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, Pl1.D., D.Sc.
`Postgraduate School ofStudies in Pharmacy, University of Bradford, Bradford, UK.
`
`
`through
`The permeability coeiheient of S-Fluorouracil
`was no consistent relationship between enhancer edict“ on
`human abdominal and hairless mouse skins was used as an
`the two skin types, and we conclude that the hairless 11101-159
`indicator of the relative effects of 12-h pretreatment of the
`model shOuld not he used to predict the eli‘ects ofpenerratlun
`enhancers in human skin. After treatment with saline, hair-
`skins with either penetration-enhancer mixtures [including
`laurocapram (Azone), decylmethylsulfoxide, oleic acid. and
`less mouse skin shat ly increased in permeability after aP'
`prOximately 50 h hydiation. suggesting that the stratum cor—
`propylene glycol} or saline (control). After treatment with
`saline. fluxes of 5-fluorouracil through the two skin types
`neum had started to disrupt, whereas the flux through human
`skin remained unchanged. )1 Invest Dermatol 90:310c3134
`were similar, but the mouse skin showed exaggerated re-
`I938
`sponses to all the penetration-enhancer formulations. There
`
`he range of drugs that can be effectively delivered via
`the percutaneous route is limited largely by the rela-
`tive irn ermeability of the stratum corneum. Various
`methocrs of increasing the absorption of poorly pene—
`tratinfg agents have been attempted, with earlier stud-
`ies concentrating o ten on the effects of occlusion and hydration and
`more recent investigations dwelling on penetration enhancers [l ,2].
`Such accelerants reduce the barrier properties of the stratum cor-
`ncum to other permeants. thereby potentially increasing the range
`of drugs that can be delivered through the slrin.
`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 of pretreatment with a range
`ofpenetration enhancers on the permeabilities of human abdominal
`and hairless mouse skins to a model permeant. 5-fluorouracil
`(S-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-stare permeability coefficient (Kr) of
`5-FU as a test for the relative effects 0F 12-11 pretreatments with
`seven potential penetration-enhancer formulations compared with
`normal saline (control). Previous work [3] has shown that such
`pretreatment optimizes penetration-enhancement effects. Effecrs
`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. 1937', accepted for publication December
`2], 198?.
`This worlt was supported by a grant From 3M Health Care, Lough-
`borough. England. Reprint requesls to: B. W. Barry, Postgraduate School of
`Studies in Pharmacy. University of Bradford. West Yorkshire. BD7 lDP.
`UJC.
`Abbreviations:
`S-FU: S-fiuorouracil
`DCMS: deeylmethylsulfoxide
`
`Skin Sources and Preparation. Four male hairless mice (C30:
`HL strain] aged 60 to 30 days were killed by spinal dislocatlorl: an
`their dorsal skins were immediately excised, any underlylllg [155“:
`being gently removed. Each mouse supplied 12 skin samples for "5‘3
`in permeation experiments.
`13
`Human midline abdominal skin From caneasian donors “'35 (it;
`tained at autopsy and stored in evacuated polythene bags at '20 1.
`until required [4] Samples were sectioned with a derlnatolue (03‘15
`Duplex '3') to approximately 420-pm-thiclt sections COflSlsnng 0
`the epidermis and a portion of the dermis. Two pieces 0? I193?“
`abdominal skin were used (males. 60 and 63 years}, each providlng
`24 samples (3 from each donor for each of the 3 pretrEatinentsl; h
`The number ofteplieates allowed for occasional cell leakagt‘ “”E
`consequent rejection of data, a common problem with in vnro 5km
`permeation work.
`
`Pretreatment Formulations. Three potentially useful penetra-
`tion enhancers ofdifi'erent chemical types—laurocaprilm (Mont.
