`
`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 humanskin. We suggest,
`therefore, that acetone-mediated facilitation of percutaneous
`determinedin vitro after exposure of the epidermal surface to
`volumes of acetone epically used in human in vivo skin
`absorption in humansis unlikely. A further conclusion ofthis
`workis that in vitro solvent-deposition penetration experi-
`penetration studies. It has been shownthat the transport of
`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
`approximately 15 zl/cm?. Submersionof the membranes be-
`Although hairless mouse skin is more permeable than its
`humancounterpart, in vitro measurementsusing the murine
`tween aqueousdonorandreceptorphases for periods greater
`than 34h, 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. J 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 fundamental knowledge
`toward our understanding of barrier function. There
`are importantdifferences, however, in the permeabil-
`ities of skin taken fromdifferent species and these
`inconsistencies have been highlighted in a numberof publications
`[1-4]. Currently, there is considerableactivity 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 workshop onin vitro methodsfor the
`purpose cfeashtahtig guidelines thatcould 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 emergenceof transdermaldrug delivery
`to provide systemic pharmacologic effect has introduced percutane-
`ous penetration measurementas a key component of the pharma-
`ceutic research and developmenteffort [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 [8] has frustrated researchers and has directed them to
`consider alternatives. Of the various models that have been studied
`the skin of the hairless mouseis probably the most
`popular. Thereis
`no doubt that this tissue has enabled a number of 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 U.S. Environmental Protection
`Agencythrough 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 includeits
`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 throughhairless 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 documentthis 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 encounteredin 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 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 ofc
`chemicals has involved
`deposition from an organic solvent, usually acetone. The question
`posed by the research
`presented here, therefore, was: “Does the
`amountofacetone coal
`as the vehicle in human skin penetration
`studies cause significant changesto 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 info
`ormation
`relevant to percutaneous absorption in humans.
`MATERIALS AND METHODS
`
`Toassess barrier functionstatus of hairless mouse skin, the perme-
`ability of tritiated water (3H,O, 0.05 Ci/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) (26)
`Thearea of skin exposed was 3.14 cm;
`the volume of the receptor phase was approximately 5 cm*. The
`flow-rate was adjusted by 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.
`IPR2018-01119
`
`0001
`
`

`

`88 HINZ ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`NY)so thatthe receptor solution was completely exchangedin 1h.
`Thereceptor phase was normalsaline in phosphatebuffer at pH 7.4.
`Perfusate 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 II] Model 6880,
`Elk Grove, IL). The diffusion cells were thermostatted at 35°C
`throughoutthe experiments; under these conditions, with the skin
`open to the laboratory atomosphere,the surface temperature of 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 mountedin 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 of cells.
`Because ofthe time involved in setting up eightdiffusion cells and
`adjusting the receptor solution flow-rate Speen an experi-
`mentwastypically started within 2hafter killing of the animals.
`0
`The experiments performed are summarized in Table I. The de-
`sign was selectedto test the effects of an acetone dose on barrier
`function andtissue constancy. The water treatments involved appli-
`cation of 1 cm? of3H,Oto the skin surface. To prevent evaporation,
`the upperhalfof the diffusion cell was then covereduntil the water
`was removed. Whenever water was not in contact with the skin, the
`surface was open to ambient conditions. Acetone treatments in-
`volved application of 50 yl of the solvent by a capillary pipet. As
`pelisenset in vivo, the acetone was distributed evenly over the skin
`surface, which, again, was open to the laboratory atmosphere.
`When water was administered subsequentto acetonetreatment(ex-
`perimentsIIIa, IVa, V) there was a 2- to 3-min time lapse between
`solventapplications. No liquid acetone remained at this point.
`ExperimentsI and II simply determined water permeation over
`24- and 48-hperiods,in the absence of acetone treatment. Experi-
`ments III and IV used short 5-h exposuresofthe 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
`tackaded three volatile solvent treatments. Experiment VI consid-
`ered the effect of a time delay postacetone application followed by
`prolonged water contact.
`
`4
`
`3
`
`ke
`=o
`o& 2
`
`xe
`
`1
`
`0
`
`10
`
`20
`
`30
`
`time (hr)
`Figure 1. Permeation (mean flux + SD, n = 8) of *H,O through hairless
`mouse skin when the membrane is sandwiched between aqueous solutions
`for 24 h (experimentI).
`
`contact. Indeed,in six outof eight cells, the membranehas been so
`damaged that 9H,Oflux decreasesatlater times due to the substan-
`tial depletion of radiolabel in the donor phase.
`Theresults ofexperimentsII] and I'V are summarized in Figures 3
`and4, respectively.It is apparent that when wateris dosed intermit-
`tently to the skin surface for 5-h
`periods, pretreatmentwith acetone
`does not cause anysignificant difference to the permeability behav-
`ior. This conclusion was substantiated by
`statistical comparisons
`(paired Wilcoxon and Student's t-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 (w > 0.2, p > 0.1) on water permeation. Figure 5, which
`contains the data from experiment V, demonstrates that repeated
`acetone administration before dosing with wateralsoelicits no sig-
`nificant derangement ofbarrier function. Although Figures 3, 4,
`and 5 appearto suggestthat, regardless of acetone treatmentor not,
`the
`permeability of 3H,O increases with time, the trendis notstatis-
`tically significant. It is perhaps reasonable, however, to conclude
`that hydration and the detrimental effects thereof, can continue
`during the water “off”periods.
`Finally, Figure6 iliustrates the results ofexperimentVI, in which
`the permeation of3H,O was followed 24 h hie acetonetreatment.
`Again, no difference from the control studies was observed al-
`
`RESULTS
`
`In experiments I and II, the skin remained sandwiched between
`aqueoussolutions throughout the measurementperiods (24 and 48
`h, respectively). Figure 1 showsthat in experiment I, 3H,O fluxis
`essentially constant over the 3- to 20-h postapplication period, cor-
`responding to a permeability coefficient of about 2.95 * 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 experimentII. Figure 2 indicates that prolonged
`and complete hydration leads to barrier breakdown after 24 h of
`
`Table I. Experimental Design Summary (n = numberofreplicates)
`
`
`
`Experiment
`n
`Treatments
`
`|
`8s
`I
`|
`s
`Ul
`t
`{
`8 *
`Illa
`fT
`1
`s
`|
`ILb
`tf
`f
`L
`iz
`*{
`Va
`Lt
`Tf
`1
`16
`|
`IVb
`“1
`of
`a
`4)
`v
`Via
`12
`*
`1
`t
`
`VIb
`12
`{
`t
`32
`28
`24
`48
`Time(hours)
`
`t
`
`T
`
`T
`t
`t
`t
`ft
`
`* = 50 pl acetone; | = water on; f = water off
`
`0
`
`4
`
`8
`
`12
`
`#16
`
`20
`
`36
`
`40
`
`44
`
`52
`
`56
`
`0002
`
`

