Topical Absorption of Dermatological Products

Topical
Absorption of
Dermatological
Products

edited by

Robert L. Bronaugh
U.S. Food and Drug Administration
Laurel, Maryland

Howard I. Maibach
University of California School of Medicine
San Francisco, California

Marcel Dekker, Inc.

New York • Basel

TM

Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.


Portions reprinted from first, second, and third editions of Percutaneous Absorption:
Drugs—Cosmetics—Mechanisms—Methodology.

ISBN: 0-8247-0626-9
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Series Introduction

Over the past decade, there has been a vast explosion in new information
relating to the art and science of dermatology as well as fundamental cutaneous
biology. Furthermore, this information is no longer of interest only to the small
but growing specialty of dermatology. Scientists from a wide variety of disciplines have come to recognize both the importance of skin in fundamental biological processes and the broad implications of understanding the pathogenesis
of skin disease. As a result, there is now a multidisciplinary and worldwide
interest in the progress of dermatology.
With these factors in mind, we have undertaken to develop this series of

books specifically oriented to dermatology. The scope of the series is purposely
broad, with books ranging from pure basic science to practical, applied clinical
dermatology. Thus, while there is something for everyone, all volumes in the
series will ultimately prove to be valuable additions to the dermatologist’s
library.
The latest addition to the series by Larry E. Millikan is both timely and
pertinent. The authors are well known authorities in the fields of cutaneous
microbiology and clinical skin infections. We trust that this volume will be of
broad interest to scientists and clinicians alike.

Alan R. Shalita
SUNY Health Science Center
Brooklyn, New York

iii


BASIC AND CLINICAL DERMATOLOGY
Series Editors
ALAN R. SHALITA, M.D.
Distinguished Teaching Professor and Chairman
Department of Dermatology
State University of New York
Health Science Center at Brooklyn
Brooklyn, New York

DAVID A. NORRIS, M.D.
Director of Research
Professor of Dermatology
The University of Colorado

Health Sciences Center
Denver, Colorado

1. Cutaneous Investigation in Health and Disease: Noninvasive Methods
and Instrumentation, edited by Jean-Luc Lévêque
2. Irritant Contact Dermatitis, edited by Edward M. Jackson and Ronald
Goldner
3. Fundamentals of Dermatology: A Study Guide, Franklin S. Glickman and
Alan R. Shalita
4. Aging Skin: Properties and Functional Changes, edited by Jean-Luc
Lévêque and Pierre G. Agache
5. Retinoids: Progress in Research and Clinical Applications, edited by
Maria A. Livrea and Lester Packer
6. Clinical Photomedicine, edited by Henry W. Lim and Nicholas A. Soter
7. Cutaneous Antifungal Agents: Selected Compounds in Clinical Practice
and Development, edited by John W. Rippon and Robert A. Fromtling
8. Oxidative Stress in Dermatology, edited by Jürgen Fuchs and Lester
Packer
9. Connective Tissue Diseases of the Skin, edited by Charles M. Lapière
and Thomas Krieg
10. Epidermal Growth Factors and Cytokines, edited by Thomas A. Luger
and Thomas Schwarz
11. Skin Changes and Diseases in Pregnancy, edited by Marwali Harahap
and Robert C. Wallach
12. Fungal Disease: Biology, Immunology, and Diagnosis, edited by Paul H.
Jacobs and Lexie Nall
13. Immunomodulatory and Cytotoxic Agents in Dermatology, edited by
Charles J. McDonald

14. Cutaneous Infection and Therapy, edited by Raza Aly, Karl R. Beutner,
and Howard I. Maibach
15. Tissue Augmentation in Clinical Practice: Procedures and Techniques,
edited by Arnold William Klein
16. Psoriasis: Third Edition, Revised and Expanded, edited by Henry H.
Roenigk, Jr., and Howard I. Maibach
17. Surgical Techniques for Cutaneous Scar Revision, edited by Marwali
Harahap
18. Drug Therapy in Dermatology, edited by Larry E. Millikan
19. Scarless Wound Healing, edited by Hari G. Garg and Michael T. Longaker
20. Cosmetic Surgery: An Interdisciplinary Approach, edited by Rhoda S.
Narins
21. Topical Absorption of Dermatological Products, edited by Robert L.
Bronaugh and Howard I. Maibach
22. Glycolic Acid Peels, edited by Ronald Moy, Debra Luftman, and Lenore
S. Kakita
23. Innovative Techniques in Skin Surgery, edited by Marwali Harahap

