In Vitro Percutaneous Penetration: Evaluation of the
`Utility of Hairless Mouse Skin
`
`Robert S. Hinz. Ph.D.. Connie D. Hodson. B.S.. Cynthia R. Lorence, 13.8.. and Richard H. Guy, Ph.D.
`Departments of Pharmacy and Pharmaceutical Chemistry. University ofCalifornia. San Francisco.- San Francisco. California. U.S.A.
`
`is used to deposit a penetrant on human skin. We suggest.
`Thc permeability barrier of hairless mouse skin has been
`therefore. that acetone-mediated facilitation of percutaneous
`determined in vitro after ex
`sure of the epidermal surface to
`volumes of acetone typica ly used in human in vivo skin
`absorption in humans is unlikely. A further conclusion ofthis
`work is that in vitro solvent-deposition
`netration experi-
`penetration studies. It has been shown that the transport of
`ments using hairless mouse skin shou :1 provide reliable
`tritiatetl water (when applied for limited S—h periods) across
`transport information for at least 48 h postadministration.
`hairless mouse skin is not aliectcd by acetone treatments of
`Although hairless mouse skin is more permeable than its
`approximately 15 til/cm”: Submersion of the membranes be-
`human counterpart, in vitro measurements using the murine
`tween aqueous donor and receptor phases for periods greater
`than 24 h. however, leads to significant and catastrophic bar-
`barrier should. therefore. provide useful and relevant guide-
`lines for
`risk assessment calculations and bioavailability
`rier impairment. The acetone dose in the experiments re-
`determinations} Invest Dermatol 93:87—91, 1939
`ported is greater than that employed in vivo when the solvent
`
`rcutaneous
`he use of animal skin in the study of
`knowledge
`absorption has provided fundamenta
`toward our understanding of barrier 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 of publications
`[1 - 4]. Currently. there is considerable activity in the area ofin vitro
`skin permeation measurement, At least three major driving forces
`for this client can be identified: 1) The US. Food and Drug Admin-
`istration recently 5
`nsored a workshop on in vitro methods for the
`purpose ofcstablis ing 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 phannacologic effect has introduced percurane-v
`ous penetration measurement as a key component of the pharma-
`reutic research and development eliort [T].
`The heightened interest in assessing percutaneous transport has
`led several investigators to use substitutes for human skin. It is
`sometimes difiicult to obtain human tissue in a regular or timely
`fashion: in addition. the high level of variability associated with
`cadaver skin [8] has frustrated researchers and has directed them to
`consider alternatives. Of the various models that have been studied
`the slrin of the hairless mouse is probably the most opular. There is
`no douht 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. 1988: accepted for publication January 6.
`1989.
`institutes of Health grants
`This work was supported by National
`GM—33395 and HD—23010 and by the US. Environmental Protection
`Agency through cooperative agreement. {IR-812474.
`Reprint requests to: Dr. R. H. Guy. School of Phannacy. University of
`California. San Francisco. CA 94143-0446.
`
`function [17 — 19]. The advantages ofhairless 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 some 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 of hydration and of organic solvents. The hydration
`issue was recently addressed by Bond and Barry [20]. who showed
`that prolonged exposure of hairless mouse skin to
`occur donor
`and receptor phases in simple difimion cells caused considerable
`derangement of barrier function. The interests of our laboratory
`have centered on in vivo evaluation of ercutaneous absorption
`[21 - 25]. T pically. topical application oiElchemicals has involved
`deposition tom 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
`involving chemical application in acetone may provide ingrmation
`relevant to percutaneous absorption in humans.
`MATERIALS AND METHODS
`
`To assess barrier function status of hairless mouse skin. the penne-
`ability of tritiated water (3H20. 0.05 yCi/ml, New En land Nu-
`clear. Boston. MA) was determined at designated timesa ter various
`acetone treatments. Permeation experiments were performed in
`conventional flow-throu h difi'usion cells (Laboratory Glass Appa—
`ratus. Berkeley. CA) [26? The area of skin exposed was 3.14 cmz'.
`the volume of the reee tor phase was approximately 5 cm’. The
`flow-rate was adjusted by a cassette pump (Manostat. New York.
