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
`
`CRCPress
`Boca Raton Ann Arbor Boston London
`
`  
`
`

 
`
`MYLAN- EXHIBIT 1026
`
`

`

` é Ue { ee, 2
`
`[Pps ee ts
`
`-
`
`<
`,
`
`/
`
`Lb} 9
`
`nnneae
`R aR
`
`>G
`
`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
`
`2. Skin absorption--Research--Methodology.
`1. Skin absorption.
`3. Skin--Cultures and culture media.
`I. Bronaugh, Robert L., 1942-
`IE. Maibach, Howard [.
`2. Models, Biological.
`[DNLM:
`1. Administration, Cutaneous.
`
`3. Skin--metabolism. 4. Skin Absorption--physiology.|WR 102 135]
`QP88.5.146
`1991
`612.7'91--dce20
`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 madeto give reliable data and information, but the author and the publisher cannot assume responsibility
`for the validity of all materials or for the consequencesoftheir 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 Bivd., 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
`
`

`

`85
`
`Chapter 8
`
`|
`
`EFFECTS OF OCCLUSION*
`
`D. Bucks, R. Guy, and H. Maibach
`
`TABLE OF CONTENTS
`
`I.
`
`Introduction... 1. cee een een eee eee een enon EEE CEE EEE EES 86
`
`Il.
`In.
`
`Percutaneous Absorption of p-Phenylenediamine (PPDA) in Guinea Pigs........ 86
`Percutaneous Absorption of Volatile Compounds in Rhesus Monkeys ........... 86
`
`IV.
`
`Percutaneous Absorption of Steroids in Man.......... 0c. cee cece eee eee e eevee 88
`
`V.
`
`Percutaneous Absorption of Phenols in Man....... 0. ccc cece eee e eens een eens 92
`
`VI.
`
`DESCUSSION 0.ee ene ene EEC EEE eRe REE EEE EN EEE Ena EE 95
`
`References... een nee nnn ee Eee eee ee EEE EEE EERE EES 113
`
`* Sections of this chapter have been adapted from the 2ndedition in this series on Percutaneous Penetration"
`and from the doctoral thesis entitled ‘‘Prediction of Percutaneous Absorption’? .!
`
`

`

`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 corneum intercellular lipid do-
`mains form a major transport pathway for penetration.1*'*?? Perturbation of these lamellar
`lipids causes skin permeation resistance to fall and has implicated their crucial role in barrier
`function. Indeed, epidermal steralogenesis appears to be modulated by the skin’s barrier
`requirements.*! Despite the fact that the skin is perhaps the most impermeable mammalian
`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
`wasfirst documented by Garb.?! Subsequently, Scholtz*® using fluocinolone acetonide, and
`Sulzberger and Witten*’ 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
`McKenzie” and McKenzie and Stoughton.*° Occlusion has also been reported to increase
`the percutaneous absorption of various other topically applied compounds,?:!*?°?” 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.?’? The extent of absorption was determined using “C radiotracer
`methodology. The *C-PPDA was formulated as 1% PPDA in petrolatum (USP) and applied
`from each test system at a skin surface dose of 2 mg/cm’. 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)
`wasfound (Table 1).
`The rate of “*C excretion following topical application of the radiolabelled PPDAin 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.
`
`Il. PERCUTANEOUS ABSORPTION OF VOLATILE
`COMPOUNDS IN RHESUS MONKEYS
`
`The in vivo percutaneous absorption of two fragrances (safrole and cinnamy! 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 Bronaughet al.* The extent of absorption following single dose administration
`was determined using “C radiotracer methodology. Each compound wasappliedat a topical
`dose of 4 j1g/cm? from a small volumeofacetone. The fragrance materials were well absorbed
`through monkeyskin. Plastic wrap occlusion of the application site resulted in large increases
`
`

