Bioavailability of Topically Administered Steroids: A
`Mass Balance" Technique
`
`6 6
`
`Daniel A.W. Bucks, M.A..James R. McMaster, M.A., Howard I. 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 "protected"
`(i.e., covered, but non-occlusive) conditions. The experi­
`mental approach, involving simple modifications of standard
`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 topical bioavailability can be inferred. In addition,
`because of 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 hydophobic analog studied) did not
`continue the trend and was at least partly rate-limited by slow
`
`I
`
`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 [t]. However, in vivo percutaneous
`absorption experiments in man arc much more difficult to perform
`than either animal model or in vitro penetration studies. Further­
`more, most of the in vivo investigations which have been carried out
`have not allowed accountability of the applied dose and, hence, have
`not produced results which can be interpreted unequivocally.
`The majority of human in vivo percutaneous absorption measure­
`ments have used indirect radiochemical methods [2-6]. Typically, a
`,4C labeled chemical is applied topically from a volatile solvent
`vehicle and penetration is evaluated from the excretion of the UC
`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.
`ipproach has some clear limitations: any conclusions are based
`The a
`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 of that fraction
`of the topical dose which is not absorbed immediately into the skin
`post-application 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
`
`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 passive transepi-
`dermal water loss at the application site) to be assessed. Oc­
`clusion significantly increased percutaneous absorption of
`estradiol, testosterone, and progesterone but did not effect
`the penetration of hydrocortisone. A mechanism is proposed
`to explain why the absorption of the more lipophilic steroids
`is enhanced 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 ot homologous or analogous
`chemicals, y Invest Dermatol 90:29-33, 1988
`
`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-
`stripping the upper layer of stratum corneum. The key improve­
`ment afforded 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 multiple-dosing regimens and mea­
`surements have been made under both 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 relationships for skin ab­
`sorption in man, and revealing quantitatively the effects of
`occlusion on the transport of compounds across the cutaneous bar­
`rier.
`
`MATERIALS AND METHODS
`The penetrants considered were four steroids: progesterone, testos­
`terone, estradiol, and hydrocortisone. The ,4C-labeled chemicals
`(RPI Corp., Mount Prospect, IL) were applied in acetone to the
`ventral forearm of healthy male volunteers (n > 5), from whom
`informed consent, approved by the UCSF Committee on Human
`Research, had been previously obtained. Chemical and radioactivity
`doses were 4 fig/cm2 and 1 /iCi/cm2, respectively ; the area of appli­
`cation was 2.5 cm2 and the dose was administered in 20 fi\ of ace­
`tone.
`After evaporation of the vehicle (<0.5 min), the application site
`was covered with a semirigid, polypropylene Hilltop* (Hilltop Re­
`search, Inc., Cincinatti, OH) chamber (HTC), which was affixed to
`
`0022-202X/88/S03.50 Copyright © 1988 by The Society for Investigative Dermatology, Inc.
`29
`
`  
`
`

