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, B.S., and Richard H. Guy, Ph.D.
`Departments of Pharmacy and Pharmaceutical Chemistry, University of California, San Francisco; San Francisco, California, U.S.A.
`
`The permeability barrier of hairless mouse skin has been
`is used to deposit a penetrant on human skin. We suggest,
`therefore, that acetone-mediatedfacilitation ofpercutaneous
`determinedinvitro after exposure of the epidermalsurface to
`absorption in humansis unlikely. A further conclusionofthis
`volumes of acetone typically used in human in vivo skin
`penetration studies. It has been shownthat the transport of
`workis that in vitro solvent-deposition
`netration experi-
`ments using hairless mouse skin should provide reliable
`tritiated water (whenapplied for limited 5-h periods) across
`transport information for at least 48 h postadministration.
`hairless mouse skin is not affected by acetone treatments of
`Although hairless mouse skin is more permeable thanits
`approximately 15 sil/cm?. Submersion of the membranesbe-
`tween aqueous donorandreceptor phases for periods greater
`human counterpart, in vitro measurements using the murine
`barrier should, therefore, provide useful and relevant guide-
`than 24 h, however,leads to significant and catastrophic bar-
`lines for
`risk assessment calculations and bioavailability
`rier impairment. The acetone dose in the experiments re-
`
`determinations. ] Invest Dermatol 93:87-91, 1989
`portedis greater than that employed in vivo when the solvent
`
`he use of animal skin in the study of
`percutaneous
`absorption has provided feudaneieallnveletee
`toward our understanding ofbarrier 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
`[1-4]. Currently, there is considerable activity in the area ofin 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 sponsored a workshopon in vitro methodsfor the
`purposeofestablishing guidelines that could be followed when new
`topical drug formulations are under development[5]. 2) Thereis a
`continuing needfor reliable, and meaningful, proceduresthat 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 pharmacologiceffect has introduced percutane-
`ous penetration measurement as a key component ofthe pharma-
`ceutic research and developmenteffort [7].
`The heightened interest in assessing percutancous transport has
`led several investigators to use substitutes for human skin. It is
`sometimes difficult to obtain humantissue in a regular or timely
`fashion; in addition, the high level of variability associated with
`cadaverskin [8] has frustrated researchers and has directed them to
`consider alternatives. Of the various models that have been studied
`the skin ofthe hairless mouse is probably the most popular. There is
`no doubrthat this tissue has enabled a number obey 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 U.S. Environmental Protection
`Agency through cooperative agreement, CR-812474.
`Reprint requests to: Dr. R. H. Guy, School of Pharmacy, University of
`California, San Francisco, CA 94143-0446,
`
`function [17-19]. The advantagesofhairless mouse skin include its
`availability and reproducibility. It is more permeable than human
`skin, too, and thisis 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 throughhairless mouseskin will not lead to an under-
`estimate of dermal exposure in humans. This higher permeability,
`however,is also considered by someto be a major disadvantage of
`the tissue, althoughthere is little evidence to documentthis con-
`cern. A moreserious question, though, pertains to the response of
`hairless mouse skin, relative to that of human skin,to situations or
`circumstancesoften encountered in percutaneous penetration work,
`e.g., the effect of hydration and oforganic solvents. The hydration
`issue was recently addressed by Bond and Barry [20], who showed
`that prolonged exposure ofhairless mouse skin to aqueous donor
`and receptor phases in simple diffusion cells caused considerable
`derangementofbarrier function. The interests of our laboratory
`have centered on in vivo evaluation of percutaneous absorption
`[21-25]. Typically, topical application of|
`chemicals has involved
`deposition from an organicsolvent, usually acetone. The question
`posed by the research
`presented here, therefore, was: “Does the
`amountof acetone i
`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)
`humanskin in vivo is not damaged by the acetone deposition and
`delivery process and 2) in vitro hairless mouse skin experiments
`involving chemicalapplication in acetone may provide fake
`ormation
`relevant to percutaneous absorption in humans.
`MATERIALS AND METHODS
`
`To assess barrier function status of hairless mouse skin, the perme-
`ability of tritiated water (7H,O, 0,05 wCi/ml, New England Nu-
`clear, Boston, MA) was determinedat designated timesafter various
`acetone treatments. Permeation experiments were performed in
`conventional flow-through diffusion cells (Laboratory Glass Appa-
`ratus, Berkeley, CA) (D6)
`Thearea of skin exposed was 3.14 cm’,
`the volume of the receptor phase was approximately 5 cm*. The
`flow-rate was adjusted ,
`y a cassette pump (Manostat, New York,
`
`0022-202X/89/$03.50 Copyright © 1989 by The Society for Investigative Dermatology,Inc.
