, surfactant, cosurfactant, and oil – water ratio is key [110] . Generally, the solid
lipid of low melting point (e.g., stearic acid) melts at a high temperature (e.g., 65 –
70 ° C), and then hot microemulsions are prepared using it. SLNs can be obtained
after the hot microemulsions are rapidly cooled by injecting them into cold water
(e.g., 2 ° C) under stirring. Emulsifi ers in a formulation typically include Tween
20/60/80, lecithin, and sodium taurodeoxycholate, and coemulsifi ers include alcohols
and sodium monooctylphosphate. The typical volume ratios of hot microemulsions
to cold water are from 1 : 25 to 1 : 50. The very low solid concentration of SLN
suspensions is the disadvantage of the microemulsion method. Rapid temperature
decrease in hot microemulsions is key to obtaining homogeneous and small - sized
nanoparticles. A high temperature gradient can also ensure rapid lipid crystallization
and prevent aggregation [94] .
The solvent emulsifi cation/evaporation method involves lipid precipitation in
O/W emulsions. Solid lipids are dissolved in a water - immiscible organic solvent
(e.g., cyclohexane) followed by emulsifi cation in an aqueous medium. Upon evaporation
of the solvent, the nanoparticle dispersion is formed due to lipid precipitation.
Residue of organic solvents is the major problem of this method [94] . However,
the microemulsion and solvent emulsifi cation/evaporation methods can be performed
conveniently in the laboratory without specifi c apparatuses.
During research of SLNs, some problems have continually appeared, for example,
very low drug loads, drug expulsion during storage, and high water content of SLN
dispersions. The . and .. crystallines of higher energy state mainly appear in conventional
SLNs manufactured by hot - homogenization technique. However, these
crystallines prefer to transform to the more ordered . modifi cation of low energy
state during storage. The high ordered degree improves the crystal imperfections,
diminishing further to lead to drug expulsion. To solve this problem, a new kind of
lipid nanoparticle was developed, called a nanostructured lipid carrier (NLC). NLCs
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1268 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
are composed of spatially very different lipid molecules, that is, solid lipids and
liquid lipids (oils). The matrix remains solid at body temperature though its melting
point is lower than one of the original solid lipids. No crystallization happens in
NLCs so that the drug loads can be increased and the expulsion during storage is
avoided [111] .
New functions can be obtained by modifi cations of SLNs. Incorporation of Tween
80 and Poloxamer 188 can stabilize SLNs to achieve long - circulating or crossing
blood – brain barrier effects [112] . Recently, novel nanoparticles called polymer –
lipid hybrid nanoparticles (PLNs) were developed [113] . They can entrap cationic
anticancer agents (e.g., doxorubicin) effectively by incorporation of an anionic
lipophilic polymer into lipids to treat multidrug - resistant (MDR) cancers.
In general, SLNs are used to entrap hydrophobic drugs due to their lipid nature,
but a few reports show that hydrophilic drugs can also be entrapped. A hydrophilic
peptide, gonadorelin, and monostearin were dissolved in acetone and ethanol at
50 ° C followed by pouring the resultant organic solution into an aqueous medium
containing 1% PVA under agitation to obtain peptide - loaded SLNs that were sub-
FIGURE 4 High - pressure homogenization for manufacturing SLNs.
Melt lipid and dissolve/disperse
drugs in lipid
Hot homogenization technique Cold homogenization technique
Disperse
drug-loaded lipid in
hot surfactant aqueous
solution
Mix thoroghly using
stirrer to form coarse
preemulsions
High-pressure
homogenization at
temperature above
melting point of
lipid to form hot o/w
nanoemulsions
Solidification of
nanoemulsions by
cooling down to room
temperature
Solid lipid nanoparticles (SLNs)
High-pressure
homogenization at
room temperature or
below
Disperse powder
in surfactant
aqueous solution rind
in powder mill
(50–100 .m)
Grind in powder
mill (50–100 .m)
Solidification of
drug-loaded lipid in
liquid nitrogen or dry
ice
sequently separated by centrifugation. Up to 69% of gonadorelin was incorporated.
The in vitro release of gonadorelin from SLNs was slow [114] . The W/O/W multiple -
emulsion technique was also used to manufacture peptide - loaded SLNs. Insulin is
a model peptide located in the inner water phase of the W/O/W emulsion, tripalmitin
is the core of SLNs, and the surfaces are modifi ed with PEG 2000 – stearate. The
insulin - loaded SLNs show good stability upon the low pH of the gastric medium
and the pancreatic enzymes in intestinal medium [115] .
Perspective of Nanoparticles As drug nanocarriers, nanoparticles have unique
advantages: for example, high dispersing, adhesive property, targeting in vivo. Like
liposomes, anticancer therapy is a major function of nanoparticles [116] . Easy modi-
fi cation of nanoparticles also makes them platforms to perform more functions, for
example, delivering drugs across the blood – brain barrier (BBB) [117] , lymphatic
targeting [118] , and gene delivery [119] .
Abraxane is a successful paradigm of nanoparticle application. It is an albumin
nanoparticle loading paclitaxel developed by American Pharmaceutical Partners
(APP) and American BioScience. The outstanding advantage of Abraxane is no
signifi cant side effects, not like the traditional paclitaxel preparation with Cremophor
EL (polyethoxylated castor oil) and ethanol. More nanoparticle products will
reach the market in the future.
Other Nanocarriers
Nanoemulsions Lipid nanoemulsions were introduced in the 1950s as parenteral
nutrition. Vegetable oils (e.g., soy oil) or middle - chain triglycerides are used, typically
occupying 10 – 20% of the emulsion. Other ingredients include phospholipids
as stabilizers and glycerol as osmolar regulation agent. In recent years this system
has been further developed to load lipophilic drugs and several formulations
are commercialized. Examples are etomidate (Etomidat - Lipuro), diazepam
(Diazepam - Lipuro and Stesolid), propofol (Disoprivan), and dexamethasone palmitate
(Lipotalon). In comparison to previous, solubilization - based formulations of
these drugs, reduction of the local and systemic side effects (e.g., pain during injection)
has been achieved. The possibility of controlled drug release from nanoemulsions
is restricted due to the small size and the liquid state of the carrier. Most drugs
show a rapid release from them. Advantages of nanoemulsions include toxicological
safety and a high content of the lipid phase as well as the possibility of large - scale
production by high - pressure homogenization [94] .
Microemulsions Microemulsions are nanoemulsions which are optically isotropic,
transparent or translucent, low - viscous, and thermodynamically stable liquid solutions,
mainly containing tiny liquid droplets less than 100 nm. The manufacturing
of microemulsions as a self - formed system is relatively simple. They are bicontinuous
systems essentially composed of water and oil with surfactant and cosurfactant
separating. A very low interfacial tension to 0 mN/m is found in microemulsions
despite the large oil – water interfacial areas. A prominent example is the Sandimmun
Optoral/Neoral preconcentrate for microemulsions. Now microemulsions are
usually limited to dermal and peroral applications due to their high surfactant
content. Because they only exist in narrow regions of phase diagrams, they are very
restricted in tolerance to quantitative formulation changes [120] .
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1270 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
Polymeric Micelles Polymeric micelles composed of amphiphilic copolymers, that
is, polymers consisting of a hydrophobic block and a hydrophilic block, are gaining
increasing attention. They show high stability both in vitro and in vivo and good
biocompatibility, and more importantly they can solubilize a broad variety of poorly
soluble drugs in their inner core. Many of these drug - loaded micelles are currently
at different stages of preclinical and clinical trials. Due to their hydrophilic shell
and small size, they prefer to exhibit prolonged circulation times in vivo and can
accumulate in tumor tissues. Polymeric micelles are formed by block copolymers
consisting of hydrophilic and hydrophobic monomer units with the length of a
hydrophilic block exceeding to some extent that of a hydrophobic one. If the length
of a hydrophilic block is too high, copolymers exist in water as unimers (individual
molecules), while molecules with very long hydrophobic block prefer to form structures
with nonmicellar morphology, such as rods and lamellae. Diblock copolymers
with an A – B structure and tri - or multiblock copolymers such as poly(ethylene
oxide) – poly(propylene oxide) – poly(ethylene oxide) (PEO – PPO – PEO) (A – B – A)
may self - organize into micelles. The amphiphilic copolymers commonly have
the critical micelle concentration (CMC) values as low as 10 . 6 M , which is about
two orders of magnitude lower than that of such surfactants as Tween 80. As potential
drug carriers, the hydrophobic core of polymeric micelles generally consists of
a biodegradable polymer such as poly( . - benzyl - l - aspartate) (PBLA), PLA, or PCL
which serves as a reservoir for an insoluble drug, protecting it from contact with the
aqueous environment. The core may also consist of a water - soluble polymer [e.g.,
poly(aspartic acid; P(Asp)], which is rendered hydrophobic by the chemical conjugation
of a hydrophobic drug or is formed through the association of two oppositely
charged polyions (polyion complex micelles). Another special group of polymeric
micelles is formed by lipid - core micelles, that is, micelles formed by conjugates of
soluble copolymers with lipids (e.g., PEG – PE) [35, 36] .
Three methods are used to manufacture polymeric micelles: dialysis, emulsifi cation,
and fi lm methods. In the dialysis method, the drug and copolymer are dissolved
together in a water - miscible solvent (e.g., ethanol) followed by dialysis against
water. During the process (possibly several days), the insoluble drugs are incorporated
into the formed micellar core. In the emulsifi cation method, an O/W emulsion
is fi rst prepared using an aqueous solution of the copolymer and the drug solution
in a water - insoluble volatile solvent (e.g., chloroform). The drug - loaded micelle is
formed as solvent evaporation. In the fi lm method, the copolymer solution and the
drug solution are dissolved separately in miscible volatile organic solvents and are
mixed followed by evaporating solvents to form a polymer/drug fi lm. The fi lm is
hydrated in water or buffers, and then the micelle is formed by intensive shaking.
If the amount of a drug exceeds the solubilization capacity of micelles, the excess
drug precipitates in a crystalline form and is removed by fi ltration. The loading
effi ciency for different compounds varies from 1.5 to 50% by weight. The major
driving force behind self - association of amphiphilic polymers is the decrease of free
energy of the system due to removal of hydrophobic fragments from the aqueous
surroundings with the formation of micelle core stabilized by hydrophilic blocks
exposed to water [35, 36] .
Various drugs, for example, diazepam and indomethacin, doxorubicin, anthracycline
antibiotics, and polynucleotides, were effectively solubilized in polymeric
micelles. Also polymeric micelles can carry various reporter (contrast) groups and
become the imaging agents. Besides targeted drug delivery due to the EPR effect
of tumor, specifi c polymeric micelles having stimuli - responsive amphiphilic block
copolymers, targeting ligand molecules, or monoclonal antibody molecules are also
manufactured [35, 36] .
Nanogels Nanogels are colloidal stable particles made from hydrogels with
nanosized hydrophilic polymeric networks. Hydrogels are the simple gels swelling
strongly in aqueous media, typically composed of hydrophilic polymer components
cross - linked into a network by either covalent (chemical cross - linking) or noncovalent
(physical cross - linking) interactions. It is the cross - linking that provides for
dimensional stability, while the high solvent content gives rise to the fl uidlike transport
properties. Cross - links are important to maintain the network structure of the
hydrogels and prevent dissolution of the hydrophilic chains [121] .
Two methods, emulsifi cation – evaporation and the micelle/nanoparticle approach,
are used to manufacture nanogels. In the former method, bis - activated PEG in
dichloromethane is added dropwise to the aqueous solution of polyethylenimine
(PEI) and then sonicated. The resulting white emulsion is evaporated in vacuum,
producing a clear, slightly opalescent solution. This solution is stirred for less than
one day at room temperature and much debris is separated by centrifugation. The
nanogel suspension is obtained after dialysis against water [51] . This procedure is
convenient except for using organic solvents, a vacuum evaporation step, and the
obtained particles with a wide size distribution. Another method involves surface
preactivated micelles or nanoparticles followed by reaction with other polymers on
the surface in aqueous media. None of the organic solvents involved are of benefi t.
For example, a Pluronic block copolymer both ends of which are activated by 1,1 . -
carbonyldiimidazole is dissolved in water at a concentration above its CMC. A
diluted aqueous solution of PEI is then added dropwise to the micellar solution,
stirring overnight. During this procedure a covalently linked cationic polymer PEI
layer is formed on the Pluronic micelles, producing nanogels with narrow size distribution.
After dialysis the resulting nanogel suspension can be further lyophilized.
Using this procedure, the nanogels based on Pluronic P85/PEG and F127/PEG are
obtained with fi nal yields of 55 and 70% by weight and average hydrodynamic
diameters of 100 and 180 nm, respectively [20, 52] .
Many drugs can be entrapped into nanogels, for example, valproic acid, nucleoside
analogues, antisense oligonucleotides, adenosine triphosphate (ATP), and small
interfering ribonucleic acid (siRNA). Because macromolecular drugs such as peptides
and proteins need to locate in a hydrophilic environment to maintain their
activity, the particular hydrophilic property of nanogels would be of benefi t. Special
functions such as cellular targeting, crossing the BBB, and controlled release may
also be achieved by the nanogel technique. In addition, the nanogel materials should
be biodegradable. In cationic nanogels, PEI and PEG are cross - linked via urethane
bonds, usually considered as stable links. However, due to the presence of highly
protonated PEI, hydrolysis of these bonds was signifi cantly accelerated, and the
polymer network of nanogels could rapidly degrade in aqueous solution at the
physiological pH during a period of about two weeks [20] .
Dendrimers Dendrimers attracted much attention after they were fi rst investigated
by Tomalia 20 years ago [122, 123] , and they have become the star molecules
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1272 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
in recent years. Dendrimers possess perfect nanoarchitectures from 1 nm to more
than 10 nm, consisting of repetitive chemical moieties with tree architecture. According
to repetitive folds, dendrimers with the same basic cores are divided into a series
of generations. The higher generation of dendrimers represents more repetitive
units. Dendrimers are hyperbranched macromolecules that can be subdivided into
three architectural parts: (a) the multivalent surface, with a high number of potential
reactive sites; (b) the “ outer shell ” just beneath the surface, having a well -
defi ned microenvironment protected from the outside by the dendrimer surface;
and (c) the core, which in higher generation dendrimers is protected from the surroundings,
creating a microenvironment surrounded by dendritic branches. Therefore,
the interior of dendrimers is well suited for entrapment of guest molecules.
The multivalent surfaces on a higher generation dendrimer can contain a very high
number of functional groups. This makes the dendritic surfaces and outer shell well
suited to host – guest interactions. Dendrimers can be tailored specifi cally for the
desired purposes, for example, as dendritic sensors, drug vehicles, or even drugs
[23] .
Dendrimers are synthesized through a stepwise repetitive reaction sequence,
wherein a convergent or divergent approach is used. On the one hand, the most
divergent dendrimer syntheses require excess monomer loading and lengthy chromatographic
separations, particularly at higher generations. On the other hand,
convergent synthesis strategies are generally limited to the construction of only
lower generation dendrimers due to the nanoscale steric issues that are encountered
when attaching the dendrons to the molecular - level core [124] . Currently much of
the work on dendrimers has been based on the commercially available Starburst
poly(amidoamine) (PAMAM) dendrimers that are extensively studied as drug carriers.
PAMAM may be synthesized from an ammonia or ethylenediamine core
(EDA) by the divergent approach, involving Michael addition followed by amidation
with methyl acrylate and resulting in the production of a dendrimer family
(G = 0 – 7), and half - generation dendrimers are carboxyl terminated and full - generation
dendrimers are amine terminated (e.g., G = 5.0, 5.3 nm in size) [122, 123] .
Dendrimers have been evaluated as drug nanocarriers, gene transfection agents
imaging agents, and nanodrugs [124] . Also many surface - modifi ed dendrimers have
been synthesized to obtain more functions such as active targeting and gene delivery.
Dendrimers may be used as drugs for antibacterial and antiviral treatment and
as antitumor agents. VivaGel, a topical water - based gel based on sulfonated naphthyl
- modifi ed poly(lysine) dendrimers, has been evaluated against human immunodefi
ciency virus (HIV) and other sexually transmitted diseases (STDs). The cationic
dendrimers prefer to destabilize cell membranes and cause cell lysis and the cytotoxicity
is generation dependent with higher generation dendrimers being the most
toxic. The degree of substitution as well as the type of amine functionality is important,
with primary amines being more toxic than secondary or tertiary amines.
Another common dendrimer, poly(propylenimine) (PPI), shows similar behavior.
However, anionic dendrimers show signifi cantly lower cytotoxicity than cationic
ones. PEG or fatty acid surface - modifi ed dendrimers can reduce the cytotoxicity of
cationic dendrimers [124] .
Carbon Nanotubes, Fullerenes, and Quantum Dots Carbon nanotubes and fullerenes
are carbon - based nanomaterials, and quantum dots are semiconductor nano
crystals. All of them show hydrophobic property. The possibility of cytotoxicity of
these materials with inorganic nature should not be ignored although low toxicity
is shown [25, 40, 125, 126] . However, these seemingly good results may be partly
attributed to their poor solubility in polar solvents, which subsequently makes
investigation of their biological properties diffi cult. They hardly load any drugs
unless the surface is modifi ed hydrophilically. Because these nanomaterials are
mainly produced in laboratories with the special devices, their modifi cations and
subsequent pharmacological investigations are limited. However, a number of functional
derivatives have been synthesized, and it is found that the modifi ed products
have potent and selective pharmacological effects on organs, cells, enzymes, and
nucleic acids [25, 37, 57] .
7.2.3.2 Nanosuspensions
Nanosuspensions of drugs are submicrometer colloidal dispersions of pure particles
of drug which are stabilized by surfactants. A surprisingly large proportion of new
drug candidates emerging from drug discovery programs are water insoluble, and
therefore poorly bioavailable, leading to development efforts being abandoned.
More than 40% of active substances during formulation development by the pharmaceutical
industry are poorly water soluble. A substantial factor that prevents the
development of such substances is the limited dissolution rate. Nanosuspensions are
promising in addressing these so - called brickdust candidates. During the process of
overcoming issues involving solubility, the additional pharmacokinetic benefi ts of
drugs formulated in nanosuspensions come to be appreciated [19, 61] .
Nanosuspensions can be used for those water - insoluble and oil - soluble compounds
(high log P ), although other lipidic carriers such as liposomes and emulsions
can be used to formulate these compounds as well. However, nanosuspensions can
be used to address other problems, such as compounds that are insoluble in both
water and oil. Nanosuspensions can maintain the drug in a preferred crystalline
state of size suffi ciently small for pharmaceutical acceptability. For reasons of convenience
to the patients, aqueous nanosuspensions can also be transformed to
tablets or capsules after spray drying or freeze drying. Moreover, utilization of the
dense, solid state confers an additional advantage of higher mass per volume loading.
This is crucial when high dosing is required, for example, low - volume intramuscular
and ophthalmic applications. Conventional approaches often attempt to solubilize
insoluble drugs with the use of excessive amounts of cosolvents, but this often brings
problems of toxicity. Besides, very large doses of drugs must be administered to
animals when acute toxicity is investigated in preclinical research. As a result, the
interference of toxic side effects caused by cosolvents cannot be ignored if using
them [19] .
Nanosuspensions are not nanocarriers so that what is emphasized during manufacturing
is not materials but the manufacturing techniques. Only drugs and stabilizers
(usually surfactants) participate in manufacturing nanosuspensions so that the
process may be simple depending on drug instincts but sometimes it is not easy.
The bottom - up and top - down approaches may be used in manufacturing nanosuspensions
depending on drug nature and in - house devices.
Antisolvent precipitation is a bottom - up method wherein two phases are involved:
the initial creation of crystal nuclei of drugs and the subsequent growth. Formation
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1274 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
of a stable suspension with the smallest particle size requires a high nucleation rate
but low growth rate. Both process rates are dependent on temperature and supersaturation.
The optimum temperature for nucleation might lie below that for growth.
A high - supersaturation condition is achieved by adding small amounts of a water -
miscible organic solution of the drug to the nonsolvent (water) under rapid mixing,
which leads to spontaneous nucleation. At high - supersaturation levels, the crystal
habit or external appearance is changed to a needlelike or dendritic morphology.
These crystals are easily broken, forming new smaller nuclei. Rapidly grown crystals
tend to be more imperfect and often incorporate impurities and dislocations. This
effect is more pronounced for fl exible molecules that have many degrees of freedom
[19] . The presence of stabilizing surfactants is generally necessary to assist in forming
submicrometer particles, and hydrophilic groups in the surfactants lead to rapid
wetting of the high - surface - area particles in aqueous media, for example, in the case
of oral administration. It is well known that the unprotective surfaces of nanoparticles
show a high energy that leads to particle agglomeration. Therefore, the
nanoparticles must be protected by, for example, steric hindrance and electrostatic
pulsion. In the case of itraconazole (ITZ) nanosuspension manufacturing, a mixture
solution of ITZ and Poloxamer (P407) in THF at room temperature was mechanically
injected into a P407 aqueous solution at 3 ° C. Magnetic stirring was utilized to
enhance heat and mass transfer. Nanosuspensions containing sub - 300 - nm particles
were obtained with drug loads as high as 86% [60] .
Top - down methods are also commonly used to manufacture nanosuspensions,
including wet milling and homogenization. In pearl/ball milling, the active agent, in
the presence of surface stabilizer(s), is comminuted by milling media. Particle size
is determined by stress intensity and the number of contact points. The drug macrosuspensions
are poured into a milling container containing milling pearls from,
for example, glass, zircon oxide or special polymers such as hard polystyrene derivatives.
The drugs are ground to nanocrystals between the pearls. The nanosus pension -
derived products, Rapamune (sirolimus tablets) and Emend (aprepitant capsules),
were approved by the FDA and launched in 2000 and 2003, respectively. They are
manufactured by Elan ’ s NanoCrytal technology using a proprietary wet - milling
technique. A general problem of pearl mills is potential erosion of materials from
the milling pearls leading to product contamination. A polymer as substitution may
minimize erosion. Scaling up with pearl mills is possible; however, there is a certain
limitation in the size of the mill due to its weight. Up to about two - thirds of the mill
volume are the pearls lead to heavy weight of the machinery, thus limiting the
maximum batch size [127] .
Homogenization can be divided into two types. One is the forcing of a suspension
under pressure through a narrow - aperture valve (microfl uidization). The other is
high - pressure homogenization of particles in water or other media (piston gap).
Microfl uidization is a jet stream principle. The suspension is accelerated and passes
a specially designed homogenization chamber with a high velocity. In the Z - type
chamber, the suspension changes the direction of its fl ow a few times, leading to
particle collision and shear forces, while in the Y - type chamber, the suspension
stream is divided into two streams colliding frontally. Sometimes it is necessary to
pass through the microfl uidizer many times to minimize particle size [127] .
