`
`In Vitro Percutaneous Penetration: Evaluation of the
`Utility of Hairless Mouse Skin
`
`Robert S. Hinz, Ph.D.. Connie D. HodsOn, 13.8.. Cynthia R. Lorence, B.S.. and Richard H. Guy, Ph.D.
`Departments of Pharmacy and Pharmaceutical Chemistry. University of California. San Francisco; San Francisco. California. USA.
`
`is used to deposit a penettant on human skin. We suggest.
`The permeability barrier of hairless mouse skin has been
`therefore. that acetone~mediated facilitation ofpercutaneous
`determined in vitro after ex
`sure ofthe epidermal surface to
`volumes of acetone typical-l: used in human in vivo skin
`absorption in humans is unlikely. A further conclusion ofthis
`work is that in vitro solvent-deposition enetration experi-
`penetration studies. It has been shown that the transport of
`ments using hairless mouse skin shou d provide reliable
`tritiated water {when applied for limited 5-h periods} across
`trans
`hairless mouse skin is not affected by acetone treatments of
`rt information for at least 48 h postadministration.
`Although hairless mouse skin is more permeable than its
`approximately 15 til/cm? Submersion of the membranes be-
`tween :1 ueous donor and receptor phases for periods greater
`human counterpart, in vitro measurements using the murine
`than 2411. however. leads to significant and catastrophic bar-
`barrier should. therefore. provide useful and relevant guide-
`lines fot
`risk assessment calculations and bioavailability
`rier impairment. The acetone dose in the experiments re-
`
`determinations] Invest Dermatol 93:37—91, 1989
`ported is greater than that employed in vivo when the solvent
`
`he use of animal skin in the study of percutaneous
`absorption has provided fundamental knowledge
`toward our understanding ofharrier function. There
`are important differences, however. in the permeabil—
`ities of skin taken from different species and these
`inconsistencies have been highlighted in a number ofpublications
`[I — 4]. Currently. there is considerable activity in the area of in vitro
`skin permeation measurement. At least three major driving forces
`for this effort can be identified: 1) The U.S. Food and Drug Admin-
`istration recently 5
`nsored a workshop on in vitro methods for the
`purpose ofcstablishihg guidelines that could be followed when new
`topical drug formulations are under development [5]. 2) There is a
`continuing need for reliable. and meaningful. procedures that can be
`used to predict the health risk resulting from dermal exposure to
`toxic substances [6]. 3) The emergence of transdermal drug delivery
`to provide systemic pharmacologic efi'ect has introduced percutane-
`ous penetration measurement as a key component of the pharma-
`ceutic research and development effort [7].
`The heightened interest in assessing percutaneous transport has
`led several
`investigators to use substitutes for human skin. It is
`sometimes difficult to obtain human tissue in a regular or timely
`fashion; in addition. the high level of variability associated with
`cadaver skin [3] has frustrated researchers and has directed them to
`consider alternatives. OFthe various models that have been studied
`the skin of the hairless mouse is probably the most
`pular. There is
`no doubt that this tissue has enabled a number 0 key studies that
`have greatly increased our understanding of the skin permeation
`process. For example,
`it has allowed fundamental research into
`structure—penetration relationships [9 12!. concurrent transport
`and metabolism [13 - 16]. and the effects of skin damage on barrier
`
`Manuscript received June 21. 1983: accepted for publication January 6.
`1989.
`Institutes of Health grants
`This work was supported by National
`GMv33395 and HD-23010 and by the U.S. Environmental Protection
`Agency through cooperative agreement. CPL—812474.
`Re rint requests to: Dr. R, H. Guy, School of Pharmacy. University of
`Call oruia. San Francisco. CA 94143-0446.
`
`function [17 7 19]. The advantages of hairless mouse skin include its
`availability and reproducibility. It is more permeable than human
`skin. too. and this is an asset for both bioavailability and risk assess-
`ment. as a result obtained with hairless mouse skin will err on the
`conservative side. In risk assessment. for instance. a permeation
`measurement through hairless mouse skin will not lead to an under-
`estimate of dermal exposure in humans. This higher permeability.
`however. is also considered by sortie to be a major disadvantage of
`the tissue. although there is little evidence to document this con-
`cern. A more serious question. though. pertains to the response of
`hairless mouse skin. relative to that of human skin. to situations or
`circumstances often encountered in percutaneous penetration work.
`e.g.. the effect ofhydration and of organic solvents. The hydration
`issue was recently addressed by Bond and Barry [20]. who showed
`that prolonged expo
`sure of hairless mouse skin to aqueous donor
`and receptor phases in simple diffusion cells caused considerable
`derangement of barrier function. The interests of our laboratory
`have centered on in vivo evaluation of
`rc-utaneous absorption
`chemicals has involved
`[21—25]. Typically. topical application or
`deposition from an organic solvent. usually acetone. The question
`posed by the research
`resented here. therefore. was: "Does the
`amount of acetone used)
`as the vehicle in human skin penetration
`studies cause significant changes to the barrier function of hairless
`mouse skin in vitro?" A negative response would imply that 1)
`human skin in vivo is not damaged by the acetone deposition and
`delivery process and 2) in vitro hairless mouse skin ex
`riments
`orrnation
`involving chemical application in acetone may provide in?6
`relevant to percutaneous absorption in humans.
`MATERIALS AND METHODS
`
`To assess barrier function status of hairless mouse skin. the perme-
`ability of tritiated water (314,0. 0.05 .uCi/ml. New En land Nu-
`clear. BOston. MA) was determined at designated times a ter various
`acetone treatments. Permeation experiments were performed in
`conventional flow-throu h diffusion cells (Laboratory Glass Appa-
`ratus. Berkeley. CA} [26%
`The area of skin exposed was 3.14 cmz'.
`the volume of the rcce tor phase was approximately 5 cm’. The
`flow-rate was adjusted E
`y a cassette pump (Manostat. New York.
`
`DOZZ-ZUZX/S‘J/Smjfl Copyright © 1989 by The Society for Investigative Dermatology. Inc.
`87
`
`Noven Pharmaceuticals. Inc.
`EX2018
`
`0001
`
`Mylan Tech., Inc. v. Noven Pharma, Inc.
`lPR2018-00173
`
`

