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`Advanced
`
`DRUG DELIVERY
`SCIenceDIrect
`Reviews
`
`Advenoed Drug Delivety Rwiews 59 (200?) 1152—1161 —mehefimmmfloesle’sddr
`
`Transdermal skin delivery: Predictions for humans
`from in viva, ex vivo and animal models“5r
`
`Biana God'm, Elka Touitou *
`W 4W, WQI'PRM Faculty .9me The Hebrew Ummy 11me mm 91120, Israel
`
`Received IOWyZW;mepwd201nly2W7
`Availableonline 16 Augmzm‘f
`
`Alum!
`
`Theassewneowfpercumnewspermesfionefmolemflesisoneoffliemainstepsinlheinifialdesignandlaterintlmevslusfionofdemulor
`Whigdefiverysyslems.’1‘heHWMWam,mfimmmmmndekmdmdmmedmgsfinpemufim
`profiles and kinetic parameters, some studies focusing on file correlation of [he data obtained using these models with the dermalfoansdennnl
`absorpfioninhumans. Thispeperreviewsworkfi'ommfimmmmmefimmmmmfingvefimmemmodds
`usedmdmmflfkansdemflmsmhmohidingtheuseofexoisedhmmwsnimalsmcultuedskinequivslentsandsnitnsls.smdiesfomsing
`anti-madam] absorptionofaeefiesofdmgmoleeifles sndvm-iousdeliverysyssemssswellssmflnemnficslmodels forskinabsomfion are
`reviewed.
`0 2007 Published by Elsevier B.V.
`
`Keywords: Trensdamsl absorption; In Woo-ht vino correlation; Animal skin; Studies in htnmns: Skin equivalents; Petroleum permeation
`
`Contents
`
`1.
`
`Intruducfim: ............................................................. 1152
`
`[ssuesrelatedmflnrionsndiumskmpermwfionsmdies .................................... 1153
`2.
`Skin immune: 1111mm Vs. minus} models .............................................. 1153
`3.
`Inw‘tmpermeatimemsshumsnskinvsanhmlnmls ..................................... 1154
`4.
`5. Theuseoffissueculwe-defivedskinequivalemsmflsnsdemalresemch ............................. 1155
`6.
`Invmosldnpermeafionsmfiiesfixusiogondelivmysym ................................... 1155
`7. Animal models for evaluation of skin absorption in hm: molecules .............................. 1156
`8. Animslmodelsforevaluafionofskinsbsorpfioninhlmms: delivery systems ........................... 1157
`9. Msthmticsi models ofskin absorpfion .............................................. 1158
`10.
`Conclusions ............................................................. 1159
`Acknowledgment ............................................................. 1160
`Refmees ................................................................. 1160
`
`
`
`1. Introduction
`
`* ThisreviewispmofmeAa'vumsdDmngayfimflwmeismm
`"11%de and]DMD??? OmbunesUmg!Wham".
`Faulty of Medicine, T'he Hebrew University of Jenisslem, p03 12065:
`Jerusalm 91120, Lsmel.'1‘el.: +972 2 6758660; fax: +972 2 6757611.
`Mam: touitou@oc.huji.sc.fl (E. 'I‘n'nituu)
`
`OJWM-mmmommmndbymsv
`Minion/immuom
`
`The assessment of permhmeous absorption ofmolecules is s
`“WW3“? "1me °fan¥m°rw
`MB ‘1de System Akey Boalmfllfldfifilgnafldopmzfimfio?
`dermal or tmisdmal dosage fmms lies in undetsumding the
`
`Noven Pharmaceuticals, Inc.
`EX2017
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`Mylan Tech., Inc. v. Noven Pharma., Inc.
`|PR2018—01 119
`
`

`

`B. Godin, E. Tactics [Weed Drug Delivery Reviews 59 (2002) 1152—116!
`
`1153
`
`factors that determine a good in viva performance. Certainly, the
`most reliable skin absorption data are collected in human studies;
`however, such studies are generally not feasible during the initial
`development of a novel pharmaceutical dosage form or consid-
`eration ofa new drug candidate. Thus, one ofthe main challenges
`ofbiopharmaceutical research is finding a correlation between ex
`vivo, animal and human studies for prediction of percutaneous
`absorption in humans. It is practically impossible to assess the
`skin pmeability of materials using in vivo experiments alone.