`

`87
`
`TABLE1
`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 ()
`34 (20)
`
`Note: Therate of “C excretion following topical application of the radiolabeled
`PPDAin 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/mm? PPDAfor 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.
`
`1.2
`
`10
`
`- HTC
`-~ TEFLON
`- SM FINN W PAPER
`08
`“© SMALL FINN
`-# LARGE FINN
`AL TEST 0.6
`
`
`
`
`%Dose/Hour
`
`0.4
`
`0.2
`
`0.0
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`Midpoint (Hrs.)
`
`Jn vivo percutaneous absorption of PPDA (2 mg/mm?) 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. 1., Contact
`Dermatitis, 17, 178, 1987.)
`
`i
`
`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
`
`(4.6)
`26.1
`(1.6)
`4.1
`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 j1g/cm? prior to soap and water washing.
`
`4
`
`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
`
`% Dose absorbed?
`
`Nonprotected
`
`Plastic wrap occlusion
`
`Glass chamberocclusion
`
`Log Ko/w
`
`Benzamide
`Benzyl alcohol
`Benzoin
`Benzyl!acetate
`Benzophenone
`Benzyl benzoate
`
`47 (14)
`32 (9)
`49 (6)
`35 (19)
`44 (15)
`57 (21)
`
`(8)
`85
`56 (29)
`43 (12)
`17
`(6)
`69 (12)
`71
`(9)
`
`73 (20)
`80 (15)
`TI
`(4)
`79 (15)
`69 (10)
`65 (20)
`
`0.64
`0.87
`1.35
`1.96
`3.18
`3.97
`
`Note: 24-h exposure at 4 g/cm? prior to soap and water washing.
`
`a
`
`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. L., Fd. Chem. Toxicol,, 28, 369,
`1990.
`
`silicone glue) capped with Parafilm, occlusion with plastic wrap, and nonprotected condi-
`tions. 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 underplastic wrap compared to the nonprotected condition, whereas glass
`chamberocclusion 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.'® In this study, the rate and extent of '4C-label excretion into the urine
`following topical application of '*C-hydrocortisone to the ventral forearm of normal human
`volunteers were measured. Radiolabeled hydrocortisone (75 wg) was applied in acetone
`solution (1000 jl) as a surface deposit over 13 cm? of skin. The authors estimated that this
`
`

`

`89
`
`was equivalent to a sparing application of a 0.5% hydrocortisone topical preparation (5.8
`wg/cm?), Thesite 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 f 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.
`Guyet al.*° investigated the effect of occlusion on the percutaneous absorptionof 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 applicationto the ventral forearm of male volunteers, was reported,
`The chemical dose was 4 j1g/cm? and application area 2.5 cm?. The '*C-labeled chemicals
`were applied in 20 wl acetone. In the occlusive studies, after evaporation of the vehicle,
`the site of application was covered with a plastic (polyethylene-vinyl acetate copolymer)
`chamber.** In ail cases, after 24 h, the site of application was washed. with soap and water
`using a standardized procedure.* 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.® Daily topical dosesof three of the steroids (testosterone,
`estradiol, and hydrocortisone) were administered over a 14 d period. The first and eighth
`doses were C-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:
`
`1.
`
`2.
`
`3.
`
`4.
`
`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 upto testosterone but declines
`for progesterone, under occluded and nonoccluded conditions.
`The occlusive procedure generally permits excellent dose accountability (Table 7).
`
`‘ T
`
`he percutaneous absorption of these same four steroids under ‘‘protected’’ (i.e., cov-
`ered, but nonocclusive) conditions has also been measured in vivo®'° 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 occlusion’®
`
`

`

`90
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`8071
`
`L9°0
`
`am!
`
`9r'0
`
`eL0
`
`PL0££°0
`
`ceo
`
`£0°0
`
`00°0
`
`£00
`
`00°0
`
`+0°0
`
`00°0
`
`700
`
`c0'0
`
`L0°0
`
`00°0
`
`soo
`
`£0°0
`
`00°0
`
`00°0
`
`cO'0
`
`£0°0
`
`$00
`
`+00
`
`S00
`
`90°0
`
`10°0
`
`00°0
`
`+00
`
`720°0
`
`L0°0
`
`50°0
`
`00
`
`£0°0
`
`O10
`
`00°0
`
`S00
`
`£0°0
`
`
`
`
`
`
`
`sUo0sH.1O.0IPAHD,,JOUouelysrmupyjesidoy10ye<),,Jooudiosqysnosuejns13gpCTV
`
`
`
`(asogJO9%)suoTIpUO,),po}Iaj}01duOK]Jepun
`
`
`
`
`
`a%TIOL
`or4eq
`
`6Ae
`
`gseg
`
`Lkeg
`
`9deg
`
`gsegpABQ¢AeqzsegHr7—ZIH7ZI--0fas
`
`810
`
`$00
`
`60°0
`
`
`
`30°0LV‘0L0°0€7'0ITOIl‘0I
`
`$00
`
`L0°060°060°0ITO900IOZz
`
`e10
`
`700
`
`€0'0ame)9Il‘L0°091-011010°000°0¢60°0+0010°0s0°0500soo+Z10Z10st‘0610910€0°0€
`€0°0€0°0+00L0°0
`
`90°0
`
`€0°090°090°0LOO50'0so'0as
`
`60°0
`
`
`
`
`
`30°060°060°0€1'0L0'0L0°0 UeIIAl
`
`
`
`
`
`
`
`26°TQJOAOJOV]WONDALOSATUBBUISHPoyETNdyedsonyeaWoNooxaAreuumyesidoy,,
`
`
`
`
`
`
`
`
`
`
`
`‘iayempurdeosWimuonesyddeisody-pzpoysemuoneorddejoons‘pajosjoiduoN
`
`
`
`
`
`
`
`
`
`
`
`“SOGT“199“16‘ormuaag“Yyouy“]“H“yoeqreyApue“y“UUeUIp[s4]Woyeed
`
`