ADDITIONAL VOLUMES IN PREPARATION

Safe Liposuction, edited by Rhoda S. Narins


Preface

This book is based on Bronaugh and Maibach’s Percutaneous Absorption: Third
Edition and In Vitro Percutaneous Absorption (Marcel Dekker, Inc.). It is a condensed entry into this complex area geared toward dermatologists, pharmacists,
toxicologists, cosmetic chemists working in the area of transdermal drug absorption, and others concerned with clinical treatment and studies involving the skin.
The chapters are divided into three parts. “Mechanisms of Absorption” contains
chapters that discuss the effects of skin metabolism, hair follicles, occlusion, and

other factors on skin absorption. Chapters in the “Methodology” part discuss the
various issues involved in measuring percutaneous absorption such as in vivo and
in vitro methodology, single versus multiple dosing, the relationship of blood flow
to absorption, and the significance of drug concentrations in the different layers of
skin. In “Drug and Cosmetic Absorption,” many updated and new chapters have
been added on topical dermatological products.
We would like to thank the authors for preparing outstanding chapters and
Sandra Beberman and Barbara Mathieu of Marcel Dekker, Inc., for their expert
editorial guidance.
Robert L. Bronaugh
Howard I. Maibach

v



Contents

Series Introduction
Preface
Contributors

Alan R. Shalita

iii
v
xi

Mechanisms of Absorption
1.

Cutaneous Metabolism During In Vitro Percutaneous Absorption
Robert L. Bronaugh, Margaret E. K. Kraeling, Jeffrey J. Yourick,
and Harolyn L. Hood

1

2.

Occlusion Does Not Uniformly Enhance Penetration In Vivo
Daniel A. W. Bucks and Howard I. Maibach

9

3.

Regional Variation in Percutaneous Absorption
Ronald C. Wester and Howard I. Maibach

33

4.

The Development of Skin Barrier Function in the Neonate
Lourdes B. Nonato, Yogeshvar N. Kalia, Aarti Naik, Carolyn H.
Lund, and Richard H. Guy

43

5.

Cutaneous Metabolism of Xenobiotics
Saqib J. Bashir and Howard I. Maibach

77

6.

Percutaneous Drug Delivery to the Hair Follicle
Andrea C. Lauer

93

7.

In Vivo Relationship Between Percutaneous Absorption and
Transepidermal Water Loss
André Rougier, Claire Lotte, and Howard I. Maibach

115
vii


viii

Contents

8. Dermal Decontamination and Percutaneous Absorption
Ronald C. Wester and Howard I. Maibach

129

Methodology
9.
10.

In Vivo Methods for Percutaneous Absorption Measurement
Ronald C. Wester and Howard I. Maibach
Determination of Percutaneous Absorption by In Vitro
Techniques
Robert L. Bronaugh, Harolyn L. Hood, Margaret E. K. Kraeling,
and Jeffrey J. Yourick

145

157

11.

Systemic Absorption of Cutaneous Material
Robert L. Bronaugh and Harolyn L. Hood

12.

Interrelationships in the Dose-Response of Percutaneous
Absorption
Ronald C. Wester and Howard I. Maibach

169

Effect of Single Versus Multiple Dosing in Percutaneous
Absorption
Ronald C. Wester and Howard I. Maibach

185

13.

14.

Blood Flow as a Technology in Percutaneous Absorption:
The Assessment of the Cutaneous Microcirculation by Laser
Doppler and Photoplethysmographic Techniques
Ethel Tur

15.

Drug Concentration in the Skin
Christian Surber, Eric W. Smith, Fabian P. Schwarb, Tatiana
Tassopoulos, and Howard I. Maibach

16.

Stripping Method for Measuring Percutaneous Absorption
In Vivo
André Rougier, Didier Dupuis, Claire Lotte, and
Howard I. Maibach

17.

18.

Interference with Stratum Corneum Lipid Biogenesis:
An Approach to Enhance Transdermal Drug Delivery
Peter M. Elias, Vivien Mak, Carl Thornfeldt, and
Kenneth R. Feingold
Methods for the Assessment of Topical Corticosteroid-Induced
Skin Blanching
John M. Haigh, Eric W. Smith, Carryn H. Purdon, and
Howard I. Maibach

163

195
227

241

261

275


Contents

19.

20.

21.

ix

Importance of In Vitro Release Measurement in Topical
Dermatological Dosage Forms
Vinod P. Shah, Jerome S. Elkins, and Roger L. Williams

283

Innovative Methods to Determine Percutaneous Absorption
in Humans: Real-Time Breath Analysis and Physiologically
Based Pharmacokinetic Modeling
Karla D. Thrall, Torka S. Poet, Richard A. Corley,
Howard I. Maibach, and Ronald C. Wester

299

Calculations of Body Exposure from Percutaneous
Absorption Data
Richard H. Guy and Howard I. Maibach

311

Drug and Cosmetic Absorption
22.

Percutaneous Penetration as a Method of Delivery to Skin
and Underlying Tissues
Parminder Singh

317

23.

