Dermal Absorption and Toxicity Assessment pot

Novel Drug Delivery Systems: Second Edition, Revised and Expanded, Yie W.. Dermal Absorption and Toxicity Assessment, edited by Michael S.. Percutaneous Absorption: Drugs–Cosmetics–Mecha

Dermal Absorption and Toxicity Assessment

A Series of Textbooks and Monographs

Executive EditorJames SwarbrickPharmaceuTech, Inc

Pinehurst, North Carolina

Advisory BoardLarry L Augsburger

University of Maryland Baltimore, Maryland

Ajaz Hussain

Sandoz Princeton, New Jersey

Yuichi Sugiyama

University of Tokyo, Tokyo, Japan

Geoffrey T Tucker

University of Sheffield Royal Hallamshire Hospital

Sheffield, United Kingdom

Jeffrey A Hughes

University of Florida College

of Pharmacy Gainesville, Florida

Vincent H L Lee

US FDA Center for Drug Evaluation and Research Los Angeles, California

Kinam Park

Purdue University West Lafayette, Indiana

Jerome P Skelly

Alexandria, Virginia

Elizabeth M Topp

University of Kansas Lawrence, Kansas

2 Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control,Sidney H Willig, Murray M Tuckerman, and William S Hitchings IV

3 Microencapsulation, edited by J R Nixon

4 Drug Metabolism: Chemical and Biochemical Aspects, Bernard Testa and

Peter Jenner

5 New Drugs: Discovery and Development, edited by Alan A Rubin

6 Sustained and Controlled Release Drug Delivery Systems, edited by

Joseph R Robinson

7 Modern Pharmaceutics, edited by Gilbert S Banker and Christopher T Rhodes

8 Prescription Drugs in Short Supply: Case Histories, Michael A Schwartz

9 Activated Charcoal: Antidotal and Other Medical Uses, David O Cooney

10 Concepts in Drug Metabolism (in two parts), edited by Peter Jenner and

Bernard Testa

11 Pharmaceutical Analysis: Modern Methods (in two parts), edited by

James W Munson

12 Techniques of Solubilization of Drugs, edited by Samuel H Yalkowsky

13 Orphan Drugs, edited by Fred E Karch

14 Novel Drug Delivery Systems: Fundamentals, Developmental Concepts,

Biomedical Assessments, Yie W Chien

15 Pharmacokinetics: Second Edition, Revised and Expanded, Milo Gibaldi andDonald Perrier

16 Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control,Second Edition, Revised and Expanded, Sidney H Willig, Murray M Tuckerman,and William S Hitchings IV

17 Formulation of Veterinary Dosage Forms, edited by Jack Blodinger

18 Dermatological Formulations: Percutaneous Absorption, Brian W Barry

19 The Clinical Research Process in the Pharmaceutical Industry, edited by

Gary M Matoren

20 Microencapsulation and Related Drug Processes, Patrick B Deasy

21 Drugs and Nutrients: The Interactive Effects, edited by Daphne A Roe and

T Colin Campbell

22 Biotechnology of Industrial Antibiotics, Erick J Vandamme

23 Pharmaceutical Process Validation, edited by Bernard T Loftus and

Robert A Nash

24 Anticancer and Interferon Agents: Synthesis and Properties, edited by

Raphael M Ottenbrite and George B Butler

25 Pharmaceutical Statistics: Practical and Clinical Applications, Sanford Bolton

26 Drug Dynamics for Analytical, Clinical, and Biological Chemists, Benjamin J.Gudzinowicz, Burrows T Younkin, Jr., and Michael J Gudzinowicz

27 Modern Analysis of Antibiotics, edited by Adjoran Aszalos

28 Solubility and Related Properties, Kenneth C James

and Expanded, edited by Joseph R Robinson and Vincent H Lee

30 New Drug Approval Process: Clinical and Regulatory Management, edited byRichard A Guarino

31 Transdermal Controlled Systemic Medications, edited by Yie W Chien

32 Drug Delivery Devices: Fundamentals and Applications, edited by Praveen Tyle

33 Pharmacokinetics: Regulatory † Industrial † Academic Perspectives, edited byPeter G Welling and Francis L S Tse

34 Clinical Drug Trials and Tribulations, edited by Allen E Cato

35 Transdermal Drug Delivery: Developmental Issues and Research Initiatives,edited by Jonathan Hadgraft and Richard H Guy

36 Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, edited byJames W McGinity

37 Pharmaceutical Pelletization Technology, edited by Isaac Ghebre-Sellassie

38 Good Laboratory Practice Regulations, edited by Allen F Hirsch

39 Nasal Systemic Drug Delivery, Yie W Chien, Kenneth S E Su, and Shyi-Feu Chang

40 Modern Pharmaceutics: Second Edition, Revised and Expanded, edited byGilbert S Banker and Christopher T Rhodes

41 Specialized Drug Delivery Systems: Manufacturing and Production Technology,edited by Praveen Tyle

42 Topical Drug Delivery Formulations, edited by David W Osborne and

Anton H Amann

43 Drug Stability: Principles and Practices, Jens T Carstensen

44 Pharmaceutical Statistics: Practical and Clinical Applications, Second Edition,Revised and Expanded, Sanford Bolton

45 Biodegradable Polymers as Drug Delivery Systems, edited by Mark Chasin andRobert Langer

46 Preclinical Drug Disposition: A Laboratory Handbook, Francis L S Tse andJames J Jaffe

47 HPLC in the Pharmaceutical Industry, edited by Godwin W Fong and

Stanley K Lam

48 Pharmaceutical Bioequivalence, edited by Peter G Welling, Francis L S Tse,and Shrikant V Dinghe

49 Pharmaceutical Dissolution Testing, Umesh V Banakar

50 Novel Drug Delivery Systems: Second Edition, Revised and Expanded, Yie W Chien

51 Managing the Clinical Drug Development Process, David M Cocchetto andRonald V Nardi

52 Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control,Third Edition, edited by Sidney H Willig and James R Stoker

53 Prodrugs: Topical and Ocular Drug Delivery, edited by Kenneth B Sloan

54 Pharmaceutical Inhalation Aerosol Technology, edited by Anthony J Hickey

55 Radiopharmaceuticals: Chemistry and Pharmacology, edited by Adrian D Nunn

Richard A Guarino

57 Pharmaceutical Process Validation: Second Edition, Revised and Expanded,edited by Ira R Berry and Robert A Nash

58 Ophthalmic Drug Delivery Systems, edited by Ashim K Mitra

59 Pharmaceutical Skin Penetration Enhancement, edited by Kenneth A Waltersand Jonathan Hadgraft

60 Colonic Drug Absorption and Metabolism, edited by Peter R Bieck

61 Pharmaceutical Particulate Carriers: Therapeutic Applications, edited by

Alain Rolland

62 Drug Permeation Enhancement: Theory and Applications, edited by Dean S Hsieh

63 Glycopeptide Antibiotics, edited by Ramakrishnan Nagarajan

64 Achieving Sterility in Medical and Pharmaceutical Products, Nigel A Halls

65 Multiparticulate Oral Drug Delivery, edited by Isaac Ghebre-Sellassie

66 Colloidal Drug Delivery Systems, edited by Jo¨rg Kreuter

67 Pharmacokinetics: Regulatory † Industrial † Academic Perspectives,

Second Edition, edited by Peter G Welling and Francis L S Tse

68 Drug Stability: Principles and Practices, Second Edition, Revised and Expanded,Jens T Carstensen

69 Good Laboratory Practice Regulations: Second Edition, Revised and Expanded,edited by Sandy Weinberg

70 Physical Characterization of Pharmaceutical Solids, edited by Harry G Brittain

71 Pharmaceutical Powder Compaction Technology, edited by Go¨ran Alderborn andChrister Nystro¨m

72 Modern Pharmaceutics: Third Edition, Revised and Expanded, edited by

Gilbert S Banker and Christopher T Rhodes

73 Microencapsulation: Methods and Industrial Applications, edited by Simon Benita

74 Oral Mucosal Drug Delivery, edited by Michael J Rathbone

75 Clinical Research in Pharmaceutical Development, edited by Barry Bleidt andMichael Montagne

76 The Drug Development Process: Increasing Efficiency and Cost Effectiveness,edited by Peter G Welling, Louis Lasagna, and Umesh V Banakar

