In Vitro Percutaneous
`Absorption:
`Principles, Fundamentals, and
`Applications
`
`Editors
`
`Robert L. Bronaugh, Ph.D.
`Supervisory Research Pharmacologist
`Division of Toxicological Studies
`U.S. Food and Drug Administration
`Washington, D.C.
`Howard I. Maibach, M.D.
`Professor of Dermatology
`School of Medicine
`University of California
`San Francisco, California
`
`CRC Press
`Boca Raton Ann Arbor Boston London
`
`  
`
`

 
`
`MYLAN - EXHIBIT 1026
`
`

`

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`
`Library of Congress Cataloging-in-Publication Data
`
`In vitro percutaneous absorption : principles, fundamentals, and
`applications / editors, Robert L. Bronaugh, Howard I. Maibach.
`p. cm
`Includes bibliographical references.
`Includes index.
`ISBN 0-8493-4748-3
`I. Skin absorption. 2. Skin absorption—Research—Methodology.
`3. Skin—Cultures and culture media.
`I. Bronaugh, Robert L., 1942-
`II. Maibach, Howard I.
`[DNLM: 1. Administration, Cutaneous. 2. Models, Biological.
`3. Skin-metabolism. 4. Skin Absorption-physiology. WR 102 135]
`QP88.5.146 1991
`612.7'91"dc20
`DNLM/DLC
`for Library of Congress
`
`90-15183
`CIP
`
`This book represents information obtained from authentic and highly regarded sources. Reprinted material is
`quoted with permission, and sources are indicated. A wide variety of references are listed. Every reasonable effort
`has been made to give reliable data and information, but the author and the publisher cannot assume responsibility
`for the validity of all materials or for the consequences of their use.
`
`All rights reserved. This book, or any parts thereof, may not be reproduced in any form without written consent
`from the publisher.
`
`Direct all inquiries to CRC Press, Inc., 2000 Corporate Blvd., N.W., Boca Raton, Florida 33431.
`
`® 1991 by CRC Press, Inc.
`
`International Standard Book Number 0-8493-4748-3
`
`Library of Congress Card Number 90-15183
`Printed in the United States
`
`

`

`Chapter 8
`
`EFFECTS OF OCCLUSION*
`
`D. Bucks, R. Guy, and H. Maibach
`
`TABLE OF CONTENTS
`
`Introduction
`
`II.
`
`Percutaneous Absorption of p-Phenylenediamine (PPDA) in Guinea Pigs
`
`III.
`
`Percutaneous Absorption of Volatile Compounds in Rhesus Monkeys
`
`IV.
`
`Percutaneous Absorption of Steroids in Man
`
`V.
`
`Percutaneous Absorption of Phenols in Man
`
`VI. Discussion
`
`References
`
`85
`
`86
`
`86
`
`86
`
`88
`
`92
`
`95
`
`113
`
`* Sections of this chapter have been adapted from the 2nd edition in this series on Percutaneous Penetration"
`and from the doctoral thesis entitled "Prediction of Percutaneous Absorption".12
`
`

`

`86
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`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 com­
`plications seen in several dermatological disorders. Stratum comeum intercellular lipid do­
`mains form a major transport pathway for penetration.1416'22 Perturbation of these lamellar
`lipids causes skin permeation resistance to fall and has implicated their crucial role in barrier
`appears to be modulated by the skin's barrier
`function.
`Indeed, epidermal sterologcnesjs
`in is perhaps the most impermeable mammalian requirements.31 Despite the fact that the ski
`
`membrane, 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 com­
`plete impairment of passive transepidermal water loss at the application site) is the simplest
`and most common method in use.
`The increased clinical efficacy of topical drugs caused by covering the site of application
`was first documented by Garb.21 Subsequently, Scholtz36 using fluocinolone acetonide, and
`Sulzberger and Witten37 using hydrocortisone, reported enhanced corticoid activity with
`occlusion in the treatment of psoriasis. The enhanced pharmacological effect of topical
`corticosteroids under occlusion was further demonstrated by the vasoconstriction studies of
`McKenzie29 and McKenzie and Stoughton.30 Occlusion has also been reported to increase
`the percutaneous absorption of various other topically applied compounds.9'18'26 27 However,
`as will be shown below, short term occlusion does not necessarily increase the percutaneous
`absorption of all chemicals.
`
`II. PERCUTANEOUS ABSORPTION OF p-PHENYLENEDIAMINE
`(PPDA) IN GUINEA PIGS
`
`The in vivo percutaneous absorption of PPDA from six occlusive patch test systems was
`investigated by Kim et al.27 The extent of absorption was determined using 14C radiotracer
`methodology. The 14C-PPDA was formulated as 1% PPDA in petrolatum (USP) and applied
`from each test system at a skin surface dose of 2 mg/cm2. Thus, the amount of PPDA was
`normalized with respect to the surface area of each patch test system (and, hence, to the
`surface area of treated skin). A sixfold difference in the level of skin absorption (p < 0.02)
`was found (Table 1).
`The rate of 14C excretion following topical application of the radiolabelled PPDA in the
`various patch test systems is shown in Figure 1. Clearly, the rate and extent of PPDA
`absorption was dependent upon the occlusive patch test system employed. It should be noted
`that a nonocclusive control study was not conducted.
`
`III. PERCUTANEOUS ABSORPTION OF VOLATILE
`'
`COMPOUNDS IN RHESUS MONKEYS
`
`The in vivo percutaneous absorption of two fragrances (safrole and cinnamyl anthranilate)
`and two chemical analogs (cinnamic alcohol and cinnamic acid) were measured under
`nonoccluded and plastic wrap (Saran Wrap®—a chlorinated hydrocarbon polymer) occluded
`conditions by Bronaugh et al.3 The extent of absorption following single dose administration
`was determined using 14C radiotracer methodology. Each compound was applied at a topical
`dose of 4 (xg/cm2 from a small volume of acetone. The fragrance materials were well absorbed
`through monkey skin. Plastic wrap occlusion of the application site resulted in large increases
`
`

