`
`Bioavailability of Topically Administered Steroids: A
`“Mass Balance” Technique
`
`Daniel AW. Bucks, M.A.. James R. McMaster, M.A., Howard 1. Maibach, M.D., and Richard H. Guy, Ph.D.
`Departments of Pharmacy. Pharmaceutical Chemistry. and Dermatology. University of California. San Francisco. San Francisco.
`California. U.S.A.
`
`The percutaneous absorption of four steroids (hydrocorti-
`sone, estradiol,
`testosterone, and progesterone) has been
`measured in vivo in man under occluded and “ rotected"
`(i.e., covered, but non-occlusive) conditions. T e experi-
`mental approach, involving simple modifications ofstandard
`radiochemical methodology. has enabled excellent “mass
`balance” and dose accountability to be achieved. Conse-
`quently, the utility of the procedure for the measurement of
`in vivo to ical bioavailability can be inferred. In addition,
`because 0 the precision and accountability of the results, the
`technique offers a potential means to establish quantitative
`structure-penetration relationships for skin absorption in
`man. It was found that steroid absorption increased with
`increasing lipophilicity up to a point, but that penetration of
`progesterone (the most hydo hobic analog studied) did not
`continue the trend and was at east partly rate-limited by slow
`
`interfacial transport at the stratum corneum-viable epidermis
`boundary. Comparison of data obtained from the occluded
`and “protected” experiments permitted the effect of occlu-
`sion (defined as the complete impairment of assive transepi-
`dcrmal water loss at the application site) to be amused. Oc-
`clusion significantly increased percutaneous absorption of
`estradiol, testosterone, and progesterone but did not effect
`the penetration of hydrocortisone. A mechanism is proposed
`to ex lain why the absorption of the more lipophilic steroids
`is en anced by occlusion but that of the most water-soluble
`(i.e., hydrocortisone) is not. It is suggested that the rate-de-
`termining role of the sequential steps involved in percutane-
`ous absorption can be revealed by experiments of the type
`described using related series of homologous or analogous
`chemicals] Invest Dermatol 90:29—33, 1988
`
`n the development of a dosage form intended for topical
`administration on the skin, an essential step is to determine
`the percutaneous absorption of the drug. Of the various al-
`ternatives available for the assessment of skin penetration,
`there is little doubt that an in vivo measurement in man is
`most appropriate and desirable [1].However. in vivo ercutaneous
`absorption experiments in man are much more difficu t to perform
`than either animal model or in vitro penetration studies. Further-
`more, most ofthe in vivo investigations which have been carried out
`have not allowed accountability of the applied dose and. hence. have
`not
`reduced results which can be interpreted unequivocally.
`T e majority ofhuman in vivo percutaneous absorption measure-
`ments have used indirect radiochemical methods [2 — 6]. Typically, a
`“C labeled chemical is applied to ically from a volatile solvent
`vehicle and penetration is evaluate
`from the excretion of the “C
`radiolabel over the next 5- 10 d. Correction for incomplete elimi»
`nation is made by performing an identical protocol after intravenous
`or intramuscular administration of the same ”C labeled material.
`The a proach has some clear limitations: any couclusions are based
`on radiolabel data, not specific information about the parent com-
`pound and its metabolites; the elimination profile after topical and
`parenteral dosing must be assumed identical; the fate ofthat fraction
`ofthe to ical dose which is not absorbed immediately into the skin
`post-app ication is not controlled so that the meaning of “dose" in
`this situation is usually poorly defined.
`
`
`
`Manuscript received September 10. 1987; accepted for publication Febru-
`ary 5, 1988.
`Reprint requests to: Dr. Richard H. Guy, School of Pharmacy. Box 0446.
`UCSF. San Francisco. CA 94143
`
`In this paper. simple modifications of the conventional in vivo
`experiment are described and the improvement in resulting data
`quality is illustrated for four steriods: progesterone. testosterone,
`estradiol. and hydrocortisone. The procedures involve i) covering
`the application site for the entire duration of the study. ii) washing
`the dosed skin surface at the end of the dosing period, and iii) on
`occasion. when monitoring of urinary excretion is terminated, tape-
`strippin the upper layer of stratum corneum. The key improve-
`ment aflgorded by these changes is that the radiolabeled dose can be
`totally accounted for, i.e.. mass balance is possible. The approach has
`been applied to both single and multi
`le-dosing regimens and mea-
`surements have been made under bot occlusive and non-occlusive
`(“protected") covering conditions. The results obtained demon-
`strate that the technique may have significant potential for estab-
`lishing quantitative structure-penetration relationshi s for skin ab-
`sorption in man. and revealing quantitatively t Ie effects of
`occlusion on the transport of compounds across the cutaneous bar-
`ricr.