`
`0022-202X/89/503.50 Copyright © 1989 by The Society for investigative Dermatology. Inc.
`
`Noven Pharmaceuticals, Inc.
`EX2018
`
`0001
`
`Mylan Tech., Inc. v. Noven Pharma, Inc.
`|PR2018-00174
`
`

`

`88 HINZ ET .tt.
`
`THE jOURNAL OF [NVESTIGATIVE DEWTOLOGY
`
`1o
`
`20
`
`an
`
`4
`
`3
`
`2
`
`1
`
`0
`
`0
`
`h E0 8
`
`'D
`
`32
`
`tlme (hr)
`Figure 1. Permeation (mean flux i SD. n - 8) of ’H,O 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 3H20 flux decreases at later times due to the substan-
`tial depletion of radiolabel in the donor phase.
`The results oFexperimenrs Ill and IV are summarized in Figures 3
`and 4, respectively. It is a parent that when water is dosed intermit-
`tently to the skin surface For 5-h
`riods, pretreatment with acetone
`does not cause any significant digit-met 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 eHect {or > 0.2, p > 0.13 on water permeation. Figure 5. which
`contains the data from ex
`timent 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
`rmeahil ity of’HZO increases with time, the trend is not statis-
`tica l
`significant. It is perhaps reasonable, however, to conclude
`that hydration and the detrimental effects thereof, can continue
`during the water “off" periods.
`rirnent VI, in which
`Finally, Figure 6 illustrates the results of ex
`the permeation of 3I-IZO was followed 24 h a ter acetone treatment.
`Again, no difference from the centrol 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.
`Perfirsate 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 111 Model 6880.
`Elk Grove. IL). The diffusion cells were thermostatted at 35°C
`throughout the experiments; under these conditions. with the skin
`open to the laboratory atomosphere, the surface temperature of the
`membrane was 32°C :I: 1 °C.
`In all experiments, full-thickness skin from hairless mice (HRS/
`hr hr, 6 - 16 wk, Simonsen Laboratories, Gilroy. CA was used. The
`skin was removed from the animal immediately a ter killing, any
`small fatty dc osits were carefully removed. and the membrane was
`then mounte
`in the diliusion 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
`th the “control" and "test" set of cells.
`Because ofthe time involved in setting up eight difFusion cells and
`adjusting the receptor solution flow-rate ap ropriately, 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 eli'ects of an acetone dose on barrier
`Function and tissue constancy. The water treatments involved appli-
`cation of 1 cm3 of’Hgo to the sltin surface. To prevent evaporation.
`the upper halfof the diffusion cell was then covered until the water
`was removed. Whenever water was not in contact with the skin. the
`surface was open to ambient conditions. Acetone treatments in-
`volved a plication of 50 ,ul of the solvent by a capillary pi
`I. As
`preformedJ 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 31-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 Ill and IV used short 5-h exposures of the tissue to water and
`ISSCSSC'CI the long—term consequences of an acetone dOse at r- 0.
`Ex erirnent V involved a greater potential insult to the tissue and
`inc uded three volatile solvent treatments. Ex eriment VI consid-
`ered the effect of a time delay postacetone app ication followed by
`prolonged water contact.
`
`RESULTS
`
`In experiments I and II, the sltin remained sandwiched between
`aqueous solutions throughout the measurement periods {24 and 43
`h. respectively}. Figure 1 shows that in experiment I, 3H20 flux is
`essentially constant over the 3- to 20‘}: postapplication period, cor-
`responding to a permeability coefficient of about 2.95 X 10“3 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 I. Experimental Design Summary (n - number of replicates)
`
`n
`Treatments
`Experiment
`
`T
`T
`T
`T
`T
`
`T
`
`T
`T
`T
`T
`T
`
`T
`
`T
`T
`T
`
`l
`8
`I
`l
`8
`][
`l
`°l
`s
`Illa
`1
`1
`3
`”lb
`J.
`1
`'i
`12
`N;
`l
`l
`i
`16
`Wt
`'1
`'T
`'l
`4
`v
`vr:
`12
`'
`l
`T
`
`wt.