Phonophoresis
Joseph Kost, Samir Mitragotri, and Robert Langer

335

24.

Facilitated Transdermal Delivery by Iontophoresis
Parminder Singh, Puchun Liu, and Steven M. Dinh

353

25.

Percutaneous Penetration as It Relates to the Safety Evaluation of
Cosmetic Ingredients
Jeffrey J. Yourick and Robert L. Bronaugh

377

26.

Percutaneous Absorption of Fragrances
Jeffrey J. Yourick, Harolyn L. Hood, and Robert L. Bronaugh

389

27.

Hair Dye Absorption
Leszek J. Wolfram

401

28.

Percutaneous Absorption of Alpha-Hydroxy Acids
Margaret E. K. Kraeling and Robert L. Bronaugh

415

29.

Optimizing Patch Test Delivery: A Model
Ronald C. Wester and Howard I. Maibach

429

30.

Nail Penetration: Focus on Topical Delivery of Antifungal Drugs
for Onychomycosis Treatment
Ying Sun, Jue-Chen Liu, Jonas C. T. Wang, and Piet De Doncker

31.

Topical Dermatological Vehicles: A Holistic Approach
Eric W. Smith, Christian Surber, Tatiana Tassopoulos, and
Howard I. Maibach

437
457


x

Contents

32.

Percutaneous Absorption of Sunscreens
Kenneth A. Walters and Michael S. Roberts

465

33.

Colloidal Drug Carrier Systems
Konstanze Jahn, Annett Krause, Martin Janich, and
Reinhard H. H. Neubert

483

34.

Alternative Therapies—Calling Up the Reserves: A Topical

Nonsteroidal Formulary for the Management of Acute, Subacute,
and Chronic Steroid-Responsive Dermatoses and Other Skin
Afflictions
Jerome Z. Litt

35.

Index

Ointments, Creams, and Lotions Used as Topical Drug Delivery
Vehicles
Christian Surber, Tatiana Tassopoulos, and Eric W. Smith

495

511

519


Contributors

Saqib J. Bashir, B.Sc., M.B., Ch.B.
Medicine, San Francisco, California

University of California School of

Robert L. Bronaugh, Ph.D. Skin Absorption and Metabolism Section, Office
of Cosmetics and Colors, U.S. Food and Drug Administration, Laurel, Maryland
Daniel A. W. Bucks, Ph.D. University of California School of Medicine, San

Francisco, and Dow Pharmaceutical Sciences Inc., Petaluma, California
Richard A. Corley, Ph.D. Molecular Biosciences, Pacific Northwest National
Laboratory, Richland, Washington
Piet De Doncker, Ph.D.
Beerse, Belgium

European Business Group, Janssen-Cilag, Europe,

Steven M. Dinh, Sc.D.
stown, New Jersey

Research and Development, Lavipharm Inc., Hight-

Didier Dupuis, Ph.D.
sous Bois, France

Department of Industrial Chemistry, L’Oréal, Aulnay

Peter M. Elias, M.D. Dermatology Service, Veterans Affairs Medical Center,
and Department of Dermatology, University of California School of Medicine,
San Francisco, California
Jerome S. Elkins Hanson Research Corporation, Chatsworth, California
xi


xii

Contributors

Kenneth R. Feingold, M.D. Medical Service, Veterans Affairs Medical Center,

and Departments of Dermatology and Medicine, University of California School
of Medicine, San Francisco, California
Richard H. Guy, Ph.D. Faculty of Sciences, University of Geneva, Geneva,
Switzerland
John M. Haigh, Ph.D. School of Pharmaceutical Sciences, Rhodes University,
Grahamstown, South Africa
Harolyn L. Hood, M.S.* Skin Absorption and Metabolism Section, Office of
Cosmetics and Colors, U.S. Food and Drug Administration, Laurel, Maryland
Konstanze Jahn Institute of Pharmaceutics and Biopharmaceutics, MartinLuther-University, Halle-Wittenberg, Halle/Saale, Germany
Martin Janich, Ph.D. Interdisciplinary Center of Material Science, MartinLuther-University, Halle-Wittenberg, Halle/Salle, Germany
Yogeshvar N. Kalia, Ph.D. Centre Interuniversitaire de Recherche et d’Enseignement, Archamps, France, and Laboratoire de Pharmacie Galénique, University of Geneva, Geneva, Switzerland
Joseph Kost, Ph.D. Department of Chemical Engineering, Ben-Gurion University, Beer-Sheeva, Israel
Margaret E. K. Kraeling, M.S. Skin Absorption and Metabolism Section, Office
of Cosmetics and Colors, U.S. Food and Drug Administration, Laurel, Maryland
Annett Krause Institute of Pharmaceutics and Biopharmaceutics, MartinLuther-University, Halle-Wittenberg, Halle/Saale, Germany
Robert Langer, Sc.D. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
Andrea C. Lauer, Ph.D. Actelion Pharmaceuticals, South San Francisco, California
Jerome Z. Litt, M.D. Department of Dermatology, Case Western Reserve University, Cleveland, Ohio