`
`MATERIALS AND METHODS
`
`The pcnetrants considered were four steroids: progesterone, testos-
`terone, estradiol, and hydrocortisone. The 1“C-labeled chemicals
`(RPI Corp., Mount Prospect. IL) were applied in acetone to the
`ventral forearm of healthy male volunteers (n 3 5). from whom
`informed consent. approved by the UCSF Committee on Human
`Research, had been previously obtained. Chemical and radioactivity
`doses were 4 Jrig/cm2 and 1 pCi/cmz, respectively; the area ofa pli-
`cation was 2.5 cm2 and the dose was administered in 20 pl 0 ace-
`tone.
`
`After eva
`ration ofthe vehicle (< 0.5 min). the application site
`was covered):)vith a semirigid.
`lypropylcne Hilltop' (Hilltop Re-
`search. Inc., Cincinatti. OH) c iamber (HTC), which was affixed to
`
`0022-202Xf88f50350 Copyright © I988 by The Society for Investigative Dermatology. Inc.
`
`29
`
`  
`
`

 
`
`MYLAN - EXHIBIT 1022
`
`

`

`30
`
`BUCKS ET M.
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`the skin with hypoallergenic adhesive tape. The cotton pads. with
`which the chambers are supplied. were removed prior to application
`on the subjects' forearms. In the occluded studies, intact chambers
`were employed; for the penetration experiments under “protected"
`conditions. the chambers were “ventilated" by boring several small
`holes through the plastic (such that about 50% of the surface area
`was expOsed). To prevent loss of surface material (squames. undis-
`solved penctrant. etc.). the roof of the chamber was covered with a
`piece of Gore-Tex‘ (W.L. Gore 8t Associates. Inc.. Elkton. MD)
`membrane (0.2 lrim pore size). It was found that Gore-Tex’ did not
`impede transepiderrnal water less to any significant extent and
`hence the objective of dosing site protection without occlusivity
`was achieved (Bucks et al, in press).
`The subjects collected their urine for 7 d. post-steroid applica—
`tion. according to the schedule: 0-4. 4—8. 8- 12. and 12—24—h;
`day 2. 3. 4. 5. 6. and 7. Urine volumes were determined gravimetri-
`cally for each time period. and duplicate 3-ml samples were ana-
`lyzed for radioactivity. ”C-Toluene was added. as an internal stan-
`dard. to a third 3-ml sample to determine quenching. The percent
`“dose" (as total radioactivity) excreted was determined for each
`time interval. At 24 [1 after dosing. the chamber (or chamber +
`Gore-Tcx') was removed. placed in scintillation fluid, and seques-
`tered 1“C was counted. An appropriate quench correction was again
`made. The application site was washed with a standardized proce-
`dure [7] using 5 cotton balls consecutively soaked in soap solution
`(Ivory Liquid Soap. Proctor and Gamble Co., Cincinatti, OH; di-
`luted 1 : 1 with water). water. soap solution. water. and water. All
`washings were collected and were processed for liquid scintillation
`counting to assay for residual surface chemical. For the remaining
`6 d of the urine collection period. the administration site was again
`covered with a (new) chamber. Finally. this chamber was also as-
`sayed for "C~chemieal: also. at this time. in the “protected" experi-
`ments, stratum corneum at the site of application was stripped 10
`times with adhesive tape (Scotch Cellophane Tape'. 3M. St. Paul.
`MN) and the skin strips were analyzed for residual radioactivity
`(once more. corrected accordingly for scintillation quenching).
`
`A parallel protocol was also performed following a multiple-dos-
`in g regimen [8] for testosterone. estradiol. and hydrocortisone
`under occluded conditions. The compounds were applied every
`24 h for 14 d at a dose of 4 rig/cm: to the same skin site. The firsr
`and eighth applications utilized “(I-labeled drug and urinary excre-
`tion for 7 d (using the collection schedule described above) after
`each ofthcse doses was followed. In these studies, the 24-h washing
`procedure was performed daily (prior to that day‘s dosing) and a
`new chamber was provided on each occasion.