`

`IN VITRO SKIN PENETRATION 89
`
`%dose/hr
`
`time (hr)
`Figure 4. Effect of acetone pretreatment (15 il/cm?) ontritiated waterflux
`(mean + SD, n = 12)across hairless mouse skin after applications from t =
`0-5 h, t= 24-29 h, and t = 48-53 h (experiment IV).
`
`clearly reveal that exposure ofthe tissue to aqueous solutions, in
`both donor and receiver compartments, for periods in excess of 24h,
`leads to substantial degeneration of the stratum corneum. Thesec-
`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 he barrier to waterto any significant degree. The
`results of experiments III, [V, and V indicate that acetone adminis-
`tration per se does not contribute to derangement of the stratum
`corndum. Thedata also suggest thatif a penetrant weredelivered in
`
`“It
`
`is
`
`|
`
`1.5
`
`1.0
`
`0.5
`
`dose/hr
`
`%
`
`0.0
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`time (hr)
`Figure 5. Tritiated water flux (mean -t SD, n = 4) across hairless mouse
`skin after applications from t= 0-5 h, t= 24-29 h, and t= 48-53 h. Before
`each administration of water, the skin was pretreated with acetoneat adose of
`15 pl/cm?*.
`
`VOL. 93, NO.
`
`1
`
`JULY 1989
`
`%dose/hr
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`time (hr)
`Figure 2. Permeation of 3H,O through hairless mouse skin when the
`membraneis sandwiched between aqueoussolutions for 48 h (experiment
`II). The results from eight separate experiments are shown. The average
`3H,O permeability coefhcient during the 5-15-h postdosing period is
`1.27 X 10 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 reported by Bond and Barry [20],
`the barrier function ofhairless mouse skin does not withstand pro-
`longed submersionin aqueoussolution. The data in Figures 1 and 2
`
`
`
`0
`
`10
`
`20
`
`30
`
`1.5
`
`1.0
`
`0s
`
`0.0
`
`k£ @w° 0
`
`ae
`
`time (hr)
`Figure 3, Effect of acetone pretreatment(15 jil/cm*) ontritiated water flux
`(mean + SD, n= 8) across hairless mouse skin after applications from
`t= 0-5 hand t= 24-29 h (experiment III),
`
`0003
`
`