In piston - gap homogenization, suspension contained in a cylinder passes a very
thin gap with an extremely high velocity. Bubbles of water vapor are produced for
compensation followed by collapsing in the valve. Cavitation - induced shock waves
occur and crack the particles. Homogenization can also be utilized to further reduce
the size of particles made by precipitation. Commonly dendritic crystals made by
precipitation are more susceptible to rupture by the subsequent mechanical shock
of homogenization. In addition, the mechanical energy supplied by the homogenizer
can change the initially formed, unstable amorphous particles to a stable state
through subsequent crystallization. The size of drug nanocrystals depends mainly
on (a) power density of the homogenizer, (b) number of homogenization cycles,
and (c) temperature. Another important determining factor for the fi nal size of drug
nanocrystals is the hardness of drugs. A relatively soft drug, paclitaxel, can reach
250 nm in size, which is less than harder drugs. The size should be homogeneous as
achieved with a homogenizer to avoid physical destabilization. Stabilizers have an
effect on long - term physical stability but not on maximum dispersity or the nanocrystal
shape. Contamination from the production equipment is typically below
1 ppm, which is within a suitable range. Besides water, water - free media and water
mixtures are used preferably due to advantages of easy evaporation or homogenization
at higher temperature (with subsequent more cavitation). Oils, propylene
glycol, and PEG with varieous molecular weights can be used. For PEG being solid
at room temperature (e.g., PEG 1000, 6000), the obtained drug nanocrystals disperse
in PEG particles at room temperature and can conveniently be put into hard
capsules [19, 127] .
The lyophilized drug nanosuspensions can be transferred to a fi nal dry oral
dosage form such as tablets or reconstituted prior to administration. Drug nanosuspensions
can be directly used as parenteral products. A shelf life of up to three years
was shown for selected nanosuspensions. Sterilization can be achieved by aseptic
processing of previously sterilized components, membrane fi ltration for parti cles
suffi ciently small or for drugs that can withstand it, steam sterilization, or
. - irradiation.
7.2.3.3 Self - Assembled Drug Nanostructures
It is well known that liposomes are composed of amphiphilic phospholipids. The
formation of liposomes is actually a procedure of molecular self - assembly. Furthermore,
great amounts of amphiphilic compounds, natural or synthesized rather than
phospholipid - like surfactants, can also self - assemble into ordered aggregates in
aqueous media or organic solvents [128 – 130] . The formed aggregates are mostly
nanoarchitectures with various shapes such as vesicle, rod, ribbon, fi ber, tube, or
helix [131 – 134] . They can remain relatively stable in certain environments. Many
of them may become drug nanocarriers, such as liposomes, or even perform as active
agents [109, 135, 136] . The research of self - assembled nanocarriers seems to go into
the fi eld of supramolecular chemistry. But these results also give us some useful
information for developing new approaches of drug delivery. A novel idea may
relate to why we do not try to construct a nanostructure from drugs themselves.
Twenty years ago the cardiovascular drug pindolol was conjugated with stearyl
glycerol via succinyl as linker followed by forming maleate salt to obtain pindolol
diglyceride. Vaizoglu and Speiser used the word “ pharmacosomes ” to describe the
colloidal dispersions prepared from drug – lipid conjugates with or without additional
surfactants [137] . Pindolol pharmacosomes (vesicle - like) were prepared from
NANOTECHNOLOGY FOR DRUG DELIVERY 1275
1276 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
pindolol diglyceride, which showed good stability and useful pharmacokinetic
parameters. Unfortunately, no more detailed research is being done about pharmacosomes,
possibly because no appropriate theory supports the new dosage form and
no proper drugs and lipids are selected.
The idea of manufacturing nanostructures from drugs may be resourced from
liposomes, pharmacosomes, and other molecular self - assemblies in supramolecular
chemistry. More importantly, this novel idea resulted from long - term efforts to
work on drug delivery and to solve the disadvantages of current drug carriers.
Almost all current delivery systems (usually called carriers) passively load drugs so
as to always lead to low entrapment effi ciency and possible drug leakage in preparation,
preservation, and transportation in vivo [30, 73] , and these carriers might have
been destroyed in vivo before reaching target sites. In addition, lipophilic biomembranes,
including cell membranes, usually prevent hydrophilic drugs from entering
into target sites. If carriers cannot override cell membranes except for endocytosis/
phagocytosis by cells/macrophages, the loaded hydrophilic drugs are probably
released only on target surfaces, not entering. In summary, a majority of drugs could
not eventually reach and get into target sites due to poor properties of carriers and
drugs.
A novel technology involving prodrug, molecular self - assembly, and nanotechnology
was developed to address the problems of drugs and classical carriers. The
nanostructures are formed by molecular self - assembly of amphiphilic prodrugs in
aqueous media generally without additional excipients. The self - assembled drug
nanostructures not only possess the amphiphilic property of monomolecular drugs,
benefi ting to cross biomembranes, but also deliver themselves in vivo without “ carriers
” and then prefer to release active parent agents with a sustained rate. They
may overcome some defi ciencies of traditional nanocarriers such as liposomes, for
example, low effi ciency of drug entrapment and loading, rapid drug leakage in
vitro/in vivo, and bad stability [22] .
Self - assembled drug nanostructure is not a proprietary term in pharmacy currently.
Herein this term is defi ned as the ordered nanosized self - aggregates of
amphiphilic drugs in aqueous media. It is abbreviated as SADN. Another term,
self - assembled drug delivery system (SADDS), introduced by Jin [22] obviously
includes SADN. Unfortunately, most current drugs do not occupy an amphiphilic
and self - assembling nature [138] , so they must be modifi ed in chemical structures
before manufacturing SADNs. Then the prodrug technique is selected.
In contrast to nanosuspension technology, SADN technology is mainly applied
to hydrophilic or polar drugs. These drugs are rationally modifi ed to their amphiphilic
prodrugs by lipid derivation. Molecular self - assembly in aqueous media is the
key to manufacturing SADNs. According to the principles of supramolecular chemistry,
the amphiphilic molecules forming self - assemblies should have proper structural
conformation. The morphology of assemblies also depends on the structure
of amphiphiles and the surrounding environment. Some parameters, including the
optimal head area ao , the volume v of fl uid hydrophobic chain, and the maximum
effi cient chain length lc , are used to describe the conformation of amphiphiles. The
critical packing parameter (CPP), equal to v / aolc , can be applied to direct self -
assembly behavior. The amphiphiles prefer to form vesicles when the CPP is – 1.
Generally, single - chain lipids with small head group areas (e.g., SDS in a low - salt
solution) are cone shaped, prone to form spherical micelles, while double - chain
lipids with large head group area and fl uid chains (e.g., phosphatidylcholine) are
truncated - cone shaped, prone to form fl exible bilayers, vesicles [139] . Therefore,
the lipids used for drug covalent conjugation are rationally selected from long - chain
alkyl lipids, for example, fatty acids, lipid alcohols, lipid amines, long - chain glycolipids,
and cholesterol. Furthermore, too large or small polar drugs are not appropriate
for preparation of self - assembling prodrugs.
Antiviral nucleoside analogues such as acyclovir, didanosine, and zidovudine
were used to prepare their long - chain glyceride or cholesteryl derivatives in Jin ’ s
laboratory [140 – 142] . All the derivatives showed amphiphilic property and some of
them self - assembled into ordered aggregates in water. Amphiphilic prodrugs were
subsequently used to manufacture self - assemblies using the bottom - up approach,
such as liposomes, and the self - assembly may be driven by a hydrophobic interaction,
hydrogen bonding, and so on [21, 143] . The monomolecular amphiphilic
prodrug is prone to incorporate into the assemblies and not to depart so that almost
no drugs leak from SADNs. The whole self - assemblies are nearly composed of
amphiphilic drugs, leading to high drug loading. When SADNs reach targets in vivo,
the continual dissociation of aggregates and the sustained degradation of prodrugs
provide controlled drug release.
Acyclovir self - assembled nanoparticles as SADNs were manufactured which
showed strong targeting effect in vivo (mainly in the MPSs) and sustained release
at target sites [22] . Based on this paradigm, a general process to manufacture
SADNs is as follows [21, 22] :
(a) To obtain an amphiphilic prodrug with proper molecular structure,
stearyl glyceride was selected to conjugate with acyclovir. Succinyl acyclovir
(SACV) was synthesized and subsequently conjugated with stearyl glyceride
by acylation reaction. The amphiphilic prodrug stearyl - glyceride - succinyl
acyclovir (SGSA) was obtained.
(b) The injection method was used to manufacture SADNs. SGSA was
dissolved in the water - miscible solvent THF. The solution containing
5 mg/mL SGSA was slowly and continually injected into vortexed water under
surface via a 100 - . L microsyringe. A homogeneous and slightly opalescent
suspension was obtained, which was acyclovir self - assembled nanoparticles
(SANs).
(c) The organic solvent was removed from the suspension through evaporation
by heating, and the suspension can further be concentrated by removing
water under heating until the appropriate prodrug concentration is obtained.
The concentrated suspension was transferred into ampoules and sealed. It
may be sterilized by autoclave.
(d) Acyclovir SANs were characterized. They were cuboidlike shaped based on
transmission electron microscopy and were nanoscale with an average size
of 83 nm based on dynamic light scattering. The zeta potential of . 31 mV
indicated the nanoparticles had negative surface charge. Hydrophobic interaction
of alkyl chains improves SGSA molecules to form bilayers, and then
cuboidlike nanoparticles were achieved by layer - by - layer aggregation based
on inter - bilayer hydrogen bonding. The gel – liquid crystalline phase transition
was about 50 ° C, and the mechanism of confi guration changes on phase
transition was analyzed [144] .
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1278 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
(e) The in vitro and in vivo behavior of acyclovir SANs was investigated. The
SANs kept the physical state stable upon centrifugation or exposure of
some common additives Autoclave and bath heat for sterilization hardly in-
fl uenced the state of SANs. SGSA in SANs showed good chemical stability
in weak acidic or neutral buffers, although they were very sensitive to alkaline
solutions and carboxylester enzymes. The SANs were rapidly removed
from blood circulation after bolus IV administration to rabbits and mainly
distributed in liver, spleen, and lung followed by slow elimination in these
tissues.
Because nucleoside analogues are important and plentiful agents in antiviral and
anticancer therapy, other polar drugs can simulate the above process to manufacture
SADNs. In addition, macrophages as the reservoirs of HIV or other viruses
prefer to carry viruses throughout the whole body even to the central nervous
system. How to deliver drugs to macrophages has become a key issue in antiviral
therapy [145] . SADNs prefer to show macrophage - specifi c distribution. Therefore,
the antiviral SADNs show the advantages of high drug loading, controlled release,
and targeting macrophage, which may provide a useful and promising way to treat
increasing viral diseases. In the future SADNs will be modifi ed to get more functions,
such as long circulating effect, pH sensitivity, and use in antiviral, anticancer,
and gene therapy.
7.2.4 NANOMEDICINE
Nanomedicine is a concept with broad implications. According to the defi nition of
the European Science Foundation (ESF), the fi eld of nanomedicine is the science
and technology of diagnosing, treating, and preventing disease and traumatic injury,
of relieving pain, and of preserving and improving human health using molecular
tools and molecular knowledge of the human body. It is perceived as embracing
fi ve main subdisciplines that in many ways are overlapping and underpinned by the
following common technical issues: (a) analytical tools, (b) nanoimaging, (c) nanomaterials
and nanodevices, (d) novel therapeutics and drug delivery systems, and
(e) clinical, regulatory, and toxicological issues. The ESF ’ s scientifi c forward look
on nanomedicine warns that nanomedicine benefi ts will be lost without major
investment and calls for a coordinated European strategy to deliver new nanotechnology
- based medical tools for diagnostics and therapeutics [146] . From a view of
narrow sense, nanomedicine can be defi ned as the use of nanoscale or nanostructured
materials in medicine that have unique medical effects according to their
structure. In addition, nanostructures up to 1000 nm in size are adopted because
from a technical point of view the control of materials in this size range not only
results in new medical effects but also requires novel, scientifi cally demanding
chemistry and manufacturing techniques [147] .
The increasing research in nanomedicine has led to many publications, accounting
for about 4% of publications on nanotechnology research (about 34,300 documents
in 2004) worldwide. Also commercialization efforts in nanomedicine are
increasing. About 207 companies (including 158 small - and medium - size enter
prises) visibly pursue nanomedicine activities and devote either all or a signifi cant
share of their business to the development of nanomedicines. A characterizing
feature of nanotechnology is its enabling function to add new functionality to existing
products, making them more competitive. For example, Ambisome (Gilead),
a liposomal formulation of the fungicide Fungizone (Bristol - Myers Squibb) that
shows reduced kidney toxicity, had total sales of $ 212 million in 2004. The total
sales of the 38 identifi ed nanomedicine products from all sectors of nanomedicine
are estimated to be $ 6.8 billion in 2004. The market is predicted to further grow
to . $ 12 billion by the year 2012. Currently, nanomedicine is dominated by drug
delivery systems, accounting for more than 75% of the total sales. Twenty - three
nanoscale drug delivery systems are available on the market, but within this group,
three polymer therapeutics alone account for sales of $ 3.2 billion: (i) Neulasta
(pegfi lagrastim; recombinant methionyl human granulocyte colony stimulating
factor and PEG), (ii) Pegasys (PEGylated interferon . 2a), and (iii) PEG - Intron
(PEGylated interferon . 2a), all protein therapeutics to which nanoscale polymer
strings of PEG have been attached to reduce immunogenicity and to prolong
plasma half - life. The most widely used nanotechnology product in the fi eld of in
vitro diagnostics is colloidal gold in lateral fl ow assays, rapid tests for pregnancy,
ovulation, HIV, and other indications. Magnetic nanoparticles are also used for
cell - sorting applications in clinical diagnostics. In the fi eld of biomaterials, the commercial
status of nanotechnology - based dental restoratives is most advanced. Furthermore,
nanohydroxyapatite - based products for the repair of bone defects have
been successfully commercialized. Nanotechnology - based contrast agents are a
market with estimated sales of about $ 12 million. All of the marketed contrast
agents consist of superparamagnetic iron oxide nanoparticles for magnetic resonance
imaging. Nanostructured electrodes are used to improve the electrode tissue
contact, and nanomaterials are used to increase the biocompatibility of implant
housings. Pacemakers with nanostructured (fractal) electrodes are the only active
implants currently on the market that contain a nanotechnology - enabled component
[147] .
In spite of the great success, the safety of nanomedicine is maintained as a
worrying issue. A new discipline appears to exploit the toxicological problem in
nanotechnology applications, called nanotoxicology. Nanotoxicology can be
defi ned as safety evaluation of engineered nanostructures and nanodevices.
Nanomaterials could be deposited in all regions of the respiratory tract after
inhalation. The small size facilitates uptake into cells and transcytosis across
epithelial and endothelial cells into the blood and lymph circulation to reach
potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and
heart. Access to the central nervous system and ganglia via translocation along
axons and dendrites of neurons has also been observed. Nanomaterials could
also penetrate the skin via uptake into lymphatic channels [41] . Although possible
damages of those biodegradable nanomaterials for drug delivery need consideration,
too much fear is needless. Usually they would be ultimately degraded
nearly without any trace. However, hard or nonbiodegradable materials, including
carbon nanotubes, fullerenes, quantum dots, polystyrene, and metal nanoparticles,
should be thoroughly investigated about their toxic effects on our body
before clinical application.
NANOMEDICINE 1279
1280 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING
7.2.5 PERSPECTIVE
Nanotechnology has had a great effect on pharmaceutical manufacturing and
strongly improves it, rapidly progressing. No one suspects the key role nanotechnology
will have in future pharmaceutical research and manufacturing. The continually
increasing achievements in nanotechnology will result in exciting changes in the
pharmaceutical industry. Now it has gone into an era of controlling the behavior of
drugs in vitro/in vivo. Although some problems such as toxicity are not addressed,
the tremendous advantages that result from nanotechnology are obvious. More and
more potent medicines will be manufactured and diseases such as cancer, HIV, cardiovascular
diseases, and nervous system diseases may well be cured or better
treated in the future by nanomedicine technology.
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145. Aquaro , S. , Calio , R. , Balzarini , J. , Bellocchi , M. C. , Garaci , E. , and Perno , C. F. ( 2002 ),
Macrophages and HIV infection: Therapeutical approaches toward this strategic virus
reservoir , Antivir. Res ., 55 , 209 – 225 .
146. European Science Foundation ( 2005 ), Nanomedicine, An ESF - European Medical
Research Council (EMRC) forward look report, European Science Foundation,
Strasbourg.
147. Wagner , V. , Dullaart , A. , Bock , A. - K. , and Zweck , A. ( 2006 ), The emerging nanomedicine
landscape , Nat. Biotechnol ., 24 , 1211 – 1217 .
1289
7.3
PHARMACEUTICAL
NANOSYSTEMS: MANUFACTURE,
CHARACTERIZATION, AND
SAFETY
D. F. Chowdhury
University of Oxford, Oxford, United Kingdom
Contents
7.3.1 Defi nition
7.3.1.1 Top - Down and Bottom - Up Approaches to Nanotechnology
7.3.2 Taxonomy of Nanomedicine Technologies
7.3.3 Nano – Pharmaceutical Systems
7.3.4 Description of Nanosystems
7.3.4.1 Polymeric Systems
7.3.4.2 Quantum Dots and Quantum Confi nement
7.3.4.3 Metal Nanoparticles and Surface Plasmon Resonance
7.3.4.4 Self - Assembled Systems
7.3.4.5 Nanostructures Based on Carbon
7.3.5 Manufacturing Technologies
7.3.5.1 Nanoscale Assembly Methods
7.3.5.2 Nano - structuring processes for polymeric materials
7.3.6 Characterization Techniques
7.3.6.1 Nanoparticle Characterization Methods and Tools
7.3.6.2 Scanning Probe Technologies
7.3.7 Toxicology Considerations
7.3.7.1 Lung Toxicity
7.3.7.2 Systemic Uptake
7.3.7.3 Skin Permeation of Nanoparticles
References
Suggested Reading
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1290 PHARMACEUTICAL NANOSYSTEMS
7.3.1 DEFINITION
Nanotechnology is an enabling technology and one which is generally manifest at
the primary level in the form of nanomaterials. The defi nition of nanotechnology
therefore focuses on materials and how manipulation at the nanoscale leads to novel
properties and therefore potentially new uses. The pharmaceutical industry has yet
to adopt strict guidelines for what falls under the remit of nanotechnology, with
numerous defi nitions in existence. For the purpose of this chapter, the current U.S.
Food and Drug Administration (FDA) defi nition for nanotechnology as applied to
pharmaceuticals is deemed most appropriate. The FDA describes nanotechnology
as technology that includes the following [1] :
1. Research and technology development or products regulated by the FDA that
are at the atomic, molecular or macromolecular levels and where at least one
dimension that affects the functional behavior of the product is in the length
scale range of approximately 1 – 100 nm
2. Creating and using structures, devices, and systems that have novel properties
and functions because of their small and/or intermediate size
3. Ability to control or manipulate at the atomic scale
Nanotechnology is therefore essentially about understanding and manipulating
materials at the atomic, molecular, and macromolecular level in a way that imparts
properties to the material that would otherwise not exist either as individual atoms
or as bulk processed macroscopic systems.
Properties that can be exploited to provide novel and unique properties to materials
include surface and quantum effects, for example, van der Waals forces; electrostatic
interaction; ionic, covalent and hydrogen bonding; and quantum confi nement.
Additionally nonconventional means of molecular assembly and atomic manipulation
can lead to novel material properties. Control and exploitation of these effects
can lead to new and useful changes to the thermal, magnetic, electrical, optical and
mechanical, and biological and physicochemical properties of materials.
7.3.1.1 Top - Down and Bottom - Up Approaches to Nanotechnology
There are generally two approaches to nanotechnology, the top - down and bottom -
up approaches. As the names suggest, the top - down approach utilizes ultraprecision
machining and nanolithographic techniques among others to achieve very high
defi nition structures with nanolevel accuracy, usually either by removing material
from the surface of a larger structure until the desired structure with desired features
is achieved or through deposition of material with almost atomic - scale precision
and control. The bottom - up approach involves the assembly of atoms, molecules,
or nanoscale components to assemble a larger structure within the nanoscale range.
There are numerous methods by which this can be achieved, including conventional
bulk chemical processing methods and exploitation of chemical and biological self -
assembly techniques. The pharmaceutical industry is primarily involved in the application
of nanomaterials rather than the discovery and development of new materials,
though as this chapter will indicate, there are often areas of overlap between what
is a new material and a construct of a novel material.
7.3.2 TAXONOMY OF NANOMEDICINE TECHNOLOGIES
A useful starting point would be to gauge the breadth of technologies falling under
the classifi cation of nanomedicine. Table 1 provides a means of classifying materials
and processes derived from nanotechnology as relating to pharmaceuticals and
medicine in general.
7.3.3 NANO – PHARMACEUTICAL SYSTEMS
Having gauged the huge scope of nanotechnology in medicine, the scope of this
chapter is limited to pharmaceuticals. The term pharmaceutical is intended in the
context of systems pertaining to drugs or dosage formulations. Nano – pharmaceutical
systems generally imply products that may in their own right or in combination
with another moiety bring about therapeutic benefi t. They may also include engineered
nanostructured systems that may act as a carrier for drugs or a delivery
vehicle or a delivery system for drugs and therapeutic agents. The defi nition is
extended to include imaging systems which may be used alone or in conjunction
with therapeutic agents given the numerous nanosystems that have found application
in diagnostics and imaging.
The various nanoscale architectures that can be achieved using nanotechnology
include spheres (solid or hollow), tubes, porous particles, solid particles, and branched
structures, and with the rapid evolution of lithographic techniques, three -
dimensional objects of almost any desired shape can be achieved from both metals
and polymers. Given the vast spectrum of materials of construction, size, shape, and
form covered by nanosystems, a simple means of classifi cation is needed for effective
differentiation between systems. Nanosystems can be classifi ed in a number of ways,
for example, according to their elemental composition, according to size, structure,
and function, or perhaps according to a structure – function relationship. It can be
said that nanosystems fall into the broader category of nanostructures, which can
be generalized into the following categories:
Particulate nanostructures
Capsular nanostructures
Crystalline nanostructures
Polymeric nanostructures
It can be readily appreciated that there will be an element of overlap between
the broad categories above and in particular with evolving complex hybrid systems.
The classifi cation is intended as a point of reference and for ease of understanding
the vast possibilities that exist with nanosystems without the need for constant
reclassifi cation as far as possible. These structures may be further differentiated
according to their primary composition:
Organic
Inorganic
Organic/inorganic hybrid
Carbon based
NANO-PHARMACEUTICAL SYSTEMS 1291
1292 PHARMACEUTICAL NANOSYSTEMS
TABLE 1 Partial Nanomedicine Technologies Taxonomy
Raw nanomaterials
Nanoparticle coatings
Nanocrystalline materials
Nanostructured materials
Cyclic peptides
Dendrimers
Detoxifi cation agents
Fullerenes
Functional drug carriers
Magnetic resonance (MR) scanning
(nanoparticles)
Nanobarcodes
Nanoemulsions
Nanofi bers
Nanoparticles
Nanoshells
Carbon nanotubes
Quantum dots
Artifi cial binding sites
Artifi cial antibodies
Artifi cial enzymes
Artifi cial receptors
Molecularly imprinted polymers
Control of surfaces
Artifi cial surfaces, adhesive
Artifi cial surfaces, nonadhesive
Artifi cial surfaces, regulated
Biocompatible surfaces
Biofi lm suppression
Engineered surfaces
Pattern surfaces (contact guidance)
Thin - fi lm coatings
Nanopores
Immunoisolation
Molecular sieves and channels
Nanofi ltration membranes
Nanopores
Separations
Biological research
Nanobiology
Nanoscience in life sciences
Drug delivery
Drug discovery
Biopharmaceuticals
Drug delivery
Drug encapsulation
Smart drugs
Synthetic biology and early nanodevices
Dynamic nanoplatform “ nanosome ”
Tecto - dendrimers
Artifi cial cells and liposomes
Polymeric micelles and polymersomes
Nanorobotics
DNA - based devices and nanorobots
Diamond - based nanorobots
Cell repair devices
Cell simulations and cell diagnostics
Cell chips
Cell stimulators
DNA manipulation, sequencing,
diagnostics
Genetic testing
Deoxyribonucleic acid (DNA)
microarrays
Ultrafast DNA sequencing
DNA manipulation and control
Tools and diagnostics
Bacterial detection systems
Biochips
Biomolecular imaging
Biosensors and biodetection
Diagnostic and defense applications
Endoscopic robots and microscopes
Fullerene - based sensors
Imaging (e.g., cellular)
Lab on chip
Monitoring
Nanosensors
Point - of - care diagnostics
Protein microarrays
Scanning probe microscopy
Intracellular devices
Intracellular assay
Intracellular biocomputers
Intracellular sensors/reporters
Implant inside cells
BioMEMS
Implantable materials and devices
Implanted bio — microelectromechanical
systems (MEMSs), chips, and electrodes
MEMS/nanomaterial - based prosthetics
Sensory aids (e.g.,)
Microarrays
Microcantilever - based sensors
Microfl uidics
Microneedles
Medical MEMS
MEMS surgical devices
Molecular medicine
Genetic therapy
Pharmacogenomics
Artifi cial enzymes and enzyme control
Enzyme manipulation and control
Nanotherapeutics
Antibacterial and antiviral nanoparticles
Fullerene - based pharmaceuticals
Photodynamic therapy
Radiopharmaceuticals
Biotechnology and biorobotics
Biological viral therapy
Virus - based hybrids
Stem cells and cloning
Tissue engineering
Artifi cial organs
Nanobiotechnology
Biorobotics and biobots
Source : From ref. 2 .