`Consequently, numerous ex vivo and in vitro models are fre-
`quently employed to assess drug skin permeation profiles and
`kinetic parameters. Hence, a method that can consistently cor-
`relatesxvimandinw’vodatatoshorten and economizethe
`
`process of drug development and minimize the number ofhuman
`studies is critically needed.
`This article begins with a short overview of various aspects
`aswellasprosandconsofinvitmandinwvoanimalmodels
`for skin permeation. Further, studies evaluating percutaneous
`absorption ofvarious drugs with or without permeation enhance-
`menttechniquesarecovered. Andfinally,fl1euseofdatatiom
`experiments in skin cultures and maflremaficaUpharmacokinetic
`models for predicting n'ansdermal absorption are critically
`discussed.
`
`2. Issues related to in via-0 and in vivo skin permeation
`studies
`
`Despite ethical concerns, the use of animals or isolated animal
`skin models to assess pucutaneous absorption of molecules is
`frequently reported. These models, guerally more available than
`human skin, are ofprime importance inbasic researchtoimprove
`our understanding of the processes, pathways and driving forces
`ofvarious agents across the skinbarrier. However, due tothe large
`number of animal species described in the literature, it is quite
`difliculttocomparethedatainthe fieldo‘fdermal andtransdermal
`drug delivay. Variations in methodology used with a specific skin
`model, such as type of diflbsion cells, skin ternperamre, receiver
`media, application dose and difl’usion area, can all significantly
`afi'ectdata [1]. Yet, it is importanttoemphssizethatin vine and
`animal models provide important tools for screening a series of
`drug formulations, evaluation of skin permeation enhancing
`properties and mechanism of action of the carrier systems and
`estimation ofrank of skin transport for a series ofdrug molecules.
`
`3. Skin structure: human vs. animal models
`
`Skin is the largest body organ, weighing approximawa 5 kg
`with a surface area of about two square meters in adult humans
`[2—4]. This multilayered organ has an essential filnction of
`protecting the body from the surrounding environment, thus
`being an efl‘icient pa'meation obstacle for exogenous molecules.
`The barrier properties ofthe skin lie mainly within its uppermost
`strata, the stratmn cornetmi (SC). This highly hydrophobic layer
`is composed of differentiated non-nucleated cells, corncocytes,
`which are filled withkemtins and embedded in the lipid domain.
`Since the rate limiting step for skin absorption ofmostmolecules
`is considered to be this non-viable layer, percutaneous per-
`0002
`
`meation of molecules is behaved to be governed by diffusion
`laws [2]. The extent of skin permeation of a compound may
`depend on the route of absorption. There are three pathways
`which can be involved in the transdermal permeation of che-
`micals: (1) through the interoellular lipid domains in SC; (2)
`through the skin appendages; and (3) through the keratin bundles
`in SC [2,5].
`The lack ofcorrelation in transdermal permeation ofmolecules
`across species or from differed application sites in the same
`animal model is due mainly to variations in skin (or SC) thickness,
`inthecompositionofirrtercellularSClipidsandinthenumberof
`skin shafts. thzlafi‘et a]. [6] have shoum that the amount offi-ee
`fatty acids and triglycerides and the density of hair follicles are
`important factors causing differences between the skin barriers
`among species. As the majority ofmolecules applied onto the skin
`permeate along the SC lipid domain, the organization of these
`regions is very important for the barrier function of the skin.
`The SC lipid composition and organization difl’a' finm that of
`other biological membranes, with long chain cermnides, free fatty
`acids, cholesterol and cholesteryl esters being the main lipid
`classes [2—4,7,8].
`To evaluate transdermal absorption of a molecule, the most
`relevant membrane is hmnan skin Skin from various sources,
`including cosmetic smgery and amputations, has been used for
`the in vine assessment of permtanews penetration [9,10].
`However, its availability is limited and animal skin is therefore
`fiequently used. A wide range of animal models has been sug-
`gested as a suitable replacement forhumsn skin and has been used
`to evaluate percutaneous penneation ofmolecules. These include
`primates, porcine, mouse, rat, guinea pig and snake models.