`

`-O%Teoyoneq6fegAegLseg9deggsdegpdeg¢AeqzfeqHpt—Z1H7ZI—0fqs
`
`99°61z01706£°06¢'0Z8°06I'T61197%00°Z09°0Pl‘z6E01170o¢"0870IvoLEO99°Tz9'TIstLET36°069'1I
`
`
`
`-L'0sro560€1'0¥60°6sto1Z0sr'0L5°0+9°080°Tscl£6'TBLTor'0ZL'0O1'61LU'0£706¢°0crotrl66'°TEST60°€Ze98198°€998°700°0€0°0oroseoz0P701Z’0701$°06080¢cos10°0oroZ€08e0+90850cel
`
`
`
`9r°6E10610ze'0sro69°0ZITBelE91rl6L°0wzava]
`
`
`
`
`
`to'sOl'0oroEroO10ero590SL'0rO'T10°T€9°0Ir'las
`
`
`
`auosHIOD0IpAHD,,JOuoneysmUMpyjesdoyryeD,,Joondiosqysnosurno1g~~
`
`
`
`
`
`S$WTavi
`
`
`
`(2s0qjJO%)SUOTIPUCD,PIPNIIIOJopun
`
`
`
`
`
`91
`
`
`
`
`
`"S961“199“16“Joruuag“Yyoty*'T“H“Yoeqreypue“y“uuelipfedwoved
`
`
`
`
`
`“o]empuedeosWIMUoTeorddeysod4-96peysemuoneroryddejoays‘pp10Jderonseldympepnyo9Qsy
`
`
`
`
`
`
`
`
`
`
`
`"6°79JOIOJOR]WONDOLIODATUeBUISNpoyepnoyessanfeauoHsIoxeAreutinfesidoy
`
`
`
`
`
`
`
`€
`

`

`92
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`+r OCCLUDED
`~@ NON-PROTECTED
`
`%DOSE/HR
`
`0
`
`48
`
`96
`
`144
`
`192
`
`240
`
`HOURS
`
`Percutaneous absorption of hydrocortisone in man, Human
`FIGURE 2.
`96 h occluded versus 24-h nonprotected exposure of hydrocortisone at 4 wg/
`cm?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)
`
`Nonprotected
`
`Occlusion
`
`24 2
`
`341
`341
`
`lia S
`
`10 +2
`tb+5
`
`13 + 33
`
`2146
`20 +7
`
`4+2
`
`42+1
`341
`
`27 + 6
`
`38 + 8
`2247
`
`46 + 15
`
`51 + 10
`50 +
`
`ll + 6
`
`33 +9
`
`Hydrocortisone
`Single application
`Multiple application:
`Ist Dose
`8th Dose
`Estradiol
`
`Single application
`Multiple application:
`Ist Dose
`8th Dose
`Testosterone
`Single application
`Multiple application:
`ist Dose
`8th Dose
`
`Progesterone
`Single application
`
`|
`
`4 Data from Feldmann, R. and Maibach, H. I., J. /nvest.
`Dermatol., 52, 89, 1969.
`
`andthat of the plastic chamber on hydrocortisone absorption,”° we repeated the measurements
`of penetration using plastic wrap (Saran Wrap®) with the experimental protocol of Guy et
`al.?° 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
`
`