`87
`
`Noven Pharmaceuticals,Inc.
`EX2018
`Mylan Tech., Inc. v. Noven Pharma., Inc.
`IPR2018-00174
`
`0001
`
`

`

`88 HINZ ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`4
`
`3
`
`2
`
`1
`
`0
`
`0
`
`10
`
`20
`
`30
`
`_ =o
`
`o o
`
`uo
`
`se
`
`time (hr)
`Figure 1. Permeation (mean flux + $D, n = 8) of 7H,O through hairless
`mouse skin when the membraneis sandwiched between aqueoussolutions
`for 24 h (experimentI),
`
`contact. Indeed, in six out ofeight cells, the membrane has been so
`damaged that 3H,O flux decreasesat later times due to the substan-
`tial depletion of radiolabel in the donor phase.
`Theresults ofexperimentsII] and IV are summarized in Figures 3
`and 4,respectively.It is apparent that when wateris dosed intermit-
`tently to the skin surface for 5-h
`periods, pretreatment with acetone
`does not cause any significant diteence to the permeability behav-
`ior. This conclusion was substantiated by
`statistical comparisons
`(paired Wilcoxon and Student'st-tests) of
`the cumulative amounts
`of water transported across the control and acetone-treated mem-
`branes, following the 5-h applications. Acetoneelicited an insignifi-
`canteffect (a > 0.2, p > oyon water permeation. Figure 5, which
`contains the data from experiment V, demonstrates that repeated
`acetone administration before dosing with wateralso elicits no sig-
`nificant derangementofbarrier function. Although Figures 3, 4,
`and 5 appear to suggest that, regardless of acetone treatmentornot,
`the
`permeability of7H,increases with time,the trendis notstatis-
`tically
`significant. It is perhaps reasonable, however, to conclude
`that fedration and the detrimental effects thereof, can continue
`during the water “off” periods.
`Finally, Figure 6 iliustrates the results of experimentVI, in which
`the permeation of 7H,O wasfollowed 24 h after acetone treatment.
`Again, no difference from the control studies was observed al-
`
`NY)so that the receptor solution was completely exchangedin 1 h.
`The receptor phase was normalsaline in phosphate buffer at pH 7.4.
`Perfusate was collected in test tubes mounted ona fraction collector
`(Gilson FC-220, Middleton, WI) and the samples were then ana-
`lyzed by liquid scintillation counting (Searle Mark II] Model 6880,
`Elk Grove, IL). The diffusion cells were thermostatted at 35°C
`throughout the experiments; under these conditions, with the skin
`opento the laboratory atomosphere, the surface temperatureof the
`membrane was 32°C + 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 after 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 treatmentvs.
`no treatment, for example), the four skins were halved so that each
`animal contributed to
`both the “control” and “test” set ofcells.
`Because ofthe time involvedin setting up cight diffusion cells and
`adjusting the receptor solution flow-rate senpeae an experi-
`ment wastypically started within 2 h after
`killing of the animals.
`The experiments performed are summarized in Table I. The de-
`sign was selected to test the effects of an acetone dose onbarrier
`function andtissue constancy. The watertreatments involved appli-
`cation of 1 cmof3H,O to the skin surface. To preventevaporation,
`the upperhalf of the diffusion cell was then covered until the water
`was removed. Whenever water was nor in contact with the skin, the
`surface was open to ambient conditions. Acetone treatments in-
`volved application of 50 yl of the solventby a capillary pipet. As
`sehr in vivo, the acetone wasdistributed evenly over the skin
`surface, which, again, was open to the laboratory atmosphere.
`When water was administered subsequentto acetone treatment(ex-
`perimentsIIIa, IVa, V) there was a 2- to 3-min timelapse between
`solventapplications. No liquid acetone remainedat 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 5-h exposures of the tissue to water and
`assessed the long-term consequences of an acetone dose at t= 0.
`Experiment V involved a greater potential insult to the tissue and
`included three volatile solvent treatments. Experiment VI consid-
`ered the effect of a time delay postacetone application followed by
`prolonged water contact.