`12
`i
`T
`U
`4
`B
`12
`16
`20
`24
`23
`32
`3t.
`40
`44
`43
`52
`5t.
`Time {hours}
`
`‘ - 5031 acetone: 1 - water on: 1 .— water of
`
`0002
`
`

`

`VOL. 93, NO.
`
`1
`
`JULY 1939
`
`[N VITRO SKIN PENETRATION 89
`
`%doselhr
`
`0
`
`1O
`
`20
`
`30
`
`40
`
`50
`
`llme(hr)
`Figure 2. Permeation of #120 through hairless mouse skin when the
`membrane is sandwiched between aqueous solutions for 48 h (experiment
`II). The results from eight separate experiments are shown. The average
`311,0 permeability coefficient during the 5—15~h posrdusing period is
`1.27 X 10" cth.
`
`though. as before. prolonged exposure to aqueous solutions did
`begin to compromise the skin.
`DISCUSSION
`
`The experiments performed in this inve ti ation lead to two im or-
`tant conclusions. First, as recently reportejby Bond and Barry 20].
`the barrier function of hairless mouse skin does not withstand pro-
`longed submersion in aqueous solution. The data in Figures land 2
`
`%dosemr
`
`time (hr)
`Figure 4. Etlect of acetone pretreatment {15 Jtil/‘cmzj on tritiated water flux
`(mean 1' SD. n a 12] acrms hairless mouse skin after applications from .I' -
`0— 5 h. r '= 24— 2‘} h. and f = 43— 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 cotneum. 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—
`vent—deposition" application procedure [22 - 25] does not appear to
`alter permanently the barrier to water to any significant degree. The
`results of experiments III, IV. and V indicate that acetone adminis-
`tration per se does not contribute to derangement of the stratum
`corneum. The data also suggest that iFa penetrant were delivered in
`
`1.5
`
`2.0
`
` 1.5
`
`E 3
`
`a?
`
`’,
`01.0
`'U
`
`0.5
`
`0.0
`
`o
`
`10
`
`20
`
`30
`
`so
`
`50
`
`so
`
` 1.0
`
`I-
`
`:E0
`6‘!
`
`D 1
`
`$
`
`: 0.5
`
`0.0
`
`0
`
`1 0
`
`2 0
`
`3 0
`
`tlme (hr)
`Figure 3. Effect ofacetonc pretreatment (I 5 Jtil/em“) on tritiatcd water flux
`{mean :I: SD. n -= 3} across hairless mouse skin after applications from
`t= 0-5 h and t I‘ 24-29 h [experiment III}.
`
`time(hr)
`Figure 5. Tritiated water flux l[mean 1 SD, n '- 4) across hairless mouse
`skin after applications from [E 0— 5 h. r= 24- 29 h. andt= 43 , 53 h. Before
`rut l1 administration of water. the skin was pretreated with acetone all a close of
`15 #lfcrnz.
`
`0003
`
`

`

`90 mm. ET AL
`
`THE jOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`larger volumes of solvent or of more structurally destructive chemi-
`cals (e.g.. penetration enhancers) [2?] will. no doubt. be greater and
`may be amplified in the less substantial stratum comeum of the
`hairless mouse.
`
`
`We thank Dr. [any I.. Hull qfrhe Environmental Prumrinn Agrnryfor his interest
`and input, Nan Spenrerfor manuscript preparation.
`
`REFERENCES
`
`l. Bartek M]. LaBadde JA: Percutaneous absorption in vitro. In: Maibach
`HI (ed). hnimal Models in Dermatology. New York. Churchill
`Livingstone. I975. pp 103—120
`2. Bronaugh RL. Stewart RF: Methods for in vitro percutaneous absorp-
`tion studies. I]. Animal models for human skin. Toxicol Appl Phar-
`nucolt'12:431—483. 1982
`3. Wetter RC. Maibaeb HI: Animal models for pcrcutaneous absorption.
`In: Maibach HI. Lowe N] (eds.). Models in Dermatology, vol. 2.
