`
`Hairless Mouse Skin is Limited as a Model for Assessing
`the Effects of Penetration Enhancers in Human Skin
`
`John Russell Bond, Ph.D., and Brian William Barry, Ph.D., D.Sc.
`Postgraduate School of Studies in Pharmacy, University of Bradford, Bradford, U.K.
`
`
`was no consistent relationship between enhancer effects on
`through
`The permeability coefficient of 5-fluorouracil
`human abdominal and hairless mouse skins was used as an
`the two skin types, and we concludethat the hairless mouse
`model should notbeused to predictthe effects ofpenetration
`indicator of the relative effects of 12-h pretreatment ofthe
`enhancers in humanskin. After treatment withsaline, hatr-
`skins with either penetration-enhancer mixtures[including
`less mouse skin sharply increased in permeability after ap-
`laurocapram (Azone), decylmethylsulfoxide, oleic acid, and
`proximately 50 h hydration, suggesting that the stratum cor-
`propylene glycol] or saline (control). After treatment with
`neumhadstarted to disrupt, whereas the flux through human
`saline, fluxes of 5-fluorouracil through the two skin types
`skin remained unchanged. J Invest Dermatol 90:810-813,
`were similar, but the mouse skin showed exaggerated re-
`1988
`sponsesto all the penetration-enhancer formulations. There
`
`he range of drugsthat can beeffectively delivered via
`the percutaneous route is limited largely by the rela-
`tive impermeability of the stratum corneum. Various
`methods ofincreasing the absorption of poorly pene-
`trating agents have been attempted, withearlier stud-
`ies concentrating often onthe effects of occlusion and hydration and
`morerecentinvestigations dwelling on penetration enhancers [1,2].
`Such accelerants reduce the barrier properties of the stratum cor-
`neum to other permeants, thereby potentially increasing the range
`of drugs that can be delivered through the skin.
`The developmentoftopical formulations containing penetration
`enhancers often involvesin vitro work withisolated skin. As human
`tissue is not always readily available, various animal models have
`beenused, with hairless mouse skin currently being popular.
`In this paper, we comparetheeffects ofpretreatmentwith a range
`of penetration enhancers on the permeabilities of human abdominal
`and hairless mouse skins to a model permeant, 5-fluorouracil
`(5-FU). We conclude that hairless mouse skin is a poor mimic of
`human skin with respect to enhanceractivity.
`MATERIALS AND METHODS
`
`We used the pseudo-steady-state permeability coefficient (K,) of
`5-FU as a test for the relative effects of 12-h pretreatments with
`sevenpotential penetration-enhancer formulations compared with
`normal saline (control). Previous work [3] has shown that such
`pretreatment optimizes penetration-enhancement effects. Effects
`on human abdominaland hairless mouse skins were compared to
`assess the suitability of the hairless mouse as. a model for human skin
`as modified by penetration enhancers.
`
`Manuscript received July 15, 1987; accepted for publication December
`23, 1987.
`This work was supported by a grant from 3M Health Care, Lough-
`borough, England. Reprint requests to: B. W. Barry, Postgraduate School of
`Studies in Pharmacy, University of Bradford, West Yorkshire, BD7 1DP,
`U.K.
`Abbreviations:
`5-FU: 5-fluorouracil
`DCMS:decylmethylsulfoxide
`
`Skin Sources and Preparation. Four male hairless mice (CBA/
`HLstrain) aged 60 to 80 days werekilled by spinaldislocation,
`their dorsal skins were immediately excised, any underlying "ssU¢
`being gently removed. Each mouse supplied 12 skin samples for use
`in permeation experiments.
`b
`Human midline abdominal skin from caucasian donors was Cc
`tained at autopsy and stored in evacuated polythenebags at —20°C
`until required [4]. Samples were sectioned with a dermatome (Davis
`Duplex 7) to approximately 420-~um-thick sections consisting ©
`the epidermis and a portion ofthe dermis. Two pieces of human
`abdominalskin were used (males, 60 and 63 years), each providing
`24 samples (3 from each donorfor each of the 8 pretreatments).
`The numberofreplicates allowed for occasionalcell leakage with
`consequentrejection of data, a common problem within vitro skin
`permeation work.