TABLE 1 Continued
7.3.4 DESCRIPTION OF NANOSYSTEMS
There is clear evidence from Table 2 that some of the nanosystems indicated are
based on conventional colloidal chemistry, and their characteristics are well established
and understood. The descriptions below deal mainly with systems deemed
nonconventional and cover some of the key novel properties derived from or utilized
as part of their construction.
7.3.4.1 Polymeric Systems
Polymer - based systems offer numerous advantages, such as biocompatibility, biodegradability,
and ability to incorporate functional groups for attachment of drugs.
Drugs can be incorporated into the polymer matrix or in the cavity created by the
polymeric architecture, from which the drug molecule can be released with an
element of temporal control, and controlled pharmacokinetic profi le with almost
zero - order release achievable.
Dendrimers are large complex globular polymeric molecules [46] with well -
defi ned chemical structure, size, and shape [47] . They consist of characteristic three -
dimensional branched structures. The key components of dendrimers are their core,
branches, and end groups, and precise control over these features is possible during
the bottom - up synthesis process, thus allowing control over size composition, and
fi nal chemical reactivity. A more advanced form of dendrimer is the hyperbranched
dendrimer, where precision control over the architectural construct is lost during
the synthesis process [48] . Dendrimers are produced from monomers through an
iterative sequence of reaction steps [49] using either convergent [50, 51] or divergent
[52, 53] step growth polymerization [54 – 56] and have potential applications in gene
and cancer therapy and drug delivery through complexation or encapsulation
[57 – 59] .
7.3.4.2 Quantum Dots and Quantum Confi nement
Quantum dots are inorganic semiconductor nanocrystals that possess physical
dimensions smaller than the exciton Bohr radius, giving rise to the unique phenom-
DESCRIPTION OF NANOSYSTEMS 1293
1294 PHARMACEUTICAL NANOSYSTEMS
enon known as quantum confi nement. Quantum confi nement is the spatial confi nement
of charge carriers (i.e., electrons and holes) within materials. It leads to unique
optical and electrical properties that are not common for bulk solids.
Quantum dots have novel and unique optical, magnetic [60] , and electronic properties,
exceptional imaging properties due to high color intensity, with up to 20 fl uorophores,
high resistance to photobleaching, and narrow spectral line widths. Their
size and composition allow for tunable emission that can be excited using a single
wavelength [61 – 64] . These properties have led to uses as fl uorescent imaging probes,
detection of cell signaling pathways, and cell targeting. Low depth of light penetration
and relatively high background fl uorescence are the key limitations of quantum
dots in in vivo clinical applications. Quantum dots are generally of the size rage 2 –
8 nm in diameter [65] and have large molar extinction coeffi cients [66] , thus making
them very bright in vivo probes.
TABLE 2 Classifi cation of Nanostructures According to Composition and Perceived
Applications
Composition Type of Nanostructure Applications
Organic Polymer micelles [3, 4] Drug delivery
Polymeric spheres [5] Drug delivery
Polymer nanoparticles [6, 7] Drug delivery
Polymer vesicles/containers [8 – 10] Drug delivery
Lipid nanovesicles [11] Drug delivery
Lipid emulsions [12] Drug delivery
Ring peptides [13] Drug delivery
Lipid nanospheres [14, 15] Drug delivery
Lipid nanoparticles [16, 17] Drug delivery
Lipid nanotubes [18] Drug delivery
Peptide nanoparticles [19, 20] Drug delivery
Nanobodies [21] Therapeutic, diagnostic
Dendrimers [22 – 24] Drug delivery, gene delivery
Inorganic Palladium/platinum nanoparticles [25] Drug delivery
Silicon nanoneedles [26] Drug delivery
Porous silicon [27, 28] Drug delivery
Gold nanoparticles [29] Drug delivery
Iron oxide nanoparticles [30] Imaging
Gold nanoshells [31] Imaging agent, thermal ablation
Quantum dots [32] Imaging, cell targeting
Metallic nanoshells [33] Imaging, thermal ablation
Nanocrystals [34, 35] Drug delivery
Organic/
inorganic
hybrid
Nanocomposites [36] Drug delivery
Nanosphere – metallic particle
composite [37]
Drug delivery, imaging
Carbon nanotube clusters [38] Drug delivery, imaging, thermal
ablation
Core – shell structures [39, 40] Imaging, thermal ablation
Carbon
based
Fullerenes [41 – 43] Drug delivery, prodrug
Carbon nanotubes [44, 45] Drug delivery, imaging, thermal
ablation
Applications of quantum dots include optical detection of genes and proteins in
animal models and cell assays and tumor and lymph node visualization through
imaging [67, 68] .
7.3.4.3 Metal Nanoparticles and Surface Plasmon Resonance
Surface plasmons, also known as surface plasmon polaritons or packets of electrons,
are surface electromagnetic waves that propagate parallel along a metal – dielectric
interface [69, 70] . Surface plasmons exist where the complex dielectric constants of
the two media are of opposite sign. The excitation of surface plasmons by light of
a wavelength matching the resonant frequency of the electrons is termed surface
plasmon resonance (SPR) for planar surfaces and localized surface plasmon resonance
where nanometer - sized metallic structures are concerned [71, 72] .
Surface plasmon effects result in useful photothermal effects [73] and have been
used to enhance the surface sensitivity of various spectroscopic measurements [74] ,
including fl uorescence, Raman scattering, and second - harmonic generation.
Metallic nanostructures exhibiting SPR are composed of a dielectric core and
metallic shell, for example, gold sulfi de dielectric core and gold shell. By varying the
core – shell thickness ratio, the surface plasmon resonance is shifted from the visible
to the infrared range [75] , spanning a range that is mostly transparent to human
tissue, that is, has a high physiological transmissivity. Additionally, control over the
particle diameter allows control over light scattering and light absorption at particle
diameters below approximately 75 nm. Potential applications of the photothermal
effects of engineered nanoparticles include the following:
Controlled drug delivery [76]
Analysis of controlled drug release from a matrix [77]
DNA sensor [78, 79]
Deep tissue tumor cell thermal ablation [80]
Real - time assessment of drug action [81, 82]
Immunosensor applications [83, 84]
7.3.4.4 Self - Assembled Systems
Molecular self - assembly is a synthetic technique that has been widely used to
produce nano - and microstructures in a quick and effi cient manner. It has become
all the more crucial to the formation of nanostructures due to the control attainable
over the end product and the relative ease with which nanostructures of defi ned
structure and function can be produced using bulk manufacturing methods.
The basic principle of self - assembly is based on the simultaneous coexistence of
two parallel forces [85, 86] , long - range repulsive forces and short - range attractive
interactions.
The types of structures attainable using molecular self - assembly are referred to
as micellar structures [87] and can take on various sizes and shapes:
Direct spherical micelles [88]
Inverse spherical micelles [89, 90]
DESCRIPTION OF NANOSYSTEMS 1295
1296 PHARMACEUTICAL NANOSYSTEMS
Lamellar sheets [91]
Vesicles (hollow or concentric) [92]
Body - centerd - cubic [92]
Hexagonally packed cylinders/tubes [92]
Gyroids [93]
Hollow spheres [94]
These systems have found widespread use as drug delivery vehicles, and the more
advanced nanosystems are termed smart nano - objects due to their ability to sense
local variations in physiological conditions, such as pH and temperature, and respond
to the stimulus accordingly.
7.3.4.5 Nanostructures Based on Carbon
Nanotubes The structure of carbon nanotubes as observed by scanning tunneling
microscopy is that of rolled grapheme sheets where endpoints of a translation vector
are folded one onto another [95] . Single - walled carbon nanotubes (SWCNTs) were
fi rst reported by Iijima and Ichihashi [96] in 1993. Enormous interest in CNTs has
centered around their unique properties, including high electrical conductivity,
thermal conductivity, high strength and aspect ratio, ultralight weight, and excellent
chemical and thermal stability.
The most common method for the production of carbon nanotubes is hydrocarbon
- based chemical vapor deposition (CVD) [97] and adaptations of the CVD
process [98, 99] , where the nanotubes are formed by the dissolution of elemental
carbon into metal nanoclusters followed by precipitation into nanotubes [100] . The
CVD method is used to produce multiwalled carbon nanotubes (MWCNTs) [101]
and double - walled carbon nanotubes (DWCNTs) [102] as well as SWCNTs [103] .
The biomedical applications of CNTs have been made possible through surface
functionalization of CNTs, which has led to drug and vaccine delivery applications
[104, 105] .
Fullerenes Fullerenes were fi rst discovered in 1985 [106] and are large molecules
composed exclusively of carbon atoms and manifest physically in the form of hollow
spherical cagelike structures. The cages are in the region of 7 – 15 A in diameter with
the most common form being C 60 , though other forms exist too, such as C 70 , C 76 , and
C84 , depending on the number of carbon atoms making up the cage. Fullerenes can
be produced using combustion [107] and arc discharge methods [108] .
Fullerenes offer numerous points of attachment and allow precise bonding of
active chemical groups in three - dimensional (3D) conformations and positional
control with respect to matching conjugated fullerene compounds with a given
target. Water - soluble fullerenes have shown low biological toxicity both in vitro
[109] and in vivo [110] . Some of the potential applications of fullerenes in pharmaceuticals
include their use in neurodegenerative and other disease conditions where
oxidative stress is part of the pathogenesis due to their powerful antioxidant properties
[111] and in nuclear medicine for binding of toxic metals ions, increasing therapeutic
potency of radiation therapy and reducing adverse events as fullerenes do
not undergo biochemical degradation within the body. Fullerene applications in
photodynamic tumor therapy have also been shown [112] .
7.3.5 MANUFACTURING TECHNOLOGIES
There are a diverse range of technologies being applied to the manufacture of
nanosystems for pharmaceutical applications. Some of these are derived from
conventional pharmaceutical technologies, such as colloidal processing, and many
have been adopted from the semiconductor industry, whereby precision spatial
control is achieved over the production of nanosystems and particles using fabrication
techniques. To add to this, new technologies are constantly evolving through
the adaptation and amalgamation of existing technologies in different fi elds or
through pure innovation leading to completely new processes. It is outside the
remit of this chapter to cover in any depth all those manufacturing technologies
that may be applied to pharmaceutical manufacturing. The summary in Table 3
provides a detailed synopsis of the different types of manufacturing processes and
types of technologies for each process. This is followed by a brief introduction to
some of the technologies, with the omission of silicon - and carbon - based fabrication
processes, which are beyond the scope of this chapter, to provide the reader
with a starting point for further detailed study and investigation into those processes
and technologies that may be most suited to their particular product or
concept.
TABLE 3 Summary of Manufacturing Processes and Technologies for Producing
Nanosystems
Manufacturing Process Technology
Nanoscale assembly Self - assembling micellar structures [113, 114]
Bio - self - assembly and aggregation [115, 116]
Nanomanipulation [117, 118]
Soft lithography [119, 120]
Molecular imprinting [121, 122]
Layer - by - layer electrostatic deposition [123, 124]
Chemical vapor deposition [125]
Nanostructuring processes for
polymeric materials
Mold replication [126, 127]
Colloidal lithography [128, 129]
X - ray lithography [130]
Interfacial polymerization [131, 132]
Nanoprecipitation [133, 134]
Emulsion solvent evaporation [135]
Nanoimprinting [136]
Electrospinning [137, 138]
Nanostructuring processes for silicon Photolithographic fabrication
X - ray lithography [130]
Electron beam lithography [139]
Chemical etching [140]
Physical and chemical vapor deposition [141]
Nanostructuring processes for carbon Electric arc discharge [142, 143]
Laser ablation [144, 145]
Chemical vapor deposition [146]
Combustion [147]
MANUFACTURING TECHNOLOGIES 1297
1298 PHARMACEUTICAL NANOSYSTEMS
7.3.5.1 Nanoscale Assembly Methods 1
Self-Assembly through Micelle Formation Self - assembly at the nanoscale is
deemed important to be able to produce commercially viable products and processes,
since it offers a mode of bulk production with control over features such as
size, shape, and morphology at the nanoscale. The basic principle of self - assembly
is based on the simultaneous coexistence of two parallel forces:
Long - range repulsive interactions between incompatible domains
Short - range attractive interactions
If we take the example of an amphiphilic diblock copolymer, the polymer is
composed of two blocks, a hydrophobic block and a hydrophilic block. When introduced
to a solvent beyond a minimum concentration, the critical micelle concentration
(CMC), the monomers begin to orientate such that the block that is soluble in
the solvent orients itself toward the periphery, in contact with the continuous media,
and the insoluble portion turns toward the core in an attempt to minimize contact
with the continuous phase, thus leading to the formation of a micelle. The long - range
repulsive forces arise from the relative solubilities of the blocks in the solvent, and
the short - range attractive forces arise from the covalent link between the two blocks.
The basic theory of micelle formation using block copolymers is outlined below
since nanosystem and nano - object self - assembly is likely to be facilitated by such
polymer systems, and similar principles will apply or aid toward developing self -
assembling systems.
Key factors that affect micelle formation are as follows:
Equilibrium constant
Solvent type
Solvent quality
Critical micelle temperature (CMT)
Critical micelle concentration
Overall molar mass of the micelle, MW
Micelle aggregation number, Z
Copolymer architecture
Relative block lengths
Relative geometries of copolymer blocks
Polymer composition
Core – corona interfacial tension
These key factors will infl uence the following micelle characteristics:
Hydrodynamic radius of micelles formed, RH
Radius of gyration, RG
1 The description in this section has been summarized and adapted from J. Rodriguez - Hernandez et al.,
Toward “ smart ” nano - objects by self - assembly of block copolymers in solution, Progress in Polymer
Science , 30 (2005), 691 – 724 [148] .
Ratio of hydrodynamic radius, RH , to radius of gyration, RH
Micelle core radius, RC
Micelle corona thickness, C
Micellar structures can be produced either by addition of the polymer solution
or addition of the powdered material to the desired solvent and stirring at the
optimum temperature and monomer concentration. The CMC can be determined
by ultraviolet (UV) absorption or light scattering techniques such as static light
scattering (SLS), dynamic light scattering (DLS), or small - angle X - ray scattering
(SAXS). At the concentration at which monomers form micelles, there will be a
radical drop in monomer concentration in the bulk.
The stability of micellar systems depends upon the ability to ensure the aggregated
monomers do not deaggregate and that individual micelles do not coalesce
to form larger aggregates. This is inevitable over a period of time, but steps can be
taken to prolong the stability of the systems through various techniques, and those
listed below are some of the methods used for spherical micellar systems:
Steric stabilization using emulsifi ers and surfactants
Shell or core cross - linking
Viscosity - enhanced stabilization
Amine cross - linked stabilization
Thermodynamic stabilization
The types of systems that can be produced using micelle formation include spheres,
shells, capsules, vesicles, clusters, and particles of various shapes and sizes, such as
spheres, rods, planar structures, and layered structures. Further processing can be
undertaken to add rate - controlling polymer membranes to the outer shell and to
incorporate different molecules to the surface (e.g., for receptor recognition).
Biological Self -Assembly Using DNA as Construction Tool This is a technique
that has been adopted to produce 2D or 3D nanosystems by utilizing the base -
pairing affi nity of DNA [149, 150] .
Biological self - assembly using DNA can be described as a process that allows the
systematic assembly of molecules with high levels of precision and accuracy without
external constraints or infl uences. This allows the construction of nanoscale objects
to the desired structure, conformation, and composition very rapidly and without
the need for complex processing techniques and conditions.
DNA is a copolymer composed of a phosphate and sugar backbone and four
types of bases that branch off from the backbone, A (adenine), G (guanine), C
(cytosine), and T (thymidine). During DNA replication two strands of DNA come
together to form a helical structure through complementary base pairing which is
highly specifi c, whereby thymidine pairs with adenine and guanine with cytosine.
When strands of DNA come together where the ends of the strands are noncomplementary,
a portion of the strand extends beyond the complementary base - paring
region leading to an overhang, otherwise known as a “ sticky end ” .
The natural mechanism of DNA base pairing can be used to assemble synthetic
sequences of DNA molecules by synthesizing DNA molecules such that they form
MANUFACTURING TECHNOLOGIES 1299
1300 PHARMACEUTICAL NANOSYSTEMS
stable branches [150, 151] with arms that form sticky ends that in turn can assemble
to form supramolecular structures [152, 152a] . This approach may be used to produce
complex 3D assemblies through sequential or layer - by - layer self - assembly and may
incorporate other materials, such as particles and proteins [153 – 156] . The advantages
of this method are as follows:
Specifi city and geometry of intermolecular interactions that can be predicted
Precision control over the fi nal structure at the nanoscale
Simple manufacturing process without external restrictions
Complex structures that can be built with defi ned topologies
Potential for creation of nanodevices
The types of structures that may be constructed are Branched planar/2D quadrilateral
structures [157] , cubes [158] , octahedrons [159] , and complex 2D and 3D
periodic structures [160] .
DNA Synthesis for Nanoconstruction Single strands of DNA, otherwise known as
oligomers, are most commonly produced using a solid - support synthesis process [161,
162] . This is a cyclic process where each nucleotide is sequentially coupled to form a
nucleotide chain (working from the 3 . end to the 5 . end). The 3 . end is initially covalently
linked to a solid support and the nucleotide monomers are added sequentially.
This is a well - established process and its key parameters and critical process steps are
well documented in the literature [163, 164] . The DNA strands can be tailored according
to the desired nanoconstruction scheme and target structure [165] .
Nano Manipulation As the name suggests, this is quite literally a technique for
physically manipulating matter at the nanoscale. Scanning probe microscopy (SPM)
techniques have been most widely used to achieve this using the scanning probe tip
as an implement for assembling atoms, molecules, or nanoparticles according to the
desired spatial conformation [166 – 169] .
Soft Lithography Lithography is essentially a process for printing features on a
planar surface. Nanolithography tools, commonly referred to as soft lithography,
allow precisely defi ned nanoscale features to be produced on a substrate, which can
be removed from the substrate as free - standing 3D nano - objects. A number of
techniques fall within the fi eld of soft lithography, primarily for construction of
micrometer - sized objects:
Replica molding
Micromolding in capillaries (MIMIC)
Microtransfer molding
Solvent - assisted microcontact molding (SAMIM)
Microcontact printing
Near - fi eld phase shift lithography is a soft lithographic technique used to produce
geometric shapes with size features at the nanoscale (approximately 40 – 80 nm). This
involves the production of a polymer mask containing the desired pattern to be
replicated on the substrate, with nanoscale features usually patterned by X - ray or
electron beam exposure. The mask is then placed on the surface of the substrate
and exposed to near - fi eld light, the intensity of which leads to replication of the
pattern on the mask on to the substrate. Complex geometries, shapes, and features
can be produced on the substrate which can subsequently be removed to give free -
standing particles or objects [170, 171] .
Molecular Imprinting Molecular Imprinting is a process used to imprint or copy
recognition sites from desired molecules on to polymer structures [172, 173] . The
recognition sites can be produced on organic or inorganic polymers and inorganic
materials such as silica and biomaterials such as proteins. A template molecule is
dissolved in solvent with polymerizable monomers which undergo bond formation
with the template molecule forming either noncovalent bonds through electrostatic
interactions, hydrogen bonds or hydrophobic interactions, or reversible covalent
bonds. The monomers are then polymerized to form a cast or semirigid polymeric
structure which maintains the steric conformation of the molecule template and its
recognition site upon removal of the template molecule. As a result, the molecular
template affi nity for molecules and analyte is mimicked by the “ imprinted ” polymer
[174] . This has applications in chromatography and drug discovery and potential
applications in targeted drug delivery.
Layer -by -Layer Electrostatic Deposition Electrostatic deposition utilizes the
electrostatic bonding affi nities of materials imparted by their surface charge to build
highly ordered multilayered fi lms or structures on a substrate. The process involves
the successive deposition of oppositely charged polyions, exploiting the Coulombic
long - range electrostatic interactions between the oppositely charged molecules,
allowing formation of multilayers over a large distance. This technique can be used
to build multilayer composite fi lms on particles incorporating molecular fragments
such as polymer – polymer, polymer – organic, polymer bimolecular, and polymer –
mineral composition [175 – 177] .
Chemical Vapor Deposition CVD is a crystal growth process whereby a solid
material is deposited from the gas phase onto a controlled substrate using a suitable
mixture of volatile precursor materials which react to produce the desired deposit
on the substrate surface (Table 4 ). Types of fi lms and structures that can be produced
include the following:
Polycrystalline
Amorphous
Epitaxial silicon
Carbon fi ber
Filaments
Carbon nanotubes
Silicon dioxide
Tungsten
Silicon nitride
Titanium nitride
MANUFACTURING TECHNOLOGIES 1301
1302 PHARMACEUTICAL NANOSYSTEMS
7.3.5.2 Nanostructuring Processes for Polymeric Materials
Numerous microfabrication techniques have been used to produce a wide range of
implantable and oral drug delivery systems using materials ranging from silicon,
glass, silicone elastomer, and plastics. Fabrication techniques have rapidly evolved
to produce nanoscale objects and therapeutic systems using polymeric materials as
the substrate due to their biodegradable nature. There are a number of different
synthetic polymer systems that have been developed for this type of application,
and the most common ones are listed below:
Poly( d - lactic acid) (PDLA)
Poly( . - caprolactone) (PCL)
Poly(vinyl alcohol) (PVA)
Polyalkylcyanoacrylates (PACA)
Poly( l - lactide) (PLLA)
Poly(lactide - co - glycolide) (PLGA)
Polymethylcyanoacrylate (PMCA)
Techniques for the production of micrometer - sized features using polymers are
well established and apply primarily to device construction. The techniques listed
TABLE 4 Chemical Vapour Deposition Methods and Their Key Features
CVD Method Key Features
Atmospheric pressure CVD [178,
179]
Operates at atmospheric pressure
Atomic layer CVD (atomic layer
epitaxy) [180, 181]
High - precision fi lm thickness and uniformity
requirements
Aerosol - assisted CVD [182, 183] For use with involatile precursors
Direct liquid injection CVD [184,
185]
High fi lm growth rates possible
Hot - wire CVD [186] High growth rate, low temperature, and use
of inexpensive materials such as plastics
as substrate
Low - pressure CVD [187] Improved fi lm uniformity
Metal organic CVD [188] Uniform and conformal deposition
Microwave plasma - assisted CVD
[189]
No external heating required
Plasma - enhanced CVD [190] Reduced substrate temperatures can be
used
Rapid thermal CVD [191] Conformal coverage over high - aspect - ratio
features is possible, i.e., improved control
of interfacial properties
Remote plasma - enhanced CVD
[192]
Excellent conformal coverage of complex
structures
Can produce multilayer and graded layers
with tailored functional group attachment
Ultrahigh vacuum CVD [193] Reduced surface contamination
below focus primarily on attaining submicrometer, nanoscale features, and geometries
using polymers such as those listed above.