77 Microparticulate Systems for the Delivery of Proteins and Vaccines, edited bySmadar Cohen and Howard Bernstein

78 Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control,Fourth Edition, Revised and Expanded, Sidney H Willig and James R Stoker

79 Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms: Second Edition,Revised and Expanded, edited by James W McGinity

80 Pharmaceutical Statistics: Practical and Clinical Applications, Third Edition,Sanford Bolton

81 Handbook of Pharmaceutical Granulation Technology, edited by Dilip M Parikh

82 Biotechnology of Antibiotics: Second Edition, Revised and Expanded, edited byWilliam R Strohl

Richard H Guy

84 Pharmaceutical Enzymes, edited by Albert Lauwers and Simon Scharpe´

85 Development of Biopharmaceutical Parenteral Dosage Forms, edited by

John A Bontempo

86 Pharmaceutical Project Management, edited by Tony Kennedy

87 Drug Products for Clinical Trials: An International Guide to Formulation † Production

† Quality Control, edited by Donald C Monkhouse and Christopher T Rhodes

88 Development and Formulation of Veterinary Dosage Forms: Second Edition,Revised and Expanded, edited by Gregory E Hardee and J Desmond Baggot

89 Receptor-Based Drug Design, edited by Paul Leff

90 Automation and Validation of Information in Pharmaceutical Processing, edited byJoseph F deSpautz

91 Dermal Absorption and Toxicity Assessment, edited by Michael S Roberts andKenneth A Walters

92 Pharmaceutical Experimental Design, Gareth A Lewis, Didier Mathieu, andRoger Phan-Tan-Luu

93 Preparing for FDA Pre-Approval Inspections, edited by Martin D Hynes III

94 Pharmaceutical Excipients: Characterization by IR, Raman, and NMR troscopy, David E Bugay and W Paul Findlay

Spec-95 Polymorphism in Pharmaceutical Solids, edited by Harry G Brittain

96 Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products, edited byLouis Rey and Joan C.May

97 Percutaneous Absorption: Drugs–Cosmetics–Mechanisms–Methodology,

Third Edition, Revised and Expanded, edited by Robert L Bronaugh and

Howard I Maibach

98 Bioadhesive Drug Delivery Systems: Fundamentals, Novel Approaches, andDevelopment, edited by Edith Mathiowitz, Donald E Chickering III, and Claus-Michael Lehr

99 Protein Formulation and Delivery, edited by Eugene J McNally

100 New Drug Approval Process: Third Edition, The Global Challenge, edited byRichard A Guarino

101 Peptide and Protein Drug Analysis, edited by Ronald E Reid

102 Transport Processes in Pharmaceutical Systems, edited by Gordon L Amidon,Ping I Lee, and Elizabeth M Topp

103 Excipient Toxicity and Safety, edited by Myra L Weiner and Lois A Kotkoskie

104 The Clinical Audit in Pharmaceutical Development, edited by Michael R Hamrell

105 Pharmaceutical Emulsions and Suspensions, edited by Francoise Nielloud andGilberte Marti-Mestres

106 Oral Drug Absorption: Prediction and Assessment, edited by Jennifer B Dressmanand Hans Lennerna¨s

107 Drug Stability: Principles and Practices, Third Edition, Revised and Expanded,edited by Jens T Carstensen and C T Rhodes

108 Containment in the Pharmaceutical Industry, edited by James P Wood

Control from Manufacturer to Consumer, Fifth Edition, Revised and Expanded,Sidney H Willig

110 Advanced Pharmaceutical Solids, Jens T Carstensen

111 Endotoxins: Pyrogens, LAL Testing, and Depyrogenation, Second Edition,Revised and Expanded, Kevin L Williams

112 Pharmaceutical Process Engineering, Anthony J Hickey and David Ganderton

113 Pharmacogenomics, edited by Werner Kalow, Urs A Meyer and Rachel F Tyndale

114 Handbook of Drug Screening, edited by Ramakrishna Seethala and

Prabhavathi B Fernandes

115 Drug Targeting Technology: Physical † Chemical † Biological Methods, edited byHans Schreier

116 Drug–Drug Interactions, edited by A David Rodrigues

117 Handbook of Pharmaceutical Analysis, edited by Lena Ohannesian and

Anthony J Streeter

118 Pharmaceutical Process Scale-Up, edited by Michael Levin

119 Dermatological and Transdermal Formulations, edited by Kenneth A Walters

120 Clinical Drug Trials and Tribulations: Second Edition, Revised and Expanded,edited by Allen Cato, Lynda Sutton, and Allen Cato III

121 Modern Pharmaceutics: Fourth Edition, Revised and Expanded, edited by

Gilbert S Banker and Christopher T Rhodes

122 Surfactants and Polymers in Drug Delivery, Martin Malmsten

123 Transdermal Drug Delivery: Second Edition, Revised and Expanded, edited byRichard H Guy and Jonathan Hadgraft

124 Good Laboratory Practice Regulations: Second Edition, Revised and Expanded,edited by Sandy Weinberg

125 Parenteral Quality Control: Sterility, Pyrogen, Particulate, and Package IntegrityTesting: Third Edition, Revised and Expanded, Michael J Akers, Daniel S.Larrimore, and Dana Morton Guazzo

126 Modified-Release Drug Delivery Technology, edited by Michael J Rathbone,Jonathan Hadgraft, and Michael S Roberts

127 Simulation for Designing Clinical Trials: A Pharmacokinetic-PharmacodynamicModeling Perspective, edited by Hui C Kimko and Stephen B Duffull

128 Affinity Capillary Electrophoresis in Pharmaceutics and Biopharmaceutics,edited by Reinhard H H Neubert and Hans-Hermann Ru¨ttinger

129 Pharmaceutical Process Validation: An International Third Edition, Revised andExpanded, edited by Robert A Nash and Alfred H Wachter

130 Ophthalmic Drug Delivery Systems: Second Edition, Revised and Expanded,edited by Ashim K Mitra

131 Pharmaceutical Gene Delivery Systems, edited by Alain Rolland and

Expanded, edited by Anthony J Hickey

135 Pharmaceutical Statistics: Practical and Clinical Applications, Fourth Edition,Sanford Bolton and Charles Bon

136 Compliance Handbook for Pharmaceuticals, Medical Devices, and Biologics,edited by Carmen Medina

137 Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products:

Second Edition, Revised and Expanded, edited by Louis Rey and Joan C May

138 Supercritical Fluid Technology for Drug Product Development, edited by Peter York,Uday B Kompella, and Boris Y Shekunov

139 New Drug Approval Process: Fourth Edition, Accelerating Global Registrations,edited by Richard A Guarino

140 Microbial Contamination Control in Parenteral Manufacturing, edited by

143 Generic Drug Product Development: Solid Oral Dosage Forms, edited by

Leon Shargel and Isadore Kanfer

144 Introduction to the Pharmaceutical Regulatory Process, edited by Ira R Berry

145 Drug Delivery to the Oral Cavity: Molecules to Market, edited by Tapash K Ghoshand William R Pfister

146 Good Design Practices for GMP Pharmaceutical Facilities, edited by

Andrew Signore and Terry Jacobs

147 Drug Products for Clinical Trials, Second Edition, edited by Donald Monkhouse,Charles Carney, and Jim Clark

148 Polymeric Drug Delivery Systems, edited by Glen S Kwon

149 Injectable Dispersed Systems: Formulation, Processing, and Performance,edited by Diane J Burgess

150 Laboratory Auditing for Quality and Regulatory Compliance, Donald Singer,Raluca- Ioana Stefan, and Jacobus van Staden