`

`87
`
`TABLE 1
`Percutaneous Absorption of PPDA from Patch
`Test Systems"
`
`Patch test system
`
`mg PPDA
`in chamber
`
`Mean % dose
`absorbed (SD)
`
`Hill Top chamber
`Teflon (control)
`Small Finn chamber
`Large Finn chamber
`AL-Test chamber
`Small Finn chamber with paper disc insert
`
`40
`16
`16
`24
`20
`16
`
`53 (21)
`49 (9)
`30 (9)
`23 (7)
`8 (1)
`34 (20)
`
`Note: The rate of 14C excretion following topical application of the radiolabeled
`PPDA in the various patch test systems is shown in Figure 1. Clearly,
`the rate and extent of PPDA absorption was dependent upon the occlusive
`patch test system employed. It should be noted that a nonocclusive control
`study was not conducted.
`
`"
`
`2 mg/mm2 PPDA for 48 h on the dorsal mid-lumbar region of the guinea
`Pig-
`
`Data from Kim, H. O., Wester, R. C., McMaster, J. R., Bucks, D. A. W.,
`and Maibach, H. I., Contact Dermatitis, 17, 178, 1987.
`
`3
`O
`I
`G>
`w o
`Q
`
`1.2 -1
`
`1.0 -
`
`0.8 "
`
`0 . 6 "
`
`0.4-
`
`0.2 "
`
`0.0
`
`0
`
`-0- HTC
`TEFLON
`SM FINN W PAPER
`SMALL FINN
`LARGE FINN
`AL TEST
`
`T
`20
`
`•a
`T
`T
`60
`40
`80
`Midpoint (Hrs.)
`
`T
`100
`
`1
`120
`
`In vivo percutaneous absorption of PPDA (2 mg/mm2) following a
`FIGURE 1.
`48 h exposure on the dorsal lumbar region of guinea pigs (Redrawn from Kim, H.
`O., Wester, R. C., McMaster, J. R., Bucks, D, A. W., and Maibach, H. I., Contact
`Dermatitis, 17, 178, 1987.)
`
`in absorption (see Table 2). The authors also presented in vitro data documenting the
`significant increase in percutaneous absorption of these chemicals under occluded compared
`to nonoccluded conditions.
`Investigation of the effect of occlusion on the percutaneous absorption of six additional
`volatile compounds (benzyl acetate, benzamide, benzoin, benzophenone, benzyl benzoate,
`and benzyl alcohol) was conducted using the same in vivo methodology. These studies
`included occlusion of the site of application with a glass cylinder (secured to the skin by
`
`

`

`88
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`TABLE 2
`In Vivo Percutaneous Absorption of
`Fragrances in Monkeys
`
`% Dose absorbed-
`Nonprotected
`Plastic wrap occlusion
`
`Cinnamyl anthranilate
`Safrole
`Cinnamic alcohol
`Cinnamic acid
`
`26.1 (4.6)
`4.1 (1.6)
`25.4 (4.4)
`38.6 (16.6)
`
`39.0 (5.6)
`13.3 (4.6)
`74.6 (14.4)
`83.9 (5.4)
`
`Note: 24-h exposure at 4 (xg/cm2 prior to soap and water washing.
`
`• Single dose application; values corrected for incomplete renal elim­
`ination. Mean ± SD (N = 4).
`
`Data from Bronaugh, R. L., Stewart, R. F., Wester, R. C., Bucks, D.,
`and Maibach, H. I., Fd. Chem. Toxicol., 23, 111, 1985.
`
`TABLE 3
`In Vivo Percutaneous Absorption of Benzyl Derivatives in Monkeys
`
`Nonprotected
`
`% Dose absorbed"
`Plastic wrap occlusion
`
`Glass chamber occlusion
`
`Log Ko/w
`
`Benzamide
`Benzyl alcohol
`Benzoin
`Benzyl acetate
`Benzophenone
`Benzyl benzoate
`
`47 (14)
`32 (9)
`49 (6)
`35 (19)
`44 (15)
`57 (21)
`
`85 (8)
`56 (29)
`43 (12)
`17 (5)
`69 (12)
`71 (9)
`
`Note: 24-h exposure at 4 fig/cm2 prior to soap and water washing.
`
`73 (20)
`80 (15)
`77 (4)
`79 (15)
`69 (10)
`65 (20)
`
`0.64
`0.87
`1.35
`1.96
`3.18
`3.97
`
`• Single dose application; values corrected for incomplete renal elimination. Mean ± SD (N = 4).
`
`Data from Bronaugh, R. L., Wester, R. C., Bucks, D. A. W., and Maibach, H. I., Fd. Chem. Toxicol., 28, 369,
`1990.
`
`silicone glue) capped with Parafilm, occlusion with plastic wrap, and nonprotected condi­
`tions.4 As shown in Table 3, occlusion, in general, enhances the percutaneous absorption
`of these compounds. However, differences in percutaneous absorption were observed be­
`tween plastic wrap and "glass chamber" occlusive conditions. The absorption of benzyl
`acetate was lower under plastic wrap compared to the nonprotected condition, whereas glass
`chamber occlusion resulted in the greatest bioavailability. This discrepancy might be due to
`compound; sequestration by the plastic wrap.
`
`IV. PERCUTANEOUS ABSORPTION OF STEROIDS IN MAN
`
`The earliest attempt to correlate the increased pharmacological effect of hydrocortisone
`under occlusive conditions with the pharmacokinetics of absorption was reported by Feld-
`mann and Maibach.18 In this study, the rate and extent of 14C-label excretion into the urine
`following topical application of 14C-hydrocortisone to the ventral forearm of normal human
`volunteers were measured. Radiolabeled hydrocortisone (75 fig) was applied in acetone
`solution (1000 |xl) as a surface deposit over 13 cm2 of skin. The authors estimated that this
`
`