 
`
`MYLAN - EXHIBIT 1022
`
`

`

`30 BUCKS ET AL
`
`THE JOURNAL OP INVESTIGATIVE DERMATOLOGY
`
`the skin with hypoallcrgcnic 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 penetrant, etc.), the roof of the chamber was covered with a
`piece of Gore-Tex* (W.L. Gore & Associates, Inc., Elkton, MD)
`membrane (0.2 /Jtn pore size). It was found that Gore-Tex" did not
`impede transepidermal water loss 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. MC-Toluenc 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 h after dosing, the chamber (or chamber +
`Gore-Tex*) was removed, placed in scintillation fluid, and seques­
`tered l4C was counted. An appropriate quench correction was again
`made. The application site was washed with a standardized proce­
`dure [71 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 uC-chemical; 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
`ing 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 jig/cm2 to the same skin site. The first
`and eighth applications utilized uC-labelcd drug and urinary excre­
`tion for 7 d (using the collection schedule described above) after
`each of these 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 myris-
`tate and water, and tctradecane and water, were determined using a
`standard technique |9|. Octanol-water partition coefficients were
`obtained from the literature [10|.
`RESULTS
`Data from the single dose experiments performed under occlusive
`conditions arc presented in Table I and should be compared to the
`corresponding results from the "protected" studies given in Table
`11. Total recoveries are, in general, high and were greater for the
`"protected" measurements. These experiments were performed
`after the occluded investigation and incorporated obligatory evalua­
`tions of i) HC-radiolabelcd sequestered on the second HTC, h)
`chemical in the second set of washings, and iii) material remaining
`in the upper layers of the stratum corneum at the end of 7 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
`effect of occlusion on the topical bioavailability of the four steroids.
`With the exception of hydrocortisone, unpaired t-tests show that
`occlusion significantly increases the percutaneous absorption (p
`<0.01) of these compounds in man. This finding is further empha­
`sized in Fig 1, which shows, for each of the four steroids, the rate of
`excretion of radiolabel following their topical application under
`both occluded and "protected" conditions. To optimize clarity, data
`for estradiol, testosterone, and progesterone are plotted semi-loga-
`rithmically because of the difference in absorption between oc-
`
`Table I. Disposition of Topically Applied ,4C-labeled Steroids Following a Single Dose under Occluded Conditions
`Percentage of Applied Dose"
`1 st Washd
`
`Steroid
`
`Absorbed1,
`
`1st HTC
`
`2nd HTC
`
`2nd Wash'
`
`Total
`
`68 ± 3.9
`87 ± 13
`90 ± 8.4
`80 ± 5.5
`
`Hydrocortisone
`Estradiol
`Testosterone
`Progesterone
`
`4.0 ± 2.4
`27 ± 6.4
`46 ± 15
`33 ± 8.9
`
`28 ± 5.6
`41 ± 10
`41 ±8.4
`46 ± 10
`
`36 ± 3.0
`18 ±7.2
`3.0 ±4.1
`1.2 ±0.8
`
`n.d."
`0.5 ±0.3
`0.3 ± 0.2
`.07 ± .02
`
`n.d.«
`n.d.'
`n.d.«
`.01 ± 0.0
`
`1 Mean ± standard deviation (n ~ 5. except for progesterone, for which n ~ 6).
`Values corrected for incomplete renal elimination [3|.
`' Material sequestered on Hilltop chamber (HTC) removed at 24 h post-dosing.
`d Chemical found in combined washings performed 24 h post-dosing.
`' Material sequestered on HTC removed at end of measurement period.
`1 Chemical found in combined washings performed at end of experiment.
`• n.d.: not determined.
`
`Table II. Disposition of Topically Applied ,4C-labelcd Steroids Following a Single Dose Under "Protected" Conditions
`
`Steroid
`
`Hydrocortisone
`Estradiol
`T estosteronc
`Progesterone
`
`Absorbed11
`
`4.4 ± 1.7
`3.4 ± 1.2
`18 ±8.6
`13 ±6.3
`
`1st HTC
`
`27± 11
`38 ± 13
`46 ± 7.5
`54 ± 7.7
`
`Percentage of Applied Dose*
`1st Washd
`2nd HTC
`
`51 ± 18
`58± 12
`30 ± 15
`27 ± 8.7
`
`3.2 ± 1.7
`0.7 ± 0.4
`1.4 ±0.4
`1.2 ±0.6
`
`• Mean ± standard deviation (n — 6).
`' Values corrected for incomplete renal elimination (3).
`' Material sequestered on HTC + Gore-Tex® removed at 24 h post-dosing.
`J Chemical found in combined washings performed 24 h post-dosing.
`' Material sequestered on HTC + Gore-Tex® removed at end of measurement period.
`' Chemical found in combined washings performed at end of experiment.
`« "C-radiolabel present in 10 tape strippings of stratum corneum (SC) removed after final washing procedure.
`k n.d.: not determined.
`
`2nd Wash'
`
`SC "strips"'
`
`Total
`
`2.7 ± 1.3
`0.3 ± 0.4
`0.1 ±.08
`0.3 ± 0.4
`
`2.5 ± 1.1
`0.1 ±0.1
`n.d.h
`n.d.'1
`
`89 ± 5.6
`100 ±0.9
`96 ± 2.0
`96 ± 3.4
`
`