`

`90 HINZ ET AL
`
`—O— control
`
`—@— acetone
`
`
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`£3
`
`S
`0)
`
`ov P
`
`S
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`larger volumesofsolventor 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 Environmental Protection Agencyfor his interest
`and input, Nan Spencer for manuscript preparation.
`
`REFERENCES
`
`1. Bartek MJ, LaBadde JA: Percutaneous absorptionin vitro. In: Maibach
`HI (ed.). Animal Models in Dermatology. New York, Churchill
`Livingstone, 1975, pp 103-120
`2. Bronaugh RL, Stewart RF: Methodsfor in vitro percutaneous absorp-
`tion studies. II. Animal models for human skin. 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 4:8224-$230, 1984
`5. Skelly JP, Shah VP, Maibach HI, Guy RH, Wester RC, Flynn G,
`Yacobi A: FDA and AAPS report of the workshop onprinciples and
`practices of in vitro percutaneouspenetration studies: Relevance to
`ioavailability and bioequivalence, Pharmcol Res Commun4:265 -
`267, 1987
`
`6. Mathias CGT, Hinz RS, Guy RH, Maibach HI: Percutaneous absorp-
`tion: interpretation ofin 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,
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`7. Guy RH, Hadgraft J: Selection of drug candidates for transdermaldrug
`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: 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 percutaneous absorption. L
`Influence of hydration on alkanol
`permeation through hairless
`mouseskin, J Invest Dermatol 25:346-352, 1980
`10. Durrheim HH, Flynn GL, Higuchi WI, Behl CR: Permeation of
`hairless mouse skin. !. Experimental methods and comparison with
`humanepidermal permeation by alkanols. J Pharm Sci 69:781-786,
`1980
`
`11. Flynn GL, Durrheim HH, Higuchi WI: Permeation of hairless mouse
`skin. I. Membrane sectioning techniques and influences on alkanol
`permeabilities. J Pharm Sci 70:52-56, 1981
`12. 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.
`I. Physical model development. J
`Pharm Sci 68:1341- 1346, 1979
`14, Yu CD, Fox JL, Ho NFH, Higuchi WI: Physical model evaluation of
`topical prodrug delivery — Simultaneoustransport and bioconver-
`sion ofvidarabine-5’-valerate, II. Parameter Aebedttonie 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: ShrootB,
`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
`
`time (hr)
`Figure 6. Permeation (mean flux + $D, n= 12) of 3H,O through hairless
`mouse skin 24 h after mounting the membranein thediffusioncell, and after
`treating theee with a solvent dose of 15 yl/cm?. For the
`subsequent 24-h period
`of observation presented here, the skin is sand-
`wiched between aqueous donor and receptor phases (experiment VI). Based
`on the essentially constant 3H,O fluxes between 5 and
`10 h, permeability
`coefhcients for the control and acetone-treated membranesare calculated to
`be 1.88 & 10~ cm/h and 1.83 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 5-h 3H,O applications were de-
`signed to test the skin permeability while minimizing hydration
`Pics In this respect, they would appear to have fulfilled their
`function adequately. Experiment V challenged thetissue further by
`considering multiple acetone treatments. Again, however, nosig-
`nificant effect of the solvent, over that observed in the controls
`(experiment
`IVb), was apparent. Furthermore, experiment VI
`showed thatair-exposureof the epidermalsurface for 24 h (acetone
`pretreated or not) did notsignificantly alter subsequent H,O per-
`meation compared with the “control”, i.c., experimentI.
`Oneramification of our researchis 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 somewhatconservative. 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 approximately 15 pul/
`cm?, does not appear to alter significantly the stratum corneum
`
`barrier function ofhaislesd mouse skin. Giventhataere topical
`
`dose of acetone in a solvent-deposition, human in vivo skin penetra-
`tion study is less than 10 yl/em? [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-
`tion study using hairless mouse skin and solvent-deposition ofpene-
`trant from acetone (at a dose of 15 44l/cm?orless), should provide a
`reasonable model experimentfor the in vivo situation. We recog-
`nize, however, that the latter two conclusionsare 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 appear to
`plas the murine barrier under measurable stress. The effects of
`
`0004
`
`

`

`VOL. 93, NO. 1
`
`JULY 1989
`
`IN VITRO SKIN PENETRATION 91
`
`18.
`
`19.
`
`20.
`
`21.
`
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`Behl CR, Flynn GL, Barrett M, Linn EE, Higuchi WI, Ho NFH,
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`Flynn GL, Behl CR, Linn EE, Higuchi WI, Ho NFH, Pierson CL:
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`Bond JR, Barry BW: Limitations of hairless mouse skin as a model for
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`Guy RH, Hadgraft J, Hinz RS, Roskos KV, Bucks DAW: In vivo
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`
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`
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`
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`0005
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`