Skin Barrier KA RG Ε R. Hans Schaefer Thomas E. Redelmeier. Principles of Percutaneous Absorption. 65 figures and 28 tables, 1996

Transcription

1 Skin Barrier

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3 Hans Schaefer Thomas E. Redelmeier Skin Barrier Principles of Percutaneous Absorption 65 figures and 28 tables, 1996 KA RG Ε R Basel Freiburg Paris London New York New Delhi 'Bangkok. Singapore 'Tokyo' Sγdney

4 Hans Schaefer Professor, PhD, Scientific Director; Centre de Recherches C. Zviak, L'Oral, 90, Rue du Général Roguet, F Clichy, France Thomas E. Redelmeier PhD, Chief Executive Officer; Jack Bell Research Centre, Northern Lipids Inc., 2660 Oak Street, Vancouver, B.C., V6H 3Ζ6, Canada This book was supported by L'Oréal, Clichy, France Library of Congress Cataloging-in-Publication Data Schaefer, H. (Hans) Skin barrier: principles of percutaneous absorption / Hans Schaefer, Thomas E. Redelmeier. Includes bibliographical references and index. 1. Skin absorption. I. Redelmeier, Thomas E. II. Title. [DILl: 1. Skin Absorption. 2. Epidermis metabolism. WR 102 S294sa 1996] QP88.5.S '91 dc20 ISBN (hardcover: alk. paper) Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Copyright 1996 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Thür AG Offsetdruck, Pratteln ISBN

5 Contents Foreword Preface XIII XV 1 Structure and Dynamics of the Skin Barrier 1 Structure of the Skin 1 Topographical Features 1 Compartments 1 Stratum corneum 4 Viable Epidermis 5 Keratinocyte Differentiation 5 Keratohyalin Granules 7 Cornified Envelopes 7 Desmosome Plaques 8 Lamellar Bodies 11 Transition Region between Stratum compactum and Stratum granulosum 12 Interstitial Fluid 13 Dermal-Epidermal Junction 13 Dermis 14 Cutaneous Circulation 15 Hypodermis 17 Appendages 18 Sweat Glands 18 Hair Follicles 18 Sebaceous Glands 20 The Skin as a Dynamic Structure 21 Keratinocyte Proliferation 21 Cytokines 22 Epidermal Responses to Skin Barrier Perturbation 23 Mediators of the Epidermal Response 25 Synthesis and Assembly of Corneocytes 26 Core Components 26 Cornified Envelope 27 Covalent Attachment of Lipids to Cornified Envelopes 28 Synthesis and Assembly of Intercellular Lipid 29 ν

6 Fatty Acids 31 Cholesterol 34 Phospholipids and Glucosylceramides 36 Formation and Secretion of Lamellar Bodies 38 Extracellular Processing 39 Desquamation 40 Summary 42 2 Composition and Structure of the Stratum corneum 43 General 43 Definitions 43 What Provides the Barrier to Percutaneous Absorption? 44 Organization of the Stratum corneum into Protein and Lipid-Rich Domains 46 Heterogeneity across the Stratum corneum 46 Composition of the Stratum corneum 47 Water 47 Low-Molecular-Weight Polar Molecules 49 Corneocytes 51 Core Proteins 51 Cornified Envelopes 52 Covalently Bound Lipid 53 Other Proteins 54 Intercellular Lipid 55 Lipid Derived from the Lamellar Bodies 55 Extraneous Lipids 60 Comparison of the Intercellular Lipid Composition to Cellular Membranes 60 Structure of the Stratum corneum 61 Corneocytes 61 Cornified Envelopes 61 Intercellular Lipid Space 62 Freeze-Fracture Electron Microscopy 63 Transmission Electron Microscopy 63 The Motion of Lipids in the Intercellular Spaces 67 The Phase Behavior of Lipid Systems 67 Differential Scanning Calorimetry 71 X-Ray Diffraction 73 Fourier Transform Infrared Spectroscopy 76 Physical Basis for Percutaneous Absorption Pathways 77 Appendagael 77 Transcellular vs. Intercellular? 79 Skin Barrier VI