`

`VOL. 91. NO. 1 JULY 1988
`
`STEROID BIOAVAILABILITY 31
`
`HYDROCORTISONE ABSORPTION
`
`ESTRADIOL ABSORPTION
`
`Table IV. Oil-W.itcr Partition Coefficients of Steroids Studied
`
`005
`
`004
`
`003
`
`0 02 •
`
`001
`
`000
`
`10'
`
`5 "•
`
`I D0-
`
`10 1
`
`20 40 80 B0 100 120 140 160
`
`10 1'
`
`20 40 60 60 100 120 140 160
`
`TIME (HRS)
`
`TIME (HRS I
`
`TesToairnoNE ABSORPTION
`
`PROGESTERONE ABSORPTION
`
`.6'
`
`5
`
`10 H
`
`2 0 4 0 a o a o
`
`t o o l a o M O l e o
`
`10 *•
`
`2 0 4 0 tO 8 0 1 0 0 < 3 0 1 4 0 I CO
`
`T I M t (MRS)
`
`TIME (MRS )
`
`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" (tilled 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
`applied 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 1 and III) (with the
`possible exception of estradiol for which marginally significant dif­
`ferences (p = 0.04) in percutaneous absorption between the first
`and eighth 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, tetradecane)
`and water are reported.
`
`Steroid
`
`log Ko/w'
`
`'"g K.,/wb _
`
`Hydrocortisone
`Estradiol
`T cstosteronc
`Progesterone
`
`1 . 6 1
`2.41
`3.32
`3.87
`
`-0.19 ±0.02
`2.33 ± 0.04
`1.98 ±0.002
`2.62 ± 0.005
`
`Ion K
`T/W
`-2.17 ±0.03
`-0.027 ± 0.003
`0.68 ± 0.02
`2.27 ±0.11
`
`'K.
`>/w " Octanol-watcr partition coefficient |9.10|.
`'' K|/w — hopropyl myristate-water partition coefficient (mean ± standard devia­
`tion; n ~ 6).
`' KT/W — Tctradccanc-watcr partition coefficient (mean ± standard deviation;
`n "• 6).
`
`DISCUSSION
`The experiments reported in this paper highlight three issues: 1) the
`accountability of the applied chemical dose and the potential utility
`of the technique for measurement of topical bioavailability; 2) the
`effect of occlusion on the in vivo skin permeation of steroids; and 3)
`the relationship between percutaneous absorptic
`in and the relative
`lipophilicity of the penetrant.
`The mass balances achieved in this work are generally high and
`often approach 100%. A conventional in vivo approach [2-6]
`would have only revealed the percent dose absorbed columns in
`Tables I - 111. Disposition of the remainder of the applied radioactiv­
`ity would remain unknown. The importance of repeating 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" (Tabic 11) and multiple-dosing (Table 111) 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 pertinent to note that in Table II hydrocortisone, the least
`lipophilic 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 percutaneous absorption.
`The persistence of hydrocortisone 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 para-substituted phenolic pe
`netrants
`has revealed the same pattern: phenols with more polar para-substit-
`uents (e.g.. —NH2, —NHCOCHj, —NHCOC2Hs) show pro­
`longed stratum corneum residence, whereas more lipophilic ana­
`logs (p-CN, p-1) do not.
`In Fig 2, the percentage dose absorbed for each steroid is plotted as
`a function of penetrant octanol/water partition coefficient; results
`obtained under occluded and "protected" conditions are compared.
`
`Table III. Disposition of Topically Applied 14C-labeled Steroids Following Multiple Dose Under Occluded Conditions
`
`Steroid
`
`Hydrocortisone
`
`Estradiol
`
`Testosterone
`
`lJoseb
`
`1st
`8th
`Ist
`8th
`1st
`8tli
`
`Absorbed'
`
`3.5 ± 1.3
`3.1 ± 1.0
`38 ± 7.9
`22 ±7.1
`51 ± 10
`50 ± 9.5
`
`Percentage of Applied Dose*
`Ist HTC
`1st Wash'
`
`23 ± 7.7
`32 ± 5.4
`47 ± 12
`37 ± 9.9
`46 ±9.1
`37 ± 9.7
`
`53± 11
`33 ± 7.5
`14 ±6.8
`21 ± 5.2
`1.7 ± 1.0
`4.3 ±5.4
`
`" Mean ± standard deviation (n ~ 5. except for hydrocortisone 8tli dose, for which n — 4).
`b The Ist and 8th doses of a daily dosing regimen, lasting 14 d. were l4C-radiolabclcd.
`* Values corrected for incomplete renal elimination (31.
`J Material sequestered on HTC removed at 24 h post-doling.
`' Chemical found in comhined washings performed 24 h post-dosing.
`1,4C-labelcd material sequestered on HTC removed at 48 h post-dosing.
`• "C-labelcd chemical found in comhined washings performed at 48 h post-dosing.
`
`2nd HTC
`
`3.5 ± 1.4
`7.4 ± 0.8
`0.6 ± 0.8
`0.4 ± 0.2
`0.2 ±0.1
`0.2 ± 0.2
`
`2nd Wash'
`
`2.6 ± 0.8
`4.8 ± 1.7
`0.5 ± 0.6
`0.5 ± 0.2
`.06 ± .06
`.06 ± .04
`
`Total
`
`85 ± 4.3
`81 ± 2.5
`100 ± 3.9
`81 ± 6.0
`99 ± 4.3
`92 ± 17
`
`