`

`89
`
`was equivalent to a sparing application of a 0.5% hydrocortisone topical preparation (5.8
`(xg/cm2). The site of application was either nonprotected or occluded with plastic wrap (Saran
`Wrap®). When the skin was unprotected, the dosing site was washed 24 h post application.
`On the other hand, when the skin was occluded, the plastic wrap remained in place for
`96 h (4 d) post application before the application site was washed. The % of the applied
`dose excreted into the urine, corrected for incomplete renal elimination, was (mean ± SD)
`0.46 ± 0.20 and 5.9 ± 3.5 under nonprotected and occluded conditions, respectively (see
`Tables 4, 5, and Figure 2). These numbers differ from those in the original paper which
`were calculated incorrectly. A paired t test of the results indicates a significant difference
`(p = 0.01) in cumulative absorption of hydrocortisone between two exposure conditions.
`Quantitatively, the occlusive condition employed increased the cumulative absorption of
`hydrocortisone by about an order of magnitude. However, note that the occlusive system
`retained the drug in contact with the skin for 96 h compared to the 24 h exposure period
`under nonprotected conditions.
`Guy et al.26 investigated the effect of occlusion on the percutaneous absorption of steroids
`in vivo following single and multiple application. The extent of absorption of four steroids
`(progesterone, testosterone, estradiol, and hydrocortisone), using radiotracer elimination into
`the urine following topical application to the ventral forearm of male volunteers, was reported.
`The chemical dose was 4 fig/cm2 and application area 2.5 cm2. The 14C-labeled chemicals
`were applied in 20 p,l acetone. In the occlusive studies, after evaporation of the vehicle,
`the site of application was covered with a plastic (polyethylene-vinyl acetate copolymer)
`chamber.34 In all cases, after 24 h, the site of application was washed with soap and water
`using a standardized procedure.5 In the occlusive studies, the administration site was then
`recovered with a new chamber. An essentially identical protocol was also performed fol­
`lowing a multiple dosing regimen.6 Daily topical doses of three of the steroids (testosterone,
`estradiol, and hydrocortisone) were administered over a 14 d period. The first and eighth
`doses were 14C-labeled and urinary excretion of radiolabel was followed. As above, the 24 h
`washing procedure was performed daily and a new chamber applied. Occlusive chambers
`and washes were collected and assayed for residual surface chemical. The results of this
`study are in Table 6. Steroid percutaneous absorption as a function of penetrant octanol-
`water partition coefficient (Ko/w) is shown in Figure 3. The studies indicate that:
`
`The single-dose measurements of the percutaneous absorption of hydrocortisone, es­
`tradiol, and testosterone are predictive of percutaneous absorption following a com­
`parable multiple dose regimen (see chapter on the effect of repetitive application),
`under both occluded and nonoccluded conditions.
`Occlusion significantly (p < 0.05) increased the percutaneous absorption of estradiol,
`testosterone, and progesterone, but not that of hydrocortisone.
`Percutaneous absorption increases with increasing Ko/w up to testosterone but declines
`for progesterone, under occluded and nonoccluded conditions.
`The occlusive procedure generally permits excellent dose accountability (Table 7).
`
`2.
`
`4
`
`( (
`
`protected" (i.e., cov-
`The percutaneous absorption of these same four steroids under
`ered, but nonocclusive) conditions has also been measured in vivo9'10 using the same meth­
`odology. The data obtained from these later experiments permitted the effect of occlusion
`to be rigorously assessed (since complete mass balance of the applied dose was possible).
`With the exception of hydrocortisone (Table 8), occlusion significantly increased the per­
`cutaneous absorption (p < 0.01) of the steroids. These results were in excellent agreement
`with the comparable nonprotected studies described above. As stated before, excellent dose
`accountability was reported (Table 9).
`To investigate the apparent discrepancy between the effect of plastic wrap occlusion18
`
`