151 Active Pharmaceutical Ingredients: Development, Manufacturing, and Regulation,edited by Stanley Nusim

152 Preclinical Drug Development, edited by Mark C Rogge and David R Taft

153 Pharmaceutical Stress Testing: Predicting Drug Degradation, edited by

Steven W Baertschi

154 Handbook of Pharmaceutical Granulation Technology: Second Edition,

edited by Dilip M Parikh

155 Percutaneous Absorption: Drugs–Cosmetics–Mechanisms–Methodology,

Fourth Edition, edited by Robert L Bronaugh and Howard I Maibach

156 Pharmacogenomics: Second Edition, edited by Werner Kalow, Urs A Meyer andRachel F Tyndale

158 Microencapsulation: Methods and Industrial Applications, Second Edition,

edited by Simon Benita

159 Nanoparticle Technology for Drug Delivery, edited by Ram B Gupta and

Uday B Kompella

160 Spectroscopy of Pharmaceutical Solids, edited by Harry G Brittain

161 Dose Optimization in Drug Development, edited by Rajesh Krishna

162 Herbal Supplements-Drug Interactions: Scientific and Regulatory Perspectives,edited by Y W Francis Lam, Shiew-Mei Huang, and Stephen D Hall

163 Pharmaceutical Photostability and Stabilization Technology, edited by

Joseph T.Piechocki and Karl Thoma

164 Environmental Monitoring for Cleanrooms and Controlled Environments,

edited by Anne Marie Dixon

165 Pharmaceutical Product Development: In Vitro-In Vivo Correlation, edited byDakshina Murthy Chilukuri, Gangadhar Sunkara, and David Young

166 Nanoparticulate Drug Delivery Systems, edited by Deepak Thassu, Michel Deleers,and Yashwant Pathak

167 Endotoxins: Pyrogens, LAL Testing and Depyrogenation, Third Edition,

edited by Kevin L Williams

168 Good Laboratory Practice Regulations, Fourth Edition, edited by

Anne Sandy Weinberg

169 Good Manufacturing Practices for Pharmaceuticals, Sixth Edition, edited byJoseph D Nally

170 Oral-Lipid Based Formulations: Enhancing the Bioavailability of Poorly

Water-soluble Drugs, edited by David J Hauss

171 Handbook of Bioequivalence Testing, edited by Sarfaraz K Niazi

172 Advanced Drug Formulation Design to Optimize Therapeutic Outcomes,

edited by Robert O Williams III, David R Taft, and Jason T McConville

173 Clean-in-Place for Biopharmaceutical Processes, edited by Dale A Seiberling

174 Filtration and Purification in the Biopharmaceutical Industry, Second Edition,edited by Maik W Jornitz and Theodore H Meltzer

175 Protein Formulation and Delivery, Second Edition, edited by Eugene J McNallyand Jayne E Hastedt

176 Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, Third Edition,edited by James McGinity and Linda A Felton

177 Dermal Absorption and Toxicity Assessment, Second Edition, edited by

Michael S Roberts and Kenneth A Walters

178 Preformulation Solid Dosage Form Development, edited by Moji C Adeyeye andHarry G Brittain

179 Drug-Drug Interactions, Second Edition, edited by A David Rodrigues

180 Generic Drug Product Development: Bioequivalence Issues, edited by

Isadore Kanfer and Leon Shargel

An-eX Analytical Services Ltd

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1 Dermatotoxicology 2 Dermatologic agents–Toxicology 3 cology 4 Skin absorption 5 Health risk assessment I Roberts, Michael S., 1949– II Walters, Kenneth A., 1949– III Series.

Cosmetics–Toxi-[DNLM: 1 Skin Absorption–physiology 2 Cosmetics–adverse effects.

3 Cosmetics–pharmacokinetics 4 Environmental Exposure 5 Pharmaceutical Preparations–adverse effects 6 Risk Assessment W1 DR893B v.117 2007/WR 102 D434 2007]

Since this book was first published in 1998, there have been significant advances inour understanding of the morphology of the skin and the properties that govern thetransport of molecules into and across the three major strata The multitude of datathat has been generated has allowed the development of predictive models for boththe rate and extent of dermal absorption and has increased our ability to predict thelikelihood of local toxic events subsequent to solute penetration and permeation Inthis second edition we have completely revised and updated many of the chaptersthat appeared in the earlier version and we have expanded the scope of the volume

to include coverage of the more recent exciting and innovative areas of research.Those chapters concerned with dermatological and cosmeceutical therapy havebeen moved to a companion publication, Dermatologic, Cosmeceutic, and CosmeticDevelopment: Therapeutic and Novel Approaches

This second edition has been divided into six parts covering skin structureand absorption, measurement of absorption, modeling of dermal absorption andrisk assessment, skin toxicity and its prevention, regulatory issues, and specificexamples of the absorption of environmental materials As in the first edition, thisbook provides an overview of the dermal absorption process, with particularemphasis on the determinants for toxicity arising from dermal exposure

A general introduction, covering the structure of human and animal skin and itsrelationship to dermal absorption, is followed by Part I, which expands on thespecific barrier properties of the skin, such as its physical structure, biosensorproperties, cutaneous metabolism, skin lipid morphology, and dermal blood andlymphatic flow The range of methods used to assess skin absorption is fullydiscussed in Part II There, the use of standard established laboratory methods,such as diffusion cell technology, are covered together with some of the newertechniques, such as the use of cultured skin equivalents Many of the techniquesused for measurement and modeling of dermal absorption and risk assessment arediscussed in Part III This section also includes the use of mathematical models,many of which have been refined to provide more realistic predictions, togetherwith structure-penetration relationships as principles for estimating dermal riskassessment In addition, this part covers the estimation of systemic exposuresubsequent to dermal absorption, pharmacodynamics and the pharmacokinetics ofskin delivery, and the use of various real life exposure scenarios in dermal riskassessment

The next two parts of the book focus on local toxicity and its prevention andregulatory initiatives Within the local toxicity section, issues such as skin damage,irritation, sensitisation, phototoxicity, and the prevention of toxicity are covered.The regulatory section provides information on the various governmental andindustrial programs concerning the issues surrounding skin permeation andtoxicity, including alternative in silico, in vitro, and in vivo strategies to conductstudies for regulatory approval

The final section provides some examples of substances absorbed through theskin, giving particular emphasis to environmental contaminants and cosmetic

iii

ingredients This section includes the U.S Environmental Protection Agency’sdefined common environmental substances, together with discussions on thepercutaneous absorption of compounds from soil and bathing water, and thepermeation of pesticides, metals, fragrances, and other cosmetic ingredients.This book is intended for scientists involved in dermal absorption and forthose concerned with the marketing of products that may be absorbed through theskin intentionally or unintentionally To this end, we have been fortunate inobtaining the agreement of many internationally recognized experts in the field

of dermal absorption and toxicity assessment to provide coverage of their specificfields of expertise To all of our authors we extend our sincere thanks for theirunreserved efforts and time

Michael S RobertsKenneth A Walters

The development and use of chemicals, especially those in the pharmaceutical,general chemical, and cosmetic fields, are associated with hazards arising fromhuman exposure For these compounds, absorption into the skin may represent amajor route of entry into the body In addition, a number of such substances areapplied to the skin for therapeutic or cosmetic purposes The rate and extent ofpenetration into and absorption through the skin are defined by a number ofvariables, including environmental conditions, skin physiology, permeantstructure, method of application, and species differences.