`donated by Nelson Research], decylmethylsulfoxide (DCMS- do-
`nated by Procter and Gamble Co}, and oleic aeid (Sigma Chemical
`(30., minimum assay 99%)-—were tested. Olcic acid was used as 2
`solution in propylene glycol. and laurocapram and DCMS were
`applied in both water and propylene glycol. Concentrations ofplell-
`etration enhancers were chosen From published data, including
`work from this department [5] Laurocapram 2% in propyltnc Ell"
`col. oleic acid 5% in propylene glycol. and DCMS 15% in pIOPYI‘
`ene glycol were used by Barry and Bennett [6]. DCMS 4% in water
`was used by Sekura and Seals [7]. and laurocaprarn 3% in 0.1%
`polysorbate 20/normal saline has also been demonstrated as ell-cc-
`tive [3,8]. As the main aim ofthe work was to compare the effects of
`a variety of enhancers on two skin types. different 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 lauroeaprani in saline. Propylene glycol was included as a
`control for the enhancer solution based on this solvent and to test {0!
`enhancement efl'eets of the solvent itself (see Table 1).
`
`Automatic Diffusion Apparatus. Skin samples were mounted
`into stainless-steel difi'usion cells (cross-sectional area 0.126 emzi
`maintained at 3] i 1°C on hollow copper arms through which
`thermostated water was pumped. Receptor fluid (0.002% aqueous
`
`0022-2023([88/50350 Copyright © 1988 by The Society for Investigative Dermatology. Inc.
`3 10
`
`0001
`
`Noven Pharmaceuticals, Inc.
`EX2019
`
`Mylan Tech., Inc. v. Noven Pharma, Inc.
`IPR2018—00174
`
`

`

`VOL. 90. NO. 6 JUNE was
`
`PENETRATION ENHANCERS IN HUMAN AND MOUSE SKINS
`
`811
`
`A
`
`1-2
`
`ea
`
`Pretreatment of Skin Samples and Permeation Studies.
`Each treatment mixture was applied to six samples of both skin
`types, consisting of 150 lttL of water-based mixtures (E 1200
`pL cm”) and 10 pL of propylene glycol-based mixtures (E 80
`pl. 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 JUL ofa radiolabelcd satu-
`rated (10.2 mg cm”) solution ofS-FU in distilled water {S-fluoro-
`6-[3Hluracil
`(Amersham International PLC) was diluted to
`0.3 mCi cm‘3]. Receptor samples were collected over 2 h intervals.
`up to 60 h, and assayed for S-FU content by liquid scintillation
`counting (Packard Tri-Carb 460C) after the addition of 10 cm-°' of
`Scintran Cocktail T (BDI-I Chemicals Ltd.)
`
`Calculation of Permeability Coeflicients. Raw data from
`scintillation counting were converted to cumulative amounts per
`unit area (mg cm‘z) and computer-plotted versus time; for exam-
`ples, see Fig 1. Steady-state penetration fluxes, 1 (mg cm—2 h“).
`were caICulated 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
`plor, with a rapid initial absorption followed by a fall in rate; fluxes
`were calculated from the initial slope 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”).
`using the relationship
`
`KP = J/C
`
`RESULTS
`
`Table 1 shows the mean permeability coefiicients (KP) calculated
`for 5-K], For both sltin 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 ratio = -;———————
`KP of S-FU after saline treatment
`
`The ratios calculated for each treatment and skin type are com~
`pared in Fig 2.
`The cumulative S-FU penetration plots for saline-pretreated
`hairless mouse skin dill-cred 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.
`
`
`
`
`
`
`
`Cumulativeweightof5-FUpenetrated[mgcnr’l
`
`04
`
`1-2
`
`C
`.
`."_.;“='—:?E=:v_v
`B
`
`.
`'
`
`.
`'
`
`.
`
`'
`
`.
`
`'
`
` 4D
`
`Time lhl
`
`60
`
`Figure 1. Sample penetration plots for 5-FU through human abdominal
`skin after pretreatment of the skin with one of the rest mixtures. A. Polysor-
`bate 20 in saline (inverted open triangles), propylene glycol (closed triangles},
`laurocapram in polysorbatc 20/saline [open circles) and laurocaprarn in pro-
`pylene glycol {closed circles). B, Normal saline {open triangles). aqueous decyl-
`rnetliylsulfoxide (open diamonds), decylmetliylsulfoxide in propylene glycol
`{rinsed 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 cm3 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 Althter et al [9].
`
`Table I. Formulas and Volumes of the Eight Pretreatments Applied to the Skin Samples and Resultant Permeability Coeflicients (KP)
`of 5-Fluorouracil Through Human Abdominal and Hairless Mouse Skins
`Human Abdomen Hairless Mouse
`
`
`
`Pretreatment Formula
`Code“
`Mean K;
`SEM‘
`tr‘
`Mean KP
`SEM
`n
`
`Normal saline (0.9% sodium chloride)
`5
`0.951
`0.451
`5
`1.0?