`Partition coefficients of the penetrants between isopropyl mytis-
`rate and water. and tetradecane and water. were determined using a
`standard technique [9]. Octanol-water partition coefiicienrs were
`obtained from the literature [10].
`RESU LTS
`
`Data from the single dose experiments performed under occlusive
`conditions are presented in Table I and should be compared to the
`corresponding results from the “protected" studies given in Table
`II. Total recoveries are. in general. high and were greater for the
`“ rotected" measurements. These experiments were performed
`afier the occluded investigation and incorporated obligatory evalua-
`tions of i) "C-radiolabeled sequestered on the second HTC. ii)
`chemical in the second set ofwashings. and iii) material remaining
`in the upper layers of the stratum corneum at the end of? d. This
`more thorough determination of penetrant disposition probably ac-
`counts for the improved mass balance in the “protected” studies.
`The percentage dose absorbed columns in Tables I and II show the
`eITeCt ofocclusion on the topical bioavailahility ofthe four steroids.
`With the exception of hydrocortisone. unpaired t-tests show that
`occlusion significantly increases the
`rcutaneous absorption ([1
`(0.01) ofthese compounds in man. T is finding is further empha-
`sized in Fig 1. which shows, for each ofthe four steroids. the rate Of
`excretion of radiolabel following their topical application under
`both occluded and “protected" conditions. To optimize clarity. data
`for cstradiol. testosterone. and‘progesterone are plotted semi-loga-
`rithmically because of the di erence in absorption between 01:-
`
`Table I. Disposition of Topically Applied I"C-labeled Steroids Following a Single Dose under Occluded Conditions
`
`Percentage of Applied Dose'
`
`
`
`
`
`
`
`
`
` Steroid Absorbed" is: HTC‘ ist Wash-I 2nd HTC‘ 2nd Wash' Total
`
`
`
`68 :I: 3.9
`n.d.l
`n.d.l
`36 i 3.0
`28 i 5.6
`4.0 i 2.4
`Hydrocortisone
`37 i 13
`n.d.l
`0.5 i 0.3
`18 :t 7.2
`41 a: 10
`27 :1: 6.4
`Estradiol
`90 1 8.4
`n.d.u
`0.3 i 0.2
`3.0 i 4.1
`41 :i: 8.4
`46 i 15
`Testosterone
`80 d: 5.5
`
`Progcstfronc
`33 i 8.9
`46 j: 10
`1.2 d: 0.3
`.07 i .02
`.01 i 0.0
`. Mean i standard deviation (n - 5. except for progesterone. for which n - 6}.
`" Values corrected for incomplete renal elimination I3].
`‘ Material sequestered on Hilltop chamber (HTC) removed at 24 h post-dosing.
`4 Chemical Found in combined washings performed 24 b post-dosing.
`' Material sequestered on HTC removed at end of measurement period.
`‘ Chemical found in combined washings performed at end of experiment.
`I rid: not determined.
`
`
`
`
`
`
`
` _ _ SCCtOid Absorbed" ‘lst HTC‘ lst \‘K/ashd 2nd HTCe 2nd \Vashr SC “strips"I Total
`
`
`
`_ Table II. Disposition of Topically Applied l“Cdabeled Steroids Following a Single Dose Under “Protected“ Conditions
`Percentage of Applied Dose‘
`
`
`
`Hydrocortisone
`4-4 2t 1-7
`2? 1: ll
`5| :l: 18
`3.2 :t 1.7
`2.7 d: 1.3
`2.5 :l: Ll
`39 :l: 5.6
`Estradiol
`3.4 i 1.2
`33 :I: 13
`53 i 12
`0.? d: 0.4
`0.3 :1: 0.4
`0.1 a: 0.1
`100 d: 0.9
`Testosterone
`18 :i: 8.6
`46 :i: 7.5
`30 :l: 15
`1.4 :l: 0.4
`0.1 i .08
`n.d.*‘
`96 :I: 2.0
`
`0.3 :l: 0.4 96 :l: 3.4 dd."
`Progesterone
`13 i 6.3
`54 :l: I?
`2? :I: 8.7
`1.2 :l: 0.6
`
`
`
`
`' Mean i standard deviation (n - 6).
`" Values corrected for incomplete renal elimination (3|.
`‘ Material sequestered on HTC + Gore-Ter remcwed at 24 h post-dosing.
