`
`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 eliects 0"
`through
`The permeability coeli'icient of 5-fluorouracil
`human abdominal and hairless mouse skins was used as an
`the two skin types, and we conclude that the hairless may“
`indicator of the relative eli'ects of 12-h pretreatment of the
`model should not be used to predict the effects ofpenetfflrlfm
`enhancers in human skin. After treatment with saline. [131"
`skins with either penetration-enhancer mixtures [including
`less mouse skin sharply increased in permeability after ap-
`laurocapram {Azone}. decylmethylsulfoxide. oleie acid. and
`propylene glycol] or saline (control). After treatment with
`proximately 50 h hydration, suggesting that the stratum cor-
`neum had started to disrupt, Whereas the flux through human
`saline, fluxes of 5-fluorouracil through the two skin types
`skin remained unchanged. j Invest Dermot-91' 90:810-313!
`were similar, but the mouse skin showed exaggerated re-
`1' 938
`
`sponses to all the penetration-enhancer formulations. There
`
`he range of drugs that can be efl—ectively delivered via
`the percuraneous route is limited largely by the rela-
`tive impenneabiliry of the stratum cornenm. Various
`methods of increasing the absorption ofpoorly pene-
`tratin agents have been attempted, with earlier stud-
`ies concentrating o ten on the after: ofocclusion and hydration and
`more recent investigations dwelling on penetration enhancers [1.2].
`Such accelerants reduce the barrier properties of the stratum cor-
`ncum to other penneants, thereby potentially increasing the range
`ofdrugs that can be delivered through the skin.
`The development of topical formulations containing penetration
`enhancers often involves in vitro work with isolated skin. As human
`tissue is not always readily available. various animal models have
`been used, with hairless mouse skin currently being popular.
`In this paper, we compare the effects ofprctreatmcnr with a range
`of penetration enhancers on the permeabilities ofhuman abdominal
`and hairless mouse skins to a model permeant, S-fluorouracil
`(fi-FU}. We conclude that hairless mouse skin is a poor mimic of
`human skin with respect to enhancer activity.
`MATERIALS AND METHODS
`
`We used the pseudo-steady—state permeability coefficient (1(9) of
`S—FU as a test for the relative efi'ecrs of 12-h pretreatments with
`seven potential penetration—enhancer formulations compared with
`normal saline (control). Previous work [3] has shown that such
`pretreatment optimizes penetration-enhancement eflects. EHects
`on human abdominal and 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. l987.
`This work was supported by a grant from 3M Health Care, Lough-
`borongh. England. Reprint requests to: B. W. Barry, Postgraduate School of
`Studies in Pharmacy, University of Bradford, Wesr Yorkshire, BD? lDP,
`U.K..
`Abbreviations:
`5—FU: S—fluorouraeil
`DCMS: decylmethylsulfoxide
`
`Skin Sources and Preparation. Four male hairless mice (CBAé
`HL strain) aged 60 to 80 days were killed by spinal dislocation: an
`their dorsal skins were immediately excised, any undcrlylng "55“:
`being gently removed. Each mouse supplied 12 skin samples for use
`in permeation experiments.
`b
`I-luman midline abdominal skin from caucasian donors “'15 O‘C-
`tained at autopsy and stored in evacuated polythene bags at '20 _
`until required [4] Samples were sectioned with a dcn‘natomf‘ in"?
`Duplex 7) to approximately 420-pm-thick sections COnSISU-ng 0
`the epidermis and a portion of the dermis. Two pieces of I191?“
`abdominal skin were used (males. 60 and 63 years), each pl'OVI‘lmg
`24 samples (3 from each donor for each of the 8 pretreatments _-
`The number of replicates allowed For occasional cell leakage WI?"
`consequent rejection ofdara, a common problem with in intro Slim
`permeation work.
`
`Pretreatment Formulations. Three potentially useful penetra-
`tion enhancers of different chemical types— laurocaprarn (Atom.
`donated by Nelson Research), decylmethylsulfoxide (DCMS. do-
`nated by Procter and Gamble Co.], and oleic acid (Sigma Clicmlcll
`Co., minimum assay 99%] —were tested. Oleic acid was used as a
`solution in propylene glycol, and laurocapram and DCMS We“
`applied in both water and propylene glycol. Concentrations ofpen-
`etration enhancers were chosen from published data,
`including
`work from this department [5]. Lauroeapram 2% in propylene gh"
`col, oleic acid 5% in propylene glycol, and DCMS 15% in prowl-
`ene glycol were used by Barry and Bennett [6]. DCMS 4% in water
`was used by Sekura and Scala ['3'], and laurocaprarn 3% in 011%
`polysorbatc 20fnormal saline has also been demonstrated as effec-
`tive [3,8]. As the main aim oFthe work was to compare the eliects Di
`a variety of enhancers (in two skin types, dilierent concentrations
`were deliberately chosen. A solution of 0.1% polysorbate 20
`(Tween 20} in normal saline was included as a control for the emul-
`sion of laurocaprarn in saline. Propylene glycol was included as :1
`control for the enhancer solution based on this solvent and to test for
`enhancement effects of the solvent itself (see Table I}.