* Current affiliation: Theradex USA, Princeton, New Jersey


Contributors

xiii

Jue-Chen Liu, Ph.D. Department of Science and Technology, Johnson & Johnson Consumer and Personal Products, Skillman, New Jersey
Puchun Liu, Ph.D. Pharmaceutical Research and Development Department,
Novartis Pharmaceuticals Corporation, Suffern, New York
Claire Lotte, Ph.D. Department of Advanced Research/Life Sciences, L’Oréal,

Aulnay sous Bois, France
Carolyn H. Lund, R.N, M.S., F.A.A.N. Intensive Care Unit, Children’s Hospital, Oakland, California
Howard I. Maibach, M.D. Department of Dermatology, University of California School of Medicine, San Francisco, California
Vivien Mak, Ph.D. Research and Development, Cellegy Pharmaceuticals Inc.,
San Carlos, California
Samir Mitragotri, Ph.D. Department of Chemical Engineering, University of
California, Santa Barbara, California
Aarti Naik, Ph.D. Centre Interuniversitaire de Recherche et d’Enseignement,
Archamps, France, and Laboratoire de Pharmacie Galénique, University of
Geneva, Geneva, Switzerland
Reinhard H. H. Neubert, Ph.D. Institute of Pharmaceutics and Biopharmaceutics, Martin-Luther-University, Halle-Wittenberg, Halle/Saale, Germany
Lourdes B. Nonato, Ph.D. Department of Biopharmaceutical Sciences, University of California, San Francisco, California
Torka S. Poet, Ph.D. Molecular Biosciences, Pacific Northwest National Laboratory, Richland, Washington
Carryn H. Purdon School of Pharmaceutical Sciences, Rhodes University,
Grahamstown, South Africa
Michael S. Roberts, Ph.D., D.Sc. Department of Medicine, University of
Queensland, Brisbane, Queensland, Australia
André Rougier, Ph.D.
Courbevoie, France

Laboratoire Pharmaceutique, La Roche-Posay,


xiv

Contributors

Fabian P. Schwarb, Ph.D. Department of Dermatology and Institute of Hospital–Pharmacy, University Hospital, Basel, Switzerland
Vinod P. Shah, Ph.D. U.S. Food and Drug Administration, Rockville, Maryland
Parminder Singh, Ph.D. Transdermal Pharmaceutical Development, Novartis

Pharmaceuticals Corporation, Suffern, New York
Eric W. Smith, Ph.D. College of Pharmacy, Ohio Northern University, Ada,
Ohio
Ying Sun, Ph.D. Product Development, Johnson & Johnson Consumer and Personal Products, Skillman, New Jersey
Christian Surber, Ph.D. Department of Dermatology and Institute of Hospital–Pharmacy, University Hospital, Basel, Switzerland
Tatiana Tassopoulos Department of Dermatology and Institute of
Hospital–Pharmacy, University Hospital, Basel, Switzerland
Carl Thornfeldt, M.D. Research and Development, Cellegy Pharmaceuticals
Inc., San Carlos, California
Karla D. Thrall, Ph.D. Molecular Biosciences, Pacific Northwest National
Laboratory, Richland, Washington
Ethel Tur, M.D. Department of Dermatology, Sourasky Medical Center, Tel
Aviv University, Tel Aviv, Israel
Kenneth A. Walters, Ph.D. An-eX Analytical Services Ltd., Cardiff, Wales
Jonas C. T. Wang, Ph.D. Sycamore Ventures, Lawrenceville, New Jersey
Ronald C. Wester, Ph.D. Department of Dermatology, University of California School of Medicine, San Francisco, California
Roger L. Williams, M.D. U.S. Pharmacopeia, Rockville, Maryland
Leszek J. Wolfram, Ph.D. Independent Consultant, Stamford, Connecticut
Jeffrey J. Yourick, Ph.D., DABT Skin Absorption and Metabolism Section,
Office of Cosmetics and Colors, U.S. Food and Drug Administration, Laurel,
Maryland


1
Cutaneous Metabolism During In
Vitro Percutaneous Absorption
Robert L. Bronaugh, Margaret E. K. Kraeling, Jeffrey J. Yourick, and
Harolyn L. Hood*
U.S. Food and Drug Administration, Laurel, Maryland