Nanomold Replication A physical mold is produced that has nanoscales on the
order of tens or a few hundred nanometers. To achieve such fi ne features with
precision and repeatability, electrodeposition is used to produce the molds,
otherwise referred to as a nanostamp [194] . The stamp is then use as a master
stamp to duplicate the image or object by casting or embossing the polymeric
material.
Colloidal Lithography Colloidal lithography is a process whereby an electrostatically
self - assembled array of monodispersed colloidal nanospheres is used as a mask
to construct nanoscale objects and features through deposition or etching processes.
The monodisperse colloidal spheres, for example, surface - charged latex, self -
organize or assemble into periodic arrays on the substrate, glass, for example, and
do not aggregate due to the surface charge repulsion. This method can be used to
produce 2D [195, 196] and 3D [197] nanostructures, arrays of rings, dots, honeycomb
structures, pillars, and chemical patterns [198] with a high level of control over
structure and conformation.
Interfacial Polymerization Interfacial polymerization is a process whereby very
thin fi lms or membranes, on the order of nanometer thickness, are produced by
reacting two monomers at the interface between two immiscible solutions [199] .
Nanoparticles [200] and aqueous core capsules with very thin membranes have been
produced using this method for drug delivery applications.
Nanoprecipitation Nanoprecipitation is a self - assembly directed nanoparticle formation
method. There are three key steps involved in this process: rapid micromixing
of the solutes, the creation of a high level of supersaturation to instigate rapid
nucleation and growth of precipitate, and the kinetic control and termination of
growth using copolymer stabilizers. One of the drawbacks using this method is the
poor incorporation of water - soluble drugs [201] . However, the main advantage
associated with the production of nanospheres for drug delivery using this technique
is the high degree of control attainable over particle size [202] .
Emulsion Solvent Evaporation The basic concept of the emulsion solvent evaporation
technique producing nanoparticles is very straightforward. The particles are
formed as an emulsion of a polymer – surfactant mixture and dispersed in an organic
solvent. The solvent is then evaporated to leave behind the individual emulsion
droplets which form stable free nanoparticles [203] . This method is far easier and
more preferable over methods such as spray drying and homogenization and operates
under ambient conditions and mild emulsifi cation conditions. The size and
composition of the fi nal particles are affected by variables such as phase ratio of
the emulsion system, organic solvent composition, emulsion concentration, apparatus
used, and properties of the polymer [204] .
Nanoimprinting This is a lithographic technique similar to soft contact lithography
discussed earlier, with the main difference being that nanoimprinting uses a
MANUFACTURING TECHNOLOGIES 1303
1304 PHARMACEUTICAL NANOSYSTEMS
hard mold to produce nanoscale features down to sub - 10 nm resolution [205] by
directly imprinting onto the polymer surface at high temperatures. More recently,
molds produced from carbon nanotubes have been used to achieve molecular - scale
resolution. Molds are generally made using electron beam lithography; however,
high - defi nition molds are produced using molecular beam epitaxy. Some of the
technical issues associated with this technique include sticking, adhesion, and material
transport during imprinting [206] .
Electrospinning Electrospinning is a process that uses electrostatic force to
produce nanofi bers from a charged polymer. An electrode is placed into a spinning
polymer solution/polymer melt and the other electrode is attached to a collector
plate. A high - intensity electric fi eld is created by applying a high voltage such that
the polymer solution is discharged as a jet, and during travel of this charged polymer
jet toward the grounded collector plate, solvent evaporation leaves a charged
polymer fi ber which deposits on the collector plate [207] . These fi bers have high
specifi c surface areas and are highly fl exible, and applications include the preparation
of controlled drug release membranes [208] .
7.3.6 CHARACTERIZATION TECHNIQUES
7.3.6.1 Nanoparticle Characterization Methods and Tools
A summary of some key properties that may be assessed as part of a characterization
schedule for nanoparticles and nanostructures and a comprehensive but not
exhaustive list of tools and techniques that may be used are presented in Table 5
[209 – 223] . The degree of characterization and method used will be determined by
the intended application of the nanomaterials.
Characterization of micellar and supramolecular structures and their counterparts
often require different or additional tools and techniques [224 – 232] and a
summary is provided in Table 6 of various characterization parameters for micellar
and supramolecular structures and components and analytical tools that may be
applied.
7.3.6.2 Scanning Probe Technologies
Scanning probe microscopy has almost become synonymous with nanomaterial
characterization [233] . This is a family of techniques that have evolved from the use
of a sharp proximal probe to scan a surface in order to ascertain its properties down
to atomic - scale resolution based on tip – surface interaction. There are two main SPM
techniques, scanning tunneling microscopy (STM) [234, 235] and AFM [236] . Near -
fi eld scanning optical microscopy (NSOM) [237 – 239] also falls within the SPM
family of techniques; however, this uses a subwavelength near - fi eld light source as
the scanning probe, achieving resolutions down to 50 nm, and is not discussed further
here.
A host of techniques have evolved from STM and AFM, primarily involving
adaptations to instrumentation depending on the material and parameter under
TABLE 5 Characterization Parameters and Tools for Nanoparticles and Nanostructures
Characterization Parameter Analytical Tool
Composition Liquid chromatography, e.g., high - performance liquid
chromatography (HPLC), size exclusion chromatography
(SLC) – HPLC
Field fl ow fractionation (FFF)
UV – visible spectrophotometry
Refractive Index
Inductively coupled plasma – optical emission spectrometry
(ICP – OES)
Fourier transform infrared spectrometry
Mass spectrometry
X - ray fl uorescence
Extended X - ray absorption fi ne structure (EXAFS)
spectroscopy
X - ray absorption near edge (XANES) spectroscopy
Particle diameter Static and dynamic laser light scattering
Scanning probe technologies
Size distribution Static and dynamic laser light scattering
Photon correlation spectroscopy
Surface area BET method (Brunauer, Emmett, and Teller method)
Porosity (pore size, volume,
and distribution)
Physical gas sorption
Chemical gas sorption
Helium picnometry
Mercury intrusion porometry (MIP)
Core – shell thickness Small - angle scattering of polarized neutrons (SANSPOL)
Surface structure and
morphology
Small - angle neutron scattering (SANA)
Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy
Scanning electron microscoscopy (SEM), atomic force
microscopy (AFM), energy dispersive X - ray (EDXA),
transmission electron microscopy (TEM), scanning probe
microscopy (SPM), auger electron spectroscopy (AES),
X - ray diffraction (XRD), X - ray photoelectron
microscopy (APS), X - ray photoelectron spectroscopy
(XPS), Vertical scanning phase shifting interferometry
Surface charge density Zeta potential using Electrostatic light scattering (ELS)
Zeta potential using multifrequencyelectro acoustics
Zeta potential using phase analysis light scattering (PALS)
Shape Electron microscopy
Scanning probe technologies
Concentration distribution Energy dispersive X - ray spectrometry (EDS) combined
with SEM or scanning
Crystallinity, Bulk X - ray diffraction
Crystallinity, Local TEM/ – selected area diffraction (SAD)
Differential scanning calorimetry (DSC)
Magnetic properties Scanning probe technologies
Electrical properties Scanning probe technologies
Optical properties UV – visible Spectroscopy
CHARACTERIZATION TECHNIQUES 1305
1306 PHARMACEUTICAL NANOSYSTEMS
TABLE 6 Characterization Parameters and Analytical Tools for Micellar and
Supramolecular Structures
Characterization Parameter Analytical Tool
Critical micelle concentration Flurimetric methods
Static light scattering
Dynamic Light Scattering
Aggregation number Fluorescence correlation spectroscopy
Radius of gyration, R G Small - angle X - ray scattering
Hydrodynamic radius, R H Photon correlation spectroscopy
Core/corona size, micelle structure, overall
micelle size
Small - angle X - ray scattering
Overall shape, cross section Small - angle neutron scattering
Size, shape, and internal structure Transmission electron microscopy
Scanning probe technologies
Average molecular weight Membrane and vapor pressure osmometry
Monitor equilibrium state, stability monitoring Light - scattering methods
Structure elucidation, polymer architecture,
polymer interactions
Nuclear magnetic resonance
investigation. It should be noted that technological advances continue unabated and
new techniques are constantly being developed within the scanning probe family to
cater for the characterization of new and novel materials and nanoscopic constructs.
Table 7 gives a current synopsis of these techniques.
Scanning Tunneling Microscopy The scanning tunneling microscope was fi rst
described by Nobel Prize winners Binnig and Rohrer in 1982 [249] and consists of
an atomically sharpened tip usually composed of tungsten, gold, or platinum – irridium.
The tip is scanned within atomic distance (about 6 – 10 A ) of the sample under
study under very high vacuum, and a bias voltage is applied between the sample
and the scanning probe tip, resulting in a quantum mechanical tunneling current
across the gap. The magnitude of the tunnelling current is related exponentially to
the distance of separation and the local density of states (i.e., electron density in a
localized region of a material) [250, 251] .
The relationship between tunneling current and separation is given as:
I C ed = ..t s
0 5 .
where I = tunnelling current
C = constant (linear function of voltage)
. t = tip electron density
. s = sample electron density
e d 0.5 = separation (governed by exponential term)
The tip is scanned across the sample surface using a piezoelectric transducer in one
of two modes, topographic mode or current mode. In the topographic mode a con
TABLE 7 Summary of Scanning Probe Technologies
SPM Technique Property Measured
Atomic force microscopy Visualisation and measurement of surface features
Noncontact AFM [240] Insulating substrates, atomic resolution
Molecular systems, atomic resolution
Biocluster and biomolecular imaging
Imaging and spectroscopic data in liquid environments
Nanoscale charge measurement
Nanoscale magnetic properties
Contact AFM [241, 242] Topographic imaging of solid substrates
Mechanical properties
Local adhesive properties
Piezoresponse Characterization and domain engineering of ferroelectric
materials
Lateral force Fine structural detail
Transitions between components on surface, e.g., polymer
composites
Scanning thermal Defects in sample based on thermal differences
Intermittent AFM (tapping
mode) [243]
Biological systems: DNA/RNA analysis, protein – nucleic acid
complexes, molecular crystals, biopolymers, ligand –
receptor binding
Phase imaging AFM Two phase polymer blends
Surface contaminants
Biological samples
Lift mode AFM techniques
[244 – 246]
Topography
Magnetic Force Magnetic properties/regions
Electrical Force Electrical properties
Surface Potential Surface potential
Scanning Capacitance Material capacitance
Force modulation Elasticity
Scanning tunnelling
microscopy [247]
Surface imaging
Three - dimensional profi ling with vertical resolutions to 0.1 A
Measurement of electronic and magnetic properties
Surface electronic state
Spin - polarized STM/STS
[248]
Mapping surface magnetism at atomic scale
stant distance is maintained between the tip and sample surface using a feedback
loop operated with the scanner. In the current mode, variations in current with
changes in surface topography are monitored by switching off the feedback loop,
thus providing a 3D image of the surface under study. The key features and limitation
of STM are as follows:
Features of STM [252, 253]
Can undertake topographical imaging of surfaces with atomic - scale lateral resolution,
down to 1 A
There - dimensional profi ling possible with vertical resolutions down to 0.1 A
Wide range of materials can be analyzed
CHARACTERIZATION TECHNIQUES 1307
1308 PHARMACEUTICAL NANOSYSTEMS
Surface electronic properties may be measured
Large fi eld of view, from 1 A to 100 . m
Vibrational isolation allows highly sensitive measurements to be undertaken
Ultrahigh vacuum (in the range 10 . 11 torr) minimizes sample contamination and
reduces oxide layer growth, thus allowing for high sensitivity measurements
Limitations of STM
Can be diffi cult to differentiate between a composite of materials on the
surface
Tip - induced desorption of surface molecules may occur
Ultrahigh vacuum requirements
Vibrational isolation requirements lead to increased installation costs
Low scanning speed
Atomic Force Microscopy Atomic force microscopy is a direct descendant of STM
and was fi rst described in 1986 [254] . The basic principle behind AFM is straightforward.
An atomically sharp tip extending down from the end of a cantilever is
scanned over the sample surface using a piezoelectric scanner. Built - in feedback
mechanisms enable the tip to be maintained above the sample surface either at
constant force (which allows height information to be obtained) or at constant
height (to enable force information to be obtained). The detection system is usually
optical whereby the upper surface of the cantilever is refl ective, upon which a laser
is focused which then refl ects off into a dual - element photodiode, according to the
motion of the cantilever as the tip is scanned across the sample surface. The tip is
usually constructed from silicon or silicon nitride, and more recently carbon nanotubes
have been used as very effective and highly sensitive tips.
In noncontact - mode AFM the cantilever is oscillated slightly above its resonant
frequency and the tip does not make contact with the sample surface but instead
oscillates just above the adsorbed fl uid layer on the surface, maintaining a constant
oscillation. The resonant frequency of the cantilever decreases due to van der Waals
forces extending from the adsorbed fl uid layer. This changes the amplitude of oscillation,
the variations of which are detected using sensitive alternating current (AC)
phase - sensitive devices, providing topographical information. In contact mode, AFM
the tip remains in contact with the sample surface, and the feedback loop maps the
vertical vibrational changes. In tapping mode, the cantilever is oscillated above the
sample surface such that it intermittently contacts the sample surface. The key features
and limitations of AFM are a follows:
Features of AFM [255 – 257]
High scan speeds
Atomic - scale resolution possible
Rough sample surfaces can be analyzed
High lateral resolutions possible
Soft samples (e.g., biological tissue) can be measured
Limitations of AFM [255 – 257]
Potential for image distortion due to lateral shear forces (in contact mode)
May be reduced spatial resolution due to sample scraping (in contact mode)
Tapping mode has lower scan speed compared to contact mode, though there is
less susceptibility for sample damage and image distortion
7.3.7 TOXICOLOGY CONSIDERATIONS
Nanomaterials may in their own right possess novel and useful properties or as a
composite of the same or different materials to form larger useful structures. Safety
consideration is therefore of paramount importance since completely inert materials
have the ability to exhibit toxic effects by virtue of a reduction in their size and
associated increase in surface area – mass ratio, let alone materials manipulated
specifi cally to impart novel properties.
Two obvious routes of human contact with nanoparticulates are the skin and via
inhalation. Given the size of the particles, there may be a propensity for absorption
into the systemic circulation. In some cases the nanosystems are engineered to
achieve enhanced systemic absorption. The established methodology for toxicological
assessment of new materials should be adhered to, and the discussion below is
intended only to touch upon some of the immediate safety concerns that should be
understood and addressed when dealing with nanomaterials.
7.3.7.1 Lung Toxicity
The safety of ultrafi ne particles remains to be clearly elucidated and requires the
collaborative input of toxicologists (animal, cellular, molecular), epidemiologists,
clinicians (pulmonary, cardiovascular, neurological), and atmospheric scientists.
There are several published studies to indicate that ultrafi ne particles pose a higher
toxicity risk compared with their larger counterparts [258 – 261] . Figure 1 outlines
the potential effects of ultrafi ne particles on respiratory mucosa, the cardiovascular
system, and central and peripheral nervous systems, upon inhalation.
Hohfeld et al. [262] hypothesized the toxic effects of ultrafi ne particles to be
attributable to the following:
• High effi ciency of deposition in the alveolar region due to particle size
• Large surface area
• Decreased phagocytosis leading to interaction of the particles with the epithelium,
resulting in the development of conditions such as chronic diffuse interstitial
fi bronodular lung disease
• Dislocation from the alveolar space, leading to potential systemic effects
The hypothesis affi rms the need to characterize the material ’ s physical and
chemical properties, including morphological analysis. The latter has signifi cant
ramifi cations on the aerodynamics of the particulate matter and hence its ultimate
disposition.
TOXICOLOGY CONSIDERATIONS 1309
1310 PHARMACEUTICAL NANOSYSTEMS
Carbon nanotubes are a class of materials fi nding increasingly widespread applications
in drug discovery and development and may be classed as a form of ultrafi ne
material. It has been shown that single - wall carbon nanotubes do not produce any
signifi cant respirable aerosol levels due to agglomeration resulting from the very
high surface area – volume ratio and associated electrostatic interaction between the
nanotubes [263] . A good deal of research has focused on developing methods for
the dispersion of nanotubes for further downstream processing for conversion to
useful applications. The liquid dispersions do not pose the same level of hazards
posed by the dry powder material, and most of the work in the pharmaceutical
industry with carbon nanotubes is focused on liquid dispersions whereby nanotubes
are being functionalized and conjugated with drugs and possibly other carriers for
therapeutic intent [264] .
However, it has at the same time been shown that dry powder carbon nanotubes
can persist in the lungs and have the potential to induce infl ammatory and fi brotic
reactions, evident in the form of collagen - rich granulomas in the bronchi and interstitium
[265] . This emphasizes the need for caution and further work to establish
the exact cause of these effects given the propensity of nanotubes to agglomerate.
7.3.7.2 Systemic Uptake
Nanoparticles, by virtue of size, have a tendency to evade phagocytosis. Uptake into
the systemic circulation is thus thought to be through diffusion and via the endo-
FIGURE 1 Potential mechanisms of effects of inhaled ultrafi ne particles. ( Reproduced with
permission from G. Oberd o rster, Inhaled nano - sized particles: Potential effects and mechanisms,
paper presented at the Symposium, Health Implications of Nanomaterials, October
2004 .)
Particle translocation
Mediators Inhalation
Neurons
Interstitium
Respiratory tract deposition
Lung
inflammation
Blood vessel
dysfunction Systemic
inflammation
Heart effects
Modifying factors: Age, gender, underlying disease, copollutants
CNS
(effects ?)
Autonomic nervous system
Circulation
Extrapulmonary
organs
Liver Heart
White blood
cell activation
REFERENCES 1311
thelial cells, the epithelium, interstitium, and blood vessels. Translocation into the
blood is thought to be through enhanced epithelial or endothelial permeability
imparted by infl ammatory mediators. Systemic hypercoagulation may be triggered
by the infl ammatory mediators in response to the diffusion of the nanoparticles
through endothelium and vasculature [260] .
7.3.7.3 Skin Permeation of Nanoparticles
The stratum corneum provides a formidable barrier to the entry of chemical and
particulate matter into human tissue and systemic circulation. It provides a fi rst - line
defence to the ingress of foreign agents. However, there are indications that particles
up to 1 . m are able to penetrate the skin ’ s barrier and deposit in the epidermis
where the antigen - presenting cells reside [266] . It follows therefore that submicrometer
particles in the nanometer range have the potential to cross the stratum
corneum and illicit an infl ammatory response. Once again, however, the tendency
of fi ne particles to agglomerate will to some extent inhibit penetration into the skin,
in particular where the agglomerates are macroscopic. A correlation must however
be drawn to establish any potential link between nanoparticle affi nity for skin penetration
and particle physical and morphological characteristics or indeed whether
the novel and unique properties of the engineered particle in any way impart
enhanced skin permeation properties and, if so, their nature and mechanisms.
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SUGGESTED READING 1323
1324 PHARMACEUTICAL NANOSYSTEMS
Nano – Pharmaceutical Materials and Structures
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SUGGESTED READING 1325
1327
7.4
OIL - IN - WATER NANOSIZED
EMULSIONS: MEDICAL APPLICATIONS
Shunmugaperumal Tamilvanan *
University of Antwerp, Antwerp, Belgium
Contents
7.4.1 Introduction
7.4.2 Generations of Oil - in - Water Nanosized Emulsions
7.4.2.1 First - Generation Emulsion
7.4.2.2 Second - Generation Emulsion
7.4.2.3 Third - Generation Emulsion
7.4.2.4 Unique Property of Third - Generation Emulsion
7.4.3 Preparation Methods for Drug - Free/Loaded Oil - in - Water Nanosized Emulsions
7.4.4 Excipient Inclusion: Oil - in - Water Nanosized Emulsions
7.4.5 Medical Applications of Oil - in - Water Nanosized Emulsions
7.4.5.1 Parenteral Routes
7.4.5.2 Ocular Routes
7.4.5.3 Nasal Route
7.4.5.4 Topical Route
7.4.6 Future Perspective
References
7.4.1 INTRODUCTION
It is estimated that 40% or more of bioactive substances being identifi ed through
combinatorial screening programs are poorly soluble in water [1, 2] . Consequently,
the drug molecules belonging to these categories cannot be easily incorporated into
aqueous - cored/based dosage forms at adequate concentrations, and thus the clinical
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
* Current address: Department of Pharmaceutics, Arulmigu Kalasalingam College of Pharmacy, Anand
Nagar, Krishnankoil, India
1328 OIL-IN-WATER NANOSIZED EMULSIONS
effi cacy of highly lipophilic drugs is being impeded. Furthermore, it is well established
that the pharmaceutical industry will face more diffi culties in formulating and
developing novel dosage forms of new chemical entities since 50 – 60% of these
molecules are lipophilic in nature and often exhibit hydrophobic character. Among
the different innovative formulation approaches that have been suggested for
enhancing lipophilic drug absorption and then clinical effi cacy, lipid - based colloidal
drug delivery systems such as nanosized emulsions recognized particularly for overcoming
the formulation and bioavailability - related problems of such drug molecules.
Other nomenclatures are also being utilized often in the medical literature to refer
to nanosized emulsions, including miniemulsions [3] , ultrafi ne emulsions [4] , and
submicrometer emulsions [5, 6] . The term nanosized emulsion [7] is preferred
because in addition to giving an idea of the nanoscale size range of the dispersed
droplets, it is concise and avoids misinterpretation with the term microemulsion
(which refers to thermodynamically stable systems). Hence, nanosized emulsions
are heterogenous dispersions of two immiscible liquids [oil - in - water (o/w) or water -
in - oil (w/o)], and they are subjected to various instability processes such as aggregation,
fl occulation, coalescence, and therefore eventual phase separation according
to the second law of thermodynamics. However, the physical stability of nanosized
emulsions can substantially be improved with the help of suitable emulsifi ers that
are capable of forming a mono - or multilayer coating around the dispersed liquid
droplets in such a way as to reduce interfacial tension or increase droplet – droplet
repulsion. Depending on the concentrations of these three components (oil – water –
emulsifi er) and the effi ciency of the emulsifi cation equipment/techniques used to
reduce droplet size, the fi nal nanosized emulsion may be in the form of o/w, w/o,
macroemulsion, micrometer emulsion, submicrometer emulsion, and double or multiple
emulsions (o/w/o and w/o/w). Preparation know - how, potential application, and
other information pertinent to w/o emulsions [8] , macroemulsions [9 – 11] , microemulsions
[12, 13] , and multiple emulsions [14] are thoroughly covered elsewhere.