`Basel. Karger. 1935. pp 159— 169
`4. Reifenrath WU. Chellquist EM. 5 i wash EA,_]ederberg WW: Eval-
`uation of animal models for pr
`icting skin penetration in man.
`Fundarn Appl Toxicol 45224-5230. 1984
`5. Skelly JP. Shah VP. Maibach HI. Guy RH. Wester RC. Flynn G.
`Yacobi IR: FDA and RAPE report oFthe workshop on principles and
`practices ofin vitro pereutaneous penetration studies: Relevance to
`bioavailability and bioequivalence. Pharmcol Res Commun 4:265-
`267. I98?
`
`6. Mathias CGT. Hinz RS. Guy RH. Maibach HI: Percutaneous absorp-
`tion: interpretation of in vitro data and risk assessment. In: Honey-
`cutt RC. Zweig G. Ragsdale N N (eds). Dermal Ex
`urc Related to
`Pesticide Use. Washington. DC. American C etnical Society.
`1935. pp 3 - I?
`7". Guy RH. Hadgraftj: Selection ofdrug candidates for transdertnal drug
`delivery. In: Hadgraft J. Guy RH (eds). Transdermal Drug Deliv-
`ery: Developmental issues and research initiatives. New York. Mar-
`cel Dekker. 1989. pp 59 — 81
`8. Barry BW: Dermatological formulations: pctcutaneous absorption.
`New York. Marcel Dekker. 1983. pp 235— 236
`9. Behl CR. Flynn CL. Kurihara T. Harper N. Smith W. Higuchi WI.
`Ho NFH. Pierson CL: Hydration arid percutaneous absorption. I.
`Influence of hydration on alkanol permeation through hairless
`mouse skin.) Invest Dermatol 25:346- 352. 1930
`10. Durrheim HH. Flynn GI... Higuchi WI. Behl CR: Permeation of
`hairless mouse skin. I. Experimental methods and comparison with
`human epidemial permeation by alkanols.] Pharm Sei69:781— 786.
`1980
`
`‘l 2.
`
`1 I. Flynn GL. Durrheirn HH. Higuchi WI: Permeation of hairless mouse
`skin. II. Membrane sectioning techniques and influences on alkanol
`pcrmeabilities.] Pharm Sci 70:52—56. 1981
`Flynn GL: Mechanism of percutaneous absorption from physicochem-
`ical evidence. In: Bronaugh 11L. Maibach HI (eds). Percutaneous
`Absorption. New York. Marcel Dekker. 1985. pp 17 -42
`13. Yu CD. FoxJL. Ho NFH. Higuchi WI: Physical model evaluation of
`topical prodrug delivery—Simultaneous transport and hioconver-
`sion of vidarabine~5'-valerate. 1. Physical model development]
`Phann Sci 68:1341—1346. 1979
`14. Yu CD. FoxJL. Ho NFH, Higuchi WI: Physical model evaluationof
`topical prodrug delivery—Simultaneous trans
`rt and bioconver-
`sion ofvidarabine-S’walerate. II. Parameter dc nitrous. ] Pharm Sci
`63:134T—1357. 19'1“)
`Fox JL. Yu CD. Higuchi WI. Ho NFH: Genera] physical model for
`simultaneous diffusion and metabolism in biological membranes.
`The computational approach for the steady-state case. Int] Pharm
`2:41—57. 19:”
`
`15.
`
`16. Sanrus G. Watari N. Him RS. Benet LZ. Guy RH: Cutaneous metabo-
`lism of transdetrnally delivered nitroglycerin in vitro. In: Shroot B.
`Schaefcr H (eds). Skin Phartnacoltinetics. Basel. Karger. 1937. pp
`240—244
`1?. Bchl CR. Flynn CL. Kurihara T. Smith W. Gatmaitan 0. Hi uchi
`WI, Ho NFH. Pierson CL: Permeability ofthermally damag
`skin.
`I. Immediate influences of 60'C scalding on hairless mouse skin.)