I. INTRODUCTION
The skin is a portal of entry and the largest organ of the body. It has been shown
to contain the major enzymes found in other tissues of the body (1). Topically
applied compounds may be metabolized in skin resulting in altered pharmacologic
or toxicologic activity.
The metabolism of benzo[a]pyrene applied to mouse skin floating in an organ culture demonstrated the potential importance of metabolism of chemicals
during percutaneous absorption (2). A flow-through diffusion cell was subsequently developed to aid in quantitating skin absorption and metabolism (3). Viability of skin in the diffusion cell, which was assessed by light microscopy, was
found to be maintained for at least 17 h (3).
The viability of pig skin was maintained in flow-through cells by using tissue culture media; skin-mediated hydrolysis of diethyl malonate was observed (4).
The suitability of these conditions to maintain skin viability was assessed in initial studies by the ability to graft the skin to nude mice (5).
II. SKIN VIABILITY
The use of viable skin in percutaneous absorption studies is essential for investigating skin metabolism of absorbed compounds. The viability of skin in flow* Current affiliation: Theradex USA, Princeton, New Jersey.

1


2

Bronaugh et al.

through diffusion cells was systematically examined (6). Viability could be conveniently determined from glucose utilization by skin by measuring lactate appearing in the receptor fluid. Viability could be assessed throughout the course of
the experiment. It was observed that a HEPES-buffered Hanks balanced salt solution was equivalent to minimal essential media in maintaining skin viability for at
least 24 h. The viability of skin was also confirmed by electron microscopy and
by the maintenance of estradiol and testosterone metabolism.

III. SKIN METABOLISM
The following summary of skin absorption/metabolism studies illustrates the
types of compounds that are metabolized in skin. In many cases the metabolites
formed from the parent compounds have been determined and therefore important
metabolic reactions in skin have been identified.

In early studies from our laboratory, the penetration and metabolism of
estradiol and testosterone (6), acetylethyl tetramethyl tetralin (AETT) and butylated hydroxytoluene (BHT) (7), benzo[a]pyrene and 7-ethoxycoumarin (8), and
azo colors (9) were examined by using viable dermatome skin sections from mice,
rats, hairless guinea pigs, and humans. These early studies are not discussed here,
but may be examined separately by the interested reader.
The percutaneous absorption and metabolism of three structurally related
compounds, benzoic acid, p-aminobenzoic acid (PABA), and ethyl aminobenzoate (benzocaine), were determined in vitro with hairless guinea pig and human
skin (10). Approximately 7% of the absorbed benzoic acid was conjugated with
glycine to form hippuric acid (Fig. 1).
Acetylation of primary amines was found to be an important metabolic step
in skin. For benzocaine, a molecule susceptible to both N-acetylation and ester hydrolysis, 80% of the absorbed material was acetylated, while less than 10% of the
absorbed ester was hydrolyzed (Fig. 1). PABA was much more slowly absorbed
than benzocaine and was also less extensively N-acetylated (Fig. 1). AcetylPABA was found primarily in the receptor fluid at the end of the experiments, but
the receptor fluid contained only 20% of the absorbed dose. Much of the absorbed
PABA remained unmetabolized and in the skin, as might be expected for an
effective sunscreen agent. The compound in skin would probably not have been
exposed to N-acetylating enzymes if it was localized primarily in the stratum
corneum. A similar pattern of benzocaine metabolism was observed in human and
hairless guinea pig skin; however, there appeared to be less enzyme activity in human skin.
The extent of metabolism of radiotracer doses of benzocaine in the studies
just cited was compared to metabolism of much larger doses of benzocaine in formulations simulating exposure from use of topical benzocaine anesthetic products


Cutaneous Metabolism During Absorption

3

Figure 1 Percutaneous absorption and metabolism of benzoic acid, p-aminobenzoic acid
(PABA), and benzocaine. All compounds were studied in hairless guinea pig skin. Benzocaine was also studied in human skin.

(11). The distribution of [14C]benzocaine and metabolites as the percentage of the
absorbed dose in receptor fluid and skin was compared by using a radiotracer dose
(2 ␮g/cm2), an intermediate dose (40 ␮g/cm2) and a therapeutic dose (200 ␮g/cm2)
(Table 1). When a therapeutic dose was applied, the metabolism of benzocaine was
reduced, presumably because of the saturation of enzyme activity in skin. However, 34% of the absorbed dose was still converted to acetylbenzocaine. Although
the percent applied dose absorbed decreased with increasing dose of benzocaine,
total absorption of benzocaine and metabolites increased as the applied dose increased (Table 2). Benzocaine and acetylbenzocaine were found to have similar potencies in blocking nerve conduction in the isolated squid giant axon.
Esterase activity and alcohol dehydrogenase activity were characterized in
hairless guinea pig skin with the model compounds methyl salicylate and benzyl
alcohol (12). Subsequently, the absorption and metabolism of the cosmetic ingredient retinyl palmitate were determined in human and hairless guinea pig skin.
The metabolism of methyl salicylate was determined in viable and nonviable hairless guinea pig skin (Table 3). In viable skin over 50% of the absorbed
compound was hydrolyzed by esterases in skin to salicylic acid. Twenty-one
percent of the absorbed compound was further conjugated with glycine to form
salicyluric acid. Greater esterase activity was observed in male skin. Esterase is a


4

Bronaugh et al.