In addition, some studies have compared the performance of different emulsifi ed
systems (macroemulsions, microemulsion, multiple emulsions, and gel emulsions)
prepared with similar oils and surfactants for applications such as controlled drug
release [15] or drug protection [16] . Similarly the state of the art of so - called oxygen
carriers or perfl uorocarbon emulsions, dispersions containing submicrometer/nanosized
fl uoroorganic particles in water, is also thoroughly covered in the literature
[17 – 19] and readers can refer to these complete and interesting articles.
Possible usefulness as carriers stems from the nanosized emulsion ’ s ability to
solubilize substantial amounts of hydrophilic/hydrophobic drug either at the innermost
(oil or water) phase or at the o/w or w/o interfaces. While hydrophilic drugs
are contained in the aqueous phase of a w/o - type emulsion or at the w/o interface
of the system, hydrophobic drugs could be incorporated within the inner oil phase
of an o/w - type emulsion or at the o/w interface of the system. It appears that the
choice of the type of emulsion to be used therefore depends, to a large extent, upon
the physicochemical properties of the drug. Between w/o and o/w types, the o/w
type of nanosized emulsions would be preferred in order to successfully exploit the
advantages of an emulsion carrier system. Additionally, within the o/w type, simple
modifi cations on surface/interface structures of emulsions can be made. For instance,
incorporating an emulsifi er molecule alone or in a specifi c combination that is
capable of producing either positive or negative charges over the emulsifi ed droplets
surface will lead to the formation of surface - modifi ed emulsions. Based on these
GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1329
surface modifi cations, the o/w - type nanosized emulsions can be divided into cationic
and anionic emulsions.
The o/w nanosized emulsions have many appealing properties as drug carriers.
They are biocompatible, biodegradable, physically stable, and relatively easy to
produce on a large scale using proven technology [20] . Due to their subcellular and
submicrometer size, emulsions are expected to penetrate deep into tissues through
fi ne capillaries and even cross the fenestration present in the epithelial lining in
liver. This allows effi cient delivery of therapeutic agents to target sites in the body.
Not only considered as delivery carriers for lipophilic and hydrophobic drugs, nanosized
emulsions can also be viewed nowadays as adjuvants to enhance the potency
of deoxyribonucleic acid (DNA) vaccine. For instance, Ott et al. [21] prepared a
cationic o/w emulsion based on MF59 (commercially termed Fluad), a potent squalene
in water and a cationic lipid, 1,2 - dioleoyl - sn - glycero - 3 - trimethylammonium
propane (DOTAP). It is shown that an interaction of cationic emulsion droplets
with DNA and the formed DNA - adsorbed emulsion had a higher antibody response
in mice in vivo while maintaining the cellular responses equivalent to that seen with
naked DNA at the same doses. Another example of o/w emulsion - based adjuvants
resulting from U.S. patent literature is the Ribi adjuvant system (RAS) [22 – 24] .
Depending on the animal species used, RAS can be classifi ed into two types: one
for use in mice, termed monophosphoryl - lipid A + trehalose dicorynomycolate
emulsion (MPL + TDM emulsion), and another for use in rabbits, goats, and larger
animals, called monophosphoryl - lipid A + trehalose dicorynomycolate + cell wall
skeleton emulsion (MPL + TDM + CWS emulsion). Strikingly, the MPL + TDM
and MPL + TDM + CWS emulsions are prepared based on 2% oil (squalene) –
Tween 80 – water. These adjuvants are derived from bacterial and mycobacterial cell
wall components that have been prepared to reduce the undesirable side effects of
toxicity and allergenicity but still provide potent stimulus to the immune system.
Another example is the syntex adjuvant formulation (SAF) that contains a preformed
o/w emulsion stabilized by Tween 80 and Pluronic L121 [25] .
Keeping in mind the potential of o/w nanosized emulsions, the purpose of this
chapter is to classify the emulsions and provide a short overview on the preparation
of the new - (second - and third - ) generation emulsions followed by a description of
the unique property of the third - generation emulsion and examples of selected
excipients used for emulsion preparation. Given a specifi c interest especially on the
parenteral route and then on the ocular topical route, o/w nanosized emulsions for
both routes share a common platform on strict criteria concerning the maximum
globule size and requirement of sterility in the fi nal emulsions. Nasal and topical
routes are also covered based on published research works with nanosized emulsions.
It is emphasized that the chapter focuses only on preformed nanosized emulsions
(having size distribution ranging between 50 and 1000 nm with a mean droplet
size of about 250 nm), which should not be confused with self - microemulsifying drug
delivery systems or preformed microemulsions that are transparent, thermodynamically
stable dosage forms.
7.4.2 GENERATIONS OF OIL - IN - WATER NANOSIZED EMULSIONS
In this chapter, the o/w nanosized emulsions are classifi ed into three generations
(see Figure 1 ) based on their development to ultimately make a better colloidal
1330 OIL-IN-WATER NANOSIZED EMULSIONS
carrier for a target site within the organs/parts of the body and eye, thus allowing
site - specifi c drug delivery and/or enhanced drug absorption.
7.4.2.1 First - Generation Emulsion
To be healthy with a quality life style is every human ’ s desire. According to documented
Indian scriptures dating back to 5000 b . c ., nutritional status has always been
associated with health [26] . Because nutritional depletion due to either changes in
the quality or amount of dietary fat intake or abnormalities in lipid metabolism
results in immunosuppression and therefore host defense impairment, favoring
increased infection and mortality rates.
Traditionally depletion in dietary fats in malnourished or hypercatabolic patients
is compensated through intravenous feeding using a solution containing amino
acids, glucose, electrolytes, and vitamins as well as nanosized emulsions. Structurally,
an o/w - type emulsion is triglyceride (TG) droplets enveloped with a stabilizing
superfi cial layer of phospholipids [27] . Emulsions for parenteral use are complex
nutrient sources composed not only of fatty acids but also substances other than
TG, such as phosphatidylcholine, glycerol, and . - tocopherol in variable amounts.
The emulsions also had a complex inner structure and consisted of particles with
different structures, namely, oil droplets covered by an emulsifi er monolayer, oil
droplets covered by emulsifi er oligolayers, double - emulsion droplets, and possibly
small unilamellar vesicles. Commercially available nanosized emulsions used as
intravenous high - calorie nutrient fl uids have particle size normally around 160 –
400 nm in diameter and, typically, their surfaces are normally negatively charged.
Larger droplets can also be detected in commercially available emulsions [28] . Lutz
et al. [29] reported that the mean diameter of particles in the 20% emulsions is
larger than in the 10% emulsions.
The TG in nanosized emulsions may be presented structurally in long or medium
chains respectively, named LCT and MCT. The mean diameter of particles in LCT
emulsions is greater than that in MCT emulsions [29] . LCT contains fatty acid chains
with 14, 16, 18, 20, and 22 carbon atoms and sometimes with double bonds. The
number of double bonds present defi nes the fatty acids in LCT as saturated, monounsaturad,
or polyunsaturated. If the fi rst double bond is on carbon number 3, 6, or
9 from the methyl end of the carbon chain, then the fatty acid is n - 3, n - 6, or n - 9,
respectively. Purifi ed soybean or saffl ower oil contains LCT with a high proportion
FIGURE 1 Flow chart of three generations of emulsion.
Nanosized emulsion, NE
First-generation ne (phospholipid based)
Second-generation NE, with PEGylation on droplet surface
Third-generation NE, with PEGylation and positive charge on droplet surface
GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1331
of n - 6 polyunsaturated fatty acids whereas olive oil has LCT with n - 9 monounsaturated
fatty acids. Fish oil includes LCT with 20 or more carbon atoms where the
fi rst double bond is located between the third and fourth carbons from the methyl
terminal of the fatty acids chain (omega - 3 or n - 3). On the other hand, MCT is
derived from coconut oil and has saturated fatty acids with chains containing carbon
atoms at the 6, 8, 10, or 12 positions. Both LCT and MCT either alone or MCT in
combination with LCT are known for their long - term commercial acceptability in
parenteral emulsions and are found in several U.S. Food and Drug Administration
(FDA) – approved products. Also in Europe emulsion containing LCT/MCT enriched
with fi sh oil became available for research. With MCT/LCT combinations in a specifi
c ratio, nanosized emulsions appear to provide a more readily metabolizable
source of energy [30] . However, LCT emulsion has been used in clinical practice for
over 40 years. But for drug solubilization purpose, MCT is reported to be 100 times
more soluble in water than LCT and thus to have an escalated solubilizing
capability.
7.4.2.2 Second - Generation Emulsion
An easy and substantial association of lipophilic bioactive compound with the MCT
or other vegetable oil – based emulsions, however, allows the emulsions to be used
as vehicles/carriers for the formulation and delivery of drugs with a broad range of
applications. These applications extend from enhanced solubilization or stabilization
of the entrapped drug to sustained release and site - specifi c delivery. Hence the
emulsions used for these applications are termed second - generation emulsions. Fittingly
the o/w - type nanosized emulsions containing either positive or negative
charge can be administered by almost all available routes, that is, topical, parenteral,
oral, nasal, and even aerosolization into the lungs [31] . Despite differences in
routes of administration, examples of commercially available emulsion - based formulations
utilized for medical and nonmedical applications purposes are given in
Table 1 .
The lipid - induced enhancement in oral bioavailability of many drugs having poor
water solubility is a well - known documented fact when the drugs are incorporated
into emulsions [32, 33] . However, direct intravascularly or locally administered
conventional fi rst - and second - generation emulsions could be taken up rapidly by
the circulating monocytes for clearance by reticuloendothelial cells (through organs
such as the liver, spleen, and bone marrow) [34] . Furthermore, the extent of clearance
is enhanced by the adsorption of opsonic plasma proteins onto emulsion surfaces.
However, the oily hydrophobic particles of the emulsions can also be taken
up by macrophages without the necessity of opsonization provided the oil phase is
liberated from the emulsion through the destabilization process occurring inside the
blood compartment immediately after emulsion administration intravascularly or
locally. Although the core of o/w emulsions is indeed hydrophobic, the emulsion
envelope is not. The exposure of the hydrophobic part to the aqueous medium will
therefore destabilize the emulsion. Moreover, Sasaki et al. [35] have assumed that
when the castor oil – based emulsions interact with the tears in the eye, the electrolytes
in the tears elicit a physical emulsion instability resulting in some release of
the oil. The electrolytes present in blood or cellular fl uids can also cause a similar
type of emulsion destabilization process, resulting in separation of the oil and water
TABLE 1 Selected Marketed Medical and Nonmedical Emulsions
Parenteral Fat Emulsions (o/w Type) for
Nutrition Registered Emulsions (o/w Type) Containing Drugs
Product Producer Product Drug Producer Application
Abbolipid/Liposyn Abbott Diazepam - Lipuro Diazepam Braun Melsungen Intravenous
Intralipid Pharmacia - Upjohn Diprivan Propofol AstraZeneca Intravenous
Lipofundin N or Endolipide B. Braun Etomidat - Lipuro Etomidate Braun Melsungen Intravenous
Lipofundin MCT/LCT
— Lipotalon (Limethason) Dexamethasone palmitate Merckle Intra - arthr.
Medialipide/Vasolipid B.
Braun Stesolid Diazepam Dumex Intravenous
Medianut B.
Braun Sandimmune Cyclosporin A Novartis Oral
Lipovenos Fresenius Neoral Cyclosporin A Novartis Oral
Ivelip/Salvilipid Clintec/Baxter Gengraf Cyclosporin A Abbott Oral
Clinoleic Clintec/Baxter Norvir Ritonavir Abbott Oral
Intralipos Green Gross Restasis Cyclosporin A Allergan Ocular topical
Kabimix Pharmacia - Upjohn Refresh Endura Drug free Allergan Ocular topical
Triv e 1000 Baxter SA Fluad (MF59) Adjuvant Chiron Parenteral
Perfl
uorocarbon Emulsions (Fluorocarbon - in - Water Emulsions)
Selected Topical Formulations Based on o/w or
w/o Emulsions
Product Producer Application Product Producer
Fluosol DA
Green Gross,
Osaka Blood supplement or O 2
carrier Daivonex cream and ointment
Laboratoire Leo
Imagent Alliance Pharmaceutical Contrast agent to image heart
Voltaren emulgel Ciba - Geigy
Oxygent Alliance Pharmaceutical Blood supplement or O 2
carrier EMLA cream
Astra,
Swedan
1332
GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1333
phases from the parenterally administered emulsions. It is thus reasonable to say
that the resultant oily hydrophobic particles of the emulsions would also be taken
up by macrophages independent of an opsonization process. An opsonization
process is the adsorption of protein entities capable of interacting with specifi c
plasma membrane receptors on monocytes and various subsets of tissue macrophages
(see Figure 2 ), thus promoting particle recognition by these cells. Classical
examples of opsonic molecules include various subclasses of immunoglobulins [36,
37] , complement proteins such as C1q and generated C3 fragments (C3b, iC3b) [38] ,
apolipoproteins [36, 37] , von Willebrand factor, thrombospondin, fi bronectin [39] ,
and mannose - binding protein. When given by other parentral routes, for example,
intraperitoneally, subcutaneously, or intramuscularly, the majority of emulsion droplets
enter the lymphatic system and eventually the blood circulation where particles
behave as if given intravenously. Liver, spleen, and bone marrow uptake is signifi -
cantly lower. Indeed, relative to the emulsion droplet size, lymph nodes take up a
much greater (over 100 - fold) proportion than any other reticuloendothelial system
(RES) tissue.
There is increasing interest in developing injectable emulsions that are not cleared
quickly from the circulation when they are designed to reach non - RES tissues in
the vascular system or extravascular sites of action or to act as circulating drug
reservoirs. Earlier approaches for making long - circulating emulsions concentrate
mainly on changes in the oil phase of the emulsion such as MCT versus LCT [40] ,
use of structured lipids (SLS) having medium - chain (C 8 – C 10 ) fatty acids (SLM) and
short - chain (C 4 ) fatty acids (SLS) [41] , addition of sphingomyelin [42 – 45] and cholesterol
[46] to the emulsion, and use of hydrogenated castor oil (HCO) with at least
20 oxyethylene units (HCO20) [47 – 52] . Using the further established formulation
approaches by which the emulsion droplet surfaces could be altered might, however,
be more realistic and even more useful for a wide array of drug - targeting purposes.
Steric barrier or enhanced hydrophilicity effect exerted by a polyoxyethylene (POE)
chain having surfactants when added as coemulsifi er into the phospholipid -
stabilized fi rst - generation emulsion allows, to some extent, the passive/inverse drug
targeting to the lung, kidneys, and areas of infl ammation [53, 54] . Addition of POE -
based surfactants into the otherwise amphipathic phospholipid - stabilized emulsion
FIGURE 2 Mononuclear phagocyte system.
Promonocyte (bone marrow)
Monocyte (blood)
Macrophages (tissues) highly phagocytic
Connective tissue (histiocyte)
Liver (Kupffer cell)
Lung (alveolar macrophage)
Spleen (free and fixed macrophages, sinusoidal lining cell)
Lymph node (free and fixed macrophage)
Bone marrow (macrophages, sinusoidal lining cell)
Serous cavity (peritoneal macrophage)
Bone tissue (osteoclast)
Nervous system (microglia)
1334 OIL-IN-WATER NANOSIZED EMULSIONS
is particularly effective against plasma protein adsorption onto emulsion surfaces
because of the hydrophilicity and unique solution properties of POE - based surfactants,
including minimal interfacial free energy with water, high aqueous solubility,
high mobility, and large exclusion volume [54] . In addition, colloidal particles presenting
hydrophilic surfaces with a low contact angle will be almost ignored by
phagocytic cells [55] , although emulsion particles are not supposed to be recognized
as foreign by the body to some extent. Examples of POE chains containing surfactants
employed so far in emulsions are Tween 80, Span 80, Brij, and Poloxamer 188
(commercially named Pluronic F - 68 or Lutrol F - 68). The effectiveness of these
polymeric surfactant molecules to intercalate at the oil – water interface with strong
bonding to the phospholipid molecules could also be checked/judged through an in
vitro monolayer experiment [56] .
In general, the modifi cation of particulate carriers using amphipathic polyethylene
glycol (PEG) – containing molecules results in a prolongation of their blood
circulation time [57, 58] . A phosphatidylethanolamine derivative with polyethylene
glycol (PEG – PE) is widely used to increase the plasma retention of particulate carriers
such as liposomes [59 – 61] , polystyrene microspheres [62] , and nanospheres
[63] . Therefore, similar to POE, the PEG – PE is also incorporated as a coemulsifi er
into emulsions (termed PEGylated emulsion) to augment its circulation half - life
[64] . Liu and Liu [53] studied the biodistribution of emulsion particles coated with
phosphatidylethanolamine derivatives with three different molecular weight PEGs
(MW 1000, 2000, and 5000). Among them, PEG - 2000 was able to prolong the circulation
time of emulsion probably due to the increased hydrophilicity of the
droplet surface and/or the formation of a steric barrier. However, Tirosh et al. [65]
assumed, while characterizing the PEG - 2000 - grafted liposome by differential scanning
calorimetry, densitometry, and ultrasound velocity and absorption measurements,
that the steric stabilization is much more than increasing hydrophilicity. In
addition, PEG - containing compounds also decrease the lipolysis of emulsion particles
[47] and prevent the uptake by the mononuclear phagocytes [66] .
A dipalmitoyl phosphatidylcholine (DPPC) – stabilized emulsion was prepared by
Lundberg et al. [67] and the effect of addition of PEG – PE, polysorbate 80, or
Pluronic F - 68 on the metabolism of DPPC - stabilized emulsion was studied. Two
different radioactive markers were used to investigate the fate of emulsion particles
following injection into the tail vein of female BALB/c inbred mice. While 14 C - triolein
(TO) is susceptible to the action of lipoprotein lipase (LPL), 3 H - cholesteryl
oleate ether (CO ether) is not. Hence the removal of 14 C - TO represents the triglyceride
metabolism, whereas the other one is a core marker to represent whole particle
removal by RES organ uptake. The emulsions with DPPC as sole emulsifi er
were rapidly cleared from the blood with only 10 – 11% of CO or TO left in circulation
after 1 h. However, addition of PEG – PE gave a prolonged clearance rate,
especially during fi rst 3 h. A further addition of cosurfactant polysorbate 80 or
Pluronic F - 68 resulted in a marked extension of the circulation lifetime during the
fi rst 6 h. The notable effects of polysorbate 80 and Pluronic F - 68 can apparently be
attributed mainly to the decrease in droplet size, although an additional infl uence
due to the increased hydrophilicity may not be ruled out.
The in vivo disposition of emulsions administered as nutrients (surface -
unmodifi ed fi rst - generation emulsion) as well as administered as drug carriers
(surface - modifi ed second - generation emulsion) would depend on the particle prop
GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1335
erties, such as the size [68 – 71] , zeta potential (see Sections 7.4.2.3 and 7.4.5 ), and
compositions of phospholipids and oil phase (see the above paragraphs), which may
vary among different products and the batches of each product. The size of particulate
carriers such as liposomes is known to infl uence both the phagocytic uptake by
the mononuclear phagocyte system (MPS) [68 – 70] and the binding of apolipoprotein
(apo) to emulsions [71] . Furthermore, the particle size is a major determinant
of the transfer to extravascular spaces from the blood compartment. The capillaries
of the vascular system can be classifi ed into three categories: continuous, fenestrated,
and discontinuous (sinusoidal) [72] . Particulate carriers including nanosized
emulsions are considered to pass through capillaries and reach extravascular cells
only in organs having discontinuous capillaries such as liver, spleen, and bone
marrow. In such tissues, the extravasation of particles should be regulated by their
size since the largest pores in the capillary endothelium is reported to be about
100 nm [73] . In addition, tumor capillaries have unique characteristics in their structures
and functions in comparison with normal tissues such as muscle [74, 75] , which
results in the enhanced distribution of particulate carriers to tumor tissues [76 – 78] .
The distribution of emulsions within a tumor tissue was also regulated by the size
of particulate carriers [79] . Obviously, because of the submicrometer size range
(175 – 400 nm in diameter) of the emulsions, the more they circulate, the greater their
chance of reaching respective targets. More specifi cally, growing solid tumors as well
as regions of infection and infl ammation have capillaries with increased permeability
as a result of the disease process (e.g., tumor angiogenesis [74] ). Pore diameters
in these capillaries can range from 100 to 800 nm. Thus, drug - containing emulsion
particles are small enough to extravasate from the blood into the tumor interstitial
space through these pores [80] . Normal tissues, by and large, contain capillaries with
tight junctions that are impermeable to emulsions and other particles of this diameter.
This differential accumulation of emulsion - laden drug in tumor tissues relative
to normal cells is the basis for the increased tumor specifi city for the emulsion - laden
drug relative to free (nonemulsion) drug. In addition, tumors lack lymphatic drainage
and therefore there is low clearance of the extravasated emulsion from tumors.
Thus, long - circulating lipid carriers, such as POE/PEG - coated nanosized emulsions,
tend to accumulate in tumors as a result of increased microvascular permeability
and defective lymphatic drainage, a process also referred to as the enhanced permeability
and retention (EPR) effect [81] . Table 2 lists various formulation factors
affecting the metabolism as lipoproteins, the recognition by the MPS, and the elimination
from the blood circulation of both second - and third - generation nanosized
emulsions after parenteral administration.
On the other hand, essential requirements of this “ active ” targeting approach
include identifi cation of recognition features (receptors) on the surface of the target
and the corresponding molecules (ligands) that can recognize the surface. Indeed,
emulsions with appropriate ligands anchored on their surface must be able to access
the target, bind to its receptors, and, if needed, enter it. Furthermore, in order to
bring the colloidal carrier closer to otherwise inaccessible pathological target tissues,
homing devices/ligands such as antibodies and cell recognition proteins are usually
linked somehow onto the particle surfaces. Various methods have been employed
to couple ligands to the surface of the nanosized lipidic and polymeric carriers with
reactive groups. These can be divided into covalent and noncovalent couplings.
Noncovalent binding by simple physical association of targeting ligands to the
1336 OIL-IN-WATER NANOSIZED EMULSIONS
nanocarrier surface has the advantage of eliminating the use of rigorous, destructive
reaction agents. Common covalent coupling methods involve formation of a disul-
fi de bond, cross - linking between two primary amines, reaction between a carboxylic
acid group and primary amine, reaction between maleimide and thiol, reaction
between hydrazide and aldehyde, and reaction between a primary amine and free
aldehyde [82] . For antibody - conjugated second - generation anionic emulsions, the
reaction of the carboxyl derivative of the coemulsifi er molecule with free amine
groups of the antibody and disulfi de bond formation between coemulsifi er derivative
and reduced antibody were the two reported conjugation techniques so far
[83 – 85] . However, by the formation of a thio - ether bond between the free maleimide
reactive group already localized at the o/w interface of the emulsion oil droplets
and a reduced monoclonal antibody, the antibody - tethered cationic emulsion was
developed for active targeting to tumor cells [86] . It should be added that the
cationic emulsion investigated for tumor - targeting purpose belongs to the third -
generation emulsion category (Section 7.4.2.3 ).