`

`02
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`
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`
`Data from Feldmann, R. and Maibach, H. L, Arch. Dermatol., 91, 661, 1965.
`
`b Nonprotected, site of application washed 24-h postapplication with soap and water.
`a Topical urinary excretion values calculated using an IV correction factor of 62.9%.
`
`0.32
`0.74
`0.33
`0.73
`0.46
`1.14
`0.67
`1.08
`
`0.02
`0.02
`0.00
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`0.12
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`0.18
`0.05
`0.09
`
`0.03
`0.08
`0.03
`0.11
`0.09
`0.12
`0.07
`0.08
`
`0.06
`0.09
`0.03
`0.07
`0.04
`0.12
`0.09
`0.17
`
`0.06
`0.09
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`0.16
`0.01
`0.15
`0.09
`0.07
`
`0.07
`0.13
`0.07
`0.11
`0.05
`0.19
`0.11
`0.23
`
`0.05
`0.07
`0.03
`0.01
`0.05
`0.16
`0.06
`0.11
`
`0.05
`0.07
`0.11
`0.00
`0.05
`0.03
`0.11
`0.11
`
`SD
`Mean
`
`6
`5
`4
`3
`2
`1
`
`Total %D
`
`Day 10
`
`Day 9
`
`Day8
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`0—12 H
`
`Sbj
`
`Percutaneous Absorption3 of 14C after Topical Administration of 14C Hydrocortisone
`
`under Nonprotected11 Conditions (% of Dose)
`
`TABLE 4
`
`

`

`so
`
`Data from Feldmann, R. and Maibach, H. I., Arch. Dermatol., 91, 661, 1965.
`
`b Occluded with plastic wrap for 4 d, site of application washed 96-h postapplication with soap and water.
`a Topical urinary excretion values calculated using an IV correction factor of 62.9%.
`
`5.52
`9.46
`19.10
`2.86
`5.65
`9.09
`9.66
`10.39
`
`0.10
`0.13
`0.17
`0.00
`0.01
`0.18
`0.21
`0.21
`
`0.10
`0.19
`0.23
`0.03
`0.10
`0.21
`0.27
`0.30
`
`0.13
`0.32
`0.39
`0.10
`0.32
`0.45
`0.39
`0.28
`
`0.10
`0.45
`0.42
`0.35
`0.38
`0.57
`0.59
`0.41
`
`0.43
`0.69
`1.44
`0.22
`0.64
`0.64
`0.82
`0.37
`
`0.65
`1.12
`1.99
`0.24
`0.58
`1.08
`1.19
`1.66
`
`0.75
`1.38
`2.53
`0.21
`1.35
`1.38
`1.19
`1.62
`
`1.04
`1.63
`3.09
`0.24
`0.74
`1.93
`2.26
`1.51
`
`1.01
`1.54
`3.12
`0.51
`0.45
`1.78
`2.00
`1.37
`
`0.63
`0.79
`1.86
`0.19
`0.95
`0.16
`0.60
`0.98
`
`1.41
`1.22
`3.86
`0.78
`0.13
`0.72
`0.14
`1.69
`
`SD
`Mean
`
`6
`5
`4
`3
`2
`1
`
`Total %D
`
`Day 10
`
`Day 9
`
`Day 8
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`0—12 H
`
`Sbj
`
`Percutaneous Absorption3 of 14 C after Topical Administration of 14 C Hydrocortisone
`
`under Occluded15 Conditions (% of Dose)
`
`TABLES
`
`

`

`92
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`0.12
`
`•
`
`0 . 1 0 "
`
`-B- OCCLUDED
`NON-PROTECTED
`
`GC
`X
`UJ
`
`w o Q
`
`a?
`
`0.08"
`
`0 . 0 6 -
`
`0.04-
`
`0.02-
`
`0.00
`
`JP
`0
`
`48
`
`144
`96
`HOURS
`
`T
`
`192
`
`240
`
`FIGURE 2. Percutaneous absorption of hydrocortisone in man. Human
`96 h occluded versus 24-h nonprotected exposure of hydrocortisone at 4 n.g/
`cm2 prior to soap and water washing. Occlusion was with plastic wrap. (Data
`from Feldmann, R. J. and Maibach, H. I., Arch Dermatol., 91, 661, 1965.)
`
`TABLE 6
`Percutaneous Absorption of Steroids in Man
`
`Mean % applied dose
`absorbed (± SD)
`Occlusion
`Nonprotected
`
`Hydrocortisone
`Single application
`Multiple application:
`1st Dose
`8th Dose
`Estradiol
`Single application
`Multiple application:
`1st Dose
`8th Dose
`Testosterone
`Single application
`Multiple application:
`1st Dose
`8th Dose
`Progesterone
`Single application
`
`2 ± 2"
`
`3 ± 1
`3 ± 1
`
`11 ± 5°
`
`10 ± 2
`11 ± 5
`
`13 ± 3a
`
`21 ± 6
`20 ± 7
`
`11 ± 6"
`
`4 ± 2
`
`4 ± 1
`3 ± 1
`
`27 ± 6
`
`38 ± 8
`22 + 1
`
`46 ± 15
`
`51 ± 10
`50 ± 9
`
`33 ± 9
`
`a Data from Feldmann, R. and Maibach, H. I., J. Invest.
`Dermatol., 52, 89, 1969.
`
`and that of the plastic chamber on hydrocortisone absorption,26 we repeated the measurements
`of penetration using plastic wrap (Saran Wrap®) with the experimental protocol of Guy et
`al.26 Under these circumstances, we found no difference between plastic wrap and plastic
`chamber occlusion on the percutaneous absorption of hydrocortisone (Table 10).
`
`V. PERCUTANEOUS ABSORPTION OF PHENOLS IN MAN
`
`We subsequently investigated the effect of occlusion on the in vivo percutaneous ab­
`sorption of phenols following single dose application. The occlusive and protective chamber
`
`