This volume provides and overview of the dermal absorption process, withparticular emphasis on the determinants for toxicity arising from dermal exposure.Part I is concerned with the structure of the skin and the underlying principlesdefining percutaneous absorption and toxicity In Part II, the concept of dermal riskassessment, predicted from epidemiological factors, physiological models, invitro/in vivo experimentation, and chemical structures, is examined by experts inthese areas This section also describes the use of mathematical models togetherwith structure-penetration relationships as principles for estimating dermal riskassessment

Parts III and IV are concerned with dermal absorption and risk assessment asapplied to specific product types: pharmaceuticals and cosmetics The individualchapters discuss specific product classes such as drugs used for pain andinflammation, fragrances, sunscreens, and hair dyes The final section, Part V,provides information on skin permeation following environmental exposure andincludes discussions on the percutaneous absorption of compounds from soil andbathing water Throughout this volume, pharmacokinetic data and models oftenused in dermal risk assessment are fully described

The book has been written for scientists interested in dermal absorption andthose concerned with the marketing of products that may be absorbed through theskin either intentionally or unintentionally We hope that this book will prove useful

to those involved in research and development in the pharmaceutical, cosmetic,agrochemical, household, and general chemical industries

Michael S RobertsKenneth A Walters

v

Preface to the Second Edition iii

Preface from the First Edition v

Contributors xi

INTRODUCTION

1 Human Skin Morphology and Dermal Absorption 1

Michael S Roberts and Kenneth A Walters

2 Animal Skin Morphology and Dermal Absorption 17

Nancy A Monteiro-Riviere, Ronald E Baynes, and Jim E Riviere

PART I: SKIN BARRIER PROPERTIES AND ABSORPTION

3 The Physical Structure of the Skin Barrier 37

Lars Norle´n

4 Morphology of Epidermal Lipids 69

Jennifer R Hill and Philip W Wertz

5 Stratum Corneum as a Biosensor 79

Peter M Elias, Kenneth R Feingold, and Mitsuhiro Denda

6 Cutaneous Metabolism 89

Simon C Wilkinson and Faith M Williams

7 Formulation Issues 117

U F Schaefer, B C Lippold, and C S Leopold

PART II: MEASUREMENT OF SKIN ABSORPTION

8 Interpretation of In Vitro Skin Absorption Studies

10 Skin Absorption as Studied by Spectroscopic Methods 161

Ulrike Gu¨nther, Siegfried Wartewig, Hendrik Metz, and

Reinhard H H Neubert

PART III: MODELING OF DERMAL ABSORPTION

AND RISK ASSESSMENT

11 Physiologically Based Pharmacokinetics and Pharmacodynamics

Yuri Dancik, Owen G Jepps, and Michael S Roberts

12 Beyond Stratum Corneum 209

Yuri Dancik, Owen G Jepps, and Michael S Roberts

13 Biophysical Models for Skin Transport and Absorption 251

Johannes M Nitsche and Gerald B Kasting

14 Mathematical Models for Different Exposure Conditions 271

Yuri G Anissimov

15 Modeling Skin Permeability in Risk Assessment 287

Dara Fitzpatrick, Darach Golden, and John Corish

16 In Vitro–In Vivo Correlations in Transdermal Drug Delivery 299

Jonathan Hadgraft and Majella E Lane

17 Estimation of Subsequent Systemic Exposure—Physiological Models 309James N McDougal

18 RISKOFDERM: Predictions Based on In Vivo Factors 323

Wim J A Meuling, Johannes J M van de Sandt, and Joop J van Hemmen

PART IV: SKIN TOXICITY AND PREVENTION

19 Quantitative Structure–Activity Relationships for Skin Corrosivity

and Sensitization 339

Mark T D Cronin, Steven J Enoch, and Judith C Madden

20 Allergic Contact Dermatitis 359

Haw-Yueh Thong and Howard I Maibach

21 Irritancy of Topical Chemicals in Transdermal Drug

Delivery Systems 371

Heidi P Chan, Cheryl Y Levin, and Howard I Maibach

22 Photosensitivity Induced by Exogenous Agents:

Phototoxicity and Photoallergy 391

Haw-Yueh Thong and Howard I Maibach

23 Systemic Toxicity Caused by Absorption of Drugs and Chemicals

Through Skin 405

Haw-Yueh Thong, Susi Freeman, and Howard I Maibach

24 Solvent and Vehicle Effects on the Skin 433

Michael S Roberts, Audrey Gierden, Jim E Riviere, and Nancy A Monteiro-Riviere

PART V: REGULATORY ISSUES

25 United States Environmental Protection Agency Perspectives on

Skin Absorption and Exposure 449

Michael Dellarco

26 Dermal Absorption of Chemicals: Some Australian

Regulatory Considerations 459

Utz Mueller, Andrew Bartholomaeus, and Mark Jenner

27 International Perspectives in Dermal Absorption 471

Janet Kielhorn and Stephanie Melching-Kollmuß

28 Structure–Activity Relationships and Prediction of Photoallergic

and Phototoxic Potential 483

Martin D Barratt

29 Potential Regulatory Use of (Q)SARs to Develop Dermal Irritation

and Corrosion Assessment Strategies 495

Ingrid Gerner, Etje Hulzebos, Emiel Rorije, Betty Hakkert, John D Walker,

Matthias Herzler, and Horst Spielmann

30 Development of (Q)SARs for Dermal Irritation and Corrosion

Assessment Using European Union New Chemicals Notification Data 507Ingrid Gerner, Etje Hulzebos, Emiel Rorije, Matthias Herzler, Manfred Liebsch,

John D Walker, and Horst Spielmann

31 Regulatory Assessment of Skin Sensitization 523

Winfried Steiling and Hans-Werner Vohr

32 Assessment of Topical Bioequivalence Using Microdialysis

and Other Techniques 537

Eva Benfeldt, Edward D Bashaw, and Vinod P Shah

33 An Industry Perspective of Topical Dermal Bioequivalence 549

Dawn McCleverty, Richard Lyons, and Brian Henry

PART VI: SPECIFIC EXAMPLES OF ABSORPTION

34 Dermal Absorption of Chemical Contaminants from Soil 563

John C Kissel, Elizabeth W Spalt, Jeffry H Shirai, and Annette L Bunge

35 Percutaneous Absorption of Pesticides 575

Jon R Heylings and David J Esdaile

36 Bathing Water: Percutaneous Absorption of Water Contaminants 593Richard P Moody

37 Percutaneous Absorption of Prodrugs and Soft Drugs 605

Kenneth B Sloan and Scott C Wasdo

38 Skin Penetration of Cosmetic Ingredients and Contaminants 623

Keith R Brain and Kenneth A Walters

39 Percutaneous Absorption of Hair Dyes 635

William E Dressler

40 Dermal Absorption of Fragrance Materials 651

Keith R Brain and Jon Lalko

Index 665

Yuri G Anissimov School of Biomolecular and Physical Sciences, GriffithUniversity, Nathan, Queensland, Australia

Martin D Barratt Marlin Consultancy, Carlton, Bedford, U.K

Andrew Bartholomaeus Drug Safety and Evaluation Branch, TherapeuticGoods Administration, Woden, Australia

Edward D Bashaw Division III, Office of Clinical Pharmacology, U.S Food andDrug Administration, Rockville, Maryland, U.S.A

Ronald E Baynes Center for Chemical Toxicology Research and

Pharmacokinetics, College of Veterinary Medicine, North Carolina State

University, Raleigh, North Carolina, U.S.A

Eva Benfeldt Department of Dermatology, Gentofte Hospital, University

of Copenhagen, Hellerup, Denmark

Keith R Brain An-eX Analytical Services, Ltd., and Cardiff University,

Cardiff, U.K

Robert L Bronaugh Office of Cosmetics and Colors, Food and Drug

Administration, College Park, Maryland, U.S.A

Annette L Bunge Department of Chemical Engineering, Colorado School

of Mines, Golden, Colorado, U.S.A

Heidi P Chan Department of Dermatology, School of Medicine,

University of California San Francisco, San Francisco, California, U.S.A

John Corish School of Chemistry, Trinity College, University of Dublin,

Dublin, Ireland

Mark T D Cronin School of Pharmacy and Chemistry, Liverpool John

Moores University, Liverpool, U.K

Yuri Dancik Department of Medicine, Princess Alexandra Hospital,

University of Queensland, Woolloongabba, Queensland, Australia

Michael Dellarco U.S Environmental Protection Agency, National Center forEnvironmental Assessment, Washington, D.C., U.S.A

Mitsuhiro Denda Shiseido Research Center, Yokohama, Japan

William E Dressler Independent Consultant, Huntington, Connecticut, U.S.A

xi

Peter M Elias Dermatology and Medical (Metabolism) Services,

Veterans Affairs Medical Center, and Departments of Dermatology and Medicine,University of California, San Francisco, California, U.S.A

Steven J Enoch School of Pharmacy and Chemistry, Liverpool John MooresUniversity, Liverpool, U.K