Table 1 Percent of Absorbed Dose of Benzocaine and Metabolites Over 24 h in
Hairless Guinea Pig Skin at Different Dose Levels
Dose level
Location and compounda
Receptor fluid
Benzocaine
AcBenz
PABA
AcPABA
Skin

Benzocaine
AcBenz
PABA
AcPABA
Totalb
Benzocaine
AcBenz
PABA
AcPABA

2 ␮g/cm2

40 ␮g/cm2

200 ␮g/cm2

9.6 Ϯ 4.2
83.8 Ϯ 4.4
1.0 Ϯ 0.3
5.1 Ϯ 1.0

50.7 Ϯ 6.6
43.8 Ϯ 5.7
0.1 Ϯ 0.1
5.8 Ϯ 0.9

54.0 Ϯ 5.2
37.9 Ϯ 3.6
0.9 Ϯ 0.5
7.2 Ϯ 1.7

40.0 Ϯ 8.5
10.3 Ϯ 10.3
4.3 Ϯ 4.3
24.7 Ϯ 14.9

4.9 Ϯ 4.9
66.6 Ϯ 8.2
12.6 Ϯ 12.6
16.0 Ϯ 16.0

62.7 Ϯ 12.2
20.9 Ϯ 11.7
1.5 Ϯ 1.3
14.9 Ϯ 1.2

10.7 Ϯ 3.3
80.5 Ϯ 3.8
1.4 Ϯ 0.2
6.5 Ϯ 1.0

49.9 Ϯ 6.5
43.6 Ϯ 5.6
0.1 Ϯ 0.1
5.9 Ϯ 1.0

57.3 Ϯ 3.7
34.3 Ϯ 3.4
0.9 Ϯ 0.5
7.6 Ϯ 2.0

Values are the mean Ϯ SE for 1-6 determinations in each of 4 animals.
a
AcBenz ϭ N-acetylbenzocaine; PABA ϭ p-aminobenzoic acid; AcPABA ϭ N-acetyl-p-aminobenzoic acid.
b
Percentages are similar to receptor fluid values, since most of the absorbed dose was in the receptor
fluid.
Source: From Ref. 11.

Table 2 Percent of Applied Dose of Benzocaine Absorbed Over 24 h in Hairless
Guinea Pig Skin at Different Dose Levels
Dose level
Location

2 ␮g/cm2

40 ␮g/cm2

200 ␮g/cm2

Receptor fluid
Skin
Total absorbed

64.2 Ϯ 8.6
3.1 Ϯ 0.5
67.3 Ϯ 8.8

75.5 Ϯ 3.5
0.9 Ϯ 0.3

76.4 Ϯ 3.3

31.5 Ϯ 4.6
3.1 Ϯ 1.2
34.0 Ϯ 3.6

Values are the mean Ϯ SE for 1–6 determinations in each of 4 animals.
Source: From Ref. 11.


Cutaneous Metabolism During Absorption

5

Table 3 Metabolism of Methyl Salicylate in Hairless Guinea Pig Skin (percent
absorbed dose metabolized)
Viable skin
Sex

Salicyluric
acid

Salicylic
acid

Total

Nonviable skin,
salicylic acid

Male
Female

20.9 Ϯ 5.4
12.5 Ϯ 3.5

35.6 Ϯ 6.5a
12.3 Ϯ 2.5 a

56.5 Ϯ 5.1a,b
24.8 Ϯ 3.0a,b

38.3 Ϯ 5.0a,b
13.4 Ϯ 2.8a,b

Values are the mean Ϯ SE of determinations in 3 animals (3–4 repetitions per animal).
a
Significant male vs. female difference by the two-tailed t-test (p Ͻ .01).
b
Significant viable vs. nonviable skin difference, same sex (p Ͻ .05).
Source: From Ref. 11.

stable enzyme, and hydrolysis of methyl salicylate also occurred in nonviable
skin. However, no conjugation of salicylic acid was observed in nonviable skin.
Oxidation of benzyl alcohol was also observed in hairless guinea pig skin
(Table 4). Approximately 50% of the absorbed benzyl alcohol was oxidized to
benzoic acid in viable skin, with a small portion of this compound being further
metabolized to the glycine conjugate, hippuric acid. As with the ester, significant
activity was also observed in nonviable skin and greater oxidation of the alcohol
was obtained with male skin.