`

`Q
`LLl
`
`m oc
`O
`U)
`ffl
`<
`LU
`ir.) o
`
`Q
`
`93
`
`60 i
`
`50 -
`
`40 "
`
`30 -
`
`20 -
`
`1 0 -
`
`0
`
`1
`
`-o- OCCLUDED
`NON-PROTECTED
`
`PG
`
`ES
`
`TS
`
`HC
`
`T
`2
`
`T
`3
`
`Log Ko/w
`
`1
`4
`
`FIGURE 3. Percutaneous absorption of 4 steroids (HC = hydrocortisone, ES
`= estradiol, TS = testosterone, PG = progesterone) in man as a function of
`penetrant octanol/water partition coefficient. Exposure period 24-h at
`4 |Ag/cm2 prior to soap and water washing. (Redrawn from Guy et al., in Skin
`Pharmacokinetics, Shroot, B. and Schaefer, H., Eds., Karger, Basel, 1987,
`70.)
`
`TABLE 7
`Accountability of Applied Dose in Occluded Studies"
`
`Observed PA % Removed from skin
`
`Total % dose
`
`Hydrocortisone
`Single doseb
`1st MD0
`8th MDd
`Estradiol
`Single doseb
`1st MDC
`8th MDd
`Testosterone
`Single doseb
`1st MDC
`8th MDd
`Progesterone
`Single doseb
`
`4 ± 2
`4 ± 1
`3 ± 1
`
`27 ± 6
`38 ± 8
`22 ± 7
`
`46 ± 15
`51 ± 10
`50 ± 9
`
`33 ± 9
`
`64 + 5
`82 ± 5
`78 ± 2
`
`60 ± 12
`62 ± 6
`59 ± 8
`
`44 ± 7
`48 ± 9
`42 ± 9
`
`47 ± 10
`
`68 ± 4
`85 ± 4
`81 ± 3
`
`87 ± 13
`100 ± 4
`81 ± 6
`
`90 ± 8
`99 ± 4
`92 ± 17
`
`80 ± 6
`
`Note: Mean (in % applied dose) ± SD
`
`• 24-h exposure at 4 |ig/cra2 prior to soap and water washing. Occlusion was
`with a plastic (Hilltop) chamber.
`b Single dose study.
`c First dose of a 14-d multiple-dose study.
`d Eighth dose of a 14-d multiple-dose study.
`
`Data from Bucks, D. A. W., et al., Unpublished observations.
`
`

`

`94
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`TABLE 8
`Percutaneous Absorption of Steroids in Man
`Single Dose Application for 24 h @ 4 (xg/cm2
`
`Mean % dose absorbed (± SD; N & 5)
`Protected"
`Occluded1"
`
`Hydrocortisone
`Estradiol
`Testosterone
`Progesterone
`
`4 ± 2
`3 ± 1
`18 ± 9
`13 ± 6
`
`• Ventilated plastic chamber.
`b Occlusive plastic chamber.
`
`4 ± 2
`27 ± 6
`46 ± 15
`33 ± 9
`
`Data from Guy, R. H., Bucks, D. A. W., McMaster, J. R., Vil-
`laflor, D. A., Roskos, K. V., Hinz, R. S., and Maibach, H. I.,
`in Skin Pharmacokinetics, Shroot, B. and Schaefer, H., Eds., Kar-
`ger, Basel, 70, 1987.; Bucks, D. A. W., Maibach, H. I., and Guy,
`R. H., in Percutaneous Absorption, Vol. 2, Bronaugh, R. and
`Maibach, H., Eds., Marcel Dekker, New York, 1989, 77.; and,
`Bucks, D. A. W., McMaster, J. R., Maibach, H. I., and Guy, R.
`H., J. Invest. Dermatol., 90, 29, 1988.
`
`TABLE 9
`Accountability of Applied Dose in Protected Studies using
`Ventilated Plastic Chambers"
`
`Observed PA % Removed from skin
`
`Total % dose
`
`Hydrocortisone
`Estradiol
`Testosterone
`Progesterone
`
`4 ± 2
`3 ± 1
`18 ± 9
`13 ± 6
`
`85 + 6
`96 ± 1
`77 ± 8
`82 ± 7
`
`89 ± 6
`100 ± 1
`96 ± 2
`96 ± 3
`
`Note: Mean % dose (± SD, N a 5).
`
`" Single dose application for 24 h at 4 (xg/cm2
`
`Data from Bucks et al.9'10
`
`TABLE 10
`Percutaneous Absorption of
`Hydrocortisone in Man
`
`% Dose absorbed"
`
`Plastic wrap occlusion
`Plastic chamber occlusion0
`"Protected" condition"1
`
`4.7 (2.1)"
`4.0 (2.4)
`4.4 (1.7)
`
`• Single dose application for 24 h at 4 |xg/cm2; values
`corrected for incomplete renal elimination.
`b Mean ± SD (N = 6)
`c Guy et al.26
`^ Bucks et al.9'10
`
`