David J Esdaile LAB International Research Centre, Szabadsa´gpuszta,

Veszpre´m, Hungary

Kenneth R Feingold Dermatology and Medical (Metabolism) Services,

Veterans Affairs Medical Center, and Departments of Dermatology and Medicine,University of California, San Francisco, California, U.S.A

Dara Fitzpatrick Department of Chemistry, University College Cork,

Cork, Ireland

Susi Freeman Department of Dermatology, School of Medicine,

University of California San Francisco, San Francisco, California, U.S.A

Ulrike Gu¨nther Institute of Pharmaceutics and Biopharmaceutics,

Martin Luther University of Halle-Wittenberg, Halle, Germany

Ingrid Gerner Weidenauer Weg, Berlin, Germany

Audrey Gierden Department of Medicine, Princess Alexandra Hospital,

University of Queensland, Woolloongabba, Queensland, Australia

Darach Golden Centre for High Performance Computing, Trinity College,University of Dublin, Dublin, Ireland

Jonathan Hadgraft The School of Pharmacy, University of London,

Safety of Substances and Preparations, Thielallee, Berlin, Germany

Jon R Heylings Research and Investigative Toxicology, Syngenta CentralToxicology Laboratory, Macclesfield, Cheshire, U.K

Jennifer R Hill Dows Institute, University of Iowa, Iowa City, Iowa, U.S.A.Etje Hulzebos National Institute for Public Health and the Environment,Expertise Centre for Substances, Bilthoven, The Netherlands

Mark Jenner Scitox Assessment Services, Kambah, Australia

Owen G Jepps Department of Medicine, Princess Alexandra Hospital,

University of Queensland, Woolloongabba, Queensland, Australia

Gerald B Kasting, James L Winkle College of Pharmacy, University of

Cincinnati Academic Health Center, Cincinnati, Ohio, U.S.A

Janet Kielhorn Fraunhofer Institute of Toxicology and Experimental Medicine,Hannover, Germany

John C Kissel Department of Environmental and Occupational Health Sciences,University of Washington, Seattle, Washington, U.S.A

Jon Lalko Research Institute for Fragrance Materials, Woodcliff Lake,

New Jersey, U.S.A

Majella E Lane The School of Pharmacy, University of London, London, U.K

C S Leopold Department of Pharmaceutical Technology, Institute of Pharmacy,University of Hamburg, Hamburg, Germany

Cheryl Y Levin Department of Dermatology, School of Medicine,

University of California San Francisco, San Francisco, California, U.S.A

Manfred Liebsch Federal Institute for Risk Assessment, Centre for

Alternative Methods to Animal Experiments—ZEBET, Diedersdorfer Weg,Berlin, Germany

B C Lippold Institute of Pharmaceutics and Biopharmaceutics, HeinrichHeine University, Duesseldorf, Germany

Richard Lyons Pfizer Global Research and Development, Sandwich, Kent, U.K.Judith C Madden School of Pharmacy and Chemistry, Liverpool John

Moores University, Liverpool, U.K

Howard I Maibach Department of Dermatology, School of Medicine,

University of California San Francisco, San Francisco, California, U.S.A

Dawn McCleverty Pfizer Global Research and Development, Sandwich,

Kent, U.K

James N McDougal Department of Pharmacology and Toxicology,

Boonschoft School of Medicine, Wright State University, Dayton, Ohio, U.S.A.Stephanie Melching-Kollmuß Fraunhofer Institute of Toxicology and

Experimental Medicine, Hannover, Germany

Hendrik Metz Institute of Pharmaceutics and Biopharmaceutics, Martin LutherUniversity of Halle-Wittenberg, Halle, Germany

Wim J A Meuling Business Unit Biosciences, TNO Quality of Life, Zeist,The Netherlands

Nancy A Monteiro-Riviere Center for Chemical Toxicology Research andPharmacokinetics, College of Veterinary Medicine, North Carolina State University,Raleigh, North Carolina, U.S.A.

Richard P Moody Healthy Environments and Consumer Safety Branch,

Environmental Health Centre, Ottawa, Canada

Utz Mueller Food Standards Australia New Zealand, Canberra BC, AustraliaReinhard H H Neubert Institute of Pharmaceutics and Biopharmaceutics,Martin Luther University of Halle-Wittenberg, Halle, Germany

Johannes M Nitsche Department of Chemical and Biological Engineering,University at Buffalo, State University of New York, Buffalo, New York, U.S.A.Lars Norle´n Medical Nobel Institute, Department of Cellular and MolecularBiology, Karolinska Institute, Stockholm, Sweden

Jim E Riviere Center for Chemical Toxicology Research and Pharmacokinetics,College of Veterinary Medicine, North Carolina State University, Raleigh,

North Carolina, U.S.A

Michael S Roberts Department of Medicine, Princess Alexandra Hospital,University of Queensland, Woolloongabba, Queensland, Australia

Emiel Rorije National Institute for Public Health and the Environment,

Expertise Centre for Substances, Bilthoven, The Netherlands

Monika Scha¨fer-Korting Institut fu¨r Pharmazie (Pharmakologie und

Toxikologie) der Freien Universita¨t Berlin, Berlin, Germany

U F Schaefer Biopharmaceutics and Pharmaceutical Technology,

Saarland University, Saarbruecken, Germany

Sylvia Schreiber Institut fu¨r Pharmazie (Pharmakologie und Toxikologie)der Freien Universita¨t Berlin, Berlin, Germany

Vinod P Shah Pharmaceutical Consultant, North Potomac, Maryland, U.S.A.Jeffry H Shirai Department of Environmental and Occupational Health Sciences,University of Washington, Seattle, Washington, U.S.A

Kenneth B Sloan Department of Medicinal Chemistry, University of Florida,Gainesville, Florida, U.S.A

Elizabeth W Spalt Integral Consulting, Inc., Mercer Island, Washington, U.S.A.Horst Spielmann Federal Institute for Risk Assessment, Centre for

Alternative Methods to Animal Experiments—ZEBET, Diedersdorfer Weg,Berlin, Germany

Winfried Steiling Henkel KGaA, Corporate SHE and Product Safety—HumanSafety Assessment, Du¨sseldorf, Germany

Haw-Yueh Thong Department of Dermatology, School of Medicine,

University of California San Francisco, San Francisco, California, U.S.A

Johannes J M van de Sandt Business Unit Food and Chemical Risk Analysis,TNO Quality of Life, Zeist, The Netherlands

Joop J van Hemmen Business Unit Food and Chemical Risk Analysis, TNOQuality of Life, Zeist, The Netherlands

Hans-Werner Vohr Department of Toxicology, Bayer HealthCare AG,

Wuppertal, Germany

John D Walker TSCA Interagency Testing Committee, Office of PollutionPrevention and Toxics, U.S Environmental Protection Agency, Washington,D.C., U.S.A

Kenneth A Walters An-eX Analytical Services, Ltd., Cardiff, U.K

Siegfried Wartewig Institute for Applied Dermatopharmacy, Halle, GermanyScott C Wasdo Department of Medicinal Chemistry, University of Florida,Gainesville, Florida, U.S.A

Philip W Wertz Dows Institute, University of Iowa, Iowa City, Iowa, U.S.A.Simon C Wilkinson Medical Toxicology Research Centre, Newcastle University,Newcastle upon Tyne, U.K

Faith M Williams Medical Toxicology Research Centre and Institute for

Research on Environment and Sustainability, Newcastle University,

Newcastle upon Tyne, U.K

1 Human Skin Morphology and

of the intercellular lipids and the process of desquamation (Fig 2)

INITIATION OF KERATINOCYTE MIGRATION AND DIFFERENTIATION

The cells of the epidermis originate in the basal lamina between the dermis andviable epidermis In this layer there are melanocytes, Langerhans cells, Merkel cells,and keratinocytes The keratinocytes of the basal lamina are attached to thebasement membrane by hemidesmosomes and focal adhesions that are comprised

of several distinct proteins The hemidesmosome complex contains two bullouspemphigoid antigens (BPAg1 and BPAg2) and several integrins (including integrins

1

Targeted delivery Release

Follicles, sweat ducts

Metabolites Urine,etc.