`
`Automatic Difl'usion Apparatus. Skin samples were mounted
`into stainless-steel diffusion cells (cross-sectional area 0.126 cm!)
`maintained at 3‘] i‘ 1°C on hollow copper arms through which
`thermostared water was pumped. Receptor fluid (0.002% aqueous
`
`0022-202XX88/50350 Copyright © 1983 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 MOUSE SKINS
`
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`Pretreatment of Skin Samples and Permeation Studies.
`Each treatment mixture was applied to six samples of both skin
`types, consisting of 150 pL of water-based mixtures (E 1200
`”L (Tn—2) and 10 Jill. of propylene glycol-based mixtures (5 80
`,uL cm'zl. Liquids remained on the skin for 12 h; then they were
`gently removed with absorbent tissue and permeation studies com-
`menced immediately.
`The donor solutions consisted of 160 iiL of a radiolabeled satu-
`rated (10.2 mg cm‘a) solution of 5«FU in distilled water [5-fluoro-
`6-[3‘Hlutacil
`(Amersham International PLC} was diluted to
`0.3 mCi em’Jl. Receptor samples were collected over 2 h intervals.
`up to 60 h, and assayed for 5-F‘U content by liquid scintillation
`counting (Packard Tri-Carb 460C) after the addition of 10 cm3 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.] (mg cm“2 11"],
`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, with a rapid initial absorption followed by a fall in rate; fluxes
`were calculated from the initial slepe after this pretreatment (r typi-
`cally 0.98). Permeability coefficients, KP (cm h“), were calculated
`from the steady-state flux and donor concentration, C (mg cm'z‘),
`using the relationship
`
`KP =j/C
`
`RESULTS
`
`Table 1 shows the mean permeability cocflicients (KP) calculated
`for S-FU. for both skin types. after each treatment. From these
`values. we calculated enhancement ratios for each enhancer treat-
`ment. and both skin types. from the formula
`.
`K of S-FU after enhancer treatment
`enhancement rano = _;_______
`KP of S-FU after saline treatment
`
`The ratios 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
`human abdominal skin (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 S-FU through human abdominal
`skin after pretreatment of the skin with one oFthe test mixtures. A. Polysor-
`bate 20 in saline (inverted open triangles), propylene glycol (closed triangles),
`laurocapram in polysorbate 20[saline (open circles) and lauroeapram in pro-
`pylene glycol (closed circles). B. Normal saline {open triangles). aqueous dccyl-
`methylsulfoxide (open diamonds). deeylmetliylsulfoxide in propylene glycol
`(closed diammids} and oleic acid in propylene glycol (closed squares).
`
`sodium azidc) flowed continuously through the receptor chamber
`and was collected in glass scintillation vials. Flow rate was
`2 crrr" h"‘, corresponding to 40 changes of receptor volume per
`hour, ensuring sink conditions. The vials were changed automati-
`cally at 2-11 intervals: a detailed descri tion of the diH-usion system
`has been published by Akhter et al [9f
`
`Table I. Formulas and Volumes of the Eight Pretreatments Applied to the Skin Samples and Resultant Permeability Coefficients (Kr)
`of S-Fluorouracil Through Human Abdominal and Hairless Mouse Skins
`
`
`
`Human Abdomen Hairless Mouse
`
`Pretreatment Formula
`Code‘
`Mean Kl,”
`SEM‘
`a“
`Mean K?
`SEM
`
`Normal saline (0.9% sodium chloride)
`5
`0.951
`0.45!
`5
`1.07
`0.45?
`
`:1
`6
`
`0.1% Polysorbate 20 in normal saline
`
`3% w/v Laurocapram in 0.1% Polysorbate/saline
`
`4% w/v Decylmethylsulfoxide in water
`
`Propylene glycol
`2% w/v Laurocapram in propylene glycol
`
`TS
`
`LTS
`
`DCAQ
`
`PG
`LPG
`
`15% w/v Decylmethylsulfoxide in prepylene glycol
`
`DCPG
`
`1.03
`
`6.48
`
`71.3
`
`2.53
`17.7
`
`2.15
`
`0.466
`
`1.14
`
`23.9
`
`0.?85
`5.12
`
`0.688
`
`5
`
`6
`
`6
`
`6
`6
`
`4
`
`3.44
`
`11.4
`
`107
`
`4.88
`142
`
`6.59
`
`0.610
`
`1.04
`
`8.18
`
`1.21
`36.2
`
`0.933
`
`5
`
`6
`
`6
`
`5
`6
`
`6
`
`5% w/v Oleic acid in propylene glycol
`OAPG
`19.3
`6.20
`4
`159
`15.5
`6
`
`Igfrii::lii1iit;nc§:%i§i:::i{13°}???Kit—tiin
`‘ Standard error of the mean?