The absorption and metabolism of retinyl palmitate were measured to see if
ester hydrolysis and alcohol oxidation occurred with this cosmetic ingredient
(Table 5). Skin absorption for this lipophilic material is the sum of the absorbed
compound in skin and in the receptor fluid at the end of the 24-h study. Most of
the absorbed radiolabel remained in the skin. Substantial amounts of the absorbed

Table 4 Metabolism of Benzyl Alcohol in Hairless Guinea Pig Skin (percent absorbed
dose metabolized)
Viable skin
Sex

Hippuric
acid

Benzoic
acid

Total

Nonviable skin,
benzoic acid

Male
Female

8.5 Ϯ 1.9
4.1 Ϯ 1.8

44.2 Ϯ 8.0a
16.0 Ϯ 8.4a

52.7 Ϯ 9.6a
20.1 Ϯ 9.6a

44.0 Ϯ 11.2b
12.2 Ϯ 6.0b

Values are the mean Ϯ SE of determinations in 3 animals (3–4 repetitions per animal).
a
Marginally significant male vs. female difference by the two-tailed t-test (p Ͻ .08).
b
Significant male vs. female difference (p Ͻ .05).
Source: From Ref. 12.


6

Bronaugh et al.

Table 5 Percutaneous Absorption and Metabolism of Retinyl Palmitate
Skin

Receptor fluid

Radiolabel absorbed Metabolized Radiolabel absorbed Metabolized
(%)b
(%)c
(%)a,b
(%)c
Male guinea

pig
Female guinea
pig
Female human

29.8 Ϯ 4.5

38.2 Ϯ 13.0

0.5 Ϯ 0.2

100

33.4 Ϯ 2.3

30.2 Ϯ 16.3

0.6 Ϯ 0.3

100

17.9 Ϯ 1.3

43.9 Ϯ 5.0

0.2 Ϯ 0.01

100

Values are the mean Ϯ SE of determinations from 2 human donors (3–4 repetitions per donor) and 3

animals (3 repetitions per animal).
a
0–24 h fractions are combined.
b
Absorption is expressed as percent of applied dose in skin and receptor fluid.
c
Metabolism is expressed as percent of the absorbed retinyl palmitate hydrolyzed to retinol.
Source: From Ref. 12.

compound were hydrolyzed to retinol, but no oxidation of the alcohol to retinoic
acid was observed. Any effects of retinyl palmitate on the structure of skin may be
due to the formation of retinol during percutaneous absorption.
Absorption values from in vitro studies with viable hairless guinea pig skin
have been found to compare closely with in vivo results for phenanthrene (13),
pyrene, benzo[a]pyrene, and di(2-ethylhexyl) phthalate (14). Also, significant
metabolism was observed in vitro during the absorption of all four compounds.
Phenanthrene was metabolized in vitro to 9,10-dihydrodiol, 3,4-dihydrodiol,
1,2-dihydrodiol, and traces of hydroxy phenanthrenes (13). After topical administration of phenanthrene, approximately 7% of the percutaneously absorbed material was metabolized to the dihydrodiol metabolites.
Numerous metabolites of benzo[a]pyrene were formed during percutaneous
absorption through hairless guinea pig skin (14). Of particular interest was the
identification of benzo[a]pyrene 7,8,9,10-tetrahydrotetrol in the diffusion cell
receptor fluid. This metabolite is the hydrolysis product of the ultimate carcinogen, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This study
demonstrates that skin metabolism is likely responsible for skin tumors formed
following topical benzo[a]pyrene administration. In the earlier phenanthrene
study (13), no known carcinogenic metabolites were formed during skin permeation. This finding is consistent with the lack of tumorigenicity of phenanthrene
in rodents.
The percutaneous absorption and metabolism of trinitrobenzene have recently been examined in human, rat, and hairless guinea pig skin (15). Rapid absorption of trinitrobenzene was observed through human and animal skin. The two


Cutaneous Metabolism During Absorption

7

major metabolities found were 1,3,5-benzene triacetamide and 3,5-dinitroaniline.
It appears that nitro groups on trinitrobenzene can be reduced in skin to amino
groups, which are sometimes further metabolized by acetylation to an acetamide
derivative.
The effect of skin metabolism on the biological response to topically applied
chemicals is only beginning to be investigated. The task is complicated since skin
metabolism is difficult to measure in vivo without interference from systemic enzymes. In addition, certain metabolic systems in skin, such as cytochrome P-450,
have relatively low activity when compared with liver. In vitro studies indicate
that significant metabolism can occur during the percutaneous absorption process.

REFERENCES
1.
2.
3.

4.
5.
6.

7.
8.

9.
10.

11.

12.