Apart from non - RES - related disease treatment through target - specifi c ligand
conjugation, the second - generation emulsions may also be useful for RES - related
disease treatment. Certain lipoprotein or polysaccharide moiety inclusion into the
emulsions would help to achieve this concept. In general, uptake of small colloidal
drug carriers by the phagocytotic mononuclear cells of RES in the liver can be
exploited to improve the treatment of parasitic, fungal, viral and bacterial diseases
such as, for example, leishmaniasis, acquired immunodefi ciency syndrome (AIDS),
and hepatitis B. The approach to use emulsions as a drug carrier against microbial
TABLE 2 Formulation Factors Affecting Metabolism as Lipoproteins, Recognition by
Mononuclear Phagocyte System (MPS), and Elimination from Blood Circulation of Second - and
Third - Generation Nanosized Emulsions after Parenteral Administration
Factor
Metabolism as
Lipoproteins Recognition by MPS
Elimination from
Blood Circulation
Poor Extensive Poor Extensive Slow Rapid
Particle size Large Small Small Large Small Large
Emulsifi er DPPC EYPC DPPC DSPC DPPC EYPC
DSPC — SM — SM DSPC
SM — — — — —
Coemulsifi er Poloxamers — Poloxamers — Poloxamers —
HCO - 60 — HCO - 60 — HCO - 60 —
PEG – PE — PEG – PE — PEG – PE —
Polysorbates — Polysorbates — Polysorbates —
Solutol — Solutol — Solutol —
Cationic lipid SA/OA — SA/OA — SA/OA —
Oil phase LCT MCT — — LCT MCT
— — — — SLS SLM
Note: DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; SM, sphingomyelin;
EYPC, egg yolk phosphatidylcholine; HCO - 60, polyoxyethylene - (60) - hydrogenated castor oil; PEG – PE, phosphotidylethanolamine
derivative with polyethylene glycol; SA, stearylamine; OA, oleylamine; LCT, long - chain
triglyceride; MCT, medium - chain triglyceride; SLS, structured lipid with short - chain fatty acids, C 8 – C 10 ; SLM,
structured lipid with medium - chain fatty acids, C 4 .
GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1337
diseases is superior to free antimicrobial agents in terms of both distribution to the
relevant intracellular sites and treating disseminated disease states effectively. As
already discussed, conventional emulsion particles are capable of localizing in liver
and spleen, where many pathogenic microorganisms reside.
Rensen et al. [87] demonstrated the active/selective liver targeting of an antiviral
prodrug (nucleoside analogue, iododeoxyuridine) incorporated in an emulsion
complexed with ligands such as recombinant apolipoprotein E (apoE) using the
Wistar rat as animal model because its apoE – receptor system is comparable to that
of humans [88] . Whereas the parent drug did not show any affi nity for emulsion
due to its hydrophilic property, derivatization with hydrophobic anchors allowed
incorporation of at least 130 prodrug molecules per emulsion particle without
imparting any effect on the emulsion structure and apoE association to emulsion
droplets. The authors did not describe where the 130 prodrug molecules reside in
the emulsion and what is the emulsion/medium partition coeffi cient of the prodrug.
The prodrug molecules might have reasonably higher solubility in the oil or o/w
interface of the emulsion possibly due to a high partition coeffi cient value. Plausibly,
this high partition value for prodrug molecules will determine the kinetic parameter
koff (desorption rate of an emulsion component from the assembly), as suggested
by Barenholz and Cohen [89] for liposomal technology. Furthermore, without being
bound by theory, the apoE component helps to disguise the emulsion particles so
that the body does not immediately recognize it as foreign but may allow the body
to perceive it as native chylomicrons or very low density lipoproteins (VLDL). The
small size and the approximately spherical shape allow the emulsion particles to
exhibit similar physicochemical properties to native chylomicrons or VLDL (hydrolyzed
by LPL) whereas the incorporated prodrug remained associated with the
emulsion remnant particles following injection into the blood circulation of the rat
[87] . Because the carrier particles are not recognized as foreign, the systemic circulation
of the drug increases, thus increasing the likelihood of drug delivery to the
target tissues (up to 700 n M drug concentration in liver parenchymal cells). Additionally,
the clearance rate of the drug decreases, thereby reducing the likelihood
of toxic effects of the drug on clearance tissues since accumulation of the drug in
another part of the clearance tissues is reduced. Thus, specifi c organs may be targeted
by using nanosized emulsion particles as described above due to target cells
comprising high levels of specifi c receptors, for example, but not limited to apoE
receptors.
To address this issue, the saccharide moieties of glycolipids and glycoproteins on
the cell surface are considered to play an important role in various intercellular
recognition processes. For instance, Iwamoto et al. [90] investigated the infl uence of
coating the oil droplets in emulsion with cell - specifi c cholesterol bearing polysaccharide,
such as mannan, amylopectin, or pullulan, on the target ability of those
formulations. They observed a higher accumulation of mannan - coated emulsion in
the lung in guinea pigs. Thus selective drug targeting through emulsion - bearing
ligands not only leads to an improved drug effectiveness and a reduction in adverse
reactions but also offers the possibility of applying highly potent drugs. Hence, the
composition of the emulsion plays an important role concerning intercellular cell
recognition processes and, indeed, cell recognizability is also being improved by
incorporation of apoproteins or galactoproteins onto the emulsion particles to
enhance their specifi city [91] .
1338 OIL-IN-WATER NANOSIZED EMULSIONS
Overall, although second - generation emulsion is usually used as a means of
administering aqueous insoluble drugs by dissolution of the drugs within the oil
phase of the emulsion, employing surface modifi cation/PEGylation by the attachment
of targeting ligands (apoE, polysaccharide, and antibody) onto the droplet
surface of emulsions may be useful for both passive and active drug - targeting purposes.
Thus receptor - mediated drug targeting using ligands attached to emulsions
seems to hold a promising future to the achievement of cell - specifi c delivery of
multiple classes of therapeutic cargoes, and this approach will certainly make a
major contribution in treating many life - threatening diseases with a minimum of
systemic side effects.
7.4.2.3 Third - Generation Emulsion
In order to increase cellular uptake, a cationization strategy is applied particularly
on the surfaces of nonviral, colloidal carrier systems such as liposomes, nano - and
microparticulates, and nanocapsules [92] . To make the surface of these lipidic and
polymeric carrier systems a cationic property, some cationic lipids/polymers are
usually added into these systems during/after preparation. But, adding only the
cationic substances in phospholipids - stabilized fi rst - generation emulsions does not
help to obtain a physically stabilized emulsion for a prolonged storage period.
However, using different cationic lipids as emulsifi er and additional helper lipids
as coemulsifi er, for example, DOTAP, 1,2 - dioleoyl - sn - glycero - 3 - phosphoethanolamine
(DOPE), and 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphoethanolamine - N -
[poly(ethylene glycol) 2000 ] (PEG 2000 PE), reports are available to prepare emulsions
with positive charges on their droplet surfaces [93, 94] . Alternatively, inclusion of
cation - forming substances such as lipids (stearyl or oleyl chain having primary
amines) [95, 96] , polymers (chitosan) [97, 98] , and surfactants (cetyltrimethylammonium
bromide) [99] during the preparation of second - generation emulsion
allows the formation of a stabilized system with positive charges over on it.
Further, the positive charge caused by stearylamine was also confi rmed by a
selective adsorption of thiocyanate. Its adsorption was correlated with increasing
stearylamine concentration [95] . So, nanosized emulsion consisting of complex
emulsifi ers, that is, phospholipid – polyoxyethylene surfactant - cationized primary
amine or a polymer combination, can conveniently be termed third - generation
emulsion.
The extemporaneous addition of the solid drug or drug previously solubilized in
another solvent or oil to the preformed fi rst - and second - generation emulsions is
not a favored approach as it might compromise the integrity of the emulsion. Since
therapeutic DNA and single - stranded oligonucleotides or small interfering ribonucleic
acid (siRNA) are water soluble due to their polyanionic character, the aqueous
solution of these compounds need to be added directly to the preformed third - generation
emulsion in order to interact electrostatically with the cationic emulsion
droplets and thus associate/link superfi cially at the oil – water interface of the emulsion
[100, 101] . During in vivo conditions when administered via parenteral, nasal,
and ocular routes, the release of the DNA and oligonucleotides from the associated
emulsion droplet surfaces should therefore initially be dependent solely on the
affi nity between the physiological anions of the biological fl uid and the cationic
surface of the emulsion droplets. For instance, the mono - and divalent anions con
GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1339
taining biological fl uid available in the parenteral route is plasma and in ocular
topical route is tear fl uid, aqueous humor, and vitreous humor. Moreover, these
biofl uids contain a multitude of macromolecules and nucleases. There is a possibility
that endogenous negatively charged biofl uid components could dissociate the DNA
and oligonucleotides from cationic emulsion. It is noteworthy to conduct, during the
preformulation development stages, an in vitro release study for therapeutic DNA
and oligonucleotide - containing emulsion in these biological fl uids, and this type of
study could be considered an indicator of the strength of the interaction that
occurred between DNA or oligonucleotide and the emulsion particles. However, it
is interesting to see what could happen when the third - generation emulsion is
applied to in vitro cell culture models in the presence of serum. The serum stability
of the emulsion – DNA complex was reported [102, 103] . Further interesting investigations
using third - generation emulsions in gene delivery purposes are briefl y summarized
in a review article [104] .
7.4.2.4 Unique Property of Third - Generation Emulsion
To enhance the drug - targeting effi cacy of colloidal carriers such as nanospheres and
liposomes, a PEGylation/cationization strategy is traditionally made over the surface
of these carriers. While surface PEGylated colloidal carriers exhibit a prolonged
plasma residence time through an escaping tendency from RES uptake following
parenteral administration, surface - cationized colloidal carriers can facilitate the
penetration of therapeutic agents into the cell surface possibly via an endocytotic
mechanism. These two facts are proved in both liposomes and nanospheres when
they possess separately the cationic and PEGylatic surface modifi cations on them.
However, a cationic emulsion colloidal carrier system developed in Simon Benita ’ s
laboratory at Hebrew University of Jerusalem, Israel, differs signifi cantly in such a
way that it holds a combination of cationic and PEGylatic surface properties on it.
Benita ’ s group have prepared a novel cationic emulsion vehicle using a combination
of emulsifi ers consisting of Lipoid E 80, Poloxamer 188, and stearylamine and have
found the formulation suitable for parenteral use, ocular application, nasal drug
delivery, and topical delivery [105] .
It has been reported in an ocular pharmacokinetic study of cyclosporin A incorporated
in deoxycholic acid – based anionic and stearylamine - based cationic emulsions
in rabbit that, when compared to anionic emulsion, the cationic emulsion
showed a signifi cant drug reservoir effect of more than 8 h in corneal and conjunctival
tissues of the rabbit eye following topical application [106] . Since cornea and
conjunctiva are of anionic nature at physiological pH [107] , the cationic emulsion
would interact with these tissues electrostatically to implicate the observed cyclosporin
A reservoir effect. This hypothesis is supported, in principle, by an ex vivo
study which showed that cationic emulsion carrier exhibited better wettability properties
on rabbit cornea than either saline or anionic emulsion carrier [108] .
Studies [109, 110] have shown that small changes in physical properties of emulsions
can infl uence the elimination rate of these formulations from the blood.
Indeed, an organ distribution study of stearylamine - based cationic or deoxycholic
acid – based anionic nanosized emulsions and Intralipid, a well - known commercial
anionic emulsion, containing 14 C - CO was carried out following injection into the tail
vein of male BALB/c mice (20 – 26 g) at a volume of 5 mL/kg [111, 112] . Since CO
1340 OIL-IN-WATER NANOSIZED EMULSIONS
(cholesteryl oleate) is one of the most lipophilic compounds used in biopharmacy
and is not prone to degradation in the body (which remains within particles even
after lipolysis of emulsion), its in vivo behavior can be regarded as refl ecting that
of the injected nanosized emulsion in the early phase [42, 113] . Following intravenous
administration of the various emulsions having 14 C - CO to BALB/c mice, the
14 C - CO was found to accumulate in organs such as lung and liver. Furthermore, it
was observed that the concentration of 14 C - CO in the lung decreased but was again
elevated over time for both the developed cationic and anionic emulsion formulations,
with a concomitant decrease in the concentration of the radiolabeled compound
in the liver. However, within the various emulsion distribution patterns
observed in liver, a lower 14 C - CO concentration was observed for stearylamine -
based cationic emulsion when compared to Intralipid while for deoxycholic acid –
based anionic emulsion the observed concentration of 14 C - CO was relatively very
low when compared to cationic emulsion and Intralipid. In addition, in comparison
to both anionic emulsions, the stearylamine - based cationic emulsion elucidated a
much longer retention time of 14 C - CO in the plasma, clearly indicating a long circulating
half - life for cationic emulsion in the blood. Thus, the cationic nanosized
emulsion can be considered a stealth long - circulating emulsion.
The above two studies clearly described the unique characteristics of third -
generation emulsion in enhancing ocular drug bioavailability; on the other hand,
the same emulsion has the property of circulating for a longer time in blood following
parenteral administration. Excess positive charge at the oil – water interface in
conjunction with the projection of highly hydrophilic POE chain (due to the presence
of Poloxamer 188) toward the aqueous phase of the o/w - type nanosized emulsion
is the main reason behind the emulsion attaining its unique property, which is
absent in fi rst - and second - generation emulsions. However, a better understanding
of the structure of the third - generation emulsion in terms of forces involved in its
formation and stabilization must ultimately be obtained in an effort to provide a
clearly understood physical basis for the uniqueness in its biological effi cacy following
parenteral and ocular administration.
It should be added that the use of stearylamine in intravenous administered
emulsion might be problematic. Stearylamine is a single - chain amphiphile having
relatively high critical micellar concentration, although the concentration used in
the studied emulsion is much higher than the critical micellar concentration. Therefore,
due to the dilution in plasma as well as plasma lipoproteins and blood cells,
there is a high probability that the emulsion will lose its stearylamine almost instantaneously.
To substantiate indirectly this issue, Klang et al. [114] showed the lack of
potential induced toxicity of stearylamine - based cationic emulsion in animal models
in vivo and Korner et al. [115] investigated the surface properties of mixed phospholipid
– stearylamine monolayers and their interaction with a nonionic surfactant
(poloxamer) in vitro. Despite the presence of the stearylamine, which may be suspected
of being an irritant in pure form, in the emulsifi er combination, the hourly
instillation of stearylamine - based cationic emulsion vehicle into rabbit eye was well
tolerated without any evidence of any toxic or infl ammatory response to the ocular
surface during the 5 days of the study (40 single - drop instillations between 8 AM
and 4 PM each day) [114] . Following 0.2 - , 0.4 - , and 0.6 - mL single - bolus injections
of the same emulsion vehicle, representing a huge single administered dose of
30 mL/kg, no animal deaths were noted over a period of 30 days, apparently indicat
ing the absence of marked acute toxicity [114] . Furthermore, the same stearylamine -
based cationic emulsion vehicle did not cause acute neurotoxicity in rats when a
continuous intravenous infusion (3.3 mL) for 2 h at a rate of 27.4 . L/min was administered
through the jugular vein [114] . An another study from Benita ’ s laboratory
suggests that long - term subchronic toxicity examination of the rabbit eye (healthy)
following thrice - daily single - drop topical instillation of the stearylamine - based
emulsion elicited an almost similar nonirritating effect to eye tissues in comparison
to the thrice - daily single - drop topical instillation of the normal saline – treated
control rabbit eyes (unpublished data). Thus, overall results clearly indicated that
the stearylamine was strongly bound at a molecular level to the mixed interfacial
fi lm formed by Lipoid E 80 and poloxamer 188 at the oil – water interface system
[115] . Such an intercalation between the emulsifi ers is responsible for emulsifi ed oil
droplet stability and, in fact, prevented the stearylamine from leaking and exerting
any intrinsic possible local or systemic adverse effects in model animals.
7.4.3 PREPARATION METHODS FOR DRUG - FREE/LOADED
OIL - IN - WATER NANOSIZED EMULSIONS
To get a better idea of how to formulate the nanosized emulsion delivery systems
suitable for parenteral, ocular, percutaneous, and nasal uses, the reader is referred
to more detailed descriptions of methods of nanosized emulsion preparation [6,
116] . A hot - stage high - pressure homogenization technique or combined emulsifi cation
technique (de novo production) is frequently employed in order to prepare
nanosized emulsions with desired stability even after subjection to autoclave sterilization.
Therefore, the steps involved in this technique in making blank anionic and
cationic emulsions were arranged in the following order:
1. Weigh the oil - and water - soluble ingredients in separate beakers.
2. Heat both oil and water phases separately to 70 ° C.
3. Add the oil phase to the water phase and continue the heating up to 80 ° C
with constant stirring to form a coarse emulsion.
4. Mix at high shear to make a fi ne emulsion.
5. Cool the fi ne emulsion formed in ice bath.
6. Homogenize the fi ne emulsion.
7. Cool the homogenized emulsion in ice bath.
8. Filter the emulsion using a 0.5 . m membrane fi lter.
9. Adjust the emulsion to 7 using 0.1 N hydrochloric acid or 0.1 N sodium
hydroxide solution.
10. Pass nitrogen gas into the vials containing the emulsion.
11. Sterilize the emulsion using an autoclave.
The traditional droplet size – reducing steps involved during the preparation include
constant mild stirring using a magnetic stirrer when initially mixing oil and water
phases, rapid Polytron mixing at high speed, and fi nal droplet size homogenization
using a two - stage homogenizer valve assembly. The initial heating is vital for the
DRUG-FREE/LOADED OIL-IN-WATER NANOSIZED EMULSIONS 1341
1342 OIL-IN-WATER NANOSIZED EMULSIONS
effective solubilization of the respective oil and water phase components in their
corresponding phases. Mixing the two phases with constant mild stirring and subsequently
raising the temperature to 85 ° C are needed to form an initial coarse
emulsion and to localize the surfactant molecules for better adsorption at the oil –
water interface, respectively. A typical formula to make anionic and cationic nanosized
emulsions is given in Table 3 .
There are three different approaches to incorporate lipophilic drugs into the oil
phase or at the o/w interface of the nanosized emulsions, namely, extemporaneous
drug addition, de novo emulsion preparation, and an interfacial incorporation
approach, which includes the recently developed SolEmul technology [117] . In
principle, the lipophilic drug molecules should however be incorporated by a de
novo process. Thus, the drug is initially solubilized or dispersed together with an
emulsifi er in suitable single - oil or oil mixture by means of slight heating. The water
phase containing the osmotic agent with or without an additional emulsifi er is also
heated and mixed with the oil phase by means of high - speed mixers. Further homogenization
takes place to obtain the needed small droplet size range of the emulsion.
A terminal sterilization by fi ltration, steam, or autoclave then follows. The emulsion
thus formed contains most of the drug molecules within its oil phase. This is a generally
accepted and standard method to prepare lipophilic drug – loaded nanosized
emulsions for parenteral, ocular, percutaneous, and nasal uses, as illustrated in
Figure 3 . This process is normally carried out under aseptic conditions and nitrogen
atmosphere to prevent both contamination and potential oxidation of sensitive
excipients.
7.4.4 EXCIPIENT INCLUSION: OIL - IN - WATER NANOSIZED
EMULSIONS
In general, nanosized emulsions should be formulated with compatible vehicles and
additives. The components of the internal and external phases of emulsion should
be chosen to confer enhanced solubility and/or stability to the incorporated biologically
active lipophilic drug. In addition, it should also be designed to infl uence
biofate or therapeutic index of the incorporated drug following administration via
parenteral, ocular, percutaneous, and nasal routes. In this section, general considerations
concerning excipient selection and optimum concentrations are comprehen-
TABLE 3 Typical Formula to Make o/w Anionic and
Cationic Nanosized Emulsions
Oil Phase Water Phase
Natural/semisynthetic oils Poloxamer 188
Phospholipid mixture Glycerol
Stearylamine/oleylamine a Double - distilled water
Deoxycholic acid/oleic acid b
Vitamin E
a Necessary ingredient for cationic emulsions.
b Necessary ingredient for anionic emulsions.
sively presented mainly in their relation to the oil phase, the aqueous phase, and
the emulsifi ers.
Prior to the formulation design of the emulsions, data are needed concerning the
drug solubility in the oil vehicle. Additionally, prerequisite information is needed
about compatibility of the oil vehicle with other formulation additives and the
established ocular/skin tissues – oil vehicle matching before the dosage form can be
prepared. Table 4 lists the common emulsion excipients and the oils suitable for
dissolving or dispersing lipophilic drugs of ocular/parenteral interests. Since oils are
triglycerides, care must be taken to minimize or eliminate oxidation. Therefore,
antioxidants such as . - tocopherol (0.001 – 0.002% w/w) should be included in a
typical emulsion formulation for medical applications. The fi nal oil - phase concentration
in emulsions meant for ocular use is now widely accepted to be at or below
5% w/w taking into account that the emulsion must be kept in a low - viscosity range
of between 2 and 3 centipoises, which also is the optimal viscosity for ocular preparations
[118] . However, for all other medical uses, the amount of oil may be varied
but generally is within 5 – 20% w/w. Sometimes, a mixture of oils rather than a single
oil is employed since drug solubilization in the oil phase is a prerequisite to exploiting
the emulsion advantages. Jumaa and M u ller [98, 119] reported the effect of
mixing castor oil with MCT on the viscosity of castor oil. The oil combination at the
ratio of 1 : 1 (w/w) led to a decrease in the viscosity of castor oil and simultaneously
to a decrease in the interfacial tension of the oil phase. This was related to the free
fatty acids contained in castor oil, which can act as a coemulsifi er resulting in lower
interfacial tension and, simultaneously, in a more stable formulation in comparison
with the other oil phases. In addition to the digestible oils from the family of triglycerides,
including soybean oil, sesame seed oil, cottonseed oil, and saffl ower oil,
which are routinely used for making medical emulsions, alternative biocompatible
FIGURE 3 Preparation of o/w nanosized emulsion (de novo method).
EXCIPIENT INCLUSION: OIL-IN-WATER NANOSIZED EMULSIONS 1343
1344 OIL-IN-WATER NANOSIZED EMULSIONS
oils such as . - tocopherol and/or other tocols were also investigated for drug delivery
purposes via o/w emulsions [120, 121] . But the emulsions formed from tocols are
often considered as microemulsion systems with few exceptions being the nanosized
emulsions [122, 123] .
Unlike spontaneously forming thermodynamically stable microemulsion systems
that require a high surfactant concentration (20% and higher), the kinetically stable
nanosized emulsions can be prepared by using relatively lower surfactant concentrations.