`Pretreatment Formulations. Three potentially useful penetra-
`tion enhancers ofdifferent chemical types —laurocapram (Azone,
`donated by Nelson Research), decylmethylsulfoxide (DCMS, do-
`nated by Procter and Gamble Co.), and oleic acid (Sigma Chemical
`Co., minimumassay 99%) — were tested. Oleic acid was used as a
`solution in propylene glycol, and laurocapram and DCMS were
`applied in both water and propyleneglycol. Concentrationsofpen-
`etration enhancers were chosen from published data,
`including
`work from this department[5]. Laurocapram 2% in propylenegly-
`col, oleic acid 5% in propylene glycol, and DCMS 15% in propy!-
`ene glycol were used by Barry and Bennett [6]. DCMS 4% in water
`was used by Sekura and Scala [7], and laurocapram 3% in 0.1%
`polysorbate 20/normalsaline has also been demonstratedas effec-
`tive [3,8]. As the mainaimofthe work was to comparetheeffects of
`a variety of enhancers on two skin types, different concentrations
`were deliberately chosen. A solution of 0.1% polysorbate 20
`(Tween 20)in normalsaline wasincluded as a control for the emul-
`sion of laurocapramin saline. Propylene glycol was included as a
`controlfor the enhancersolution based onthis solvent andtotest for
`enhancementeffects of the solventitself (see Table I).
`
`Automatic Diffusion Apparatus. Skin samples were mounted
`into stainless-steel diffusion cells (cross-sectional area 0.126 cm’)
`maintained at 31+ 1°C on hollow copper arms through which
`thermostated water was pumped. Receptorfluid (0.002% aqueous
`
`0022-202X/88/$03.50 Copyright © 1988 by The Society for Investigative Dermatology,Inc.
`810
`
`0001
`
`Noven Pharmaceuticals, Inc.
`EX2019
`Mylan Tech., Inc. v. Noven Pharma., Inc.
`IPR2018-01119
`
`

`

`VOL. 90, NO. 6 JUNE 1988
`
`PENETRATION ENHANCERS IN HUMAN AND MOUSESKINS 811
`
`6
`
`‘ome
`
`A
`
`12
`
`08
`
`€Oo
`
`a £
`
`04
`
`2
`oeCy
`.
`g 12 ao6
`©
`B
`
`i 6£o
`
`s=
`&
`8
`al
`E=
`
`3 40
`
`ere
`
`©
`
`60
`
`20
`Time (h)
`
`Pretreatment of Skin Samples and Permeation Studies.
`Each treatment mixture was applied to six samples of both skin
`types, consisting of 150 uL of water-based mixtures (= 1200
`pL cm) and 10 wL of propylene glycol-based mixtures (= 80
`iL cm7). Liquids remained on the skin for 12 h; then they were
`gently removed with absorbent tissue and permeation studies com-
`menced immediately.
`The donorsolutions consisted of 160 uL of a radiolabeled satu-
`rated (10.2 mg cm73)solution of 5-FU indistilled water [5-fluoro-
`6-[>H]uracil
`(Amersham International PLC) was diluted to
`0.3 mCi cm74]. Receptor samples werecollected over 2 h intervals,
`up to 60 h, and assayed for 5-FU contentby liquid scintillation
`counting (Packard Tri-Carb 460C) after the addition of 10 cm? of
`Scintran Cocktail T (BDH Chemicals Ltd.).
`Calculation of Permeability Coefficients. Raw data from
`scintillation counting were converted to cumulative amounts per
`unit area (mg cm~?) and computer-plotted versus time; for exam-
`ples, see Fig 1. Steady-state penetration fluxes, J (mg cm~? h7),
`were calculated by regression analysis from the linear regions of the
`plots (r typically equaled 0.998). Pretreatment with aqueous
`DCMS, however, consistently produced an atypical penetration
`plot, witha rapidinitial absorption followedbya fall in rate; fluxes
`were calculated from theinitial slope after this pretreatment(r typi-
`cally 0.98). Permeability coefficients, K, (cm h~'), were calculated
`from the steady-state flux and donor concentration, C (mg cm™~),
`using the relationship
`
`K,=J/C
`
`RESULTS
`
`Table 1 shows the mean permeability coefficients (K,) calculated
`for 5-FU, for both skin types, after each treatment. From these
`values, we calculated enhancementratios for each enhancertreat-
`ment, and bothskin types, from the formula
`.