`
`RESULTS
`
`In experiments I and II, the skin remained sandwiched between
`aqueous solutions throughout the measurementperiods (24 and 48
`h, respectively). Figure 1 shows that in experimentI, 3H,Oflux is
`essentially constant over the 3- to 20-h 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 experimentIL Figure 2 indicates that prolonged
`and complete hydration leads to barrier breakdownafter 24 h of
`
`Table I. Experimental Design Summary (n = numberofreplicates)
`
`Experiment
`n
`‘Treatments
`
`t
`
`|
`8
`I
`|
`8
`Il
`a
`.-*{
`a
`Illa
`L
`|
`s
`IIb
`tf
`4
`*|
`12
`IVa
`1
`if
`}
`|
`a
`IVb
`«|
`t
`*|
`4 |
`Vv
`Via
`12°
`|
`T
`
`VIb
`12
`J
`t
`0
`4
`8
`12
`16 2 24
`28
`32
`36
`40
`44
`48
`52
`56
`Time (hours)
`
`t
`t
`1
`t
`t
`
`t
`
`ji
`Wf
`Tl
`Tf
`t
`
`* = 50 wl acetone; 4 = water on; | = water off
`
`0002
`
`

`

`VOL. 93, NO.
`
`1
`
`JULY 1989
`
`%dose/hr 0
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`time (hr)
`Figure 2. Permeation of 9H,O through hairless mouse skin when the
`membraneis sandwiched between aqueous solutions for 48 h (experiment
`II). The results from eight separate experiments are shown. The average
`4H,O permeability coefficient during the 5-15-h postdosing period is
`1.27 X 107 cm/h.
`
`though, as before, prolonged exposure to aqueous solutions did
`begin to compromisethe skin.
`DISCUSSION
`
`The experiments performedin this investigation lead to two impor-
`tant conclusions. First, as recently reorelby Bond andBarry [20],
`the barrier function of hairless mouse skin does not withstand pro-
`longed submersionin aqueoussolution. The data in Figures 1 and 2
`
`1.5
`
`1.0
`
`0.5%dose/hr
`
`0
`
`10
`
`20
`
`30
`
`IN VITRO SKIN PENETRATION 89
`
`%dose/hr
`
`time(hr)
`Figure 4, Effect of acetone pretreatment (15 l/cm?) on tritiated waterflux
`(mean + SD, n = 12) across hairless mouse skin after applications from t =
`0-5 h, r= 24-29 h, and t= 48-53 h (experiment IV).
`
`clearly reveal that exposure of the tissue to aqueous solutions, in
`both donorandreceiver compartments, for periodsin excess of 24 h,
`leads to substantial degeneration ofthe stratum corneum. Thesec-
`ond key finding revealed by this study is that treatment of hairless
`mouse skin with acetone, in a fashion that mimicsa typical “‘sol-
`vent-deposition” application procedure [22-25] does not appearto
`alter permanently the barrier to waterto any significant degree. The
`results of experimentsIII, IV, and V indicate that acetone adminis-
`tration per s¢ does not contribute to derangementof the stratum
`cornéum. The data also suggest thatif a penetrant weredelivered in
`
`“ft
`
`1.5
`
`0.5
`
`0.0
`
`0
`
`<
`
`|
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`time (hr)
`Figure 3. Effect of acetone pretreatment (15 yl/cm?) ontritiated water flux
`(mean + SD, n= 8) across hairless mouse skin after applications from
`t= 0-5 h and t= 24-29 h (experiment III).
`
`time (hr)
`Figure 5. Tritiated water flux (mean + SD, n = 4) across hairless mouse
`skin after applications from t= 0-5 h, t= 24-29 h, and r= 48-53 h. Before
`each administrationof water, the skin was pretreated with acetoneat a dose of
`15 yl/cm*.
`
`0003
`
`

`

`90 HINZ ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`larger volumesofsolvent or of morestructurally 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.
`
`
`Wethank Dr. Larry L. Hall ofthe Environmental Protection Agencyfor his interest
`and input, Nan Spencerfor manuscript preparation.
`
`REFERENCES
`
`1. Bartek MJ, LaBadde JA: Percutaneousabsorption in vitro. In: Maibach
`HI (ed.). Animal Models in Dermatology. New York, Churchill
`Livingstone, 1975, pp 103-120
`2. Bronaugh RL, Stewart RF: Methods forin vitro percutaneous absorp-
`tion studies. II, Animal models for humanskin. Toxicol Appl Phar-
`macol 62:481- 488, 1982
`3. Wester RC, Maibach HI: Animal models for percutaneous absorption.
`In: Maibach HI, Lowe NJ (eds.). Models in Dermatology,vol. 2.
`Basel, Karger, 1985, pp 159-169
`4. Reifenrath WG, Chellquist EM, Shipwash EA,Jederberg WW:Eval-
`uation of animal models for predicting skin penetration in man.