`

`88 HINZ er AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`10
`
`20
`
`so
`
`a
`
`3
`
`2
`
`1
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`52
`
`tlma (hr)
`Figure 1. Permeation (mean flux :l: SD, n = 8) of 31-120 through hairless
`mouse skin when the membrane is sandwiched between aqueous solutions
`for 24 h [experiment I).
`
`contact. Indeed. in six out of eight cells, the membrane has been so
`damaged that 3H245) flux decreases at later times due to the substan-
`tial depletion of radiolabel in the donor phase.
`The results of experiments “I and IV are summarized in Figures 3
`and 4. respectively. It is apparent that when water is dosed intermit-
`tently to the skin surface for 5-h
`riods, pretreatment with acetone
`does not cause any significant di erence to the permeability behav-
`ior. This conclusion was substantiated b
`statistical comparisons
`(paired Wilcoxon and Student’s t—tests) o the cumulative amounts
`of water transported across the control and acetone-treated mem-
`branes. following the 5-h ap lications. Acetone elicited an insignifi-
`cant effect {a > 0.2. p > 0.1 on water permeation. Figure 5. which
`contains the data from ex
`riment V. demonstrates that repeated
`acetone administration be ore dosing with water also elicits no sig-
`nificant derangement of barrier function. Although Figures 3. 4.
`and 5 appear to suggest that, regardless of acetone treatment or not.
`the
`rmcability of 31-120 increases with time, the trend is not statis-
`ticall; significant. It is perhaps reasonable, however. to conclude
`that hydration and the detrimental ellects thereof. can continue
`during the water “ofF” periods.
`Finally. Figure 6 illustrates the results ofex eriment VI, in which
`the permeation of “I120 was followed 24 h allier acetone treatment.
`Again, no difl'erence from the control studies was observed al»
`
`NY) so that the receptor solution was completely exchanged in 1 h.
`The receptor phase was normal saline in phosphate buffer at pH 7.4.
`Pcrfusate was collected in test tubes mounted on a fraction collector
`(Gilson FC-220. Middleton, WI) and the samples were then ana-
`lyzed by liquid scintillation counting {Searle Mark 11] Model 6880.
`Elk Grove, IL). The difi‘usion cells were thermcistatted at 35°C
`throughout the experiments; under these conditions. with the skin
`Open to the laboratory atomosphcre, the surface temperature of the
`membrane was 32°C 1 1°C.
`
`In all experiments, full-thickness skin from hairless mice (HRS/
`hr h r. 6 — 16 wk. Simonsen Laboratories, Gilroy, CA was used. The
`skin was removed from the animal immediately a tct killing. any
`small fatty deposits were carefully removed, and the membrane was
`then mounted in the diffusion cell. Typically. eight diffusion cells
`were used in each experiment. requiring skin from four mice.
`When comparisons were made within a run (acetone treatment vs.
`no treatment. for exam le). the four skins were halved so that each
`animal contributed to oth the “control” and “test” set of cells.
`Because of the time involved in setting up eight diffusion cells and
`adjusting the receptor solution flow-rate appropriately, an experi-
`ment was typically started within 2 h after
`illing of the animals.
`The experiments performed are summarized in Table I. The de-
`sign was selected to test the effects of an acetone dose on barrier
`function and tissue constancy. The water treatments involved appli-
`cation ofl cmJ of’HZO to the skin surface. To prevent evaporation.
`the upper half of the diffusion cell was then covered until the water
`was removed. W henevet water was not in contact with the skin, the
`surface was open to ambient conditions. Acetone treatments in-
`volved a plication of 50 p] of the solvent by a capillary pi et. As
`pcformed in vivo. the acetone was distributed evenly over t e skin
`surface, which. again. was open to the laboratory atmosphere.
`When water was administered subsequent to acetone treatment (ex-
`periments Illa, lVa, V) there was a 2- to 3-min time lapse between
`solvent applications. No liquid acetone remained at this point.
`Experiments I and II simply determined water permeation over
`24— and 48-h periods, in the absence of acetone treatment. Experi-
`ments III and IV used short S-h exposures of the tissue to water and
`assessed the long-term consequences of an acetone close at r '= 0.
`Ex
`riment V involved a greater potential insult to the tissue and
`inclfided three volatile solvent treatments. Ex eriment VI consid-
`ered the effect ofa time delay postacetone app ication followed by
`prolonged water contact.
`
`RESU LTS
`
`In experiments I and II. the skin remained sandwiched between
`aqueous solutions throughout the measurement periods (24 and 48
`h. respectively). Figure 1 showr that in experiment I. 311,0 flux is
`essentially constant over the 3- to 20-h postapplication period, cor-
`responding to a permeability coefficient ofabout 2.95 X 10"" cm/h
`{in good agreement with recently published data [20]). Increased
`permeation. however, is suggested by the later time points. an infer—
`ence confirmed by experiment II. Figure 2 indicates that prolonged
`and complete hydration leads to barrier breakdown after 24 h of
`
`
`
`Table 1. Experimental Design Summary (n = number of replicates)
`
`
`Experiment
`n
`Treatments
`
`T
`T
`T
`T
`T
`
`1
`
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`T
`T
`T
`l
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`T
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`1
`T
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`32
`28
`24
`48
`Time (hours)
`
`" V Sllyl acetone: l I water on; I - water rslT
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`