`“ Chemical found in combined washings performed 24 b post-dosing.
`' Material sequestered on HTC + Gore-Tex® removed at end of measurement period.
`‘ Chemical found in combined washings performed at end of experiment.
`I "C-radiolabel present in 10 tape strippings of stratum eorneum (SC) removed after final washing procedure.
`" n.d.: not determined.
`
`

`

`VOL. 91. N0.
`
`1
`
`jULY 1938
`
`STEROID uioameamuTv 31
`
`HYDRDCORTISONE IBBOHl’flON
`
`ESTRIDIOL ABSORPTION
`
`DDS
`
`Table IV. Oil-Water Partition Coefficients of Steroids Studied
`
` Steroid log Koyw' log hm." Ion Krrw'
`
`
`Hydrocortisone
`1.61
`-0.19 :l: 0.02
`—2.1? :l: 0.03
`Estradiol
`2.49
`2.33 :l: 0.04
`—0.027 :l: 0.003
`Testosterone
`3.32
`1.98 i 0.002
`0.68 i 0.02
`Progesterone
`3.87
`2.62 d: 0.005
`2.27 i 0.11
`
`
`
`
`
`094
`
`0.0:!
`
`002
`
`most-Horn
`
`00! 000
`
`o
`
`”I
`
`1D M W ‘00 ‘39 "‘0 ‘W
`IIME {HHS}
`TESTOSTERONE IEMPW
`
`o
`
`20
`
`an
`
`to I00 ‘201‘0 too
`on
`TIME lHHSl
`PRO-DE STEM IBSOHPMH
`
`' K9,“, - Octanol—warer partition coefficient [9.10].
`" KUW - Isopropyl myristatc-watet partition coelhcient (mean i standard devia-
`tion: n - 6).
`‘ K1,“. - Tetradecane-watet partition coefficient
`I1 "'= 6}.
`
`(mean :l: standard deviation:
`
`DISCUSSION
`
`The experiments reported in this paper highlight three issues: I) the
`accountability ofthc applied chemical time and the potential utility
`of the technique for measurement of topical bioavailability'. 2) the
`effect ofocclnsion on the in vivo skin permeation ofsteroids; and 3)
`the relationshi between percutaneous absorption and the relative
`lipophilicity olPthe pcnetrant.
`The mass balances achieved in this work are generally high and
`often a proach 100%. A conventional
`in vivo approach [2-6]
`would
`ave only revealed the percent dese absorbed columns in
`Tables I _ III. Disposition ofthe remainder ofthe applied radioactiv-
`ity would remain unknown. The importance ofrepeating the wash-
`ing procedures and chamber analysis at the end of the 7-d experi-
`mental period is indicated in the improved accountabilities observed
`in the “protected“ (Table II) and multiple-dosing (Table III) stud-
`ies. Further support for this contention has recently been observed
`in our laboratory for a series of para-substituted phenols [11]. for
`which. again. essentially complete mass balance has been recorded.
`It
`is
`ertinent to note that in Table II hydrocortisone. the least
`lipopl'lilic steroid. is significantly measurable in the stratum cor-
`neum at 7-d post-dosing. The amount recovered is clearly relevant
`when considered in relation to the level of percutancous absorption.
`The persistence of hydroeortisone in the stratum corneum for this
`prolonged period suggests chemical-tissue interaction of appreci-
`able strength. Although the nature of this “binding” phenomenon
`is not revealed by these experiments. the effect clearly goes beyond
`simple depot behavior. This hypothesis is reinforced by the fact that
`the more lipophilic estradiol is barely detectable in the stratum
`corneum at the end of the experiment (Table II). In addition. the
`recent investigation [12] using ara-substituted phenolic penetrants
`has revealed the same pattern: p enols with more polar para-substit-
`uents (e.g.. — NH}, — NHCOCHg. —NHCOC2H5) show pro-
`longed stratum corneum residence. whereas more lipophilic ana-
`logs (p-CN. p-I) do not.
`In Fig 2. the percentage dese absorbed for each steroid is plotted as
`a function of penetrant octanol/water artition coefficient: results
`obtained under occluded and “protectedj” conditions are compared.
`
`5 in W'W-Yfi—‘I—Vq—V-‘Y—‘fi’
`
`‘00
`
`1.00655“
`
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`
`IIME (HRS:
`
`rim; magi
`
`Figure 1. Urinary excretion rates (mean % dose per hour} as a function of
`time following topical application of four steroids under occluded {open
`square) and “protected“ (filled square) conditions. A, hydrocortisone; B. es-
`tradiol: C. testosterone; D. progesterone.