`0.457
`6
`
`0.1% Polysorbate 20 in normal saline
`
`3% w/v Laurocaprarn in 0.1% Polysorbate/saline
`
`4% w/v Dccylmetliylsulfoxide in water
`
`Propylene glycol
`% w/v Laurocaptam in propylene glycol
`
`TS
`
`LTS
`
`DCAQ
`
`PG
`LPG
`
`15% wfv Decylmethylsulfoxide in propylene 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
`
`0.938
`
`5
`
`6
`
`6
`
`5
`6
`
`6
`
`5% wfv Oleic acid in propylene glycol
`OAPG
`19.3
`6.20
`4
`159
`1.5.5
`6
`
`:Codes used in Fig 2 to denote treatment type.
`Permeability eoeflicient (KP) X 10“ em 11“.
`‘ Standard error of the mean.
`Number of replicates.
`
`0002
`
`

`

`812
`
`liOND AND nanny
`
`150
`
`100
`
`
`\“““\““\‘\\‘
`
`L“\\\\‘\\‘\\\.\\“‘
`
`
`
`Enhancementratio
`
`rs
`
`LTS ocao PG
`
`LPG DCPG OAPG
`
`Figure 2. Enhancement ratios For 5—FU through human abdominal skin
`(opt-u bars) and hairless mouse skin fliatrlrrrl bars) after 12-11 pretreatment
`with the enhancer mixtures. Enhancement ratios are calculated by the equa-
`tion.
`
`K of 5-FU after enhancer treatment
`.
`enhancement ratio = -J’—-———————
`KP of 5~FU after saline treatment
`
`Codes are defined in Table 1.
`
`DISCUSSION
`
`Efi'ects of Penetration Enhancers on Human Skin. Statistical
`analysis was erformed using the Wilcoxon — Mann— Whitney rank
`sum test [10 , taking a level ofsignificancc (or) of0.05. In testing for
`effects of 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 effects ofpenetration enhancers shown by human abdom-
`inal skin agree with previous studies. Laurocapram was effective
`when used as an emulsion (e.g.. [3,8]), but other workers found that
`its action was heightened by propylene glycol [11]. We discovered a
`near 7-fold rise in skin permeability after treatment with the emul-
`
`0'6
`
`Curmla‘liveweiglnoiS-FUpenetrated
`
`0
`
`20
`
`Time lhl
`
`40
`
`60
`
`Figure 3. Comparison of S—FU penetration plots through human abdomi-
`nal
`(open irimrqles) and hairless mouse (inverted closed triangles) skins afte0003
`saline pretreatment.
`
`THE jOUllNAL 0F 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 ell'ect, increasing permeability to
`S-FU some 2.6 times (or (0.025). The polysorbate 20 used to
`emulsify laurocapram in water insignificantly changed human skin
`permeability to S-FU {ct > 0.05), in agreement with previous work
`that showed that nonionics are the least damaging class of surfac-
`tants (e.g., [12,13]).
`DCMS in aqueous solution initially produced a high flux of
`S-FU, the elicct being reversible as the DCMS was washed out of
`the skin [14]. DCMS in propylene glycol, in contrast, exerted very
`little cliect 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.
`Olcic acid is an effective penetration enhancer for lipo hilic
`compounds, when used as a solution in propylene glycol [15 . We
`have found it to he as edectivc as laurocapram in promoting perme-
`ation of 5-FU (a polar drug) when applied in this way.
`
`Comparison of Hairless Mouse and Human Skins. The per-
`meability coefiicients for S-FU through human abdominal and
`hairless mouse skins pretreated with saline were similar, suggesting
`that the mouse model may have $0?th validity in simple, idea] situa-
`tions; however, after penetration-enhancer pretreatment, the hair-
`les-s mouse model was misleading. A plication of aqueous polysor-
`bate 20, which had no significant e ect on human abdominal skin
`(ct >005), increased the permeability ofliairless mouse skin 34:0ch
`(.52 «1.01).