`Since the use of primates in research is highly restricted, the
`most relevant animal model for human skin is the pig. Porcine
`skin is readily obtainable from abattoirs and its histological and
`biochemical properties have berm repeatedly shown to be
`similar to human skin [11—15]. Porcine ear skin is particularly
`well-suited for permeation studies and gives comparable results
`to human skin. Studies examining thickness of various skin
`layers have shown that the SC thickness in pigs is 21—26 urn
`[10,12] which is comparable to human skin [10,16]. The viable
`epidermis in porcine ear skin is 66—72 pm thick [10,12], which
`is very similar to the human epidermal thicloiess of 70 um
`(shoulder) [17]. The follicular structure of pig skin also resem-
`bles that of humans, with hairs and infundihula extending
`deeply into the dermis. An average of 20 hairs are present per
`1 cm2 of porcine ear skin as compared to 14—32 hairs (except
`the forehead area) in humans [12]. Moreover, the vascular
`anatomy and collagen fiber arrangement in the dermis, as well
`as the contents of SC glycosphingolipids and ceramides are
`similar in man and in the domestic pig [18].
`Due to its availability, skin of rodents (mice, rats and guinea
`pigs) is the most commonly used in in vine and in viva pa-
`cutaneous permeation studies. The advantages of these animals
`are their small size, uncomplicated handling and relatively low
`cost. There are a number of hairless species (nude mice, hairless
`rats) in which the absence of hair coat mimics the human skin
`betterthanhairyskin[l9].h1these animalsthere is no needfor
`hair removal (clipping or shaving) prior to the experiment, thus
`
`

`

`1154
`
`B. Godia, E. Ihar'tau I Advanced Drug Delivery Reviews 59 (2007) 1152—1161
`
`avoiding the risk ofinjury to cutaneous tissue. Other models have
`a disadvantage of an extremely high dmsity of hair follicles and
`require hair removal. Since both issues may affect percutaneous
`absorption ofmolecules, hairy rodent skin is usually not used info
`vitra pennestion studies, although in viva studies are still
`performed on these species. Among rodents, rat skin has more
`structural similarities to human tissue (Table 1).
`Except for rat skin, rodent skin generally shows higher
`permeation rates than human skin [20—21]. Regarding the rat
`skin, permeation kinetic parameters are fi'equently comparable
`with human skin.
`
`Snakesldnwasalsoproposedasamembraneinsldnper—
`meation experiments. Difi'erential scanning calorimetry (DSC)
`thermograrns and infra-red (IR) spectra showed that the SC of
`snake, porcine and human skins have some similarities in
`structure and components [22]. The distinguishing feature ofthe
`shed snake membrane is its lack offiollicles.
`
`4. In w'r‘ro permeation across human skin vs. animal models
`
`Various studies have been carried out in an attempt to
`correlate in vim: permeation data in animal and human skin.
`Some of them are reviewed here. Most of reports substantiate
`thevalue ofthepigasananimalmodelformaninskin
`permeation studies. Singh et al. [23] evaluated skin permeability
`coefficients (Kp) and SC reservoir of three hydrocarbons in
`porcine ear compared to human skin. They reported that pig
`skin was slightly more permeable to the Substances with the
`ratios Kpporcine skianphumanskin cf1.71, 1.28 and 1.16 for
`heptane, hexadecane and xylene, respectively. The permeation
`profiles of heptane across human and porcine skin are presented
`in Fig. 1. SC binding of the hydrocarbons to porcine and human
`skins was also comparable. The skin permeability (Kp) of
`nicorandil was investigated by Sato and eta-authors [21] using
`excised skin samples from hairless mouse, hairless rat, guinea-
`pig, dog, pig, and human. Among the tested skins, the Kp values
`ot‘nicorandil in pigs and humans were in good agreement The
`authors also found that comparable porcine and human skin
`permeation could be attributed to similar surface lipids, barrier
`thickness, and morphological aspects of the excised pig skin
`samples and human tissue. In another series of experimta, the
`in vitro permeability of pig ear skin was compared with human
`(abdominal) skin and rat (dorsal) skin using both hydrophilic
`(water, mannitol, paraquat) and lipophilic (aldrin, carbaryl,
`fluazifop-butyl) penetrants [13]. Pig skin was found to have a
`closer permeability character than rat skin to human skin,
`particularly for lipopbilic penetrants. The authors suggested that
`electrical conductivity measurements across pig skin mern-
`branes could be a valuable tool for evaluating the integrity of
`
`Table 1
`'I'hirlrnessofskinstrstsinratmiceaodhmnans[10]
`
`Rat
`Mouse
`Human
`
`SC, um
`18
`9
`1’?