Clearance Distribution

Tissues

Action

Systematic blood concentration Dermal blood flow

Epidermis and upper dermis concentration

Epidermal and demal metabolism

Stratum corneum penetration

Toxicity

Therapeutic activity

Product/device solute vehicle

FIGURE 1 Dermal absorption; sites of action and toxicity Abbreviation: SC, stratum corneum.

FIGURE 2 Major events in epidermal differentiation.

a6b4 and a3b1), together with multiple laminins (13–16), all of which are involved

in securing the cell to the basement membrane BPAg2 is a collagen that has anamino terminus within the hemidesmosome and a carboxy terminus within thelamina lucida of the basement membrane and may form part of the anchoringfilament The fundamental importance of these basal membrane-anchoring proteins

is reflected in the devastating skin disorders associated with inherited and acquiredencoding gene mutations such as dystrophic epidermolysis bullosa (type VIIcollagen) and bullous pemphigoid (integrin a6b4) (14)

In addition to hemidesmosome binding, another site for adhesion of the cells ofthe epidermal basal layer and the basal membrane is the adherens junction (17).The adherens junction expresses a different protein profile to desmosomes andhemidesmosomes, containing talin, vinculin, and cadherins; whereas, the hemi-desmosomes are linked to cytoplasmic keratin, the proteins of the adherens junctionsare linked to cytoplasmic actin microfilaments and appear to play an important role

in cell migration (18) and may also have a functional role in nuclear signaling (19).Although a secure link between the basal keratinocytes and the basal matrix isessential for skin integrity, these cells must also be capable of detaching andmigrating to begin the process of terminal differentiation that will form thestratum corneum Among the proteins implicated in detachment is CD151, atransmembrane protein of the tetraspanin family (20) CD151 is localized withintegrin a6b4 at cell–matrix interfaces and it is suggested that dissociation of thiscomplex allows remodeling of the cell–matrix attachment and subsequent cellmigration Another important link in the chain is integrin-linked kinase, whichconnects integrins to actin fibers and is thought to be highly involved in cell–matrixand cell–cell adhesion and migration (21) Similarly a role has been suggested forthe calcium/manganese-ATPase, ATP2C1, in cell migration where it may act as aregulator of the integrin-linked basal attachment (22)

The relatively short-lived adherens junctions and the more stable somes regulate cell–matrix adhesion Both types of adhesion must be disrupted toallow the keratinocyte to detach from the basal membrane Rear detachment isaccompanied by membrane ripping and the loss of cellular material in keratino-cytes Rigort and colleagues (23) showed that migrating keratinocytes leave behind

hemidesmo-“migration tracks” of cellular remnants that were anchored to a meshwork ofextracellular matrix proteins consisting of collagen type IV, fibronectin, laminin,and laminin 5 These tracts were classified on the basis of their size, distribution,and molecular composition Type I macroaggregates appeared as spherical andtubular structures with a diameter of about 50 to 100 nm These structures appeared

to be derived from fragmentation of long tubular extensions, the retracting fibers, atthe cell rear and contained high amounts of a3b1 integrin, a component offibronectin and laminin receptors in migrating keratinocytes usually found infocal adhesions Type II macroaggregates were spherical structures with a diameter

of about 30 to 50 nm that were arranged in clusters and scattered over the gapsbetween type I, macroaggregates Type II macroaggregates contained high amounts

of a6b4 integrin and probably derived from former hemidesmosomes Theirobservations support the concept that the release of macroaggregates represents adistinct cellular mechanism of rear detachment based on the loss of adhesivereceptors embedded in membrane-covered cellular remnants

The role of the non-neuronal cholinergic system of human epidermis has beenreviewed recently (24) The system is known as the keratinocyte acetylcholine axisand is composed of the enzymes mediating acetylcholine synthesis and degradation,

and two classes of acetylcholine receptors, the nicotinic and muscarinic receptors.Regulation of keratinocyte cell–cell and cell–matrix adhesion is one of the importantbiological functions of cutaneous acetylcholine The targets include both theintercellular adhesion molecules, such as desmosomal cadherins, and the cell–matrix adhesion molecules (integrins) The signaling pathways include activation

or inhibition of kinase cascades resulting in either up- or down-regulation of theexpression of cell adhesion molecules or changes in their phosphorylation status, orboth For example, it has been proposed that the muscarinic M3 receptor activation islinked to up-regulation of a2b1-integrin and a3b1-integrin-mediated cell adhesion,whereas activation of the muscarinic M4 receptor stimulates keratinocyte motility byup-regulating the migratory integrins a5b1, avb5, and avb6 (25)

Nguyen and colleagues investigated the role of the non-neuronal acetylcholineaxis in the control of cell–cell adhesion of human epidermal keratinocytes (26).Cholinergic effects on the expression of desmoglein 1 and 3 were measured usingsemiquantitative immunofluorescence and Western blot assays Keratinocyte mono-layers were treated with the cholinergic agonist carbachol or the acetylcholinesteraseinhibitor pyridostigmine bromide Both compounds increased the relative amounts

of desmoglein 1 and 3 The role for cholinergic receptor-mediated phosphorylation ofdesmoglein molecules in the assembly and disassembly of keratinocyte desmosomeswas investigated by evaluating the effects of a cholinergic antagonist, atropine, onkeratinocyte adhesion and desmoglein phosphorylation status Atropine induced arapid detachment of cells from each other and increased phosphorylation ofdesmoglein 3 The atropine-dependent phosphorylation of desmoglein 3 wasinhibited by carbachol It was concluded that keratinocyte cholinergic receptorsregulate desmosomal adhesion of keratinocytes by altering the level of expression ofboth desmoglein 1 and 3 and the phosphorylation state of desmoglein 3

The same group also investigated the roles of the muscarinic M3, the nicotinica3 and the mixed muscarinic–nicotinic a9 acetylcholine receptors in the physiologiccontrol of keratinocyte adhesion (27) Both muscarinic and nicotinic antagonistscaused keratinocyte detachment and reversibly increased the permeability ofkeratinocyte monolayers Phosphorylation of adhesion proteins is known to play

an important role in the assembly and disassembly of intercellular junctions Theauthors found that the phosphorylation levels of E-cadherin, b-catenin, andg-catenin increased following pharmacological blockage of muscarinic receptors.Long-term blocking of all three receptor-signaling pathways with antisenseoligonucleotides resulted in cell–cell detachment and changes in the expressionlevels of E-cadherin, b-catenin, and g-catenin in cultured human keratinocytes.Overall, the data indicated that the three acetylcholine receptors played keysynergistic roles in regulating keratinocyte adhesion, most likely by modulatingthe levels and activities of cadherin and catenin

The keratinocyte cholinergic axis may prove to be a very interestingbiochemical pathway to probe in the search for new therapeutic entities for thetreatment of skin adhesion malfunction, such as Hailey–Hailey and Darier’sdiseases

THE INTERCELLULAR LIPIDS OF THE STRATUM CORNEUM

The development of the stratum corneum from the keratinocytes of the basal layerinvolves several steps of cell differentiation that have resulted in a structure basedclassification of the layers above the basal layer (the stratum basale) Thus the cells

progress through the stratum spinosum, the stratum granulosum, the stratumlucidium to the stratum corneum Cell turnover, from stratum basale to stratumcorneum, is about 21 days The stratum spinosum (prickle cell layer), which liesimmediately above the basal layer, consists of several layers of cells that areconnected by desmosomes and contain prominent keratin tonofilaments In theouter cell layers of the stratum spinosum, membrane-coating granules appear andthis region forms the border with the overlying stratum granulosum The mostcharacteristic feature of the stratum granulosum are the many intracellularmembrane-coating granules, the assembly of which appears to take place in theendoplasmic reticulum and Golgi regions (28) Lamellar subunits are observedwithin these granules and these are the precursors of the intercellular lipid lamellae

of the stratum corneum In the outermost layers of the stratum granulosum, thelamellar granules migrate to the apical cell surface where they fuse and eventuallyextrude their contents into the intercellular space At this stage, in the differentiationprocess, the keratinocytes lose their nuclei and other cytoplasmic organelles, becomeflattened and compacted to form the stratum lucidum, which eventually forms thestratum corneum The extrusion of the contents of lamellar granules is a fundamentalrequirement for the formation of the epidermal permeability barrier (29) anddisturbances in this process have been implicated in various dermatologicaldisorders (30–32)