`

`95
`
`methodology described by Bucks et al.7,10 was utilized. Nine 14C ring labelled para-substituted
`phenols (4-aminophenol, 4-acetamidophenol, 4-propionylamidophenol, phenol, 4-cyano-
`phenol, 4-nitrophenol, 4-iodophenol, 4-heptyloxyphenol and 4-pentyloxyphenol) were used.
`As in the earlier steroid studies, the site of application was the ventral forearm of male
`volunteers and the area of application 2.5 cm2. Penetrants were applied in 20 (xl ethanol
`(95%). The chemical dose was 2 to 4 |ig/cm2. After vehicle evaporation, the application
`site was covered with either an occlusive or protective device. After 24 h, the patch was
`removed and the site washed with a standardized procedure.5 The application site was then
`recovered with a new chamber of the same type. Urine was collected for seven days. On
`the seventh day: (1) the second chamber was removed, (2) the dosing site was washed with
`the same procedure, and (3) the upper layers of stratum comeum from the application site
`were removed by cellophane tape stripping. Urine, chambers, washes, and skin tape strips
`were collected and assayed for radiolabel. Percutaneous absorption of each compound under
`protected and occluded conditions is presented in Tables 11 through 19 and Figures 4 through
`12. Phenol percutaneous absorption as a function of the penetrant octanol-water partition
`coefficient (Ko/w) is shown in Figure 13. Phenol percutaneous absorption is summarized
`in Table 20. The methodology permitted excellent dose accountability (Tables 21 and 22).
`The studies indicate that:
`
`Occlusion significantly increased (unpaired t test, p < 0.05) the penetration of phenol,
`heptyloxyphenol and pentyloxyphenol.
`Occlusion did not enhance the absorption of aminophenol, 4-acetamidophenol, pro-
`pionylamidophenol, cyanophenol, nitrophenol, and iodophenol.
`The methodology employed again permitted excellent dose accountability.
`VI. DISCUSSION
`
`A predominant effect of occlusion is to increase hydration of the stratum comeum,
`thereby swelling the comeocytes, and promoting the uptake of water into intercellular lipid
`domains. The normal water content of stratum comeum is 5 to 15%, a value which can be
`increased up to 50% by occlusion.1,33 Upon removal of a plastic occlusive dressing after
`24 h of contact, transepidermal water loss values are increased by an order of magnitude;10
`the elevated rate then returns rapidly (~ 15 minutes) to normal with extraneous water
`dissipation. With occlusion, skin temperature generally increases from 320C to as much as
`370C.28 Faergemann et al.17 showed that occlusion: (1) increases the transepidermal flux of
`chloride and carbon dioxide, (2) increases microbial counts on skin, and (3) increases the
`surface pH of skin from a preoccluded value of 5.6 to 6.7. Anhidrosis results from occlu­
`sion.32'23 Plastic chamber occlusion can also cause skin irritation (personal observation).
`Occlusion-induced increases in mitotic rate of skin and epidermal thickening have been
`documented by Fisher and Maibach.20
`With respect to percutaneous absorption, occlusion (or a protective cover) prevents loss
`of tfie surface deposited chemical by friction and/or exfoliation; bioavailability may, thereby,
`be increased. However, comparison of the data in Tables 6 and 8, for the percutaneous
`absorption of steroids under nonprotected and protected conditions, shows clearly that the
`potential increase in bioavailability from protection of the site of application does not explain
`the increase in steroid absorption under occluded conditions.
`Occlusion does not necessarily increase percutaneous absorption. Hydrocortisone ab­
`sorption under occluded conditions was not enhanced in single dose or multiple dose ap­
`plication studies (Table 23).
`This lack of penetration enhancement under occluded conditions has been observed with
`certain para-substituted phenols. However, a trend of occlusion-induced absorption enhance-
`
`