`
`Epidmruia, pm
`32
`29
`47
`
`Whole skin, mm
`2.09
`0.70
`2.97
`
`I200
`
`sEE§
`
`§
`
`
`
`amountofhepaticpenncatcd
`
`{mammal}
`
`0
`
`5
`
`It!
`
`Ii
`
`20
`
`25
`
`Time (Ill
`
`Fig. 1. In who permeation profiles of heptane across human (squares) and
`porcine (mambo) skin (motioned with permission from Ref. [23]).
`
`membranes. Sekkat et at. [24] reported that difl‘erentially tape-
`stripped, porcine skin could serve as an in firm model for the
`evaluation of transdermal drug delivery to pranature neonates.
`In this study the passive permeation of caffeine, phenobarbital,
`and lidocaine and the iontophoretic delivery of lidocaine across
`tape-stripped porcine skin barriers were tested. The barrier
`fimction of the tissue was monitored by measuring the trans-
`epiderrnal water loss (TEWL). For all tested drugs, the per-
`meation behavior correlated well with the skin ban-let filnction
`
`[24]. The results were sustained by a study on diamorphine in
`w’vo absorption in premature neonates [25]. lontophoretic lido-
`caine delivery was precisely controlled,
`independent of the
`barrier capability. Lin et al [22] compared in via-a penetration of
`theophylline, sodium diclofenac and benzoic acid through
`artificial cellulose membrane, animal skin (frog, snake with or
`without scales, nude mice, Sprague—Dawley rat and porcine)
`and human skin. The fastest permeation of substances was
`observedthroughcellulosemembraneandfiogsldnandthe
`slowest through human skin, with bmzoic acid being the fastest
`penchant
`through all skin types.
`In the case of sodium
`diclofenac the transderrnal permeation flux in porcine SC was
`33 timeshigherthaninintactskin,butinsnake andhumanskin,
`the rate through SC was only 2.2 and 1.6 times higher than
`through intact ones.
`Afocusofseveralreportswastocomparetrausdennal
`permeation kinetics between rodent- and human skin. In a study
`by Roy et al. [26] permeability coeflicients ofmorphine, fentauyl,
`and sufentanil across hill-thickness hairless mouse skin were in an
`
`order ofmagni’mde higher than those found for human epidermis.
`There was no correlation between the enhancement in parents-
`neous transport caused by SC removal in hairless mice and human
`epidennis. Another study examined permeation characteristics of
`htnnan skin fiom various sites compared to animal skins, and
`fomrdthatshedsnakeandhaifiessratskinshowedsimflar
`
`permeability to humanbreostand thigh skin, while Wistarrat and
`nude mouse performed similarly to human cheek, neck, and
`inguinal skin [27]. Ravenzwaay et al [28] evaluated transport of
`compounds with various lipophilicities across rat and human
`skinsinvirroandinwvoinratslnallcasestheinwoodennal
`
`penetration throughratskinwasbigherflranin vivoandratskin
`was approximately 11-fold more permeable than human skin.
`These authors suggested the use of the following equation
`0003
`
`

`

`B. Godin, E. Tauitau [Weed Drug Delivery Reviews 59 (2002) 1152—116!
`
`1155
`
`(Eq. (1)) to estimate nansdermal transport through human skin,
`based on thecomhiuedusc ofin viva andin vitro data:
`
`% Percutaneous absorptionm
`= %Percutaneous absorptiomn X (Jim/Jm)
`
`(1)
`
`whereJis the percutaneous penneationflux.