`d Number of replicates.
`
`0002
`
`

`

`812 BOND AN!) nanny
`
`150
`
`100
`
`L“\\\s\“\\\\.\\\\‘
`L
`\\\\\\\\\\\\\‘\‘
`
`
`
`Enhancementratio
`
`Figure 2. Enhancement ratios for S-FU through human abdominal skin
`{open bars} and hairless mouse skin (hatched liars) after 12-h pretreatment
`with the enhancer mixtures. Enhancement ratios are calculated by the equa-
`tion.
`
`K of 5-FU after enhancer treatment
`.
`enhancement rant) = _L—_..__—
`KP of S-FU after saline treatment
`
`Codes are defined in Table 1.
`
`DISCUSSION
`
`Effects of Penetration Enhancers on Human Skin. Statistical
`
`analysis was erformed using the Wilcoxmi — Mann — Whitney rank
`sum testf[10 , taking a level ofsignificanca (er) of0.05. in testing for
`effects 0 the penetration enhancers (compared with saline control)
`a one-tailed test was used, but in comparing human abdominal and
`hairless mouse skins we used a two-tailed test.
`
`All the cifects ofpenetration enhancers shown by human abdom-
`inal skin agree with previous studies. Laurocaprarn was effective
`when used as an emulsion (cg, [3,8]), but other workers found that
`its action was heightened by propylene glycol [11]. We disc0vered a
`near 7-fold rise in skin permeability after treatment with the emul-
`
`0‘6
`
`THE jOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`sion oflaurocapram (0: < 0.005), increasing to 18-fold when a solu-
`tion in propylene glycol was used (or <0.0005)_ Propylene glycol
`alone had a moderate enhancing effect, increasing permeability to
`S-FU some 2.6 times (a (0.025). The polysorbate 20 used to
`emulsify lauroeapram in water insignificantly changed human skin
`permeability to 5-FU (a >005), in agreement with previous work
`that showed that noniemics are the least damaging class of surfac-
`tants (cg, [12,13]).
`DCMS in aqueous solution initially produced a high flux of
`S-FU. the effect being reversible as the DCMS was washed out of
`the skin [14]. DCMS in propylene glycol, 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 corneurn.
`Oleie acid is an effective penetration enhancer for lipo hilic
`compounds, when used as a solution in propylene glycol [15]: We
`have found it to be as effective as laurocapram in promoting perme-
`ation of S-FU (a polar drug) when applied in this way.
`
`Comparison of Hairless Mouse and Human Skins. The per-
`meability coefficients For S-FU through human abdominal and
`hairless mouse skins pretreated with saline were similar, suggesting
`that the mouse model may have some validity in simple, ideal situa-
`tions; however, after penetration-enhancer pretreatment, the hair-
`less mouse model was misleading. A plication of aqueous polysor-
`bate 20, which had no significant e cct on human abdominal skin
`(or > 0.05], increased the permeability of hairless mouse skin 3-fold
`(a (0.01).
`Figure 1 demonstrates that all pretreatments modified hairless
`mouse skin more than they did human skin. The relative effect of
`each enhancer formulation on the two skins was not consistent.
`
`Thug, 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 emulsion oflaurocapram was only 1.6. As there was
`no consistent relationship between penetration-enhancement ef-
`fects on the two skin types, we conclude that hairless mouse skin
`cannot be used as a reliable model for human pcrcutaneous absorp-
`tion as modified by accelerant treatment. The enhancement ratios
`found for the accelerants used here were calculated with respect to
`S-FU. It is likely that enhancement effects will change accurding to
`the chemical nature ofthe permeant used [6,16], and this would add
`additional variability and therefore potential inaccuracy to use of the
`hairless mouse model.
`
`Previous work explains the rise in permeability after 50 h hydra-
`tion of hairless mouse skin pretreated with saline [17]. Prolonged
`hydration completely disrupts hairless mouse skin and the rise in
`permeability seen in the present work probably coincided with the
`start of stratum corneum breakdown, which would allow rapid
`permeation of S-FU through weakened regions ofthe horny layer.