`

`93
`
`+r OCCLUDED
`-> NON-PROTECTED
`
`ABSORBED
`%DOSE
`
`ES
`
`Log Ko/w
`
`Percutaneous absorption of 4 steroids (HC = hydrocortisone, ES
`FIGURE 3.
`= estradiol, TS = testosterone, PG = progesterone) in man as a function of
`penetrant octanol/water partition coefficient. Exposure period 24-h at
`4 «g/cm? prior to soap and water washing. (Redrawn from Guyet 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 dose
`Ist MD°
`8th MD¢
`Estradiol
`Single dose®
`ist MD*
`8th MD?
`Testosterone
`Single dose’
`ist MD°
`8th MD#
`Progesterone
`Single dose”
`
`4+2
`4+ 1
`3+1
`
`27 +6
`38 + 8
`2247
`
`46 + 15
`51 + 10
`50 +9
`
`33 +9
`
`64 + 5
`82 + 5
`78 +2
`
`60 + 12
`62 +
`59 + 8
`
`4447
`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
`
`a
`
`24-h exposure at 4 j.g/cm?prior to soap and water washing. Occlusion was
`with a plastic (Hilltop) chamber.
`Single dose study.
`>
`First dose of a 14-d multiple-dose study.
`°
`4 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
`
`TABLE8
`Percutaneous Absorption of Steroids in Man
`Single Dose Application for 24 h @ 4 g/cm?
`
`Mean % dose absorbed (+ SD; N = 5)
`Protected*
`Occluded®
`
`Hydrocortisone
`Estradiol
`Testosterone
`Progesterone
`
`4+2
`3+1
`18 +9
`13 +6
`
`4 Ventilated plastic chamber.
`> Occlusive plastic chamber.
`
`4+2
`27 + 6
`46 + 15
`33 +
`
`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
`341
`18 +9
`13 + 6
`
`85 + 6
`96 + Il
`77+ 8
`82 +7
`
`89 + 6
`100 + 1
`96 + 2
`96 + 3
`
`Note: Mean % dose (+ SD, N = 5).
`
`4
`
`Single dose application for 24 h at 4 j.g/cm?
`
`Data from Buckset al.?!°
`
`TABLE 10
`Percutaneous Absorption of
`Hydrocortisone in Man
`
`% Dose absorbed*®
`
`|
`
`Plastic wrap occlusion
`Plastic chamber occlusion®
`‘“*Protected’’ condition®
`
`4.7 (2.1)
`4.0 (2.4)
`4.4 (1.7)
`
`«Single dose application for 24 h at 4 j4g/cm?; values
`corrected for incomplete renal elimination.
`+ Mean + SD (N = 6)
`*
`Guyet al.
`4
`Buckset al.%!°
`
`

`

`95
`
`methodology described by Buckset al.”!° was utilized. Nine “4Cring labelled para-substituted
`phenols (4-aminophenol, 4-acetamidophenol, 4-propionylamidophenoi, 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 cm?. Penetrants were applied in 20 jl ethanol
`(95%). The chemical dose was 2 to 4 wg/cm’. 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.° 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 corneum 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:
`
`1.
`
`2.
`
`3.
`
`Occlusion significantly increased (unpaired ¢ 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 corneum,
`thereby swelling the corneocytes, and promoting the uptake of water into intercellular lipid
`domains. The normal water content of stratum corneum is 5 to 15%, a value which can be
`increased up to 50% by occlusion.'** Upon removal of a plastic occlusive dressing after
`24 h of contact, transepidermal water loss values are increased by an order of magnitude;'°
`the elevated rate then returns rapidly (~ 15 minutes) to normal with extraneous water
`dissipation. With occlusion, skin temperature generally increases from 32°C to as much as
`37°C.*8 Faergemannet al.'? 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.*?3 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.”°
`With respect to percutaneous absorption, occlusion (or a protective cover) prevents loss
`of the surface deposited chemicalbyfriction 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, showsclearly 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-
`
`