`In a separate study evaluating in vino percutaneous absorption
`offour antihypertensive drugs in mice and human cadaver skin,
`Ghoshetal reportedthatthepermeationmteinmice skin was
`muchhigherthanthatinhuman skin [29]. Van de Sandi etal. [30]
`reported a mold-center skin permeation trial, comparing the in
`vitro absorption of benzoic acid, caffeine, and testosterone com-
`pounds through human skin (nine laboratories) and rat skin (one
`laboratory) in ten European laboratories. All laboratories ranked
`the absorption ofbcnzoic acid through human skin as the highest
`of the three molecules (overall mean flux of 16.54i11.87 pg!
`311-13th while the absorption ofcafi'eine and testostmone through
`human skin was comparable (2.24s: 1.43 and 1.633: 1 .94 ug/
`cm2 X h, respectively). In this study, no difl‘ercnces were observed
`between the mean absorption through human skin and the one rat
`study for benzoic acid and testosterone, however for caffeine, the
`fluxvalue andthetotalquantitypeimeatedacrosstherat skinwere
`higher than the correspondent values in human skin.
`
`5. The use of tissue culture-derived skin equivalents in
`transdermai research
`
`A number of tissue culture derived skin equivalents such as
`living skin equivalent models ('LSEs) and human reconstructed
`epidermis (HIRE) have be- used to measure percutaneous
`absorption. These models generally are comprised of human
`cells grown as tissue culture and matrix equivalents normally
`present in skin, and are utilized as alternatives to animal skins.
`LSEs resemble human skin, having a dermis, epidermis and
`partially-difi'erentiated stratum comeum, but are deficient in skin
`appendages including pilosebaceous units, hair follicles and
`sweat glands [31]. These tissues provide much lower barrier
`properties than the whole skin due to their snuenire and lipid
`composition. For this reason, the kinetic parameters of skin
`permeation obtained when using LSEs usually highly overes-
`timate flux across human skin. For example, in a study by
`Schmook et 31., the permeation characteristics ofhuman, porcine
`andratskinswiththeGrailslcinfi’LiEiEandtheSkinethic®
`
`l-[RE models were compared using four low molecular weight
`dermatological drugs with various hydrophilicities [32]. The
`permeation of more hydrophobic compounds (clotrimazole and
`tcrbinafine) through the skin equivalents resulted in an 800—900
`fold higher flux than through split-thickness human skin. 0n the
`other hand, transdennal flux of a less hydrophobic compound,
`salicylic acid, was in the same order of magnitude as fluxes
`obtained with human skin. In this study porcine skin performed
`as the most appropriate model for human skin and they
`concluded that reconstimted skin models are not suitable for in
`
`vitro penetration studies [32]. A similar conclusion was drawn
`from results of another study in which Roy et a1. [33] evaluated
`the in vino permeabilitics of alkyl p-aminobenzoates through
`0004
`
`LSE and human cadaver skin. In the case ofcadavcr skin, the
`permeability coefficient increased as the carbon chain length
`increased. However, This relationship was not observed in the
`permeability coefficients of these esters across LSE. Moreover,
`LSE showed very low resistance to flux compared to cadaver
`skin as the permeability coefficients of these esters through LSE
`were an order of magnitude highm' than through cadavm skin.
`0n the other hand, numerous reports support the use ofskin
`equivalents for evaluation of skin irritation [31,34]. In a study by
`Monteiro-Riviere and colleagues [35], EpiDerm LSE ‘9 was
`found to be morphologically and biochemically comparable to
`normal human epidermis, providing a model in toxicological and
`skin metabolism studies. Ponec and Kempenaar [36] reportedthat
`architecture, homeostasis and lipid composition of recmstructed
`humanskinmodels (Epmerm®,SkinEtiiic @,Episkin®andRE-
`DED ‘3’) were comparable to native human tissue. It is noteworthy
`that Colipa, the European Trade Assocation for cosmetic and
`toiletry industry, recommends the use of in vim: reconstructed
`skin equivalents as the profound testing model for skin irritation
`studies [34]. However, the overall use of skin cultures is likely
`to be limited due to questionable pm'formance as a. barrier in
`skinpermeation smdies,aswellasduetotheircostanddata
`reproducibility.