`VOL. 93. NO.
`
`I
`
`jULY I989
`
`[N w‘rno SKIN PENETRATION 89
`
`%doselhr
`
`0
`
`1O
`
`20
`
`30
`
`40
`
`50
`
`time(hr)
`Figure 2. Permeation of 31120 through hairless mouse skinI when the
`membrane is sandwiched between aqueous solutions for 48 h (experiment
`[I]. The results from eight separate experiments are shown. The average
`3H20 permeability coefficient during the 5—IS-l1 postdosing period is
`1.2}I X10" cth.
`
`though. as before. prolonged exposure to aqueous solutions did
`begin to compromise the skin.
`DISCUSSION
`
`r-
`The experiments performed in this investi ation lead to two irn
`tant conclusions. First, as recently reporte by Bond and Barry 20].
`the barrier function of hairless mouse skin does not withstand pro-
`longed submersion in aqueous solution. The data in Figures 1 and 2
`
`_l "I
`
`1.0%doseihr
`
`time (hr)
`Figure «I. Effect of acetone pretreatment {I S Jnl/cm’) on tritiatcd water flux
`(mean i SI), 11 a 12:] across hairless mouse skin after appiications from t =
`0—5 h. t = 24—29 h. and I = 48—53 h (experiment IV).
`
`clearly reveal that exposure of the tissue to aqueous solutions, in
`both donor and receiver compartments. for periods in excess of24 h,
`leads to substantial degeneration of the stratum corneum. The sec-
`ond key finding revealed by this study is that treatment of hairless
`mouse skin with acetone, in a fashion that mimics a typical "sol-
`ventqieposition" a plication procedure [22-— 251does not appear to
`alter permanently the barrier to water to any significant degree. The
`results ofexpetiments III, IV, and V indicate that acetone adminis-
`tration Pct sc does not contribute to derangement of the stratum
`corneum. The data also suggest that ifa penetrant were delivered in
`
`1.5
`
`2.0
`
`1.0
`
`%dosefhr
`
`O
`
`1 0
`
`2 O
`
`3 O
`
`tlme (hr)
`Figure 3. Effect of acetone pretreatment (15 til/cm?) on tritiated water flux
`(mean i SD, n -= 3) across hairless mouse skin after applications from
`t= 0—5 h and t = 24—29 h {experiment III}.
`
`dose!hr
`
`%
`
`
`
`20
`30
`no
`so
`so
`
`
`time (hr)
`Figure 5. Tritiated water flux (mean i 51‘), n :- 4} across hairless mouse
`skin after applications from r= 0— 5 h. := 24— 29 h. and! =43 -- 53 h. Before
`mull administration of water. the skin was pretreated with acetone at a dose of
`15 plfcm’.
`
`0003
`
`