`Invest Dermatol 25:340—345. 1980
`
`—-0— control
`
`+ acetone
`
`5
`
`1 D
`
`1 5
`
`2 U
`
`2 5
`
`2.0
`
`1.5
`
`LG
`
`0.5
`
`0.0
`
`0
`
`E3
`
`"I
`O
`'U'
`
`3‘2
`
`time (hr)
`Figure.- 6. Permeation {mean flux 1: SD. n - 12) of 311.0 through hairless
`mouse skin 24 h after mounting the membrane in the diffusion cell. and after
`treating the ”acetone" 5 ecimens with a solvent dust: of 15 .uI/cm’. For the
`subsequent 2441 periodPoF observation presented here. the skin is sand-
`wiched between aqueous donor and receptor phases (experiment VI). Based
`on the essentially constant 3H2!) fluxes between 5 and 10 h. permeability
`coefficients for the control and acetone-treated membranes are calculated to
`be 1.88 X 10" crn/I'l and 1.33 X 10'" cm/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 S-h 31-130 applications were de-
`signed to rest the skin permeability while minimizing hydration
`effects. In this respect, they would a
`at to have Fulfilled their
`Function adequately. Experiment V cha lenged the tissue further by
`considerin multiple acetone treatments. Again. however. no sig-
`nificant e ect of the solvent. over that observed in the controls
`(experiment
`IVb). was apparent. Furthermore. experiment VI
`showed that air-exposure of the epidermal surface for 24 h (acetone
`pretreated or not) did not significantly alter subsequent 31-120 per-
`meation compared with the “control“. i.e.. experiment 1.
`One ramification ofour research is that the warning of Bond and
`Barry [20].
`that " .
`.
`. hairless mouse skin should not be
`used .
`.
`.
`in .
`.
`. permeation studies inco orating long-term
`hydration. as erroneous results can be ex ecte after .
`.
`. 3 days.“
`appears somewhat conservative. On the asis of our 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/
`cmz, does not ap car to alter significantly tiie stratum corneum
`barrier function thairless mouse skin. Given that a
`ical topical
`dose ofacetone in a solvent-d
`sition, human in vivo 5 in penetra-
`tion study is less than 10 .rtl/cmz [22—25]. it seems reasonable to
`suggest that no acetone—mediated Facilitation of transport will be
`evident. In addition. one may also deduce that an in vitro penetra-
`tion study using hairless mouse skin and solvent-deposition ofpene-
`tranr from acetone (at a dose of 15 til/cm: or less). should provide a
`reasonable model cx
`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
`application of small volatile solvent volumes does not ap at to
`p ace the murine barrier under measurable stress. The e ects of
`
`0004
`
`

`

`VOL. 93. NO.
`
`I
`
`JULY 1989
`
`IN VITRO SKIN PENETRA'HON 91
`
`18. Bchl CR. Flynn GL. Barrett M. Linn EE. Higuchi WI. Ho NFH.
`Pierson CL: Permeability oftl'ierl'nall}.r damaged skin. IV. Influence
`of branding iron temperature on the mass transfer of water and
`n-alkanols across hairless mouse skin. Burns 8:86—93. 1981
`19. Flynn GI... Bchl CR. Linn EB. Higuchi WI. Ho NFH. Pierson CL:
`Permeability of thermally damaged skin. If Permeability over the
`course of maturation of a deep partial thickness burn. Burns 8:196 —
`202, 1931
`20. Bond JR, Ban-y BW: Limitations ofhairless mouse skin as a model far
`in vitro permeation studies through human skin: hydration damage.
`J Invest Den-natal 90:486— 489. 1988
`21. Guy RH. Hadgtaft j. Hinz R5. Roskos [{V. Bucks DAW: In vivo
`evaluations of transdermal drug delivery.
`In: Chien YW (ed).
`Transdermal controlled systemic medications. New York. Marcel
`Dekker. 1937, pp 179—224
`22. Bucks DAW. Maibaeh H1. Guy RH: Percutaneous absorption of
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`1935
`
`23.
`
`24.
`
`25.
`
`26.
`
`27.
`
`Guy RH. Carlstmm EM. Bucks UAW. Him R5. Maihach HI: Percu-
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`
`Gummer CL, Hinz RS. Maibach HI: The skin penetrarion cell: A
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`
`0005
`
`