`Fundam Appl Toxicol 4:5224-—$230, 1984
`5. Skelly JP, Shah VP, Maibach HI, Guy RH, Wester RC, Flynn G,
`Yacobi A: FDA and AAPSreport of the workshop on principles and
`practices ofin vitro percutaneous penetration studies: Relevance to
`bioavailability and bioequivalence. Pharmcol Res Commun 4:265-
`267, 1987
`
`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 NN (eds.). Dermal Exposure Related to
`Pesticide Use, Washington, DC, American Chemical Society,
`1985, pp 3-17
`7. Guy RH, Hadgraft J: Selection of drug candidates for transdermal drug
`delivery. In: Hadgraft J, Guy RH (eds.). Transdermal Drug Deliv-
`ery: Developmentalissues and research initiatives. New York, Mar-
`cel Dekker, 1989, pp 59-81
`8. Barry BW: Dermatological formulations: percutaneous absorption.
`New York, Marcel Dekker, 1983, pp 235-236
`9, Behl CR, Flynn GL, Kurihara T, Harper N, Smith W, Higuchi WI,
`Ho NFH, Pierson CL: Hydration and percutaneousabsorption.I.
`Influence of hydration on alkanol permeation through hairless
`mouse skin. J Invest Dermatol 25:346-352, 1980
`10. Durrheim HH, Flynn GL, Higuchi WI, Behl CR: Permeation of
`hairless mouse skin. 1. Experimental methods and comparison with
`humanepidermal permeation by alkanols. J Pharm Sci 69:781-786,
`1980
`
`12.
`
`11. Flynn GL, Durrheim HH, Higuchi WI: Permeationofhairless mouse
`skin, [I], Membranesectioning techniques and influences on alkanol
`permeabilities. J Pharm Sci 70:52-56, 1981
`Flynn GL: Mechanism ofpercutaneousabsorption from physicochem-
`ical evidence. In: Bronaugh RL, Maibach HI (eds.). Percutaneous
`Absorption. New York, Marcel Dekker, 1985, pp 17-42
`13. Yu CD, Fox JL, Ho NFH, Higuchi WI: Physical model evaluation of
`topical prodrug delivery — Simultaneous transport and bioconver-
`sion of vidarabine-5’-valerate. 1. Physical model development. J
`Pharm Sei 68:1341- 1346, 1979
`14. Yu CD, Fox JL, Ho NFH, Higuchi WI: Physical model evaluation of
`topical prodrug delivery — Simultaneoustransport and bioconver-
`sion of vidarabine-5’-valerate. II. Parameter definitions. J Pharm Sci
`68:1347-1357, 1979
`Fox JL, Yu CD, Higuchi WI, Ho NFH: General physical model for
`simultaneous diffusion and metabolism in biological membranes.
`The computational approach for the steady-state case. Int J] Pharm
`2:41-57, 1979
`
`15.
`
`16. Santus G, Watari N, Hinz RS, Benet LZ, Guy RH: Cutaneous metabo-
`lism of transdermally delivered nitroglycerin in vitro. In: Shroot B,
`Schaefer H (eds.). Skin Pharmacokinetics. Basel, Karger, 1987, pp
`240-244
`17. Behl CR, Flynn GL, Kurihara T, Smith W, Gatmaitan O, Higuchi
`WI, Ho NFH, Pierson CL: Permeability of thermally damaged
`skin.
`I. Immediate influences of 60°C scalding on hairless mouse skin. J
`Invest Dermatol 25:340-345, 1980
`
`2.0
`
`1.5
`
`10
`
`0.5
`
`0.0
`
`0
`
`—O— control
`
`—@— acetone
`
`5
`
`10
`
`15
`
`20
`
`25
`
`£a
`
`0°G
`
`o
`
`ae
`
`time (hr)
`Figure 6. Permeation (mean flux + SD, n= 12) of 7H,O through hairless
`mouse skin 24 h after mounting the membranein the diffusion cell, and after
`treating the “acetone” specimenswith a solventdose of 15 yl/cm?. For the
`subsequent 24-h patio’of observation presented here, the skin is sand-
`wiched between aqueous donor and receptor phases (experiment VI). Based
`on the essentially constant “HO fluxes between 5 and 10 h, permeability
`coefficients for the control and acetone-treated membranesare calculated to
`be 1.88 X 10-5 cm/h and 1.83 X 10-3 cm/h, respectively.