`
`6. In vino skin permeation studies focusing on delivery
`systems
`
`Correlation of permeation between animal and human skin
`studies from drug delivery systems and pharmaceutical dosage
`forms has attracted significant attention from the pharmaceu-
`tical industry, academia, and regulatory sectors. Design and
`optimization of carriers for active agents is a time- and resource-
`consuming process that is an integral part of the development of
`any drug delivery system. In vitro tests reflecting bioavailahility
`data are required to prove that a new delivery carrier is bio-
`equivalent with or superior to the standard Mechanistic studies
`with sophisticated caniers are performed in animal and human
`skin to try to predict the futureperfonnnnoe of the drug delivery
`systems in hmnans horn in via-o data.
`Among the drug delivery systems tested were carriers based
`on chemical skin permeation enhancers, specially designed
`vesicles, physical and microinvasive techniques. Touitou et a1.
`[37] tested transport of tetrahydrocannabinol m an enhancing
`carrier containing 10% wlw oleic acidfpropylene glycol!
`polyethylene glycol 4000fethanol mixture In this study drug
`permeation across Sabra-strain rat skin was fetmd to be about
`12.8-fold higher than across human skin. Differing lag times,
`11.5 vs 8.5 h for the rat and human skin, respectively, may point
`toward different diffusion pathways for this drug across the skin
`of these two species. Priborsky and Muhlbachova [3 8] assessed
`the effect of chemical permeation enhancers on the fit-WW
`hansport across human skin as compared to animal models. Rat
`skin was ~3.3—4 times more permeable than human tissue.
`Using rat skin, the least potmt enhancer was dimethylsulph—
`oxide and the maximum permeation enhancement was observed
`with sodium laniylsulphate. In contrast all the tested enhancers
`performed comparably to human skin. In this study, human and
`
`

`

`1156
`
`B. Godin, E. Ibm‘tau .’ Advanced Drug Delivery Reviews 59 (2007) 1152—1161
`
`guinea-pig skins were not significantly difi‘erent in the per-
`meation of N-methyl-Z-pyrrolidone. In another study, transder-
`mal delivery of 6-beta—naltrexol,
`the active metabolite of
`nalh'exone, across human skin and guinea pig skin in vitro
`and in hairless guinea pigs in viva was assessed fi'om a
`propylene glycol} buffer mixture [39]. In w‘rm flux ofnain-exone
`was about 2.3 and 5.6 times higher than 6-beta-naltrexol across
`guinea pig and human skin, respectively, and 6-beta-naltr'exol
`lag times were longer in both skin types (Fig. 2). In vino studies
`in guinea pigs showed that the steady-state plasma level of
`nalh'exone was twofold greater than 6-beta-nalirexol, which
`correlated well with in vino data in guinea pig skin. Rigg and
`Barry [40] investigated the skin permeability of two species of
`snake (Elaphe obsolete, Hilton melons) compared to is vice
`experimental results for human skin and for hairless mouse.
`The effect of typical enhancers on the permeabilities of the
`membranes to a model penchant 5-fluorouracil (S-FU) was
`evaluated. The studied enhancers were 3% Amne in Tween 20!
`
`Interestingly, the amount oftrmolol transported during iontopho-
`resis (2 h) was significantly difi’ermt among the various skin
`species, but the final quantity of tirnolol crossing the skin during
`24 h (2 h iontophoresis and 22 h post-iontophoretic passive
`diffusion) was comparable in the difi'erent species. According to
`this data, iontophoresis may diminish intaspecies variations in in
`vitro skin pmmeatiorr studies. Microinvasive techniques (micro-
`needles, RF skin ablation, etc.) represent another means of skin
`permeation enhancement. Recently Wang et a].
`[42] imaged
`infirsion of dye molecules, insulin, polymer microparticles, and
`cells into the skin by brightfield and fluorescence microscopy
`following the insertion of hollow glass microneedles into hairless
`ratskint‘afivoandhuman cadaver skin in Woo. Studyingthe
`flow mechanin the authors reported that using both models,
`partial rehaction of the needle by wiflrdrawing 100—300 u or
`vibrating the microneedle array dramatically increased infirsion
`flow rate.
`
`saline, propylene glycol (PG), 2% Azone in PG, and 5% oleic
`acid in PG. The data from snake membranes showed minor
`
`7. Animal models for evaluation of skin absorption in
`humans: molecules
`
`efi‘ects of the enhancers, while for hairless mouse skin, the
`
`enhancer effects were significant. None ofthe membranes was a
`completely reliable model for human percutaneous absorption
`in assessing the effect of skin permeation enhancers. The
`authors concluded that human skin should be used in skin
`
`permeation studies and not hairless mouse or snake skin;
`otherwise, misleading results may be obtained.