`

`32 BUCKS ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`SO
`
`40
`
`30
`
`1
`
`10
`
`•c
`
`1 . 0
`
`2 0
`
`TST
`
`ESI
`
`ISI
`
`WG
`
`WG
`
`EST
`
`3 0
`
`4.0
`
`log Ko/w
`
`Figure 2. Percutaneous absorption of four steroids (mean % dose absorbed)
`as a function of octanol/water partition coefficient (KOJ,w) under occluded
`(open square) and "protected" (filled 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 percutaneous absorption,
`quantification of the effect in vivo is scant [13,14]. It is also believed,
`on the whole, that occlusion increases transdermal 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, hydrocortisone, 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 skin 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 steps
`involved in percutaneous penetration. Following application, the
`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) diffuse through the
`epidermis and upper dermis, and iv) encounter the cutaneous mi-
`crovasculature and gain access to the systemic pool. Occlusion leads
`to hydration of the stratum corneum and must, therefore, exert its
`cffect(s) on one or both of the first two steps. If hydration simply
`decreased the viscosity of the stratum corneum transport pathway
`(now believed to involve the intercellular lipid-filled 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 coefficient
`of the penetrant between the stratum corneum and viable epidermis
`(because the two tissue phases now appear more similar). The effect
`of this decrease will be to increase the kinetics of transfer of pene­
`trant from stratum corneum to viable epidermis, a change that
`should become progressively more aoparent as the lipophilicity of
`the absorbing molecule increases (20).
`The importance of the partitioning step discussed above is further
`implied by the dependence of percutaneous absorption on steroid
`lipophilicity (Fig 2, Table IV). Penetration does not continue to
`increase with increasing lipophilicity. This attenuation in absorp­
`tion implies a shift in the rate-determining step from stratum cor­
`neum diffusion to transfer across the stratum corneum-viable epi­
`dermis interface, a process which should become slower as
`penetrant lipophilicity increases. Once more, results with a series of
`
`para-substituted phenols are comparable (11). The possibility thai
`parabolic form of percutaneous absorption versus log K is caused the
`
`by decreased surface availability as a result of increased association
`between the penetrant and the HTC has been considered. We be­
`lieve that this explanation is not valid for two reasons: First, the
`dependency of HTC-recovered dose on penetrant lipophilicity is
`weak. Second, literature data for the absorption of the four steroids
`under open application, i.e., non-protected, conditions [3] show >
`similar trend: hydrocortisone 1.9 ± 1.6%; estradiol, 10.6 ± 4.9%;
`testosterone, 13.2 ± 3.0%; progesterone, 10.8 ± 5.8%. In this case,
`no consistently available absorptive surface was accessible to the
`applied compounds. Interestingly, only the result for estradiol in
`this earlier study is significantly different from the corresponding
`absorption values in Table II ("protected" conditions).
`In summary, this paper presents evolving improvements in in
`vivo percutaneous absorption methodology. The approach is com­
`plementary to the recently described experiments of Rougier et »1
`[21-26]. The results demonstrate mass balance and dose account­
`ability, a means to study the effects of occlusion on skin penetration,
`and, in the long term, the potential to define chemical structure-
`percutaneous absorption relationships in man.
`
`This research was supported by grams from the National Institutes of Health
`(GM-33J95 and HD-23010) to RUG, who is the recipient of a Special Emphasif
`Research Career Award (KOI-0H000I7) from CDC/NIOSH We thank the
`Dermatopharmacy group at UCSF for helpful discussions and input, Allen R
`Guuzetti for supplying the Gore-Tex' membrane, and Andrea Maze! for manu­
`script preparation.
`
`REFERENCES
`1. Guy RH. Guy AH. Maibach HI. Shah VP: The bioavailability of
`dermatological and other topically administered drugs. Pharm Res
`3:253-262. 1986
`2. Feldmann RJ. Maibach HI: Regional variation in percutaneous pene­
`tration of "C Cortisol in man. J Invest Dermatol 48:181 -183.1967
`3. Feldmann RJ. Maibach HI: Percutaneous penetration of steroids in
`man. J Invest Dermatol. 52:89-94. 1969
`4. Feldmann RJ. Maibach HI: Absorption of some organic compounds
`through the skin in man. J Invest Dermatol 54:399-404. 1970
`5. Feldmann RJ. Maibach HI: Percutaneous penetration of some pesti­
`cides and herbicides in man. Toxicol Appl Pharmacol 28:126-132.
`1974
`6. Feldmann RJ. Maibach HI: Percutaneous penetration of HC hydrocor­
`tisone in man. II. Effect of certain bases and pretreatments. Arch
`Dermatol 94:649-651, 1966
`7. Ducks DAW. Marty JPL, Maibach HI: Percutaneous absorption of
`malathion in the guinea pig: effect of repeated skin application.
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