`

`90 umz ET AL
`
`THE jOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`doselhr
`
`%
`
`
`
`time {hr)
`Figure 6. Permeation (mean flux i SD. n = 12) of 3H20 through hairless
`mouse skin 24 h after mounting the membrane in the difl'usion cell. and after
`treating the “acetone';:lpecimens with a solvent dose of 15 ,ul/cmz. For the
`subsequent 24-h peri
`of observation presented here. the sltin is sand-
`wiched between aqueous donor and receptor phases (ex
`riment VI). Based
`on the essentially constant 31120 fluxes between 5 an
`10 h. permeability
`coefficients for the control and acetone-treated membranes are calculated to
`be 1.83 X 10 ’ cm/h and 1.83 X 10" em/h. respectively.
`
`an acetone vehicle under similar circumstances. one may expect the
`barrier function of hairless mouse skin to remain reasonably con-
`stant for at least 48 h. The short 5-h snzo applications were de«
`si ned to test the skin permeability while minimizing hydration
`cigars. In this respect. they would ap ear to have fulfilled their
`function adequately. Experiment V cha lenged the tissue further by
`considerin multiple acetone treatments. Again. however. no sig-
`nificant eigeet of the solvent, over that observed in the controls
`(experiment
`lVb). was ap arcnt. Furthermore. experiment VI
`showed that air-exposure o the epidermal surface for 24 h [acetone
`pretreated or not) did not significantly alter subsequent 3H20 per-
`meation compared with the “control". i.e.. experiment 1.
`One ramification of our research is that the warning of Bond and
`Barry [20].
`that “ .
`.
`. hairless mouse skin should not be
`used .
`.
`.
`in .
`.
`. permeation studies incorporating long~term
`hydration. as erroneous results can be expected after .
`.
`. 3 days."
`appears somewhat conservative. On the basis ofour data. we would
`be reluctant to draw conclusions from any flux measurements made
`after 24 h submersion. More important. though. we have shown
`that administration of acetone. at a dose of a proximately 15 ul/
`c1112. does not ap ear to alter significantly t be stratum corneum
`
`barrier function ofhairless mouse skin. Given that a typical tapical
`
`in penetra—
`dose ofacetone in a solvent-deposition. human in vivo 5
`tion study is less than 10 till/cm2 [22-25]. it seems reasonable to
`sag est that no acetone-mediated Facilitation of transport will be
`evitfcnt. In addition. one may also deduce that an in vitro penetra-
`tion study using hairless mouse skin and solvent-deposition ofpene-
`trant from acetone {at a dose of 15 til/Cm: or less). should provide a
`reasonable model ex
`riment for the in vivo situation. We recog-
`nize. however. that t e latter two conclusions are based on observa-
`tions that use water as the model permeant. It remains to be seen
`whether these deductions are sustained when the penetating mole-
`cule is lipophilic in character. Finally. although hairless mouse skin
`is generally more permeable than its human counterpart [20]. the
`a plication of small volatile solvent volumes does not 9.13
`at to
`place the murine barrier under measurable stress. The e ects of
`
`larger volumes of solvent or of more structurally destructive chemi-
`cals (e.g.. penetration enhancers) [27] will. no doubt, be greater and
`may be amplified in the less substantial stratum corneum of the
`hairless mouse.
`
`We thank Dr. Larry L. Hall ofthe Environmenmi Pruterti'on Agencyfilr his interest
`and input. Nan Spencerfar manuscript preparation.
`
`REFERENCES
`
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`

`

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`
`JULY 1989
`
`IN VITRO SKIN PENETRATION 91
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`