For example, a 20% o/w nanosized emulsion may only require a surfactant
concentration of 5 – 10%. Traditionally, lecithins or phospholipids are the emulsifi ers
of choice to produce nanosized emulsions. However, emulsifi ers of this kind are not
suitable to produce submicrometer – sized emulsion droplets or to withstand the heat
during steam sterilization. Therefore, additional emulsifi ers preferably dissolved in
the aqueous phase are usually included in the emulsion composition. A typical
example of the aqueous soluble emulsifi ers is nonionic surfactants (e.g., Tween 20)
after taking into consideration their nonirritant nature when compared to ionic
surfactants. The nonionic block copolymer of polyoxyethylene – polyoxypropylene,
Pluronics F68 (Poloxamer 188), is included to stabilize the emulsion through strong
steric repulsion. However, amphoteric surfactants Miranol MHT (lauroamphodiacetate
and sodium tridecethsulfate) and Miranol C 2 M (cocoamphodiacetate) were
also used in earlier ophthalmic emulsions [124] . It should be added that commercially
available cyclosporin A – loaded anionic emulsion (Restasis) contains only
polysorbate 80 and carbomer 1342 at alkali pH to stabilize the drug - loaded anionic
emulsion. To prepare a cationic emulsion, cationic lipids (stearyl and oleylamine)
or polysaccharides (chitosan) are added to the formulation. Strikingly, if chitosan is
a choice of cation producing polysaccharide emulsifi er molecules, there is no need
to add amphoteric or nonionic surfactants to the phospholipid or lecithin - stabilized
TABLE 4 Excipients Used for Formulation of o/w Nanosized Emulsions
Oils Emulsifi ers
Cationic Lipids and
Polysaccharide Miscellaneous
Sesame oil Cholesterol Stearylamine . - Tocopherol
Castor oil Phospholipids (Lipoid) Oleylamine Glycerin
Soya oil Polysorbate 80 and 20
(Tween 80 and 20)
Chitosan Xylitol
Paraffi n oil Transcutol P Sorbitol
Paraffi n light Cremophor RH Thiomersal
Lanolin Poloxamer 407 EDTA
Vaseline Poloxamer 188 Methyl paraben
Corn oil Miranol C 2 M and MHT Propyl paraben
Glycerin monostearate Tyloxapol TPGS
Medium - chain
monoglycerides
Medium - chain
triglycerides
Squalene
Vitamin E
Note: TPGS, . - tocopheryl - polyethylene glycol - 1000 - succinate; EDTA, ethylenediamine tetraacetic
acid.
emulsion [125] . Conversely, a cationic emulsion based on an association of poloxamer
188 and chitosan without lecithin was prepared and also showed adequate
physicochemical properties regarding stability and charge effects [97, 98] . Oil - in -
water emulsion compositions based on a tocopherol (or a tocopherol derivative) as
the disperse phase have been described in a patent granted to Dumex [126] . The
emulsion is intended for use with compounds that are sparingly soluble in water.
Interestingly, the emulsifying agent used to make tocol - based emulsions are restricted
to vitamin E TPGS.
Additives other than antioxidants such as preservatives (e.g., benzalkonium chloride,
chlorocresol, parabens) are regularly included in emulsions to prevent microbial
spoilage of multidose medical emulsions. . - Tocopherol is a good example of an
antioxidant used to obtain a desirable stabilized emulsion under prolonged storage
conditions. The presence of components of natural origin such as lecithin or oils
with high calorifi c potential renders the emulsion a good medium to promote microbial
growth when it is packed in multidose containers. Pharmaceutical products
when distributed into multidose containers, especially for parenteral and ocular
administrations, should be properly preserved against microbial contamination and
proliferation during storage in normal conditions and proper use. Incorporation of
preservatives in single - dose vials is also a common procedure if fi ltration is used as
a sterilization method. Sznitowska et al. [127] studied the physicochemical compatibility
between the lecithin - stabilized emulsion and 12 antimicrobial agents over two
years of storage at room temperature. Preliminary physicochemical screening results
indicate that addition of chlorocresol, phenol, benzyl alcohol, thiomersal, chlorhexidine
gluconate, and bronopol should be avoided due to the occurrence of an unfavorable
pH change followed by coalescence of lecithin - stabilized droplets of the
emulsion. Furthermore, the effi cacy of antimicrobial preservation was assessed using
the challenge test according to the method described by the European Pharmacopoeia.
Despite good physicochemical compatibility, neither parabens nor benzalkonium
chloride showed satisfying antibacterial effi cacy in the emulsion against the
tested microorganisms and consequently did not pass the test. Therefore, higher
concentrations of antimicrobial agents or their combination may be required for
effi cient preservation of the lecithin - stabilized emulsion probably because of unfavorable
phase partitioning of the added antimicrobials within the different internal
structures of the emulsion. It is interesting to note that benzalkonium chloride, a
highly aqueous soluble drug, did not pass the standard challenge test even when
incorporated in a cationic emulsion, particularly the third - generation category
(unpublished data). This fi nding clearly indicates that the possible electrostatic
attraction between the negatively charged lipid moieties of the mixed emulsifying
fi lm formed around the anionic emulsifi ed oil droplets [127] and the quaternary
cationic ammonium groups of the preservative is not the plausible cause for the
reduced activity of the benzalkonium chloride. Thus, the possible intercalation of
this surfactant in either the cationic or anionic interfacial mixed emulsifying fi lm is
likely to occur, preventing benzalkonium chloride from eliciting its adequate preservative
action. Overall, it is preferable to formulate nanosized emulsions devoid
of preservative agents and fi ll it in sterile single - dose packaging units to prevent
potential contamination due to repeated use of multidose packaging. It should be
pointed out that the two available ocular emulsion products (Refresh Endura and
Restasis, Allergan, Irvine, CA) on the market are preservative free and packed in
EXCIPIENT INCLUSION: OIL-IN-WATER NANOSIZED EMULSIONS 1345
1346 OIL-IN-WATER NANOSIZED EMULSIONS
single - use vials only. Currently there is no commercial parenteral emulsion which
contains preservatives and research concerning the problem of preservation of
nanosized emulsion is very limited [128 – 131] .
7.4.5 MEDICAL APPLICATIONS OF OIL - IN - WATER
NANOSIZED EMULSIONS
It has been shown in a number of studies that the incorporation of drug in o/w
nanosized emulsions signifi cantly increased the absorption of the drug when compared
with the equivalent aqueous solution administered orally [132 – 135] . However,
the use of emulsions for oral application is limited since other attractive alternatives,
such as self - emulsifying oil delivery systems, which are much less sensitive and easy
to manufacture, are available [136, 137] . Thus the potential of nanosized emulsions
after administration with parenteral and traditional nonparenteral topical routes
such as ocular, percutaneous, and nasal is covered in this section.
7.4.5.1 Parenteral Routes
The o/w nanosized emulsion formulations of lipophilic drugs, such as propofol,
etomidate, dexamethasone palmitate, and diazepam, were already developed and
marketed (Table 1 ). Furthermore, various research groups across the world are currently
undertaking projects to exploit the potential of o/w emulsions for parenteral
delivery of a myriad of investigational drugs as well as other lipophilic drugs by
receptor - mediated targeting to cancer cells. The important technical and clinical
points to keep in mind before the use of the emulsion systems for this kind of work
are given below.
It has to be clear that, once diluted and injected (or administered in ocular and
other routes), the emulsion stability and fate are determined by three measurable
parameters. The fi rst is the partition coeffi cient of each emulsion component (including
added drugs and agents) between the emulsion assembly and the medium. To
some extent this partition coeffi cient is related to oil – water and/or octanol – water
partition coeffi cients. For example, it was well demonstrated that per component of
which log P is lower than 8, the stability upon intravenous (IV) injection is questionable
[42, 138] . The other two parameters are koff , a kinetic parameter which describes
the desorption rate of an emulsion component from the assembly, and kc , the rate
of clearance of the emulsion from the site of administration. This approach is useful
to decide if and what application a drug delivery system will have a chance to
perform well [89] .
Stability in plasma is an important requirement for IV emulsions as fl occulated
droplets may result in lung embolism. It was found that tocol - based emulsions stabilized
by sodium deoxycholate/lecithins fl occulated strongly when mixed with
mouse, rat, and sheep plasma and serum, whereas soya oil – based emulsions with
the same emulsifi ers did not [123] . It was hypothesized that this effect was caused
by the adsorption of plasma proteins onto the tocol droplets (opsonization). Indeed,
the steric stabilization of emulsions by incorporation of emulsifi ers like poloxamer
188 or PEGylated phospholipids such as PEG 5000 PE proved to be effective in the
stabilization of tocol - based emulsions in plasma. Conversely, in vitro studies were
conducted on plasma protein adsorption onto the blank second - and third -
generation emulsion droplets [37, 139] to mimic the in vivo opsonization phenomenon
responsible for the rapid clearance of the emulsion droplets from the blood.
According to these authors, the adsorption of many protein species such as apoAs,
apoCs, apoE albumin, fi brinogen, and gamma globulin onto the emulsion droplet
surfaces is detectable by two - dimensional polyacrylamide gel electrophoresis.
7.4.5.2 Ocular Routes
For the eye, the method of drug delivery is important. However, when nanosized
emulsion is used as a vehicle for ocular drug delivery purposes, both topical/local and
intraocular routes of administration can be possible (though no data concerning
intraocular drug delivery through emulsion are currently available). The o/w nanosized
emulsions having both anionic and cationic charges provide a liquid - retentive
carrier for ocular active agents, particularly when topically instilled into the eye. It is
interesting to add here that thermodynamically stable and optically isotropic colloidal
systems such as the w/o microemulsion is also designed nowadays for ocular
topical. As delivery the w/o microemulsion system comprises both aqueous and oily
components into its multistructure, it has the ability to incorporate considerable
amounts of both hydrophilic and lipophilic drugs [140] . In fact, in comparison to
ocular inserts or implants and semisolid ocular preparations, the liquid - retentive
nature gives impetus to investigating further the emulsion - based ophthalmic drug
delivery as it has the benefi t of being comfortable to use for both ophthalmologists
and patients. In addition, through topical instillation of emulsions possessing ocular
active substances, the delivery of drug molecules even to the posterior portion of the
eye might be of possible. In this context, the third - generation emulsion is being
designed by adsorbing electrostatically the therapeutic oligonucleotides onto its
surface for modulating functions of retinal pigment epithelium (RPE) cells effectively
in order to treat blindness associated with age - related macular degeneration
(AMD), proliferative vitreoretinopathy, retinal and choroidal neovascularization,
and retinitis pigmentosa. To achieve this, it becomes necessary to know fi rst the ocular
protective mechanisms and other concomitant factors to be faced by emulsion droplets
following ocular topical application. This point is further developed below.
Considerations of ocular drug delivery are not detailed in this section. Pertinent
information concerning factors affecting drug permeation or retention as well as
eye anatomy and physiology can be found in several reviews [141 – 146] . From a
medical point of view, o/w nanosized emulsions for ophthalmic use aim at enhancing
drug bioavailability either by providing prolonged delivery to the eye or by facilitating
transcorneal/transconjunctival penetration. Drugs incorporated in o/w nanosized
emulsions are lipophilic in nature, and depending on the extent of lipophilicity,
either the corneal or the conjunctival/scleral route of penetration may be favored
[147] . For the more lipophilic drugs the corneal route was shown to be the predominant
pathway for delivering drugs to the iris, whereas the less lipophilic drugs
underwent conjunctival/scleral penetration for delivery into the ciliary body [147] .
Thus, transcorneal permeation has traditionally been the mechanism by which topically
applied ophthalmic drugs are believed to gain access to the internal ocular
structures. Relatively little attention has been given to alternate routes by which
drugs may enter the eye. It was reported that drugs may be absorbed by the
MEDICAL APPLICATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1347
1348 OIL-IN-WATER NANOSIZED EMULSIONS
noncorneal route and appeared to enter certain intraocular tissues through the
conjunctiva/sclera [148 – 150] . Indeed when compared to the cornea, drug penetration
through the conjunctiva has the advantage of a larger surface area and higher
permeability, at least for drugs which are not highly lipophilic. Furthermore, the
lasting presence of drug molecules in the lower conjunctival cul - de - sac of the eye
could result in a reservoir effect. Nevertheless, the o/w nanosized emulsions more
or less physically resemble a simple aqueous - based eye drop dosage form since
more than 90% of the external phase is aqueous irrespective of the formulation
composition. Hence, following topical administration, nanosized emulsions would
probably face almost similar ocular protective events as encountered with conventional
eye drops into the eye. The o/w nanosized emulsions are likely to be destabilized
by the tear fl uid electrolytic and dynamic action. Because of constant eyelid
movements, the basal tear fl ow rate (1.2 . L/min), and the refl ex secretion induced
by instillation (up to 400 . L/min depending on the irritating power of the topical
ocular solutions [35] ), topical eye drop dosage forms are known for being rapidly
washed out from the eye. Therefore, the water phase of the emulsion is drained off
while, probably, the oil phase of the emulsion remains in the cul - de - sac for a long
period of time and functions as a drug reservoir [35] . If the volume of instilled emulsion
in the eye exceeds the normal lachrymal volume of 7 – 10 . L, then the portion
of the instilled emulsion (one or two drops, corresponding to 50 – 100 . L) that is not
eliminated by spillage from the palpebral fi ssure of conjunctiva is drained quickly
via the nasolacrimal system into the nasopharynx. In other words, the larger the
instilled volume, the more rapidly the instilled emulsion is drained from the precorneal
area. Hence the contact time of the emulsion with the absorbing surfaces
(cornea and conjunctiva) is estimated to be a maximum of a few minutes, well
beyond the short residence time of conventional eye drops. In order to verify the
extension of the residence time of the emulsion in the conjunctival sac, Beilin et al.
[151] added a fl uorescent marker to the formulations. One minute after the topical
instillation into eye, 39.9 ± 10.2% of the fl uorescence was measured for the nanosized
emulsions whereas only 6.8 ± 1.8% for regular eye drops. In addition a study
was carried out in male albino rabbits to compare the corneal penetration of indomethacin
from Indocollyre (a marketed hydro - PEG ocular solution) to that of
negatively and positively charged emulsions [108] . By this comparison, it was
intended to gain insightful mechanistic comprehension regarding the enhanced
ocular penetration effect of the emulsion as a function of dosage form and surface
charge. The contact angle of one droplet of the different dosage forms on the cornea
was measured and found to be 70 ° for saline, 38 ° for the anionic emulsion, and 21.2 °
for the cationic emulsion. Respectively, the values of the calculated spreading coef-
fi cient were . 47, . 8.6, and . 2.4 mN/m. It can clearly be deduced, owing to the
marked low spreading coeffi cient values elicited by the emulsions, that both nanosized
emulsions had better wettability properties on the cornea compared to saline.
The emulsion may then prolong the residence time of the drop on the epithelial
layer of the cornea, thereby enabling better drug penetration through the cornea to
the internal tissues of the eye, as confi rmed by animal studies [108] . It is therefore
believed that drug is not released from the oil droplet and equilibrates with the
tears but rather partitions directly from the oil droplets to the cell membranes on
the eye surface. Therefore, it is reasonable to consider that nanosized emulsions
have a real advantage since they elicit an increased ocular residence time in com
parison to conventional eye drops and will signifi cantly improve the ocular drug
bioavailability [152] . This is also confi rmed in numerous cited papers that are listed
in Table 5 .
In spite of a relatively rapid removal of conjunctivally absorbed emulsion from
the eye by local circulation, direct transscleral access to some intraocular tissues
cannot be excluded, especially if an electrostatic attraction does occur between the
cationic oil droplets of emulsion and anionic membrane moieties of the sclera, as
shown by some authors [108] . There is no doubt that the drug absorption from emulsion
through the noncorneal route needs to be investigated further as it may elicit
useful information on the potential of nanosized emulsions to promote drug penetration
to the posterior segment through a mechanism which bypasses the anterior
chamber. In addition to the above - described protective and elimination mechanisms
of the eye, nanosized emulsions remaining in the precorneal area may be subject to
protein binding and to metabolic degradation in the tear fi lm. In conjunction with
blood plasma, although low, tear fi lm, aqueous humor, and vitreous humor also have
varying amounts of relatively detectable proteins such as albumin, globulin, and
immunoglobulins (e.g., IgA, IgG, IgM, IgE) and the enzyme lysozyme. Additional
studies (at least in vitro) are necessary to understand clearly the nanosized emulsion
interaction with the ocular fl uid components. Although it is unlikely to happen
because of the low emulsion volume remaining in the conjunctival sac, the fl uid
dynamics may be moderately altered by the physical and chemical properties of
nanosized emulsions, which include tonicity, pH, refractive index, interfacial charge,
viscosity, osmolality, and irritant ingredients. Thus, formulations of ophthalmic drug
products must take into account not only the stability and compatibility of a drug in
the emulsion but also the infl uence of the emulsion on precorneal fl uid dynamics.
All of the concepts exposed in this section may ultimately result in transcorneal/conjunctival
absorption of 1 – 2% or less of the drug applied topically through the emulsions.
In summary, the rate of loss of drug/emulsion from the eye can be 500 – 700
times greater than the rate of absorption into the eye. Thus, conventional topical
delivery using emulsions cannot achieve adequate intracellular concentrations of
drugs or other substances such as oligonucleotides or genes for the treatment of
endophthalmitis or other sight - threatening intraocular diseases (e.g., AMD).
TABLE 5 Selected List of o/w Nanosized Emulsions for Ocular Topical Drug Delivery
Emulsion Type Drug Used Reference
Anionic emulsion .8 - THC 154
Pilocarpine base and indomethacin 163
Adaprolol maleate 161, 162
Indomethacin 159, 160
Synthetic HU - 211 and pilocarpine base 124, 155, 156
Pilocarpine base 157, 158, 164
Cyclosporin A 168, 165 – 177
Cationic emulsion Piroxicam 178
Indomethacin 108
Miconazole 112
Cyclosporin A 106, 179
Note: .8 - THC and synthetic HU - 211 are derivatives of Cannabis sativa.
MEDICAL APPLICATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1349
1350 OIL-IN-WATER NANOSIZED EMULSIONS
In order to achieve a high concentration of drug within the eye using an emulsion
delivery vehicle, an approach that bypasses physiological and anatomical barriers
(e.g., blood – ocular) of the eye is a more viable and attractive option. One such
approach is to administer emulsion through direct intraocular injections such as
periocular (subconjunctival and sub - Tenon), intracameral, intravitreal, intracapsular,
or subretinal. Moreover, it is likely that intraocularly administered emulsion is
able to both signifi cantly increase drug half - life and minimize intraocular side effects
that appear following intraocular injection of drug alone. In general, drugs encapsulated
within emulsion are less toxic than their free counterparts. Additionally,
there is a possibility of obtaining slow drug release from an intraocularly injected
emulsion. Taking into account the nonphagocytic character of neural retinal cells
and the ability of RPE cells to take up large molecules, including oligonucleotides,
the third - generation emulsion for intravitreal or subretinal injections is more likely
to be a successful approach in future. Moreover, intravitreally administered drug
molecules are able to bypass the blood – ocular barrier to achieve constant therapeutic
levels in the eye while minimizing systemic side effects. However, the hyalocytes,
the main cellular components of the vitreous, have been classifi ed in at least
one report [153] as macrophages and thus may play a role in the uptake of intravitreally
injected emulsion. It should be added that no studies are focused so far on
injecting emulsion intraocularly and signifi cant work should be devoted to generate
this novel idea into a fruitful solution in ophthalmic drug delivery applications.
Over the last decade, o/w nanosized emulsions containing either anionic or cationic
droplets have been recognized as interesting and promising ocular topical
delivery vehicles for lipophilic drugs. Complete details are available elsewhere [117] .
As an overview of this topic, important results on emulsion - based ocular topical
drug delivery are covered below and are listed in Table 5 .
The in vivo data obtained from studies of early formulations confi rm that o/w
nanosized anionic emulsions can be effective topical ophthalmic drug delivery
systems [154] with a potential for sustained drug release [155] . Naveh and co -
workers [156] have also noted that the intraocular pressure (IOP) – reducing effect
of a single, topically administered dose of pilocarpine - loaded anionic emulsion
lasted for more than 29 h in albino rabbits whereas that of the generic pilocarpine
lasted only 5 h. Zurowska - Pryczkowska et al. [157] studied how nanosized emulsion
as a vehicle infl uences the chemical stability of pilocarpine and the effect the drug
has on the physical stability of nanosized emulsions. In a subsequent paper [158]
from the same group on in vivo evaluation using normotensive rabbits, it was shown
that the nanosized emulsion formulated with pilocarpine hydrochloride at pH 5.0
could be indicated as a preparation offering prolonged pharmacological action
(miotic effect) together with satisfactory chemical stability. However, the ocular
bioavailability arising from such a formulation is not signifi cantly improved when
compared to an aqueous solution. Calvo et al. [159, 160] observed an improvement
in indomethacin ocular bioavailability when the drug was incorporated in an emulsion
dosage form with respect to the commercial aqueous drops following topical
application into rabbit eye.
In order to verify the extension of the residence time of the emulsion in the
conjunctival sac, Beilin et al. [151] added a fl uorescent marker to the formulations,
as mentioned previously. From that observation, it is reasonable to consider that an
emulsion formulation has the real advantage of increasing ocular residence time in
comparison to eye drops. Anselem et al. [161] and Melamed et al. [162] prepared a
nanosized emulsion containing adaprolol maleate, a novel soft . - blocking agent, and
observed a delayed IOP depressant effect in human volunteers. A similar pharmacological
effect was also observed in human volunteers by Aviv et al. [163] using
pilocarpine base - loaded emulsion. Another randomized human trial conducted by
Garty et al. [164] compared the activity of the pilocarpine base - laden nanosized
emulsion instilled twice daily with a generic dosage form instilled four times a day
to 40 hypertensive patients for seven days. No local side effects were observed. The
IOP decreased 25% in both formulations during this time period. No signifi cant
difference was noticed between the two treatments. These results proved that the
anionic emulsion extended the action of the drug and two daily administrations have
the same result as four instillations of regular eye drops.
A novel nanosized anionic emulsion incorporating the immunomodulatory agent
cyclosporin A was developed and the clinical effi cacy was investigated for the treatment
of moderate to severe dry - eye disease in humans [165 – 167] . The novel cyclosporin
A ophthalmic dosage form represents a real breakthrough in the formulation
of a complex, highly lipophilic molecule such as cyclosporin A within an o/w nanosized
emulsion. Following thorough validation of this formulation through several
clinical studies in various countries [165 – 175] , an anionic o/w emulsion containing
cyclosporin A 0.05% (Restasis, Allergan, Irvine, CA) was approved for the fi rst time
by the FDA, on December 23, 2002. In addition, this anionic emulsion having cyclosporin
A is now available at pharmacies in the United States for the treatment of
chronic dry - eye disease (available at www.restasis.com and www.dryeye.com ). Furthermore,
an over - the - counter (OTC) product that features an emulsion formula,
Refresh Endura, is already launched in the U.S. market for eye - lubricating purposes
in patients suffering from moderate to severe dry - eye syndrome.
The effect of Restasis on contact lens comfort and reducing dry - eye symptoms
in patients with contact lens intolerance was evaluated in comparison to rewetting
drops (carboxymethylcellulose 0.5%, Refresh Contacts) [176] . Both formulations
were applied twice per day before and after lens wear. Symptoms were assessed by
lens wear time, use of rewetting drops during lens wear, subjective evaluation of
dryness, and completion of the ocular surface disease index questionnaire. The
results of this pilot study indicate that cyclosporin 0.05% is benefi cial for contact
lens wearers with dry eye and reduces contact lens intolerance [176] . Furthermore,
Sall et al. [177] have recently evaluated the effi cacy of marketed artifi cial tears
(Systane and Restasis) in relieving the signs and symptoms of dry eye when used
as supportive therapy to a cyclosporin - based ophthalmic emulsion (i.e., Restasis +
Systane vs. Restasis + Refresh). Signifi cant differences were seen in favor of Restasis
+ Systane versus Restasis + Refresh for less ocular burning, stinging, grittiness, and
dryness. Systane was better than Restasis + Refresh for less burning, dryness, and
scratchiness. Results indicate that the choice of concomitant therapy used with
Restasis has signifi cant effects on outcome measures and both supportive therapies
were compatible with Restasis [177] .
When compared to either saline or anionic emulsions, the nanosized cationic
emulsions were shown to enhance the ocular bioavailability of indomethacin [108] ,
piroxicam [178] , and cyclosporin A [106, 179] following one single - drop dose instillation
into the rabbit eye (Figure 4 ). A signifi cant drug reservoir effect was noted
in the cornea and conjunctiva even for more than 8 h following the instillation [106] .