`

`96
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`a%RIOL
`Lseq
`
`9seq
`
`tro
`
`ST9
`
`BLIT
`
`$66
`
`Loa
`
`80°8
`
`9°
`
`SP0
`
`cr0
`
`oro
`
`STO
`
`0z°0
`
`020
`
`FIO
`
`o¢'0
`
`£c'0
`
`810
`
`8700L70
`
`£b'0
`
`0%FOL
`LAvg
`
`9keg
`
`66°L
`
`Ile
`
`68°C
`
`09°7
`
`Oo'L
`
`0S°6
`
`ses
`
`8Le
`
`670
`
`8c°0
`
`510
`
`£10
`
`610
`
`0c0z10
`
`L0°0
`
`62°0
`
`rr'0
`
`cc'0
`
`Sc0
`
`210
`
`9¢°0
`
`97°0
`
`110
`
`Ir'0
`
`cZ'0
`
`sP0
`
`cro
`
`gseq
`
`pseg
`
`€seq
`
`sO
`
`8¢0
`
`sro
`
`8£°0
`
`se0
`
`cr'0
`
`80°0
`
`L9°0
`
`65°0
`
`Te0
`
`88°0
`
`cs0
`
`09°0
`
`Ico
`
`60°T
`
`vS'T
`
`BLT
`
`SéT
`
`96°0
`
`LY
`
`ero
`
`gseg
`
`pseq
`
`¢4eq
`
`c£0
`
`IFO
`
`65°0
`
`oro
`
`890
`
`9r'0
`
`cs0
`
`65°0
`
`ev0€L°0
`
`LS'0
`
`c10
`
`6r'T
`
`69°0
`
`L9°0
`
`S80
`
`891
`
`CLT
`
`stl
`
`0s'0
`
`89°T
`
`99°T
`
`OLTe
`
`£9°€
`
`86'T
`
`PET
`
`
`
`
`
`
`
`(asogJo%)Ue]UI,SHONIPUODpo}Ie}01gJepupjousydowuryjouondiesqysnosueNIIIg
`
`
`
`
`
`
`
`
`
`
`
`(asogJO%)UR]UISUOKIPUSDpepnpIOJopup,jousydoumuryJouoydiosqysnosuENd.10g
`
`
`
`
`
`H8—?
`
`-H.p—-0
`
`spsfqns
`
`80°0
`
`£0°0
`
`90°0
`
`ce0
`
`910
`
`c10
`
`00
`
`£00
`
`00°0
`
`z0°0
`
`00°0
`
`00
`
`c0'0
`
`“amuAn
`
`UBS
`
`
`
`
`
`‘uomeredoadwr‘YY“y‘Angpue“TH‘qoeqreW“W897“AAW“Gd“syongWoLy
`
`
`
`
`
`‘Joyepuedeosyumuoneorddejsody--zpoysemuonesrddeJoaig.
`
`
`
`
`
`HS—?
`
`Ht—?d
`
`syalqns
`
`¥0°0
`
`920
`
`£0°0
`
`LTO
`
`100
`
`00
`
`S00
`
`700
`
`S00
`
`90°0
`
`<moamn dp
`
`VILWav
`
`ailWIaVL
`
`¢Avg
`H¢¢—?T
`
`EHCI—8
`
`96°C
`
`Ole
`
`919
`
`Tee
`
`Ore
`
`Loe
`
`Tg"0
`
`so'l
`
`soz
`
`ee?
`
`80°T
`
`Os'T
`
`68°0
`
`610
`
`80°0
`
`L10
`
`ce0
`
`310
`
`610
`
`60°0
`
`zAvg
`Ht?—cl
`
`H¢I-8
`
`L6C
`
`£0
`
`850
`
`09°1
`
`soo
`
`170
`
`£9°0
`
`Sol
`
`ble
`
`86°0
`
`30
`
`6c°0
`
`£0°0
`
`L0°0
`
`610
`
`0c'0
`
`or'0
`
`ceo
`
`910
`
`cl0
`
`v0°0
`
`L0°0
`
`£10
`
`£10
`
`Il0
`
`80°0
`
`