Pannatier A, Jenner P, Testa B, Etter JC. The skin as a drug metabolizing organ. Drug
Metab Rev 1978; 8:319–343.
Smith LH, Holland JM. Interaction between benzo[a]pyrene and mouse skin in organ
culture. Toxicol. 1981; 24:47–57.
Holland JM, Kao JY, Whitaker MJ. A multisample apparatus for kinetic evaluation
of skin penetration in vitro: the influence of viability and metabolic status of skin.
Toxicol Appl Pharmacol. 1984; 72:272–280.
Chellquist EM, Reifenrath WG. Distribution and fate of diethyl malonate and diisopropyl fluorophosphate on pig skin in vitro. J Pharm Sci. 1988; 77:850–854.
Hawkins GS, Reifenrath WG, Influence of skin source, penetration cell fluid, and partition coefficient on in vitro skin penetration. J Pharm Sci. 1986; 75:378–381.
Collier SW, Sheikh NM, Sakr A, Lichtin JL, Stewart RF, Bronaugh RL. Maintenance
of skin viability during in vitro percutaneous absorption/metabolism studies. Toxicol
Appl Pharmacol. 1989; 99:522–533.
Bronaugh RL, Stewart RF, Strom JE. Extent of cutaneous metabolism during percutaneous absorption of xenobiotics. Toxicol Appl Pharmacol. 1989; 99:534–543.
Storm JE, Collier SW, Stewart RF, Bronaugh RL. Metabolism of xenobiotics during
percutaneous penetration: role of absorption rate and cutaneous enzyme activity. Fundam Appl Toxicol. 1990; 15:132–141.
Collier SW, Storm JE, Bronaugh RL. Reduction of azo dyes during in vitro percutaneous absorption. Toxicol Appl Pharmacol. 1993; 118:73–79.
Nathan D, Sakr A, Lichtin JL, Bronaugh RL. In vitro skin absorption and metabolism
of benzoic acid, p-aminobenzoic acid, and benzocaine in the hairless guinea pig.
Pharm Res. 1990; 7:1145–1151.
Kraeling MEK, Lipicky RJ, Bronaugh RL. Metabolism of benzocaine during percutaneous absorption in the hairless guinea pig: acetylbenzocaine formation and activity. Skin Pharmacol. 1996; 9:221–230.
Boehnlein J, Sakr A, Lichtin JL, Bronaugh RL. Characterization of esterase and alcohol dehydrogenase activity in skin: metabolism of retinyl palmitate to retinol (vitamin A) during percutaneous absorption. Pharm Res. 1994: 11:1155–1159.


8
13.

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Ng KME, Chu I, Bronaugh RL, Franklin CA, Somers DA. Percutaneous absorption/metabolism of phenanthrene in the hairless guinea pig: comparison of in vitro
and in vivo results. Fundam Appl Toxicol. 1991; 16:517–524.
14. Ng KME, Chu I, Bronaugh RL, Franklin CA, Somers DA. Percutaneous absorption
and metabolism of pyrene, benzo[a]pyrene, and di(2-ethylhexyl) phthalate: comparison of in vitro and in vivo results in the hairless guinea pig. Toxicol Appl Pharmacol.
1992; 115:216–223.
15. Kraeling MEK, Reddy G, Bronaugh RL. Percutaneous absorption of trinitrobenzene:
animal models for human skin. J Appl Toxicol 1998; 18:387–392.


2
Occlusion Does Not Uniformly
Enhance Penetration In Vivo
Daniel A. W. Bucks
University of California School of Medicine, San Francisco, and Dow
Pharmaceutical Sciences Inc., Petaluma, California

Howard I. Maibach
University of California School of Medicine, San Francisco, California

I. INTRODUCTION
Mammalian skin provides a relatively efficient barrier to the ingress of exogenous materials and the egress of endogenous compounds, particularly water.
Loss of this vital function results in death from dehydration. Compromised function is associated with complications seen in several dermatological disorders.
Stratum corneum intercellular lipid domains form a major transport pathway for
penetration (1–4). Perturbation of these lamellar lipids causes skin permeation
resistance to fall and has implicated their crucial role in barrier function. Indeed,
epidermal sterologenesis appears to be modulated by the skin’s barrier requirements (5).
Despite the fact that the skin is perhaps the most impermeable mammalian
membrane, it is permeable to a degree, that is, it is semipermeable; as such, the
topical application of pharmaceutical agents has been shown to be a viable route
of entry into the systemic circulation as well as an obvious choice in the treatment

of dermatological ailments. Of the various approaches employed to enhance the
percutaneous absorption of drugs, occlusion (defined as the complete impairment
of passive transepidermal water loss at the application site) is the simplest and perhaps one of the most common methods in use. In this chapter we have summarized
the literature to evaluate the effect of occlusion on the percutaneous absorption of
9