MEDICAL APPLICATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1351
1352 OIL-IN-WATER NANOSIZED EMULSIONS
This long residence time may help reduce evaporation of the limited volume of
natural tears present in patients with dry eye. This was probably due to the adhesion
of the positively charged oil droplets to the negatively charged corneal surface
moieties as a result of electrostatic attraction. This hypothesis was supported by data
from an ex vivo study which showed that cationic emulsion exhibited better wettability
properties on albino rabbit eye cornea than either saline or anionic emulsion
[108] . Associated with Poloxamer and phospholipids, a cationic primary amine,
stearylamine, has been used to obtain the above - described third - generation cationic
emulsions. Additionally, a cationic emulsion based on an association of Poloxamer
188 and chitosan was prepared and also showed interesting physicochemical properties
on stability and charge effects [97, 98] . Moreover, the stability and ocular tolerance
following topical instillation into the eye of these cationic emulsion vehicles
were investigated [98, 114] . The overall studies hence stress the effectiveness of
nanosized cationic emulsion, which promotes ocular drug absorption via internalization
possibly through an endocytic process [112] .
7.4.5.3 Nasal Route
The nasal route is still receiving great attention due to a number of advantages over
parenteral and oral administration [180] , particularly when fi rst - pass metabolism
makes the drug ineffective. The approach of an o/w emulsion formulation of the
drug may increase absorption by solubilizing the drug in the inner phase of
the emulsion and prolonging contact time between emulsion droplets and nasal
mucosa.
One of the fi rst examples for systemic delivery of peptides concerned a lipid -
soluble rennin inhibitor [181] . The peptide was solubilized in the oil phase of an o/w
emulsion containing membrane adjuvants such as oleic acid and mono - and diglyc-
FIGURE 4 Infl uence of emulsion surface charges cyclosporin A (CsA) concentrations in
ocular surface tissues (cornea and conjunctiva) following one single - dose (50 - . L) instillation
of positively (cationic) and negatively (anionic) charged CsA - loaded nanosized emulsions
into albino rabbit eye.
0
400
800
1200
1600
2000
2400
2800
15 30 60 120 180 480
Time (min)
Cornea positive emulsion
Cornea negative emulsion
Conjunctiva positive emulsion
Conjunctiva negative emulsion
CsA concentration, ng/g
erides. Emulsion formulations have been proposed to simultaneously increase the
solubility of the peptide and to enhance membrane permeability through interaction
between the membrane and the oil components. Enhanced and prolonged in
vivo nasal absorption of the rennin inhibitor was observed in emulsion formulation
compared to aqueous suspension. From morphological studies, the emulsions did
not provoke any signifi cant changes to the nasal mucosa [181] . Such a formulation
approach was also used for the administration of a steroidal male sex hormone testosterone
[182] . The steroid was solubilized in the oil phase of the o/w emulsion and
the ionic composition of the aqueous phase was modifi ed in order to produce electrically
positive, negative, and neutral droplets. Droplets with a surface charge led
to better bioavailability than neutral droplets, but contrary to the above - described
topical applicabilities of cationic emulsions over anionic emulsions, positively
charged droplets did not provide the best results [182] . However, the emulsion
approach was advantageous since it helped to overcome the solubility problem of
the hydrophobic compounds.
In another study which does not involve peptide drugs, various emulsion formulations
were prepared in order to modulate the partitioning of the drug between the
aqueous phase and the oil phase [183] . The disappearance of a drug from the nasal
cavity was determined by an in situ perfusion technique. When the drug was solubilized
in the aqueous phase, the formulation did not have a signifi cant effect on
the drug disappearance rate. However, partitioning of the drug in the oil phase
resulted in delaying absorption. It was suggested that oil droplets containing
medium - chain triglycerides formed a pseudo – oily layer on the mucous membrane,
which slowed down the drug disappearance from the nasal cavity [183] . Another
interesting study reported nasal delivery of insulin formulated in both o/w and w/o
emulsions [184] . As insulin partitions into the aqueous phase of the emulsion, the
peptide is either incorporated within the continuous phase of the o/w emulsion or
encapsulated in the aqueous droplets of the w/o emulsion. Following nasal perfusion
experiments, plasma insulin concentration profi les showed enhanced insulin absorption
when the peptide was formulated as an o/w emulsion compared to an aqueous
solution. However, a w/o emulsion did not cause any signifi cant increase in plasma
insulin concentration. Delivery of insulin by administration of nasal drops also
revealed a large dose - dependent increase in plasma insulin concentration. It also
needs to be pointed out that the emulsifi er mixture alone did not promote any
absorption. It was suggested that insulin molecules probably were adsorbed at the
surface of the oil droplets. Adhesion of the oil droplets on the mucosal membrane
then induced a local increase in insulin concentration at the membrane surface.
However, the number of droplets in contact with the surface had to be small enough.
Otherwise, a stagnant oil layer is formed which acts as an additional barrier to the
transport, as was observed with the w/o emulsion [183] .
Other recent applications of emulsion formulation involve mucosal gene and
vaccine delivery [185 – 187] and the preparation of polymeric microspheres by the
w/o emulsifi cation solvent extraction technique [188] .
7.4.5.4 Topical Route
Many drugs exhibit low skin penetration, which results in poor effi cacy. As opposed
to common chemical skin penetration enhancers, organic solvents, which are
MEDICAL APPLICATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS 1353
1354 OIL-IN-WATER NANOSIZED EMULSIONS
generally associated to some degree with skin irritation, toxicity and sensitization,
a solvent - free topical vehicle based on drug entrapment in o/w emulsion droplets
of submicrometer size is more effi cacious in terms of percutaneous absorption and
possibly devoid of adverse effects. In addition, the uniqueness of the large internal
hydrophobic oil core of o/w nanosized emulsion droplets allows high solubilization
capacity for water - insoluble topically active medicaments and also aids in carrying
water, an excellent softener, to the skin.
The concept of using anionic nanosized emulsion vehicles for enhanced percutaneous
absorption of nonsteroidal anti - infl ammatory drugs (NSAIDs) and diazepam
was clearly proven [189, 190] . NSAIDs and diazepam in a nanosized emulsion
vehicle also demonstrated noticeable systemic activity. The o/w emulsion was tested
for primary irritation in humans in a 48 - h trial. Low irritancy and excellent human
acceptance were observed, subsequently making the further development of a nanosized
emulsion vehicle very attractive.
Even though emulsion vehicles increase dermal drug delivery of lipophilic drugs
in humans, one of the problems for topical drug delivery is the diffi culty of applying
these vehicles to the skin because of their fl uidity. Rheological properties are studied
in transdermal formulations and different results are given. Realdaon and Ragazzi
[191] have investigated different mechanical emulsifying conditions on o/w emulsion
formulations containing methyl nicotinate. The infl uence of these procedures
on rheological properties and in vivo availability of methyl nicotinate was evaluated.
Even if various viscosities were obtained, differences between batches did not compromise
drug availability. On the contrary, Welin - Berger and co - workers [192, 193]
concluded in their study on nanosized emulsions containing model compounds that
both release and permeation rates decrease when the apparent yield stress (i.e., the
macroviscosity) increases by addition of gelling polymers. Because a topical anesthetic
agent will induce a pain - suppressing anesthesia, the eutectic mixture of local
anesthetics (EMLA) has proven to be a useful medication for children. It is an
emulsion containing a mixture of lidocaine and prilocaine. This cream gives an effective
deep sedation and can be applied half an hour to 1 h prior to the procedure.
Local side effects with this emulsion are very mild [194, 195] . Systemic activity of
nanosized emulsions containing diazepam was compared with regular creams or
ointments by Schwarz et al. [190] . Their effi ciency was tested on protection against
pentamethylenetetrazole, which induces convulsive effects in mice. Diazepam
applied topically in emulsion creams was strongly dependent on oil droplet size.
Furthermore, nanosized emulsions increased transdermal drug delivery and prolonged
protective activity for up to 6 h.
Many formulations for topical emulsions are available in the scientifi c literature,
in patents, and on the market. Progresses made in the last years in this fi eld are
concentrated on the various aspects of drug release and the infl uence of droplet
size.
Third - generation cationic emulsions were suggested as drug carriers for topical
use in the skin. It was found that . - tocopherol - loaded cationic emulsion was able
to prevent oxidative damage of cultured fi broblast cells [196] . In addition, the same
cationic formulation was able to protect rat skin against oxidative stress induced by
ultraviolet (UV) irradiation signifi cantly better than either the corresponding
anionic emulsion or the cationic blank emulsion, as measured with a noninvasive
evaluation of the lipid hydroperoxidation process of the rat skin. However, no difference
was found between cationic or anionic nanosized emulsions of . - tocopherol
as assessed by endogenous peroxyl radical scavenging ability. Taken together, these
results suggest that the cationic emulsion allows the . - tocopherol to remain on the
surface of the skin because of electrostatic interactions between the negatively
charged sites of the superfi cial layers of the skin and the positively charged oil
droplets. Although the extent of . - tocopherol incorporation into the skin is similar
for both cationic and anionic emulsions, the prolonged skin surface residence time
of the cationic emulsion allows an enhanced protective effect against oxidative
stress. In contrast to these results, an in vitro percutaneous absorption study on
hairless rat skin found that the antifungal drugs econazole and miconazole nitrate
incorporated into a similar cationic emulsion formulation were more effective
in terms of skin penetration than the corresponding anionic emulsion [197] .
The enhanced rate of diffusion of these antifungal drugs through the skin by the
cationic emulsion suggests a new approach for dermal drug penetration
enhancement [197] .
7.4.6 FUTURE PERSPECTIVE
Based on the performances in previous and present decades, o/w - type nanosized
emulsions can conveniently be classifi ed into three generations. First - generation
emulsions are considered primarily as nutrient carriers to be administered via
intravenous routes to bed - ridden patients. Second - generation emulsions start initially
as drug carrier systems by solubilizing considerable amounts of lipophilic
drugs at the oil phase or at the oil – water interface of the emulsion. This particular
merit of emulsions is specifi cally exploited even commercially for both ocular and
parenteral active drugs. Modifi cations made either in the oil phase or at the o/w
interfacial fi lm forming emulsifi er molecules allow the emulsions to be able to
escape from lipolysis by lipoprotein lipase, apo adsorption, and liver uptake. Such
a surface - modifi ed emulsion would prolong the circulation time in plasma and
thereby an alteration in in vivo disposition of incorporated drugs following parenteral
administration. Attachment of homing devices such as antibody and apoE to
the surfaces of emulsions makes the selective/active delivering of emulsion -
incorporated drugs to target sites such as a tumorized organ or hepatic system.
Active targeting increases the affi nity of the carrier system for the target site, while
passive targeting minimizes the nonspecifi c interaction with nontargeted sites by
the RES. Having together a positive charge and a steric stabilizing effect led to
the development of third - generation emulsions that contain a unique property:
plasma half - life prolongation and electrostatic adhesion to ocular surface tissues
after topical instillation into eye. Furthermore, the third - generation emulsion shows
a potential of carrying a wide range of lipophilic, amphiphilic, and polyanionic
compounds, including DNA and oligonucleotides for transdermal and nasal routes.
Accumulating knowledge thus suggests that constant progress in better understanding
the principles and processes governing the various issues related to o/w
nanosized emulsions has surely brought major improvements in the effi cacy of
parenteral or nonparenteral drug delivery systems.
FUTURE PERSPECTIVE 1355
1356 OIL-IN-WATER NANOSIZED EMULSIONS
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164. Garty , N. , Lusky , M. , Zalish , M. , Rachmiel , R. , Greenbaum , A. , Desatnik , H. , Neumann ,
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165. Ding , S. , Tien , W. , and Olejnik , O. ( 1995 ), Nonirritating emulsions for sensitive tissue,
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167. Acheampong , A. A. , Shackleton , M. , Tang - Liu , DD - S. , Ding , S. , Stern , M. E. , and Decker ,
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168. Stevenson , D. , Tauber , J. , and Reis , B. L. ( 2000 ), Effi cacy and safety of cyclosporin A
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169. Sall , K. , Stevenson , O. D. , Mundorf , T. K. , and Reis , B. L. ( 2000 ), Two multicenter, randomized
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170. Turner , K. , Pfl ugfelder , S. C. , Ji , Z. , Feuer , W. J. , Stern , M. , and Reis , B. L. ( 2000 ), Interleukin
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171. Kunert , K. S. , Tisdale , A. S. , Stern , M. E. , Smith , J. A. , and Gipson , I. K. ( 2000 ), Analysis
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174. Galatoire , O. , Baudouin , C. , Pisella , P. J. , and Brignole , F. ( 2003 ), Flow cytometry in
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176. Hom , M. M. ( 2006 ), Use of cyclosporine 0.05% ophthalmic emulsion for contact lens -
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177. Sall , K. N. , Cohen , S. M. , Christensen , M. T. , and Stein , J. M. ( 2006 ), An evaluation of the
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178. Klang , S. H. , Siganos , C. S. , and Benita , S. ( 1999 ), Evaluation of a positively - charged
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179. Tamilvanan , S. , Khoury , K. , Gilhar , D. , and Benita , S. ( 2001 ), Ocular delivery of cyclosporin
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180. Hussain, A. A. (1998), Intranasal drug delivery , Adv. Drug Deliv. Rev. , 29 , 39 – 49 .
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1367
INDEX
Abortifacients, 850
Acyclovir, 1037–1042
Adjuvants, 635–637
ADMET, 8–9
AERx, 709–710
AIDS, 4
Alginate nanoparticles, 540–541
Alginic acid, 295
Anticancer drug delivery, 485–506
Antimicrobials, 845–846
API, 5
Artifi cial neural networks, 1016
Aseptic compounding, 107–108
Auxiliary excipients, 894–895
Avonex, 47
Aztirelin, 620
Bentonite, 295
Benzodiazepines, 623–626
Betaseron, 47
Bioadhesion, 305–306
Bioburden considerations, 26
Bioconversion, 566, 572–574
Biodegradable microspheres, 419–426
Biodegradable polymeric nanoparticles,
536–543
Biodrug, 565–566
Biogenerics, 35
Biological half-life, 356
Biopharmaceuticals Classifi cation System
(BCS), 237–238, 961
Boron Neutron Capture Therapy (BNCT),
489
Breast cancer, 492–497
Breath actuation, 698–699
CaCo-2, 960
Caclyx, 497
Calcitonin, 613–617
Cancer therapy, 1238–1240
Capillary Aerosol Generator (CAG),
710–711
Carbomer, 295–296
Carbxymethyl cellulose, 655
Carnauba wax, 274
Carr index, 908
Carrageenan, 296, 833
Challenges in ocular drug delivery,
730–737
Characteristics of radiopharmaceuticals,
60–61
Chemical penetration enhancers, 803
Chemically induced release, 384–385
Chitosan nanoparticles, 541
Chitosan, 608, 636, 655, 657, 658, 661–662,
665–666, 833
Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad
Copyright © 2008 John Wiley & Sons, Inc.
1368 INDEX
Cholera toxin, 637
Ciliotoxicity, 668
Classifi cation of hygroscopicity, 912
Climatization, 1085–1086
CMV, 481
Coated tablets, 244–245
Colloidal silicon dioxide, 296
Colorants, 243
Compactibility, 1138–1141
Compendial gels, 307
Compendial ointments, 289–291
Compressibility, 917–918
Container closure systems, 17–18
Controlled-release delivery systems, 11
Cryogenic spray drying, 401–402
Cytotoxicity, 1240
Defi nitions of density terms, 1178
Dendrimers, 1272
Depyrogenation, 117–120
Diluents, 240–241
Diphtheria Toxoid (DT), 420–421
DirectHaler, 602–604
Disintegration, 920–922
DPI, 689
Drug product stability, 21–25
Drug-excipient compatibility, 969–970
Dry Powder Inhalers (DPIs), 684,
700–706
Effective half life, 61
Effervescent tablets, 251–252
Electroresponsive release, 381–383
Ethylcellulose, 297
Excipients, 1344, 19–21, 239–244, 243–244,
410–412, 412–419, 695, 822–823, 883–
896, 884–885, 897
Exubera, 704, 705–706
Fair Packaging and Labeling Act,
190–195
FDA-approved transdermal patches, 794
First-uterine-pass effect, 821
Fluidized-bed coating, 1102–1103
FluMist, 592
Formulation approaches to improve ocular
bioavailability, 737–753
Formulation assessment, 7–8
Formulation development, 15–16, 238
Fortical, 52–53, 55
Friability, 928
Fullerenes, 1272, 1296–1297
Gas and vapor sterilization, 119
Gastrointestinal tract and absorption,
356–357
Gelatin capsules, 245–251
Gelatin, 539
Gelling agents, 293–301
GELS, 288–310
Giladin, 540
Glucose-responsive insulin release device,
384–385
Glycerol behenate, 298–299
Group B streptococcus vaccine, 420
Guar gum, 297
Hammer Mill, 1169
Hausner ratio, 908
Herpes simplex virus, 481
HFA reformulation, 690–692
High-throughput Screening (HTS), 934
Human Growth Hormone (HGH), 34
Hyaluronic acid, 499, 655,657, 833
Hydralazine, 627
Hydrogels, 291–292
Hydrophilic matrix tablets, 1210–1211
Hydroxyethyl Cellulose (HEC), 297–298
Hydroxypropylmethyl Cellulose (HPMC),
298
Immunity after intranasal immunization,
634–635
Immunogenicity, 50, 53–54
Inhalation drug products, 179
Injectable microspheres, 407–408
Insulin, 424–426
Ionophoresis, 804
Japanese Encephalitis Virus (JEV),
423–424
Kurve Technology, 601
Labor inducers, 850
Lanolin, 271
Lipinski Rule of Five, 934
Liposomal drugs approved for clinical
application, 1263
Liposome-based products currently under
clinical testing, 484
Liposomes, 365–367, 636, 747–748
Liquid dosage forms, 338
Low-molecular-weight heparins, 617–620
Lozenges, 252–253
INDEX 1369
Lung cancer, 497–502
Lung toxicity, 1309–1310
Lyophilization, 127–128
Magnetically induced release, 383–384
Marked medical and nonmedical
emulsions, 1332
MDI, 689
Mechanisms of protein and peptide
degradation, 22–23
Metal as packaging material, 170–171
Metered-dose Inhalers (MDIs), 684,
690–700
Microbicides, 843–845
Microbiological quality, 334–335
Microcrystalline cellulose (MCC), 653, 655
Microemulsions, 1267, 748–750
Microencapsulation, 358
Microneedles, 803–804
Milestones in early biologics regulation, 38
MLVs (multilamellar vesicles), 444
Mononuclear phagocyte system, 1333
Mucoadhesion, 840
Mucoadhesive microspheres, 657
Mucoadhesive polymers, 744
Mucosal toxicity screening method using
the slug arion lusitanicus, 667
Mucosal-associated lymphoid tissue
(MALT), 635
Musciliary clearance, 596
Nanocapsules, 363
Nanocarriers, 1258–1273
Nanoemulsions, 1269
Nanogels, 1271
Nanomaterials in pharmacy, 1253–1254
Nanomaterials, 1250–1252
Nanomedicine technologies taxonomy,
1292–1293
Nanomedicine, 1278–1279
Nanoparticles, 1231–1236, 1264–1269, 536,
746–747
Nasal delivery, 481–482
Nasal delivery of nonpeptide molecules,
622–630
Nasal delivery of vaccines, 633–637
Nasal dry powder formulations, 652–655
Nasal route, 1352–1353
Nasal vaccination delivery systems,
636–637
Nasal vasculature, 594–595
Nebulizers, 706–707
Niosomes, 367, 748
Nitroglycerin, 627–628
Noncovalent binding of ligands, 465–466
Nose-associated Lymphoid Tissue
(NALT), 635
Nose-to-Brain Delivery, 632
Ocular delivery, 477–481
Ocular drug delivery, 738–741, 784–785
Ocular routes, 1347–1352
Offi cial creams, 282
Offi cial gels, 304
Oil-in-water nanosized emulsions,
1329–1341
Ointments and creams, 269–270
Omnitrope, 51, 53–56
OptiNose, 601–602
Oral drug delivery, 781–782
Oral ER formulations, 1193–1195
Orally disintegrating tablets, 259–262
Organogels, 292
Ovarian cancer, 502–506
Pan coating, 1102
Parenteral drug delivery, 783–784
Parenteral routes, 1346–1347
Partition coeffi cient, 352, 956–957
PEGylated liposomes, 469–472
Percolation theory, 1013–1016, 1030–1042
Permeability enhancement methods, 964
Preservatives, 20–21
PET radiopharmaceuticals, 83
Petrolatum, 272
Photostability, 23
pH-sensitive polymeric nanoparticles, 547
Physiochemical properties of liposomes,
449–456
Plastic additives, 164
Plastic as packaging material, 166–170
Poloxamer, 299
Poly (lactic acid), 543–544
Polyethylene oxide, 299–300
Polymorphism, 936–942
Polysaccharides, 539–540
Polyvinyl Alcohol (PVA), 300
Povidone, 300
Preformulation approaches for tablet
production, 883
Principles for extended drug release,
1196–1197
Principles of radiation protection, 63–64
Production of radionuclides, 75–76
1370 INDEX
Production of radiopharmaceuticals, 78–88
Propylene Glycol Alginate (PGA), 300
Quantum dots, 1293–1295
Radiation sterilization, 119
Radioactive decay, 61–63
Radiochemical purity, 90–91
Radionuclides, 65
Reaction calorimetry, 141–142
Respmat, 708–709
Route of administration, 8–10
Salmon calcitonin, 52
Salt selection, 952–956
Scanning tunneling microscopy, 1306–1308
Selected drugs administered in vagina, 853
Selection guideline of pharmaceutical
excipients, 895–896
Selection of microemulsion ingredients,
773
Sodium alginate, 300–301, 538, 655
Soft Mist Aerosols, 707–708
Solubility characteristics, 950–965
Sonophoresis, 804
Spermicides, 849–850
Stability, 336–337
Stability-indicating methodologies, 14–15
Stability of liposomes, 455–456
Sterile products, 169–170
Sterilization, 117–120
Sterilization by fi ltration, 119–120
Sterilization of radiopharmaceuticals,
73–74
Surface hydrophilicity, 550
Synthesis of PET radiopharmaceuticals, 86
Synthetic cervical mucus, 816
Systemic uptake of nanoparticles,
1310–1311
Tablet coating methods, 1102–1103
Tablet tooling terminology, 1147
Tableting machines, 1058–1067
Tableting process, 1055–1056
Tetanus toxoid, 421–423
Thermoresponsive drug release dosage
forms, 379–381
Thermosensitive polymeric nanoparticles,
546–547
TNO gastrointestinal tract model, 569–571
Topical route, 1353–1355
Toxicological effects of dry powder
formulation, 666–667
Tragacanth, 301
Transdermal drug delivery, 368, 782–783
Ultrasonic atomization, 403
Ultrasound-assisted tableting, 1043–1045
United States Pharmacopoeia Center for
the Advancement of Patient Safety, 195
U.S. Pharmacopoeia, 177
USP, 281–282, 304, 903
Vaccines, 420–424, 851–852
Vaginal and uterine controlled-release
dosage forms, 371
Vaginal fi lms, 831
Vaginal fl uid stimulant, 816
Vaginal foams, 831
Vaginal rings, 826–830
Vaginal sponges, 832
Vibrio cholerae vaccine, 423