`

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`
`From Bucks, D. A. W., Lee, M., Maibach, H. L, and Guy, R. H., in preparation.
`
`a Site of application washed 24-h postapplication with soap and water.
`
`2.78
`5.95
`9.50
`7.60
`4.60
`2.89
`3.11
`7.99
`
`0.07
`0.20
`0.12
`0.19
`0.13
`0.16
`0.28
`0.29
`
`0.11
`0.26
`0.26
`0.12
`0.25
`0.22
`0.44
`0.29
`
`0.12
`0.40
`0.59
`0.45
`0.22
`0.41
`0.41
`0.32
`
`0.12
`0.57
`0.43
`0.73
`0.59
`0.52
`0.46
`0.68
`
`0.50
`1.18
`1.72
`1.68
`0.85
`0.67
`0.69
`1.49
`
`1.34
`1.98
`3.63
`2.70
`1.66
`0.58
`0.33
`2.97
`
`0.82
`0.98
`2.14
`1.25
`0.63
`0.21
`0.05
`1.60
`
`0.16
`0.22
`0.46
`0.30
`0.19
`0.07
`0.03
`0.29
`
`0.08
`0.11
`0.13
`0.13
`0.07
`0.04
`0.26
`0.04
`
`0.06
`0.05
`0.02
`0.05
`0.02
`0.01
`0.17
`0.03
`
`SD
`Mean
`
`F
`E
`D
`c
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`8—12 H
`
`4—8H
`
`0—4H
`
`Subjects
`
`Percutaneous Absorption of Aminophenol Under Protected Conditions3 in Man (% of Dose)
`
`TABLE 11B
`
`2.63
`8.08
`6.07
`9.95
`11.78
`6.15
`6.43
`
`0.14
`0.20
`0.20
`0.15
`0.10
`0.12
`0.45
`
`0.13
`0.28
`0.22
`0.27
`0.18
`0.23
`0.50
`
`0.08
`0.42
`0.35
`0.38
`0.48
`0.38
`0.54
`
`0.21
`0.60
`0.52
`0.88
`0.31
`0.59
`0.67
`
`0.43
`1.47
`0.96
`1.95
`1.78
`1.54
`1.09
`
`1.68
`3.27
`2.40
`3.31
`6.16
`2.10
`2.36
`
`0.89
`1.50
`1.08
`2.33
`2.55
`1.05
`0.51
`
`0.09
`0.19
`0.18
`0.32
`0.17
`0.08
`0.19
`
`0.12
`0.13
`0.16
`0.32
`0.06
`0.03
`0.08
`
`0.02
`0.02
`0.00
`0.02
`0.00
`0.03
`0.04
`
`SD
`Mean
`
`E
`D
`C
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`8—12 H
`
`4—8H
`
`0—4 H
`
`Subjects
`
`Percutaneous Absorption of Aminophenol Under Occluded Conditions3 in Man (% of Dose)
`
`TABLE 11A
`
`

`

`5
`
`From Bucks, D. A. W., Lee, M., Maibach, H. I., and Guy, R. H., in preparation.
`
`a Site of application washed 24-h postapplication with soap and water.
`
`2.64
`3.74
`6.62
`1.13
`1.45
`4.77
`1.71
`6.79
`
`0.48
`0.66
`1.34
`0.19
`0.32
`0.70
`0.29
`1.14
`
`0.40
`0.59
`1.10
`0.21
`0.24
`0.73
`0.28
`0.97
`
`0.33
`0.52
`0.84
`0.18
`0.23
`0.60
`0.29
`0.96
`
`0.62
`0.80
`1.37
`0.20
`0.23
`1.13
`0.32
`1.55
`
`0.43
`0.51
`0.91
`0.18
`0.16
`0.56
`0.13
`1.12
`
`0.31
`0.43
`0.57
`0.11
`0.13
`0.76
`0.24
`0.77
`
`0.12
`0.15
`0.36
`0.02
`0.08
`0.14
`0.14
`0.20
`
`0.02
`0.03
`0.07
`0.01
`0.04
`0.01
`0.02
`0.02
`
`0.03
`0.04
`0.03
`0.01
`0.01
`0.08
`0.01
`0.06
`
`0.02
`0.01
`0.02
`0.01
`0.00
`0.06
`0.00
`0.00
`
`SD
`Mean
`
`F
`E
`D
`C
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`8—12 H
`
`4—8H
`
`0—4H
`
`Subjects
`
`Percutaneous Absorption of Acetaminophen Under Protected Conditions* in Man (% of Dose)
`
`TABLE 12B
`
`2.28
`3.19
`5.80
`2.60
`1.11
`6.00
`0.61
`3.01
`
`0.28
`0.42
`0.65
`0.35
`0.15
`0.79
`0.09
`0.48
`
`0.27
`0.40
`0.73
`0.39
`0.11
`0.68
`0.11
`0.40
`
`0.31
`0.44
`0.84
`0.30
`0.16
`0.77
`0.09
`0.47
`
`0.36
`0.47
`0.66
`0.48
`0.16
`1.06
`0.09
`0.37
`
`0.52
`0.61
`0.77
`0.56
`0.17
`1.52
`0.09
`0.56
`
`0.42
`0.49
`1.16
`0.41
`0.17
`0.77
`0.02
`0.42
`
`0.11
`0.14
`0.27
`0.09
`0.07
`0.12
`0.01
`0.26
`
`0.16
`0.13
`0.43
`0.02
`0.08
`0.19
`0.01
`0.05
`
`0.09
`0.09
`0.27
`0.00
`0.06
`0.09
`0.09
`0.01
`
`0.01
`0.01
`0.02
`0.00
`0.00
`0.01
`0.02
`0.00
`
`SD
`Mean
`
`F
`E
`D
`C
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24H
`
`8—12 H
`
`4—8H
`
`0—4H
`
`Subjects
`
`Percutaneous Absorption of Acetaminophen Under Occluded Conditions3 in Man (% of Dose)
`
`TABLE 12A
`
`