`
`eluded and “protected" measurements; for hydrocortisone. on the
`other hand. a linear graph is presented and the occluded and “pro-
`tected" results essentially superimpose.
`The multiple-dose measurements. which were performed under
`occlusion. are summarized in Table III. Again. total recoveries of
`a plied radioactivity were good. An analysis of variance showed
`that for each of the steroids there was no significant difference (p
`> 0.05): a) in the percentage dose absorbed dermally between the
`first and eighth doses under occlusion and b) between the multidose
`absorption figures and the percentage dose absorbed following a
`single dose under occluded conditions (Tables I and III) with the
`ssible exception ofestradiol for which marginally signi cant dif-
`Fdi'ences
`= 0.04) in percutaneous absorption between the first
`and eighthP doses of the multidose regimen and between the first
`dose of the multiple application study and the single acute dose
`study were found].
`Finally. in Table IV. partition coefficients of the steroids between
`each of three oil phases (octanol. isopropyl myristate. tetradecanc)
`and water are reported.
`
`Table III. Disposition of Topically Applied “‘C-labcled Steroids Following Multiple Dose Under Occluded Conditions
`Percentage of Applied Dose”
`‘lst HTC‘."
`‘Ist Wash”
`Steroid
`Dose“
`Absorbed“
`2nd H'I‘Cr
`2nd Wash!t
`
`
`
`Total
`
`HydrOCOrtisone
`
`Estradiol
`
`Testosterone
`
`
`lst
`8th
`lst
`3th
`lst
`8th
`
`3.5 :l: 1.3
`3.l i 1.0
`38 :l: 7.9
`22 :l: 74
`51 i 10
`50 i 9.5
`
`23 i '17
`32 :l: 5.4
`4? :l: 12
`3? i 9‘9
`46 i 9.l
`37 i 9.7
`
`53 i 11
`33 :l: 7.5
`14 :l: 6.8
`21 :l: 5.2
`1.7 i 1.0
`4.3 :l: 5.4
`
`3.5 :l: 1.4
`7.4 i 0.8
`0.6 i 0.8
`0.4 :l: 0.2
`0.2 :l: 0.1
`0.2 i 0.2
`
`2.6 :l: 0.8
`4.3 1: L7
`0.5 :l: 0.6
`0.5 i 0.2
`.06 :l: .06
`.06 :l: .04
`
`85 i 4.3
`81 :l: 2.5
`100 i 3.9
`81 :l: 6.0
`99 :l: 4.3
`92 :l: I?
`
`‘ Mean It standard deViation (n 'I' 5. except for bydrocortisotie 3th dose. for which n - 4).
`5 The lst and 8th doses ofa dailyr dosing regimen. lasting H d. were “C-radiolabcled.
`‘ Values corrected for incomplete renal elimination [Bl
`‘ Material sequestered on HTC removed at 24 h post-dosing.
`‘ Chemical found in combined washings performed 24 h port-dosing.
`‘ "C-labeled material sequestered on HTC removed at 48 h post—dosing.
`I 1“C-labeled chemical found in combined washings perforated at 48 h post-dosing.
`
`

`

`32
`
`BUCKS ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`Absorbed
`
`5913056
`
`log Komr
`
`Figure 2. Percutaneous absorption of four steroids {mean % dose absorbed)
`as a Function of octanol/water
`rtition coefficient (Kay...) under occluded
`(open square} and “protected"
`tied square) conditions.
`
`With the exception of hydrocortisone. unpaired t-tests reveal that
`there is significantly {p < 0.01) more penetrant absorbed under oc-
`clusion than under protected conditions. Although it is generally
`accepted dogma that occlusion increases
`ercutaneous absorption.
`quantification ofthe efl'ect in vivo is scant 13.14]. It is also believed.
`on the whole. that occlusion increases transdetmal penetration for
`all compounds. but that. in partiCular. more water-soluble materials
`will exhibit greatest enhancement. However. our results show that
`the least lipophilic steroid. hydro-cortisone. appears unaffected by
`occlusion. This observation also contradicts an earlier study [15]
`which showed a clear promotion of absorption for hydrocortisone
`when the application site was occluded with thin plastic film {Saran
`Wrap). However, in this previous experiment. the skin site was not
`washed until 4 d post-dosing. during which time the occlusive pro-
`tection remained continuously in place. There is, in addition. some
`evidence to suggest that continued Frictional contact combined with
`skin flexing produces a “rubbing" effect which may cause an eleva-
`tion in percutaneous absorption [16.17]. While the plastic film re-
`mains in direct contact with the sltin surface. the HTC does not.