`

`97
`
`@%IOL
`LAvg
`
`9Avg
`
`10°¢
`
`19°0
`
`00°9
`
`It
`
`09°C
`
`08°s
`
`ore
`
`807
`
`870
`
`60°0
`
`6L°0
`
`STO
`
`seo
`
`$9°0
`
`cr'O
`
`8c'0
`
`Or'0
`
`ITO
`
`39°0
`
`ITO
`
`6e°0
`
`£L°0
`
`Or'0
`
`L770
`
`A%TOL
`Lkeg
`
`9Aeq
`
`6L°9
`
`LT
`
`LLY
`
`SVT
`
`el
`
`c9°9
`
`PLe
`
`v9"?
`
`FIT
`
`67°0
`
`0L'0
`
`ce0
`
`610
`
`vet
`
`99°0
`
`8v0
`
`L6°0
`
`8c'0
`
`£L°0
`
`FeO
`
`1c0
`
`OT
`
`650
`
`oro
`
`SEO
`
`gseg
`
`pseq
`
`Lr0
`
`60°0
`
`LLO
`
`STO
`
`0¢°0
`
`+3'0
`
`rr'0
`
`TEO
`
`Le0
`
`60°0
`
`90°T
`
`910
`
`870
`
`99°0
`
`L¥'0
`
`9¢°0
`
`gseq
`
`pdeg
`
`96°0
`
`67°0
`
`09°0
`
`£70
`
`$80
`
`cS"0
`
`£e°0
`
`gel
`
`ce0
`
`tc0ell
`
`02°0
`
`LET
`
`08°0
`
`79°0
`
`950
`
`60°0
`
`LT0cS
`
`95°0
`
`L£L'0
`
`19°0
`
`cso
`
`crt
`
`£10
`
`9¢°0
`
`910
`
`810
`
`160
`
`ep0gO
`
`£10
`
`ITO
`
`LS°0
`
`£r'0
`
`TO
`
`
`
`
`
`(2s0q]JO%)UR,UTSHONIPUODpe}de}01gJopuy,UeydourmeLja0yJoUORdIosqYsnosUYNIIIg
`
`
`
`
`
`
`
`
`
`(asogJo%)ue],UISUOIPUODPopUpPIGsJ9puyQUeYydouTME}I0VJoHONdIOsqYsnosUENIIAg
`
`
`
`
`
`H&8—?
`
`Hr—?
`spefqns
`
`10°0
`
`60°0
`
`90°0
`
`£70
`
`00°0
`
`00
`
`To°O
`
`00°0
`
`00°0
`
`00
`
`10°0
`
`100
`
`tmuQqmM&
`
`ues
`
`H8—?
`
`Ht—-0
`
`sypefqng
`
`90°0
`
`10°0
`
`80°0
`
`10°0
`
`10°0
`
`£0°0
`
`00
`
`£0°0
`
`00°0
`
`00°0
`
`90°0
`
`00°0
`
`T0°0
`
`coo
`
`T0°0
`
`coo
`
`<muamm ds
`
`
`
`“uoneredaidut‘YY‘Andpur“TH‘qoeqreyl“WW‘297MAWC‘SyongWoL
`
`
`
`
`
`“1ayeMpuedeosmimuoneorddeysody-pzpeysemuoneoyddejoag
`
`
`
`
`
`VelWIV
`
`¢stg¢stg
`H*—7I
`
`H@I—8
`
`
`
`GelATAVL
`
`cro
`
`00
`
`LT0LL0
`
`Iv'0
`
`OTT
`
`60
`
`cro
`
`920
`
`100
`
`cro
`
`L0°0
`
`60°0
`
`Leo0
`
`+T0
`
`Tt0
`
`S00
`
`T0°0
`
`65-0
`
`80°0
`
`ZOO
`
`cro
`
`€10
`
`910
`
`60°0
`
`00°0
`
`60°0
`
`60°0
`
`¢segZzseg
`H¢?—?1
`
`H7I-—-8
`
`LLO
`
`ro0
`
`9L°0
`
`02°0
`
`FTO
`
`P10
`
`80°0
`
`Z0°0
`
`9€°0
`
`sT0
`
`cro
`
`70°0
`
`20°0
`
`10°0
`
`+00
`
`10°0
`
`L0°0
`
`€0°0
`
`70'0
`
`

`

`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`98
`
`VelWIGve.
`
`
`
`
`
`
`
`
`
`(esogjoa)WeyUT,SUOTIPUO-)papnpPIOAepuy,jousydopruesAuoido1gJouondsosqyshosueNII0g
`
`
`
`
`
`A%TEIOLLAeggseqgseqpyseg¢Ae@degHv~—7H7Zi—8H8—?“Htspafqng
`
`PSIE69°08706I'T90°+0'r611LUEaSProst6£°0aSo'86£°0+90750ZO'TIgZPLZILZ0
`€1°090°000°0a£r'6850€"0ertcr09°ttTero80°0ITO00°0dSB°SZ
`667107ooEs'€16°€95°¢ZI6719¢'0Or'03CLO16°0951c8'T13'ZZLtryvant61070°00°0qSTIz86°0L£9°0SLI9'¢OsLVLPLZL770Z0°000°0Vv
`
`Il'6L¢0LOILOT60°198°099°¢an|vil00'T020as
`
`GelWIV
`
`
`
`
`
`83°8176°0gl81ZtreIScesLol80°195°0€1'0ues]
`
`
`
`
`
`(ssoqjo2%)Ue],ULSWORIPUO.pejd0}01gJepuy,jousydoprnmeAuoidolgJouondiosqysnesurns1eg
`
`
`
`
`
`
`
`
`
`0%TeIOLZLAeq9seqgseqpseg¢degzAeqHvv—ZHZI—8HS—PHr—ospafqns
`
`£0°68L°0BoLLO€3'T8o°Tv1Z6£°090°0coo+00VW
`
`
`
`LLO1€7'0T€-0ISO€0'Tc6'ToCLVZIllZ1010°0a80°EZFIT6L'1Sut96°+e9zS°9OCT1Z'000°000°0dLLgoO99°090°TselgolZO'TLr'0oro710L0°03ogeCI€L'0ZS°096°09E°1LL‘0LV090°0Z0°0£0°0qd
`
`L£6°9sro850ISO73°0TETPET90°Tsrosoo€0°0as
`
`
`
`OTLPLOS60760Eo8S°Z9LZIVE1090°0€0°0weaj
`
`
`
`
`
`‘uonemedaidul‘"y-y‘Andpue‘|“Y‘yorqreyyl“WW‘ea]“AAOVOG‘syongwo1y
`
`
`
`“iayemMpuedrosWIMuoyeorddejsody-pzpoysemuoneoyddejoaig.
`
`
`
`
`
`
`
`