`

`as
`
`rs
`sr:
`Ss
`
`ft-
`&> 3
`S sr
`as
`2
`a
`g
`
`2.
`2.
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`^3
`-t
`05
`to
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`Oa
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`3
`a
`S
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`j*.
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`^5 o
`
`s
`
`00
`VC
`
`From Bucks, D. A. W., Lee, M., Maibach, H. L, and Guy, R. H., in preparation.
`
`a Site of application washed 24-h postapplication with soap and water.
`
`6.97
`11.10
`10.77
`23.08
`6.77
`5.85
`9.03
`
`0.45
`0.74
`0.23
`1.14
`0.34
`1.23
`0.78
`
`0.58
`0.95
`0.31
`1.79
`0.66
`0.73
`1.28
`
`0.51
`0.92
`0.51
`1.75
`1.06
`0.52
`0.77
`
`0.82
`1.63
`1.03
`2.96
`1.35
`0.96
`1.83
`
`2.12
`2.58
`1.93
`6.34
`1.58
`1.36
`1.68
`
`2.34
`2.76
`3.36
`6.52
`1.02
`0.77
`2.14
`
`1.06
`1.11
`2.17
`2.36
`0.47
`0.17
`0.39
`
`0.45
`0.31
`1.11
`0.21
`0.10
`0.06
`0.06
`
`0.05
`0.06
`0.12
`0.00
`0.12
`0.02
`0.05
`
`0.03
`0.03
`0.01
`0.00
`0.07
`0.03
`0.04
`
`SD
`Mean
`
`E
`D
`C
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`8—12 H
`
`4—8H
`
`0—4H
`
`Subjects
`
`Percutaneous Absorption of Propionylamidophenol Under Protected Conditions* in Man (% of Dose)
`
`TABLE 13B
`
`9.11
`18.88
`31.54
`8.55
`9.43
`25.85
`16.73
`21.15
`
`0.57
`0.92
`0.65
`0.39
`0.58
`1.99
`0.91
`0.98
`
`1.07
`1.18
`0.48
`0.64
`0.53
`3.20
`1.56
`0.67
`
`2.07
`2.18
`1.19
`0.54
`1.43
`6.30
`1.85
`1.75
`
`1.09
`2.42
`2.06
`1.02
`1.45
`3.53
`2.81
`3.64
`
`0.86
`3.51
`4.04
`2.31
`2.60
`3.91
`3.72
`4.50
`
`3.66
`5.33
`11.9
`2.74
`2.22
`3.56
`4.42
`7.17
`
`1.23
`1.57
`3.77
`0.71
`0.43
`1.12
`1.25
`2.14
`
`1.74
`1.08
`4.52
`0.13
`0.08
`1.29
`0.19
`0.27
`
`1.00
`0.56
`2.55
`0.06
`0.11
`0.56
`0.02
`0.02
`
`0.20
`0.13
`0.39
`0.00
`0.00
`0.40
`0.00
`0.00
`
`SD
`Mean
`
`F
`E
`D
`C
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`8—12 H
`
`4—8H
`
`0—4 H
`
`Subjects
`
`Percutaneous Absorption of Propionylamidophenol Under Occluded Conditions* in Man (% of Dose)
`
`TABLE 13A
`
`

`

`V6
`sc
`
`From Bucks, D. A. W., Lee, M., Maibach, H. I., and Guy, R. H., in preparation
`
`a Site of application washed 24-h postapplication with soap and water.
`
`Note: Data corrected for 21.2% of the applied dose evaporating from the skin surface upon application.
`
`6.31
`23.59
`28.59
`21.89
`23.42
`21.26
`14.09
`32.27
`
`0.07
`0.12
`0.08
`0.10
`0.08
`0.10
`0.25
`0.10
`
`0.04
`0.15
`0.11
`0.15
`0.09
`0.15
`0.21
`0.16
`
`0.05
`0.12
`0.15
`0.07
`0.08
`0.11
`0.20
`0.11
`
`0.07
`0.19
`0.24
`0.13
`0.15
`0.19
`0.32
`0.13
`
`0.14
`0.41
`0.20
`0.39
`0.52
`0.34
`0.38
`0.61
`
`1.83
`4.01
`2.57
`4.62
`2.85
`4.29
`2.48
`7.26
`
`2.66
`6.79
`7.51
`6.99
`6.57
`6.99
`2.20
`10.5
`
`1.47
`4.36
`5.04
`3.34
`5.34
`4.25
`2.07
`6.11
`
`2.85
`4.68
`8.49
`3.41
`5.63
`1.30
`2.14
`7.08
`
`1.47
`2.76
`4.20
`2.69
`2.10
`3.53
`3.85
`0.22
`
`SD
`Mean
`
`F
`E
`D
`C
`B
`A
`
`Total %D
`
`Day 7
`
`Day 6
`
`Day 5
`
`Day 4
`
`Day 3
`
`Day 2
`
`12—24 H
`
`8—12 H
`
`4—8H
`
`0—4H
`
`Subjects
`
`Percutaneous Absorption of Phenol Under Protected Conditions11 in Man (% of Dose)
`
`TABLE 14B
`
`4.04
`33.90
`32.93
`32.78
`27.14
`35.47
`35.99
`39.08
`
`0.04
`0.04
`0.00
`0.04
`0.02
`0.