There has been some debate on the physical structure and state of theintercellular lipid regions A structural model of the skin barrier was proposed

by Norlen (33), who postulated that the stratum corneum intercellular lipid existed

as a single and coherent lamellar gel phase The proposed intercellular structurewas stabilized by the unique mixture of lipids and their chain length distributionsand had virtually no phase boundaries The intact gel phase was suggested to belocated mainly in the lower half of stratum corneum In the outermost regions of thestratum corneum, crystalline segregation and phase separation may be the result ofthe desquamation process This single gel phase model differed significantly fromearlier models in that it predicted that no phase separation was present in theunperturbed barrier structure Norlen and colleagues went on to show, usingatomic force microscopy on Langmuir–Blodgett films composed of extractedhuman stratum corneum ceramides, cholesterol, free fatty acids, cholesterolsulfate, and cholesteryl oleate, that the saturated long-chain free fatty aciddistribution of human stratum corneum prevented hydrocarbon chain segregation(34) More recently, this group used differential scanning calorimetry, fluorescencespectroscopy, and two-photon excitation and laser scanning confocal fluorescencemicroscopy to show that, at normal skin temperatures, the phase state of hydratedbilayers made from human stratum corneum lipids corresponded microscopically

to a single gel-phase at pH 7 There was coexistence of different gel-phases between

pH 5 and 6, and no fluid phase at any pH [(35), see also Chapter 3 of this volume]

It is not surprising that the biologically unique stratum corneum lipidcomposition, mainly long chain ceramides, free fatty acids and cholesterol [(36),see also Chapter 4 of this volume], results in lipid phase behavior that is differentfrom that of other biological membranes The extensive work of Bouwstra and hercolleagues suggests that crystalline phases are predominantly present in thestratum corneum intercellular lipid, but also that there is probably a subpopulation

of lipids that form a liquid phase, probably promoted by the presence of free fattyacids (37) The authors pointed out that mixtures prepared only with ceramides andcholesterol formed a lamellar phase with a 13 nm periodicity When free fatty acids

were present the lattice density of the structure increased The presence of ceramide

1 is essential to the formation of the 13 nm lamellar phase Bouwstra’s groupproposed a molecular model for the structural organization of the 13 nm lamellarphase (the sandwich model), in which crystalline and liquid domains coexisted (38).The discussion on the precise nature of the stratum corneum intercellularlipid state seems set to continue (34,39,40), but, whatever the actual state is, thepossibility of using the collective knowledge of the structure of the skin barrier toformulate vesicles for improved drug delivery across the skin has been the subject

of intense investigation For example, the Leiden group focused on differencesbetween the effects of gel-state vesicles, liquid-state vesicles, and elastic vesicles(41–43) The in vivo and in vitro interactions between elastic-, rigid vesicles andmicelles with human skin were investigated (42) Following application of thesolutions containing the vesicles and micelles, the stratum corneum was tape-stripped and subsequently visualized by freeze fracture electron microscopy Therewere no ultrastructural changes in skin treated with rigid vesicles Elastic vesiclesappeared to rapidly partition intact into the deeper layers of the stratum corneum,where they accumulated in channel-like regions Since only small amounts ofvesicle material was found in the deepest layers of the stratum corneum, it wasconcluded that partitioning of intact vesicles into the viable epidermis was unlikely.There was excellent in vitro/in vivo correlation It was concluded that elasticvesicles were superior to rigid vesicles for interaction with skin and drug delivery.Distribution profiles in the stratum corneum were obtained for the elastic andrigid vesicle material and for the model drug ketorolac (43) As suggested

by earlier work (42), the elastic vesicle rapidly entered the deeper layers of thestratum corneum The rigid vesicle material did not penetrate deeply intothe stratum corneum As expected, the elastic vesicles delivered more ketorolacinto the stratum corneum than the rigid vesicles Distribution of ketorolac in thedeeper layers of the stratum corneum was different than that of the vesicle material,suggesting that the ketorolac was released from the vesicles in the skin

Similarly, deformable or elastic liposomal formulations have proved to besuperior to rigid and traditional liposomes in the delivery of many compounds intoand across the skin, including estradiol (44), diclofenac (45), and propranolol (46).The use of vesicles such as liposomes to modulate drug delivery into and throughthe skin has become reasonably accepted but the mechanism(s) of action remainsunclear As recently pointed out by El Maghraby and colleagues (47), vesicles varyconsiderably with respect to size, lamellarity, charge, membrane fluidity orelasticity, which allows for multiple functions ranging from local to transdermaleffects and this may result in multiple modes of action

DESQUAMATION

During the process of terminal differentiation, keratinocytes migrate from the basallayer toward the stratum corneum where they ultimately detach in the process ofdesquamation Since the keratinocytes are linked by desmosomes, it is perhaps notsurprising that it is the degradation of these links that signals the initiation ofdesquamation The major adhesive molecules in the desmosome are cadherins.These are CaCC-dependent molecules and they cooperate to make up the adhesivecore of the desmosome The adhesion molecules may have differentiation-specificfunctions over and above their roles in cell adhesion (for review see Ref 48)

The most important cadherins located in the desmosome are desmoglein 1,desmocollin 1, and corneodesmosin, and it is thought that, while desmoglein 1promotes the formation of the adhesive link, it is the relative level of desmogleinand desmocollin expressed at the cell surface that regulates the adhesive process(49) For desquamation to occur, it is necessary for the desmosomal link to degrade.Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secretedserine proteases (KLK1–KLK15) that have been identified in normal stratumcorneum, and are candidate desquamation-related proteases Two serine proteases

of the kallikrein family have been implicated in this process: the stratum corneumchymotryptic enzyme (SCCE/KLK7/Hk7) and the stratum corneum trypticenzyme (SCTE/KLK5/Hk5) The capacity of these enzymes to cleave desmoglein

1, desmocollin 1, and corneodesmosin has been investigated (50) At acidic pHssimilar to that of the stratum corneum, SCCE cleaved corneodesmosin anddesmocollin 1 but was unable to degrade desmoglein 1 On the other hand,incubation with SCTE induced degradation of all three proteins The data suggestedthat SCTE was able to activate the proform of SCCE and that both kallikreins wereinvolved in desquamation More recently, it was found that the epidermal pHgradient regulates the activity of KLK5 with acidic conditions being required for itsactivation in the superficial layers of the stratum corneum (51)

There are several more serine proteases (apart from KLK5 and KLK7) that arelocated in the epidermis Borgono and colleagues (52) investigated the contribution

of KLK1, KLK5, KLK6, KLK13, and KLK14 to the desquamation process byexamining their interaction with a colocalized serine protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI) and their ability to digest desmo-glein 1 Apart from KLK13, all kallikreins digested the ectodomain of desmoglein 1within cadherin repeats, CaCC-binding sites, or in the juxtamembrane regionsuggesting that multiple kallikreins participate in desquamation

As is quite common in biological systems, evolution has resulted in anextremely complex mechanism for shedding a layer of skin each day It is asimple matter to accept that this happens for a reason but not so simple to figureout just what that reason (or reasons) is Why does the stratum corneum keeprenewing itself? Milstone (12) puts forward the concept that continuous desquama-tion might reflect “a first line of defense against a myriad of known as well asunanticipated or novel physical, chemical or toxic assailants.” The argument isintriguing, the discussion is continuing

INTERRELATIONSHIP BETWEEN SKIN PHYSIOLOGY

AND KINETICS OF DERMAL ABSORPTION

As some of the key concepts concerning these inter-relationships are covered in thelater chapters “Physiologically-based pharmacokinetics and pharmacodynamics ofskin” and “Beyond stratum corneum,” this section is limited to a summary of thekey principles and implications

Principles of Dermal Absorption

The amount of a compound absorbed (Q) into the body depends on effective fluxthrough the epidermis Js, the area of application (A), the effective lag time (lag), andthe exposure time (T):