`K,, of 5-FU after enhancer treatment
`enhancement ratio = ———________—_____
`K,of 5-FUafter saline treatment
`Theratios calculated for each treatment and skin type are com-
`pared in Fig 2.
`The cumulative 5-FU penetration plots for saline-pretreated
`hairless mouse skin differed markedly from those obtained with
`humanabdominalskin (Fig 3). Fluxes through hairless mouse skin
`increased dramatically after 35 to 40 h permeation, corresponding
`to 47 to 52 h hydration.
`
`Figure 1. Sample penetration plots for 5-FU through human abdominal
`skin after pretreatmentof the skin with oneofthe test mixtures. A. Polysor-
`bate 20 in saline (inverted opentriangles), propylene glycol (closed triangles),
`laurocapram in polysorbate 20/saline (opencircles) and laurocapram in pro-
`pylene glycol(closed circles). B. Normalsaline (opentriangles), aqueous decyl-
`methylsulfoxide (open diamonds), decylmethylsulfoxide in propylene glycol
`(closed diamonds) and oleic acid in propylene glycol (closed squares).
`
`sodium azide) flowed continuously through the receptor chamber
`and was collected in glass scintillation vials. Flow rate was
`2 cm? h~', corresponding to 40 changes of receptor volume per
`hour,ensuring sink conditions. The vials were changed automati-
`cally at 2-h intervals; a detailed description of the diffusion system
`has been published by Akhteretal (oi.
`
`Table I, Formulas and Volumesof the Eight Pretreatments Applied to the Skin Samples and Resultant Permeability Coefficients (K,)
`of 5-Fluorouracil Through Human Abdominal and Hairless Mouse Skins
`
`Pretreatment Formula
`
`Normalsaline (0.9% sodium chloride)
`
`Human Abdomen Hairless Mouse
`
`Mean K,!
`SEM‘
`nd
`Mean K,
`SEM
`n
`Code*
`
`Ss
`0.951
`0.451
`5
`1.07
`0.457
`6
`0.1% Polysorbate 20 in normalsaline
`TS
`1.03
`0.466
`5
`3.44
`0.610
`
`5
`
`3% w/v Laurocapram in 0.1% Polysorbate/saline
`
`4% w/v Decylmethylsulfoxide in water
`
`Propylene glycol
`2% w/v Laurocapram in propylene glycol
`
`15% w/v Decylmethylsulfoxide in propylene glycol
`
`LTS
`
`DCAQ
`
`PG
`LPG
`
`DCPG
`
`6.48
`
`71.3
`
`2.53
`17.7
`
`2.15
`
`1.14
`
`23.9
`
`0.785
`5ole
`
`0.688
`
`6
`
`6
`
`6
`6
`
`4
`
`11.4
`
`107
`
`4.88
`142
`
`6.59
`
`1.04
`
`8.18
`
`1.21
`36.2
`
`0.938
`
`6
`
`6
`
`5
`6
`
`6
`
`5% w/v Oleic acid in propylene glycol
`
`OAPG
`
`19.3
`
`6.20
`
`o
`
`159
`
`15.5
`
`6
`
`
`bPermesltycocficent()%10am
`€ Standard error of the mean.
`Numberofreplicates.
`
`0002
`
`

`

`812 BOND AND BARRY
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`150 100
`
`
`
`Enhancementratio
`
`sion of laurocapram(@ < 0.005), increasing to 18-fold when a solu-
`tion in propylene glycol was used (a@ <0.0005). Propylene glycol
`alone had a moderate enhancingeffect, increasing permeability to
`5-FU some 2.6 times (@ <0.025). The polysorbate 20 used to
`emulsify laurocapramin water insignificantly changed humanskin
`permeability to 5-FU (@ > 0.05), in agreement with previous work
`that showed that nonionics are the least damaging class of surfac-
`tants (e.g., [12,13]).