`

`
`
`
`
`%TOL,Lseg9deggsseqpseq¢seqzseqH¢7—ZIHZI—8HS—?Hr—0spafqns
`
`
`
`
`
`topO€'€aPLZZ0°0+006009106£°066°Fcc'soss9e'¢SLTtdLYSE00°030°0s0°0STO910867v7TELboL00°€366°SE500L0°0
`
`€Z7'9OTLaBLTE+00€0°0L0°0910ZET€8'Lg'O1L6O¥
`
`
`€6°ZE00°0L0°060°06108r'0LI'sge9SOP
`
`€0°090°0ZO6E°%SSIPL’S76OL'Tq80°6£ITO60097070SL‘0Vil871618oCSwVv
`
`$07+0°0Z0°030°090°0Sr’LOE13268°TZ6'T91%as
`
`
`
`06°+0090°060°0910€S°0go9IL'01oP'9€r'9PIEues}
`
`GelWIGVL
`
`
`
`
`
`(esogJO%)URIUISUOHIPUODpepnysI»0OJepuyjousygJouoydiosqysnosueNIIag
`
`
`
`
`
`VlAIAVL
`
`
`
`
`
`(asogJo%)UeyUL,sSuOVIPUE?ps}d9}01g19puyjousygJouondiosqysnosueMIIRg
`
`
`
`
`
`
`
`
`
`a%oyLseq9keggdegpseq¢seqzAgHtz—21HZIHS—?tH?—0spafqns
`
`
`ILOS1070070LOZISL+0°S6r'807'Fa68°1ZO10STO40°0
`6S°8Z80°0
`
`€l06£°079'F66°9PEEIv697atr’€Z30°060°080°0Sl‘0zo'0C8ZLg°9Pes69'SOLza9ZIZO10cl‘011061°0+£'060466°9Scro¢'lEse360°FTSZ'01z'0070ze08e'0Br077LO°%PZSseaLZZEO10910110€L'0190OTLcolIl'980°L20Vv
`
`1g°9L0°0+00co0L0°0+10EST99°CLvlC87Lr'las
`
`
`
`6S°€7Z10SEOZ1'0610Ir'010'r6L°99C'F89°F9L'Zues,
`
`99
`
`
`
`‘uonesedoiduf‘H“Y“Andpue“]“H“qoeqrey“WW“OoT“AA“V“Cd‘syongwoLy
`
`
`
`
`
`‘ayempuedeosTIMuoneoddejsody-pzpoysemuogeoyddejoaig.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`“uogeslddesoepmsurysoy}WoySunvIodeasasoppeyddeayyJo97°17JO}payoaoseyeq.-ajoyyuodn
`
`
`
`
`
`
`
`
`
`