`
`an acetone vehicle under similar circumstances, one may expectthe
`barrier function of hairless mouse skin to remain reasonably con-
`stant for at least 48 h. The short 5-h °H,O applications were de-
`signed to test the skin permeability while minimizing hydration
`effects. In this respect, they would appear to have fulfilled their
`function adequately. Experiment V challenged thetissue further by
`considering multiple acetone treatments. Again, however, no sig-
`nificant effect of the solvent, over that observed in the controls
`(experiment
`IVb), was apparent. Furthermore, experiment VI
`showedthat air-exposure of the epidermalsurface for 24 h (acetone
`pretreated or not) did not significantly alter subsequent >H,O per-
`meation compared with the “control”, i.c., experimentI.
`One ramification ofour 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 erroneousresults can be expected after .
`.
`. 3 days,”
`appears somewhat conservative. Onthebasis of our data, we would
`be reluctant to draw conclusions from anyflux measurements made
`after 24 h submersion. More important, though, we have shown
`that administration of acetone, at a dose of approximately 15 yl/
`cm?, does not appear to alter significantly is stratum corneum
`barrier function odhuiless mouseskin. Given thata typical topical
`dose of acetoneina solvent-deposition, human in vivo skin penctra-
`tion study is less than 10 ul/cm? [22-25], it seems reasonable to
`suggest that no acetone-mediatedfacilitation of transport will be
`evident. In addition, one may also deduce that an in vitro penetra-
`tionstudyusing hairless mouse skinand solvent-deposition ofpene-
`trant from acetone(at a dose of 15 yl/cm?or less), should provide a
`reasonable model experiment for the in vivosituation. We recog-
`nize, however, that the latter two conclusions are based on observa-
`tions that use water as the mode! permeant. It remains to be seen
`whetherthese deductions are sustained whenthepenetating mole-
`cule is lipophilic in character. Finally, although hairless mouse skin
`is generally more permeable than its human counterpart [20], the
`<een of small volatile solvent volumes does not appear to
`place the murine barrier under measurable stress. The effects of
`
`0004
`
`

`

`VOL, 93, NO.
`
`1
`
`JULY 1989
`
`IN VITRO SKIN PENETRATION 91
`
`Behl CR, Flynn GL, Barrett M, Linn EE, Higuchi WI, Ho NFH,
`Pierson CL: Permeability of thermally damaged skin. IV. Influence
`of branding iron temperature on the mass transfer of water and
`n-alkanols across hairless mouse skin. Burns 8:86-98, 1981
`Flynn GL, Behl CR, Linn EE, Higuchi WI, Ho NFH, Pierson CL:
`Permeability of thermally damaged skin. V Permeability over the
`course of maturation of a deep partial thickness burn. Burns 8:196 —
`202, 1981
`Bond JR, Barry BW: Limitations of hairless mouse skin as a modelfor
`in vitro permeation studies through human skin: hydration damage.
`J Invest Dermatol 90:486-489, 1988
`Guy RH, Hadgraft J, Hinz RS, Roskos KV, Bucks DAW: In vivo
`evaluations of transdermal drug delivery.
`In: Chien YW (ed.).
`Transdermal controlled systemic medications. New York, Marcel
`Dekker, 1987, pp 179-224
`Bucks DAW, Maibach HI, Guy RH: Percutaneous absorption of
`steroids: effect of repeated application. J Pharm Sci 74:1337- 1339,
`1985
`
`19,
`
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`27.
`
`Guy RH, Carlstrom EM, Bucks DAW, Hinz RS, Maibach HI: Percu-
`taneous penetration of nicotinates: in vivo and in vitro measure-
`ments. J Pharm Sci 75:968-972, 1986
`Roskos KV, Maibach HI, Guy RH: Percutaneous absorption in the
`aged. In: Gilchrest B (ed.). Dermatologic Clinics, vol 6: The aging
`skin. Philadelphia, W.B. Saunders, 1986, pp 455-465
`Bucks DAW, McMaster JR, Maibach HI, Guy RH: Bioavailability of
`topically administered steroids: a ‘*mass balance” technique. J Invest
`Dermatol 90:29 -33, 1988
`Gummer CL, Hinz RS, Maibach HI: The skin penetration cell: A
`design update. Int J Pharm 40;101-104, 1987
`Mirejovsky D, Takruri H: Dermal penetration enhancement profile of
`hexamethylenelauramideandits homologues: in vitro versus in vivo
`behavior of enhancers in the penetration of hydrocortisone. ] Pharm
`Sci 75:1089- 1093, 1986
`
`0005
`
`