`
`0 is
`
`Curr'ulativeweightof5FUpei'ietrated oM
`
`["190le
`
`O
`
`20
`
`Time lhl
`
`40
`
`60
`
`Figure 3. Comparison of S—FU penetration plots through human abdomi-
`nal
`(open triangles) and hairless mouse {inverted rinsed triangles) skins afteooos
`saline pretreatment.
`
`REF EREN CBS
`
`1. Barry 13W: Properties that influence pereutaneous absorption, in Der-
`matological Formulations:
`l’ercutaneous Absorption. Marcel
`Dekker, New York, 1983, pp 127-233
`2. Woodford R. Barry BW: Penetration enhancers and the percutaneous
`absorption ofdrugs: An update. j Toxicol Cutaneous Ocular Toxi-
`col 5:165-175. 1986
`
`3. Sngibayashi K, l-Iosoya KI, Morimoto Y, Higuchi WI: Efi'ect of the
`absorption enhancer, Azone, on the transport of S-llnorouracil
`across hairless rat skim] l’harm l’liarmacol 3?:578—580, 1985
`4. Harrison SM. Barry 13W, Dugard l’H: Effects of freezing on human
`skin perrneability._l Pharm Pharmacol 36:261—262, 1984
`5. Goodman M, Barry 11W: Action of skin permeation enhancers Azonet
`oleic acid and decylmethyl sulphoxide: Permeation and DSC stud-
`ies._] I’harm I’harniaco] 38:71]). 1986
`6. Barry “W, Bennett SL1 Effect ofpcnetration enhancers on the perme-
`ation ofmannitol, hydrocortisone and progesterone through iuman
`skim] l’harm l’harmaeol 39:535-546. 1987
`
`

`

`VOL. 90, NO. 6 ]UNE 1938
`
`PENETRATION ENHANCERS IN HUMAN AND MOUSE SKINS
`
`813
`
`7. Sekura DL. Scala]: The percutaneous absorption of alkyl methyl sul-
`foxides, in Advances in Biology of the Skin. vol 12. Edited by W
`Montagna. E] Van Scott. RB Stoughton. Appleton—Century—
`Crofts. New York, 1972, pp 257 —269
`8. Shannon WM. Westbrook L, Higuehi WI. Sugibayashi K, Baker DC.
`Kumar SD. Foij. Flynn GL, Ho NFH. Vaidyanathan R: Influence
`of 1-dodecylazacycloheptan-Z-one {Azone} on the to ical therapy
`of cutaneous herpes simplex virus type 1 infection in hairless mice
`with 2’, 3’-di-O-acetyl-9-fl-o-arabinofuranosyladenine and 5’-O-
`valeryl-9-fl-D-arabinofuranosyladenine. ] Pharrn Sci 74:1 157 —
`1161,1935
`
`9. Akhter SA. Bennett 81., Waller IL. Barry BW: An automated diffusion
`apparatus for studying skin penetration. Int ] Pharm 21:17 — 26.
`1984
`
`1 0.
`
`Iman RL. Conover W]: A Modern Approach to Statistics. ]ohn Wiley,
`New York. 1983,1319 230—237
`'1 1. Wotton PK, Mollgaard B. Hadgraft]. Hoelgaard A: Vehicle effect on
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`ation of metronidazole and propylene glycol. Int] Pharm 24:19—
`26. 1985
`
`12. Lansdown ABG. Grasso P: Physico-chemical factors influencing epi-
`dermal damage by surface active agents. Br] Detmatol 86:361 - 373.
`1972
`
`13. Dalvi UG. Zatz ]L: Efl'ect of nonionic surfactants on penetration of
`dissolved benzocaine through hairless mouse skin. ] Soc Cosmet
`Chem 32:87—94. 1981
`
`14. Cooper ER: Effects of decylmethylsulfoxitle on skin penetration, in
`Solution Behaviour of Surfactants. Theoretical and Applied Aspects.
`vol 12. Edited by KL Mittel, E] Fendler. Plenum Press. New York.
`1982. pp 1505—1516
`15. Cooper ER: Increased skin permeability for lipophilic molecules]
`Pharm Sci 73:1153-1156. 1984
`
`16. Bennett SL, Barry BW: Effectiveness of skin penetration enhancers
`propylene glycol. Azone. decyltnethylsulphoxide and oleie acid
`with model polar (tnannitol) and nonpolar {hydroeottisone} pene-
`trants.] Pharm Pharmacol 37:84P, 1985
`17. Bond ]R. Barry BW: Long term hydration efl'eces on permeability of
`hairless mouse skin.] Pharm Pharmacol 37:77P. 1985
`
`0004
`
`