`Kanikkannan and colleagues [41] evaluated the effect of
`species variation (rat, rabbit, mouse, guinea pig and human)on
`the transdermal iontophoretic permeation of timolol maleate.
`
`In studies conducted in the 1970s and 1980s, transdermal
`absorption of various radio-labeled molecules in human volun-
`teers and animals was assessed [43—45]. In these studies, the same
`concentration ofsubstance (4 ug/cmz) was applied on the forearm
`of subjects in an attempt to standardize the application conditions,
`and pencutaneous absorption was quantified by following the
`excretion of the tracer for 5 days. Bartok et a]. [45] undertook a
`comparative suldy of percutaneous absorption of haloprogin,
`acetylcystein, cortisone, cafi'eine and testosterone in visa in
`various animal species (rats, rabbits, miniature swine) and
`humans. The highest extent of percutaneous absorption was
`observed with haloprogin, with complete absorption in rats and
`rabbits but not in humans and pigs. In rats and rabbits the absorbed
`fraction of applied dose followed the order: acetylcys-
`tein<cortisone<caifeine=wstostuone<haloprogin In vim data
`from man and pigs indicated that the ordn' ofabsorption was:
`scetylcystein < cortisone <haloprogin <testostemne < cafi'eine.
`The authors concluded thatthe transdermal absorption in rats and
`rabbits was not predictive for human data, while results obtained
`in porcine model and humans were comparable.
`Using the same technique, Wmter and Mafliach [46,47] com-
`paredthepercutaneousabsorptionofvruiousmoleculesbetween
`rhesus monkey and humans. They found that
`the in viva
`percutaneous absorption of hydrocortisone,
`testosterone and
`hurzoic acid was similar forrhesus monkey and man. For example,
`when hydrocortisone, testosterone andbeneoic acid were applied at
`a dose of4 uglcmZ, the absorbed dose was 2.9, 18.4 and 59.2% vs.
`1.9, 13.2 and42.6% inmonkeyvs.humans, respectively. Bronaugh
`and Maibach [48] measured the percutaneous absorption extent of
`five nitroaromatic compormds (p-nitroaniline, 4-amino-2-nitrophe-
`no], 2,4-dinitrochlorobenzene, 2-nilro-p-phmylenediamine, nitro-
`henzene) inhrnnansandmonkeysusingboflir‘n vitm andr'n vim
`techflqumhwasfounddratexceptforflrehighlyvolatile
`nitrohenzene, no significant differaroes were observed in the four
`grmmsofdaflAndersenetafqusedthesamemethodology
`to evaluate percutaneous absorption of 14C ring-labelled
`0005
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`Fig. 2. Cumulative amount of nalu'exone (squares, n-7) and 6-beta-ns1trexol
`(thumbs, n=8) pcrmeatedtbroughthe human skin (A) and guincepigskinm)
`(reproduced with permission iron: Ref. [39]).
`
`

`

`.8. Mil, E. Tauitou [Advanced Drug Delivery Reviews 59 (2032) 1152—116!
`
`1157
`
`
`
`hydrocortisone,mstosteroneandbenzoicacidinviminguineapigs
`andcompaletheobtainedresultstopuevioushumandata[49].lhe
`absomtion ofhydrocortisone and benzoic acid was similar to the
`publishedhmmnabsorpfiondmturttestostaonewasabsorbedto
`agreaterexteutinguineapigsmaninrnan. lutemstinglxinthis
`work a tbioglycollate based depilatory cream significantly
`increased the extmt of transdennal permeation ofWe
`Although the above studies [43—49] used radiolabeled molecules
`(whose weakness is the accurate detection of the original
`compormd), the clear advantage ofthese early works was their
`abilitytocornpmeskinabsorptionofalargeseries ofmoleculcs
`using the same experimental protocol.