`

`100
`
`In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications
`
`0%IOL
`LAvg
`
`9seg
`
`Lor4LTV6E
`
`OS'S
`
`66OF
`
`88°0S
`
`bLSP
`
`06°S
`
`L410
`
`1Z0
`
`00
`
`9T0ITO
`
`St'0
`
`810
`
`90°0
`
`veo
`
`170
`
`£10
`
`Oro
`
`STO
`
`T€0
`
`1c0
`
`60°0
`
`%TI0OL
`ZLAeq
`
`9seq
`
`09°6€
`
`seg
`
`GLLE
`
`LO'EE
`
`98°07
`
`819s
`
`L608
`
`9C°91
`
`80°0
`
`STO
`
`00°0
`
`90°0
`
`10-0
`
`80°0
`
`90°0
`
`S00
`
`L0°0
`
`FI0tc0
`
`£0°0
`
`ITO
`
`10°0
`
`oro
`
`80°0
`
`LT0
`
`90°0
`
`I10
`
`gkeg
`
`pdeg
`
`¢seq
`
`610
`
`Or'0
`
`$70
`
`tT0z10
`
`£c0
`
`co0
`
`oro
`
`FIO
`
`8£0
`
`62°0
`
`ee'0
`
`se0
`
`10
`
`Le0
`
`O10
`
`£0
`
`6¢°0
`
`bro
`
`00
`
`tv0
`
`sco
`
`ce0
`
`60°0
`
`geq
`
`pdeg
`
`¢deg
`
`L0°0
`
`020
`
`60°0
`
`00
`
`L0°0
`
`80°0
`
`ve-0
`
`0c'0
`
`oro
`
`IT0
`
`90°0
`
`£10
`
`40°0
`
`870
`
`06°0
`
`6c0
`
`910
`
`c10
`
`00°0
`
`670
`
`ce0
`
`LET
`
`OETIve
`
`
`
`
`
`(asogJO9%)UB;UISUORIPUO-)PapPNpPIOJopuyg[ousydoued>JouoNdIosqysnosueENdI9g
`
`
`
`
`
`
`
`
`
`(asogJO%)UR],UTSUONIPUO?)p9}d2}01gJopuyjousydoues>JouondsiosqysnosuLyNsIeg
`
`
`
`
`
`H8—?
`
`Hb—6
`
`syafqng
`
`89°0
`
`Sit
`
`06°€
`
`87ST
`
`1S°6
`
`css
`
`FeO
`
`L9°0
`
`£0°€
`
`£31
`
`ScT
`
`£0°0
`
`0¢'T
`
`orl
`
`<moamu dg
`
`H8—?
`Heo
`spafqns
`
`€ST
`
`CLI
`
`Vel
`
`69¢
`
`cri
`
`LEC
`
`9s°¢
`
`Se
`
`XLT
`
`ell
`
`ce€
`
`OCI
`
`ell
`
`“amuouome
`
`ues
`
`
`
`‘uoneredardur‘YH“yf‘Andpue‘]“H“yoeqre“WW“eeT“MV“Cd‘SyongWoL
`
`
`
`
`
`“Jayempuedeosyimuonesyddeysody-pzpoysemuoneonddejoag,
`
`
`
`
`
`
`
`WSTATAVL
`
`
`
`astATAVL
`
`zfeq
`Heb?—?er
`
`HCi--8
`
`6SPSTT80°¢
`
`csTcel
`
`1@?
`
`Set
`
`ET
`
`608
`
`core
`
`00°6
`
`£s°9
`
`ges
`
`COL
`
`0c€
`
`L7l
`
`Vt
`
`61
`
`L470
`
`Lvs
`
`TOL
`
`Svs
`
`Ly'v
`
`OCT
`
`zseq
`Ht@—71
`
`HctI—s
`
`98F06°C
`
`9o°P
`
`61e
`
`61+
`
`8£°0
`
`619
`
`LL6
`
`SOL
`
`70°38
`
`Bor
`
`PCr
`
`FCT
`
`700
`
`8L9
`
`FOr
`
`OP
`
`IT86SI
`
`gog
`
`SIT
`
`C68
`
`8TE
`
`108
`
`org
`
`£78
`
`

`

`
`
`
`
`%TRIO],Lkeg9deggseqpydeg¢seqzAvgHpv—7IHZI—8H8—?HP—ospefqns
`
`
`
`OESZ-0'040°060°060°0r1060SBs°¢IrsylivanW
`
`
`
`60°0P00cooc0'0670LLY+09SL'900°F10°0gdLS'8E60°0soo60°0E10€1°0SLT96°LOr'6EST997380°61
`
`
`
`€0°090°0L0°0z10SelnS6FIS9ESITa79'8E80°080°0S00LV0LV080°779LOSSEI66°6a7TELV‘060°0Z10+1061°0vara65Pfe€91oroda
`+9°OL80°0
`
`¥0'81¥0'0ZOO€0°0¥0'090°0Lr0IVTESOlSusas
`
`
`
`PTLE60°090°030°0I1‘08r°060°10°908oorofruray]
`
`G90WIAVL
`
`
`
`
`
`(esoqiJO9%)URIUT,SHONIPUODpa}sa};01g1apuy,joueydoINJouoNdiosqysnosuENIIag
`
`
`
`
`
`
`
`
`
`0%FIOLLseq9seqgkeqpseq¢feqzseqH¢7—ZIHTI—8sH&8—?H>—0spefqns
`
`ZL67sto1Z°0610I¥'01S°086766°83aororsort2oSSPPlo620910LEO66'0IL'8rLI671619OTa88°Sr50S70Or'0S9065°0SPPIzTHIL9°9L0°0Vv
`
`IT‘00c0S10OPTL'07S16ZOLetd69°LE810ZEO910SZ'0LetZS766£6100°098°7aTE6EL10L10Ez'0670Ee95°¢eps6cosr0'0dq
`76'bb6700z°0
`
`OCITSTO90°0oroF10Sr'061739°9ers9C'€OLTas