Q Z JsAðTKlagÞ (1)The time required to reach a steady-state flux JsA is w2 times the lag time.Figure 3 shows an illustration of equation (1) for release of nicotine from a patch invivo and in vitro It is apparent that in vivo there is an additional pharmacokineticlag and the JsA is only slightly less consistent, with release from the patch being therate-determining step in the absorption process The effective flux into a target site

in the epidermis or dermis in turn is dependent on the first pass availability of thesolute through the skin Fs, recognizing that this may be less than one through lossfrom removal on clothing, evaporation, adsorption to outermost layers in thestratum corneum, vaporization, desquamation or by skin first pass metabolismprior to reaching the site In general, the in vivo flux:

Wester et al (53) estimated Fsto be 0.57 for nitroglycerin in the monkey usingthe AUC ratio for unchanged nitroglycerin after transdermal and intravenousadministration A higher Fsof 0.77 was obtained when the AUC of total radio-activity was used implying about 20% of the nitroglycerin had been metabolizedduring the topical absorption process

Solute flux through the stratum corneum may occur by a number of pathwaysand be retarded by various barriers and removal by the cutaneous blood supply, asdiscussed later Of practical interest, is the maximum flux Jmaxas this should applyacross all vehicles and may be used to estimate Jsfor a given vehicle if the fractionalsolubility in that vehicle is known The precise determinant of Jmax is unclear, asshown in Figure 4, although solute size can be shown to be a dominant determinant(54,55) It is evident that the Jmax, for steroids at approximately the same molecularweight, is similar except for the most polar one for both infinite (55) and finite (56)dosing (Fig 4) The maximum flux concept does, however, have limitations in that

FIGURE 3 Nicotine release from patches in vitro (&) and in vivo (B) Source: Adapted from Ref 63.

solutes in high concentrations may exhibit nonlinear concentration dependenciesdue to effects on the stratum corneum or association in the vehicle An idealmaximum flux may be estimated as a product of aqueous solubility and per-meability coefficient kp, where kpmay be defined in terms of octanol–water partitioncoefficients log Koctand molecular weight (MW) by an expression similar to thatdeveloped by Potts and Guy (57):

Epidermal Reservoir and Dermal Clearance

Lipophilic solutes may also accumulate in the skin to form a so-called skin reservoir(58) Rehydration of the skin may cause such solutes to be released An example ofthe historical evidence of this effect is illustrated by the work of Vickers (59) in

(C)

FIGURE 4 (A C) Determinants of maximum flux for a large set of solutes, (D) steroids from an infinite dosing situation, and (E) steroids from finite dosing for occluded (open symbols) and non- occluded (closed symbols) conditions Abbreviations: Mpt, melting point; MW, molecular weight Source: From Refs 54 56.

which vasoconstriction was achieved by occluding an area of skin with plastic film

12 to 14 days after an initial application of steroid under occlusion and a fading ofthe vasoconstriction on removal of the film at 16 hours The clearance of a solutefrom the epidermis is also an important determinant of dermal absorption andtoxicity and, if impaired e.g., by vasoconstriction, may lead to higher levels of thesolute in the stratum corneum and in the viable epidermis Altered topicalabsorption due to changing blood flow can also occur as a consequence of elevatedtemperature or exercise or coadministration of vasodilating drugs

Systemic Concentrations of Solutes

Figure 5 shows serum plasma concentrations of testosterone obtained on theapplication of a patch and on its removal It is noted that there is a lag prior toreaching maximal levels and on in returning to baseline on patch removal Further,after reaching a maximum, the serum levels slowly decline with time, consistentwith a reduction in flux due to a gradual depletion in the amount of testosterone inthe patch Topical products, especially transdermal patches, often seek to provideconstant therapeutically effective plasma concentrations Css The release flux Jsideally needed to reach such concentrations is given by

systems based on the substitution of desired plasma concentrations and bodyclearances into equation (4).

Physiological and Pathological Determinants of Barrier Function

Age, gender, race, environment, species, and application site can affect neous absorption (60) The interrelationship between skin pathology and dermalabsorption and toxicity assessment is variable and, usually, related to the severity ofthe pathology Transepidermal water loss (TEWL) is one measure of skin barrierfunction that is either unaffected or compromised by the disease process In general,percutaneous absorption appears to show an association with TEWL (61)

percuta-Various strategies to impair and enhance skin permeability are detailedelsewhere in the companion to this book (62) Technologies considered include:chemical enhancement, iontophoresis, ultrasound, microneedles, and pressurewaves Prodrugs are considered later in this volume

There are also a range of strategies that can be used to assess topicalbioavailability and bioequivalence as summarized in Scheme 1

TOXICITY ASSESSMENT

In general, toxicity after topical exposure may be classified as (i) accidental arisingfrom environmental, occupational or recreational exposure or (ii) cosmetic ortherapeutic related In this second edition, a greater emphasis has been placed ontoxicity issues, with a section devoted specifically to regulatory issues

Blood and/or urine levels

Target tissue levels (e.g., biopsy, microdialysis)

Stratum corneum tape stripping

Remainder in topically applied product

Direct: Clinical trial

Indirect: Related response (e.g., vasoconstriction, TEWL)

Cadaver skin percutaneous absorption

Membrane rate of release

Drug concentrations across human skin in vivo

Pharmacodynamic response in vivo

In vivo animal studies

In vitro pharmacokinetic studies

SCHEME 1 Strategies used to define percutaneous absorption Abbreviation: TEWL, dermal water loss.

Dermal absorption and toxicity may be defined by the interrelationship of theanatomy and physiology of the skin with the physicochemical properties of thesolutes and the conditions under which they are used A number of structure–transport/activity relationships and pharmacokinetic models have been developed

to be able to predict both absorption and toxicity under a range of conditions Onechallenge is to be able to quantify solute concentrations and effects in the lowerlayers of the epidermis in vivo in dynamic studies

6 Elias PM Stratum corneum defensive functions: an integrated view J Invest Dermatol 2005; 125:183–200.

7 Di Nardo A, Gallo RL Cutaneous barriers in defense against microbial invasion In: Elias PM, Feingold KR, eds Skin Barrier New York: Taylor & Francis, 2006:363–77.

8 Bouwstra JA, Pilgram GSK, Ponec M Structure of the skin barrier In: Elias PM, Feingold KR, eds Skin Barrier New York: Taylor & Francis, 2006:65–95.

9 Roberts MS, Cross SE, Pellett MA Skin transport In: Walters KA, ed Dermatological and Transdermal Formulations New York: Marcel Dekker, 2002:89–195.

10 Bronaugh RL, Maibach HI Percutaneous Absorption 4th ed New York: Taylor & Francis, 2005.

11 Cross SE, Roberts MS Dermal blood flow, lymphatics, and binding as determinants of topical absorption, clearance, and distribution In: Riviere JE, ed Dermal Absorption Models in Toxicology and Pharmacology New York: Taylor & Francis, 2006:251–81.

12 Milstone LM Epidermal desquamation J Dermatol Sci 2004; 36:131–40.

13 Borradori L, Sonnenberg A Structure and function of hemidesmosomes: more than simple adhesion complexes J Invest Dermatol 1999; 112:411–8.

14 Fassihi H, Wong T, Wessagowit V, et al Target proteins in inherited and acquired blistering skin disorders Clin Exp Dermatol 2006; 31:252–9.

15 McMillan JR, Akiyama M, Nakamura H, et al Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane J Histochem Cytochem 2006; 54:109–18.

16 Aumailley M, El Khal A, Knoss N, et al Laminin 5 processing and its integration into the ECM Matrix Biol 2003; 22:49–54.

17 Kaiser HW, Ness W, Jungblut I, et al Adherens junctions: demonstration in human epidermis J Invest Dermatol 1993; 100:180–5.

18 Haftek M, Hansen MU, Kaiser HW, Kreysel HW, Schmitt D Interkeratinocyte adherens junctions: immunocytochemical visualization of cell–cell junctional structures, distict from desmosomes, in human epidermis J Invest Dermatol 1996; 106:498–504.