`later reports used more advanced analytical methods for
`evaluation and comparison of percutaneous absorption in
`animals and humans. Wester et al. [50] employed inductively
`coupled plasma-mass spectrometry for quantitation of biolog-
`ical samples of boric acid, betas and disodium octaborate
`tetrahydrate after-their applicationonthe skin. Theycompared
`the usefulness of finite and infinite dose permeation method-
`ologies across human skin to absorption data in humans. The
`results firmr the finite dose model were much closer to the in
`
`viva absorption data, while the infinite dose methodology
`differed by 10-fold from the in viva results. Cnubben and
`colleagues [51] measured the percutaneous absorption ofortho-
`phenylphenol, a fimgicide, in rats, humans and a perfused pig
`ear model. The drug was applied in a hydroethanolic vehicle
`and samples from in viva studies were evaluated using capillary
`gas chromatography with MS detector. In viva results indicated
`that in human volunteers, approximately 27% of the applied
`dose was excreted with urine within 48 h versus 40% excreted
`
`in rats. Among the in vitra parameters tested, the daemon of
`applied dose most accurately predicted human in viva
`percutaneous absorption of the drug (Fig. 3). With respect to
`the other parameters studied, considerable differences were
`observed between the various in vitra models.
`
`Skin permeation studies using inadequate protocols will
`generate inaccurate data. Currently used sunfilters are lipophilic
`substances with relatively low molecular weight, thus posses-
`sing a good potential to be systemically absorbed across the
`skin. In fact, for a long period of time scientists have been aware
`of the issues of potential toxicity caused by the percutaneous
`absorption of chemical sunscreens. Recently these concerns
`have been confirmed in numerous reports [52—54]. However,
`the experimental conditions, such as a hydrophilic receiver fluid
`that is used in many in via-a skin permeation experiments with
`sunscreens, generally do not permit a good clearance of these
`molecules hen: the skin. For example, one study compared the
`skin penetration of benzophenone-3 (BPH), ethylhexyl meth-
`cxycinnamate, butyl methoxydiberrzoyl methane, ethylhexyl
`salicylate and homosalate, from two vehicles, an oil-in-water
`(01W) emulsion gel and petrolaturn jelly, both in vine and in
`viva. The receptor fluid used in in vitro experiments was saline
`containing 1.5% BSA and at these conditions none oftbe filter
`agents permeated through the skin and negligible amounts were
`detectedin various skin layers after 6 h of product application.
`Also, the effect of the vehicle was minimal in the in vim)
`permeation experimental setup. On the other hand,‘in humans
`0006
`
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`
`—9— Human epidermis
`
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`Human fol skin
`
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`
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`Flat splcannls
`
`0 Human lull skln
`
`n Human sober-mks
`
`0 masochistic-r
`
`Vfiel
`
`Fig. 3. Teamdennal absorption or ["Clorthc-phenylphenol, in vino-in vivo
`correlation:
`(A) Cumulative amount of [“C]ortho-phenylphenol (n=6)
`permeated in vice through human viable sldn, rat viable skin. human epidermal
`mernbranes.ratepidermslmembranesandperfilsedpigears;(]3) Factor-of
`difiersnco (FOB) between in vitro and in viva shin absorption of ["Clortiro-
`phenylphenol based on the systemically available (SA) amour.“ at4, 3, 24, and
`48 h after a 4-h ertpostu'e period of 120 ugl'cmz, the permeability coeficient
`(Kp),andthepmtially absorbeddoeefPAJinhumanaandratafieproduced
`with pamissiou fi'omRef. [51]).
`
`the amount of sun filtering agents accumulated in the SC was
`significantly higher (around 3 times) with the OIW emulsion gel
`than with the petroleum jelly, which was reflected also in SPF
`measured in vivo 30 min after application of the products [55].
`Yet, when an appropriate receiver fluid was used, a large
`amount [9% from the applied dose] of octylmethoxycinnamate
`(OMC) permeated the skin [56]. In this study, the OMC skin
`permeation flux was 2'? uglemzh. [tis importantto keep inmind
`that sunscreen formulations are applied to a large skin area
`(> 1.5 m2) and for a long period, producing a constant and high
`inputofthechemicalintotheviablesldnstrataandtothe
`systemic circulation. These in vino results are supported by data
`from a number of human and animal in viva studies. Hayden
`et a]. [57] reported that BPH has been detected in human urine