7 Unresolved Questions 80 Do Intercellular Lipid Lamellae Penetrate the Stratum corneum? 81 Is the Intercellular Lipid Homogeneous? 82 What Material Properties of the Intercellular Lipid Are Relevant to Skin Barrier Function? 84 Summary 85 3 Relationship between the Structure of Compounds and Their Diffusion across Membranes 87 Theoretical Aspects of Diffusion 87 Fick's Laws 87 Energetic Barriers to Diffusion across Membranes 89 Permeability Coefficient 89 Partition Coefficient 90 Model Membrane Systems 91 Water 92 Nonelectrolytes 95 Ions 96 Transmembrane ph Gradients 97 Transmembrane Electrical Potentials 98 Ions across Membranes 98 A General Model for Solute Diffusion across Membranes 99 Material Properties of Membranes Relevant to Diffusion 100 Permeability Properties of the Stratum corneum 101 Water 101 Nonelectrolytes 105 Mathematical Models for the Prediction of Percutaneous Absorption 108 Hydrogen Bonding of Penetrants 109 Gases 110 Electrolytes 111 Protons 111 Weak Acids or Weak Bases 112 Other Charged Compounds 113 Large Molecules 114 Model Membranes vs. the Stratum corneum 114 Summary 116 Contents VII

8 4 Prediction and Measurement of Percutaneous Absorption 118 Definitions of Terms 118 Limits to Topical Exposure 119 In vivo Measurement of Percutaneous Absorption 120 Recommendations for an in vivo Standard Protocol 120 Species 120 Purity of the Compound 121 Labeling Procedure for Detection of Compounds 121 Physical and Chemical Characterization of a Formulation 122 Preparation and Housing of Animals 122 Dose 122 Urine, Fecal and Serum Concentrations 123 Mass Balance Analysis 123 Data Reporting and Analysis 123 Alternative in vivo Procedures 124 Disappearance from Site of Application 124 Transepidermal Water Loss 124 Localization of Compounds within the Skin (Slicing Method) 125 Biological Endpoints 128 Grafted Human Skin 128 The Rat-Human Skin Flap System 128 In vitro Approaches for Predicting Percutaneous Absorption 129 Recommendation for in vitro Standard Protocols 129 Species 129 Reconstituted Skin 130 Isolation and Preparation of Intact Epidermal Sheets 133 Franz Cell 134 Completeness 136 Receptor Fluid 137 Skin Integrity 137 Application of Dose 137 Recovery 138 Reporting and Calculation of Results 138 Analysis 138 In vitro/ín vivo Comparisons of Percutaneous Absorption 141 Cutaneous Metabolism 142 Alternative Approaches 145 Isolated Porcine Skin Flap 145 Qualitative Structure-Activity Relationship (QSAR) 146 Stripping Method 147 Spectroscopic Approaches 149 Microdialysis 150 Summary 151 Skin Barrier VIII

9 5 Factors Effecting Percutaneous Absorption 153 Application of Compounds to the Skin Surface 154 Formulations and Vehicles 154 Vehicles 155 Kinetics of Compound Release from Formulations 156 Interaction of Compounds/Formulations with the Skin Surface 159 Evaporation 160 Dose 161 Application Thickness 161 Duration of Exposure 162 Stratum corneum as Reservoir 162 Repeated Applications 163 Skin Decontamination 163 Strategies to Alter Skin Barrier Properties 165 Enhancers 165 Occlusion 167 Chemical Enhancers 168 Physical Approaches 172 Strategies to Augment Barrier Function 176 Retarders 176 Skin Barrier Creams 177 Recovery of Barrier Function after Extraction of the Intercellular Lipid 178 Surfactant-Induced Dry Skin 182 Viable Tissue 183 Skin Metabolism 183 Resorption 185 Strategies to Target Compounds to Skin Compartments 186 Compounds 187 Vehicles 187 Short Contact Therapy 188 Vasoactive Drugs 188 Biological Factors which Influence Percutaneous Absorption 189 Interindwiduql Variation in Skin Barrier Function 189 Age, Gender and Race 191 Temperature and Humidity 193 Anatomical Site 193 Appendages 195 Scalp 201 Pathological Processes 201 Ichthyosis 202 Psoriasis 204 Atopic Dermatitis 205 Contents IX

10 Ultraviolet Radiation 206 Contact Dermatitis 207 Summary The Practical Application of Techniques to Measure Percutaneous Absorption 213 Transdermals 214 General Requirements 214 Rationale 216 Development 217 Selection of Drug Candidates 217 Bioavailability 218 Release Rate from the Device 219 In vitro Percutaneous Absorption 219 Rate Control 220 Skin Surface Metabolism 221 Cutaneous Metabolism 221 In vivo Percutaneous Absorption 222 Buprenorphine as a Candidate for a Transdermal Drug 223 Dermatological Preparations 224 Rationale 225 Steps Required for Drug Development 225 Selection of a Compound 227 Formulation 227 In vitro Release Studies 228 In vitro Percutaneous Absorption Measurements 228 In vivo Animal Studies 229 In vivo Human Studies 229 Examples of Dermatological Drugs which Are Not Efficacious Topically 233 Cyclosporin 233 Methotrexate 234 Cyproterone Acetate 235 A Strategy to Target Compounds to Hair Follicles 235 Particle Targeting to Hair Follicles 235 Safety Assessment of Cosmetics 237 Steps Required in the Safety Assessment of a Cosmetic Ingredient 238 Identification of Ingredients 238 Toxicity 238 In-Use Conditions 239 Initial Safety Estimate 242 In vitro Investigations 243 Skin Barrier X