`Figure 1 demonstrates that all pretreatments modified hairless
`mouse skin more than they did human skin. The relative cliect of
`each enhancer formulation on the two skins was not consistent.
`
`Thus, laurocapram in propylene glycol was 7 times more active in
`promoting S-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-cnhancemcnt ef-
`Fects on the two skin types, we conclude that hairless mouse skin
`cannot be used as a reliable model for human percutaneous absorp-
`tion as modified by accelerant treatment. The enhancement ratios
`Found for the accelerants used here were calculated with respect to
`5-FU. It is likely that enhancement effects will change according to
`the chemical nature of the permeant used [6,16], and this would add
`additional variability and therefore potential inaccuracy to use ofthe
`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 5-FU through weakened regions ofthe horny layer.
`
`REFERENCES
`
`1. Barry 15W: Properties that influence percutaneous absorption, in Der-
`matological Formulations: Pcrcntaneous Absorption. Marcel
`Dekker, New York, 1983, pp 127—233
`2. Woodford Ii. Barry 15W: Penetration enhancers and the percutaneous
`absorption ofdrugs: An update.) Toxicol Cutaneous Ocular Toxi-
`col5:165—1?5.1986
`
`3. Sugibayaslli K, l-losoya KI, Morimoto Y, Higuchi WI: Eli'ect of the
`absorption enhancer, Acone, on the transport of 5-iluorouracil
`across hairless rat skin.) l’harm Pharmacol 37:578-580. 1935
`4. Harrison SM, Barry BW, Dugard l’H: Effects of freezing on human
`skin permeability] Pharni l’harniacol 36:261-262. 1984
`S. Goodman M, Barry 15W: Action of skin penneation enhancers Azonc.
`oleie acid and deeyhnethyl sulphoxide: Permeation and DSC stud-
`ies] l’harm Pharmacol 38:7!1", l986
`6. Barry “W, Bennett SL: Elliott ofpenetration enhancers on the perme-
`ation of mannirol, hydrocortisonc and progesterone through mural:
`skin.) l’liarm l’harmacol 39:535- 546, I987
`
`

`

`VOL. 90. NO. 6 JUNE 1988
`
`PENETRATION ENHANCE-.115 IN HUMAN AND MOUSE SKINS
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`
`7. Sekura DL, Scala J: The percutaneous absorption of alkyl methyl 5qu
`fmiides. in Advances in Biology of the Skin. vol 12. Edited by W
`Montagna. EJ Van ScottI RB Stoughton. Appleton—Century—
`Crofts, New York, 1972, pp 257—269
`3. Shannon WM. Westbrook L, Higuchi W1. Sugibayashi K, Baker DC.
`KumarSD. FoxJL. Flynn GL. H0 NFH. Vaidyanathan R: Influence
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`valeryl-9-fl-D-arabinofuranosyladenine. J Phann Sci 74:1 15'?—
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`
`9. Akhter SA. Bennett SL, Waller ILI Barry BW: An automated diffusion
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`10.
`
`Iman RL. Conover WJ: A Modern Approach to Statistics.John Wiley,
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`1 1. Wotton PK, Mollgaard B. Hadgraft]. Hoelgaard A: Vehicle effect On
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`12. Lansdown ABG, Grasso P: Physico-ehemical factors influencing epiw
`dermal damage by surface active agents. BrJ Dermarol 86:361 - 373.
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`
`13. Dalvi UG. Zatz JL: Effect of nonionie surfactants on penetration of
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`Chem 32:87 - 94, 1981
`
`14. Cooper ER: EfTeets of decvlrnethylsulfoxide on skin penetration, in
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`vol 12. Edited by KL Mittel. EJ Fendler. Plenum Press. New York.
`1982.13131505-1516
`15. Cooper ER: Increased skin permeability for lipophilie molecules. J
`Pharm Sci 73:1153L1156. 1934
`
`16. Bennett SL. Barry BW: Effectiveness of skin penetration enhancers
`propylene glycol, Azone. decylmethylsulphoxide and oleic acid
`with model polar (mannitol) and nonpolar (hvdrocortisone) pene-
`rrants. J Pliarm Pharmacol 37:84P. 1985
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`hairless mouse skin. J Pharm Pharmacol 37:77P, 1985
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`0004
`
`