`The occlusion-induced enhancement in absorption seen for the li-
`pophilic steroids may be understood by a consideration of the ste s
`involved in percutaneous penetration. Following application. tlEC
`chemical must i) diffuse from the skin surface through the stratum
`corneum, ii) partition from the stratum corneum into the much
`more aqueous in nature viable epidermis, iii) did-use through the
`epidermis and up er dermis. and iv) encounter the cutaneous mi-
`crovasculature an gain access to the systemic pool. OCclusion leads
`to hydration of the stratum corneum and must. therefore. exert its
`efl'ect(s} on one or both of the first two steps. If hydration simply
`decreased the viscosity of the stratum corneum trans ort pathway
`(now believed to involve the intercellular lipid-fl led channels
`[18,19]). then the penetration of all chemicals should be equally
`enhanced by occlusion. An alternative possibility is that the stratum
`corneum-viable epidermis partitioning step is altered. Hydration of
`the stratum corneum will reduce the effective partition coeflicient
`of the penetrant between the stratum corneum and viable epidermis
`(because the two tissue phases now a pear more similar). The efiect
`of this decrease will be to increase t e kinetics of transfer of pene-
`trant from stratum corneum to viable epidermis. a change that
`should become progressively more a parent as the lipoPhilicity of
`the absorbing molecule increases [20)
`The importance ofthe partitioning step discussed above is further
`implied by the dependence of percutaneous absorption on steroid
`lip0philicity (Fig 2. Table IV). Penetration does not continue to
`increase with increasing lip0plii1icity. This attenuation in absorp-
`tion im lies a shift in the rate-determining step from stratum cor-
`neum iffusion to transfer across the stratum corneum-viable epi-
`dermis interface.
`a process which should become slower
`as
`penetrant lip0philicity increases. Once more. results with a series of
`
`para-substituted phenols are comparable [11]. The possibility tllit
`the arabolic form ofpercutaneous absorption versus log K is caused
`by {recreased surface availability as a result of increased association
`between the penetrant and the HTC has been considered. We b6-
`lieve that this explanation is not valid for two reasons: First. tl1‘c
`dependency of HTC-recovered dose on penetrant lipophilicityr lS
`weak. Second. literature data for the absorption ofthe four steroid-5
`under open ap lication. i.e.. non-protected. conditions [3] showr 3
`similar trend: Eydrocortisone 1.9 i 1.6%; estradiol. 10.6 :t 4.9%:
`testosterone. 13.2 i 3.0%;
`rogesterone. 10.8 :i: 5.8%. In this cash
`no consistently available absorptive surface was accessible to the
`a plied compounds. Interestingly. only the result for estradiol i!1
`tll'ijis earlier study is significantly different from the corresponding
`absorption values in Table 11 (“protected" conditions).
`_
`In summary. this paper presents evolving improvements in In
`vivo percutaneous absorption methodology. The approach is com-
`lemcntary to the recently described ex eriments of Rougier et 11
`21 —26[. The results demonstrate mass balance and dose account-
`ability. a means to study the effects ofocclusion on skin penetration.
`and. in the long term. the potential to define chemical structure-
`percutaneous absorption relationships in man.
`
`This research was supported by grants from the National Institutes of Hank
`(GM—33395 and HD-ZJGIOJ to RHC. who is the recipient oft: Sperial' Emphasii
`Research Career Award (K01-0HOOOI7)fmm CDC/NIOSH. We thank Ill!
`Dermaropharmary group at UCSF for helpful discussions and input. Ailen R
`Gm'zzetti for supplying the ComTex' membrane, and Andrea Mazel for maflfl‘
`stripr preparation.
`
`REFERENCES
`
`1. Guy RH. Guy AH. Maibach HI. Shah UP: The bioavailability 0f
`dermatological and other topically administered drugs. Pharm Res
`31253—262. i986
`
`2.
`
`Feldmann R). Maibach HI: Regional variation in percutaneous pent'
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`jULY 1988
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`STEROID BIOAVAILABILITY 33
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