`DCMSin aqueous solution initially produced a high flux of
`5-FU,the effect being reversible as the DCMS was washed out of
`the skin [14]. DCMSinpropyleneglycol, in contrast, exerted very
`little effect on skin permeability, slightly less than that ofpropylene
`glycol alone. The effect of DCMS may have been reduced here
`because propylene glycol was a good solvent for the enhancer and
`inhibited its partitioning into the stratum corneum.
`Oleic acid is an effective penetration enhancer for lipophilic
`compounds, whenusedasa solution in propylene glycol [15]. We
`have foundit to beas effective as laurocapramin promoting perme-
`ation of 5-FU (a polar drug) when applied in this way.
`Comparison of Hairless Mouse and Human Skins. Theper-
`meability coefhcients for 5-FU through human abdominal and
`hairless mouse skins pretreated withsaline weresimilar, suggesting
`that the mouse model may have somevalidity in simple,idealsitua-
`tions; however, after penetration-enhancer pretreatment,the hair-
`less mouse model was misleading. Application of aqueouspolysor-
`bate 20, which had nosignificant effect on human abdominal skin
`(a > 0.05), increased the permeability of hairless mouse skin 3-fold
`(a <0.01).
`Figure 1 demonstrates that all pretreatments modified hairless
`K,, of 5-FU after enhancer treatment
`;
`mouse skin more than they did human skin. Therelative effect of
`enhanceméiit- ratio =—————————_
`each enhancer formulation on the two skins was not consistent.
`K, of 5-FUaftersaline treatment
`Thus, laurocapram in propylene glycol was 7 times more active in
`promoting 5-FU penetration through hairless mouse skin than
`through human abdominal skin, whereas the corresponding ratio
`for the aqueous emulsionof laurocapram wasonly 1.6. As there was
`no consistent relationship between penetration-enhancement ef-
`fects on the two skin types, we conclude that hairless mouse skin
`cannotbe used as a reliable model for humanpercutaneous absorp-
`tion as modified by accelerant treatment. The enhancementratios
`found for the accelerants used here werecalculated withrespect to
`5-FU. It is likely that enhancementeffects will change according to
`the chemical nature of the permeantused |6,16], and this would add
`additionalvariability and therefore potential inaccuracy to use ofthe
`hairless mouse model.
`Previous work explains therise in permeability after 50 h hydra-
`tion of hairless mouse skin pretreated withsaline [17]. Prolonged
`hydration completely disrupts hairless mouse skin and therise in
`permeability seen in the present work probably coincided with the
`start of stratum corneum breakdown, which would allow rapid
`permeation of 5-FU through weakened regions of the horny layer.
`
`Figure 2. Enhancementratios for 5-FU through human abdominal skin
`(open bars) and hairless mouse skin (hatched bars) after 12-h pretreatment
`with the enhancer mixtures. Enhancement ratiosare calculated by the equa-
`tion.
`
`Codesare defined in Table I.
`
`DISCUSSION
`
`Effects of Penetration Enhancers on Human Skin. Statistical
`analysis was performedusing the Wilcoxon-Mann- Whitney rank
`sum eu , taking a levelofsignificance (a) of0.05.In testing for
`effects of
`the penetration enhancers (comparedwithsaline control)
`a one-tailed test was used, but in comparing human abdominal and
`hairless mouse skins we used a two-tailed test.
`All the effects ofpenetration enhancers shown by human abdom-
`inal skin agree with previous studies. Laurocapram waseffective
`whenused as an emulsion (e.g., [3,8]), but other workers found that
`its action was heightened by propyleneglycol[11]. We discovered a
`near 7-fold rise in skin permeability after treatment with the emul-
`
`06
`
`Cumulativeweightof5-FUpenetrated(mgcm?)
`
`o4
`
`Qo
`
`20
`
`Time(h)
`
`40
`
`60
`
`Figure 3, Comparison of 5-FU penetrationplots through human abdomi-
`nal
`(opentriangles) and hairless mouse (inverted closed triangles) skins afteQgg3
`saline pretreatment.