11 In vivo Investigations 246 Examples of Safety Assessment of Cosmetic Formulations 247 Ethyl Alcohol 247 Hair Dyes 248 Sun Screens 249 Risk Assessment of Environmental and Occupational Hazards 249 Skin Denotation 250 Risk Assessment and Risk Management 251 Hazard Identification 251 Dermal Exposure 252 Hazard Assessment 253 Model-Based Risk Assessment 254 In vitro Approaches 256 In vivo Estimates of Exposure 256 Skin Stripping 257 Animal Studies 258 Summary 259 In vitro versus in vivo Investigations 259 Transdermal Delivery Systems 260 Dermatologicals 260 Cosmetics 261 Environmental and Occupational Hazards 261 References 263 Subject Index 305 Contents XI

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13 Foreword The views on the permeability of the skin barrier are burdened with misconceptions, ranging from `the barrier is impermeable' to `everything can pass through'. For an outsider it is almost impossible to base on classical literature reading a competent position on a specific problem of a topical drug, in the safety assessment of a pesticide or a cosmetic ingredient, on any kind of material which can come in contact with the skin. It is up to textbooks like this one to not only assemble the wealth of knowledge, but to analyze its complexity and to evaluate its importance for day-to-day problems. The most pertinent task of such a book is to point out the general rules, to indicate their limits and more importantly to convey the scientific basis for such rules in order to facilitate their understanding, even more so since skin permeability is a multifactorial, hence multidisciplinary area. Here are the major principles which I personally find worth pointing out after having browsed through the book: Firstly the authors emphasize that the skin barrier, although often termed `dead', is in reality an excellent paradigm of an active function of the body. It is based on biometabolic processes which are unique to the skin and which assemble sophisticated lamellar structures in a very thin albeit sturdy layer. The horny layer barrier is an `all-purpose' membrane which limits the passage of any kind of material, be it water, chemicals or proteins. Α healthy barrier is as tight to the entrance of foreign material as it prevents loss of body constituents to the outside, both processes do occur but such a passage is small under `normal' conditions. The passage can be modulated on purpose or accidentally, i.e. it can be increased or reduced, first of all by design of vehicles in the most general sense, secondly by the exposure conditions (surface area, duration, etc.), and thirdly by the state of the barrier (pathologic disturbance, its importance and extent). The authors underline that contrary to resorption organs and other application routes, the maximal dosage or exposure to the skin is limited and depends strictly on the exposed area. This particular feature of the skin as barrier and as site for absorption can be taken advantage of when dealing with material which should be prevented from entering to a significant extent into the body (cosmetics, agrochemicals, etc.) as well as in the treatment of diseases via the topical route. In the latter case a targeting of drugs to specific sites in the skin can be designed, thereby avoiding a significant passage into the body and increasing considerably the therapeutic ratio of drugs.

14 Any estimate on percutaneous absorption has to take the very practical aspects of close-to-life exposure into account, that is the in-use conditions for a drug, a chemical, a cosmetic product. As pointed out by the authors, the upper and lower limits of passage through the skin barrier can be estimated, i.e. a window for a specific problem defined within the confines of which experimental studies on percutaneous absorption are recommended. Hierarchies of procedures to the practical approach to different categories of problems and classes of compounds and products are derived from these rules and given in the book in an excellent way of description. A major part of the book is dedicated to the methods in the barrier assessment. These are of two kinds, those which improve our knowledge about the underlying principles, but which are mostly too sophisticated for routine investigations, and those which underwent the scrutiny of everyday problems and thereby have been standardized. Among the latter, in vitro methods for percutaneous absorption take an everincreasing place, since they allow to quantify the barrier towards a particular compound directly, and to circumvent confounding factors encountered in vivo like systemic metabolism, deep compartments, excretion mechanisms, etc. Should I summarize the essence of this book in one sentence, I might say that the authors show that the skin barrier behaves differently according to the nature and concentration of the compound, the vehicle in which this compound is present as well as the exposure conditions, and that they explain how to make choices and why. This book will help the reader to better understand the basic mechanisms, to make valid predictions and to plan reasonable experiments rather than to make vague assumptions in a field as complex as percutaneous absorption through the skin barrier. This book benefits from the life-long experience of one of its authors, H. Schaefer, who got his scientific profile in years of University research on fundamental principles of dermatology and pathophysiology of the skin. It is therefore full of insights on the pivotal role of the skin barrier when it comes to the rationale underlying the sometimes subtle difference between a cosmetic and a drug, as well as on the risk assessment of topically applied products, providing the reader with all the up-to-date scientific knowledge necessary to weigh the numerous intervening factors. I am pleased to congratulate the authors for their major contribution to this important topic of the skin studies. July 16, 1996 Fritz Kemper, Dr. med. Dr. h.c. mult. Univ.-Prof. em. for Pharmacology and Toxicology President of the EU Scientific Committee Cosmetology Munster, Germany Foreword XIV