`
`REFERENCES
`
`1. Barry BW:Properties that influence percutaneous absorption,in Der-
`matological Formulations: Percutaneous Absorption. Marcel
`Dekker, New York, 1983, pp 127-233
`2. Woodford R, Barry BW: Penctration enhancers and the percutaneous
`absorption of drugs: An update, J Toxicol Cutaneous Ocular Toxi-
`col 5:165-175, 1986
`3. Sugibayashi K, Hosoya KI, Morimoto Y, Higuchi WI: Effect of the
`absorption enhancer, Azone, on the transport of 5-fluorouracil
`across hairless rat skin. ] Pharm Pharmacol 37:578- 580, 1985
`4. Harrison SM, Barry BW, Dugard PH: Effects of freezing on human
`skin permeability. J Pharm Pharmacol 36:261-262, 1984
`5, Goodman M,Barry BW:Action ofskin permeation enhancers Azone,
`oleic acid and decylmethy! sulphoxide: Permeation and DSC stud-
`ics. ] Pharm Pharmacol 38:71P, 1986
`Barry BW, Bennett SL: Effect ofpenetration enhancers on the pene
`ation ofmannitol, hydrocortisone andprogesterone through human
`skin. J Pharm Pharmacol 39:535 - 546, 1987
`
`6.
`
`

`

`VOL. 90, NO. 6 JUNE 1988
`
`PENETRATION ENHANCERS IN HUMAN AND MOUSE SKINS 813
`
`7.
`
`10.
`
`11.
`
`Sekura DL, Scala J: The percutaneousabsorption of alkyl methyl sul-
`foxides, in Advances in Biology of the Skin, vol 12. Edited by W
`Montagna, EJ Van Scott, RB Stoughton, Appleton-Century-
`Crofts, New York, 1972, pp 257-269
`Shannon WM, Westbrook L, Higuchi WI, Sugibayashi K, Baker DC,
`KumarSD,FoxJL, Flynn GL, Ho NFH,Vaidyanathan R:Influence
`of 1-dodecylazacycloheptan-2-one (Azone) on the topical therapy
`of cutancous herpes simplex virus type 1 infection in Taiietaes mice
`with 2’, 3‘-di-O-acetyl-9-f-p-arabinofuranosyladenine and 5’-O-
`valeryl-9-f-p-arabinofuranosyladenine. J Pharm Sci 74:1157-
`1161, 1985
`Akhter SA, Bennett SL, Waller IL, Barry BW: An automateddiffusion
`apparatus for studying skin penetration. Int J Pharm 21:17-26,
`1984
`
`Iman RL, Conover WJ: A Modern ApproachtoStatistics. John Wiley,
`New York, 1983, pp 280-287
`Wotton PK, Mollgaard B, Hadgraft J, Hoelgaard A: Vehicleeffect on
`topical drug delivery, III. Effect of Azone on the cutaneous perme-
`ation of metronidazole and propylene glycol. Int J Pharm 24:19-
`26, 1985
`
`12,
`
`13.
`
`14.
`
`ao;
`
`16.
`
`17.
`
`Lansdown ABG,Grasso P: Physico-chemicalfactors influencing epi-
`dermal damageby surface active agents. Br] Dermatol 86:361-373,
`1972
`Dalvi UG, Zatz JL: Effect of nonionic surfactants on penetration of
`dissolved benzocaine through hairless mouse skin. J] Soc Cosmet
`Chem32:87-94, 1981
`Cooper ER:Effects of decylmethylsulfoxide on skin penetration, in
`Solution BehaviourofSurfactants, Theoretical and Applied Aspects,
`vol 12. Edited by KL Mittel, EJ Fendler. Plenum Press, New York,
`1982, pp 1505-1516
`Cooper ER: Increased skin permeability for lipophilic molecules. J
`Pharm Sci 73:1153-1156, 1984
`Bennett SL, Barry BW: Effectiveness of skin penetration enhancers
`propylene glycol, Azone, decylmethylsulphoxide and oleic acid
`with modelpolar (mannitol) and nonpolar (hydrocortisone) pene-
`crants. J Pharm Pharmacol 37:84P, 1985
`Bond JR, Barry BW: Longtermhydration effects on permeability of
`hairless mouse skin. J Pharm Pharmacol 37:77P, 1985
`
`0004
`
`