15 Preface The skin of terrestrial animals is essential for their survival. Though providing a degree of physical protection and integrity, the principal role of the skin is to keep harmful agents out and water in. In order to accomplish this seemingly trillial task, nature has developed an elaborate structure, termed the stratum corneum, which provides to the skin its barrier function. The almost complete impermeability of the skin barrier is all the more astonishing if one considers that it resides in a very thin and elastic layer. Percutaneous absorption deals firstly with the quantification of small amounts, often traces of material diffusing through the barrier, secondly with measures to overcome the barrier for therapeutic purposes, and thirdly its deficiency in skin diseases. This book intents to provide a general overview of the principles of the structure and the function of the skin barrier. Within this broad subject, we focus on several different aspects, the nature of the barrier, a description of the properties governing the diffusion of compounds across the skin and methods for measuring percutaneous absorption. The subject area is of fundamental importance to widely divergent fields of scientific investigation. This includes, for example, the delivery of drugs across the skin to treat either local (skin) diseases or those which can be treated systemically. Also, it is fundamental to the safety assessment of cosmetics and the risk assessment of environmental hazards. Given the nature of this subject, it is not surprising to find that there is a relatively rich history of scientific endeavor which has focused on these particular problems dating back, in some cases, more than several decades. However, we have chosen to primarily focus on studies published in the last years, without disregarding the importance of some of this earlier work. The broad nature of the subject coupled with the length of the book has necessitated that we focus our remarks and only occasionally have we allowed ourselves the luxury of discussing particular subjects in detail. In order to provide the reader and especially students with access to more detailed information, we have included a comprehensive listing of references published over the last 10 years. A major stimulus for writing this book at this time has been the recent clarification of ideas on how to measure percutaneous absorption in vitro and when. The notion of a window of three to four orders of magnitude, in the frame of which rates of percutaneous absorption can be reasonably quantified and the xv

16 results interpreted, provides the dimension for such studies. It helps to avoid investigations on rates at or below detection limits or on rates close to complete absorption. Though there were a large number of laboratories involved in measuring percutaneous absorption years ago, it is only recently that there has been a general consensus in the scientific field regarding the development of standard protocols. We have recognized the pivotal importance of this consensus by devoting two chapters to the measurement of percutaneous absorption; chapter 4 focuses on general protocols and chapter 6 on the application of these techniques to solving problems related to percutaneous absorption. The interpretation of these measurements and in particular the extrapolation of information obtained in vitro to the in vivo situation requires a relatively broad understanding of the factors which can influence the flux of compounds across the skin. For this reason, we have summarized the relationship between the physico-chemical properties of compounds and their propensity to undergo absorption (chapter 3) and how the bioavailability of compounds is influenced by processes occurring on or within the skin (chapter 5). However, to the biologist, what is remarkable about the skin is the extraordinary nature of the stratum corneum. The composition, structure and material properties of this layer are not only unique compared to other structures within the body but also are particularly suited for their prime role in maintaining barrier function. In chapter 1, we discuss the general organization of the skin compartments as well as the processes leading to synthesis and assembly of the precursor molecules to form the skin barrier. Chapter 2 provides an ín-depth description of the stratum corneum and focuses our attention on how the composition and structure provide the material properties required for retention of water by terrestrial animals. Together, we hope that this material provides a convenient reference for researchers and students interested in percutaneous absorption. We gratefully acknowledge the helpful comments and in some cases access to unpublished material from Brian Barry, Florence Benech-Kieffer, Joke Bouswtra, Rudgero Caputo, Richard Guy, Peter Elias, Ken Feingold, Manigé Fartasch, Gordon Flynn, Neil Kitson, Howard Maibach, Russel Potts, Martin Rosdy and André Rougier. Hans Schaefer Thomas E. Redelmeier Preface XVI