Review Article Onychomycosis: Potential of Nail Lacquers in Transungual Delivery of Antifungals

13 Pages·2016·1.26 MB·English

Hindawi Publishing Corporation
Scientifica
Volume 2016, Article ID 1387936, 12 pages
http://dx.doi.org/10.1155/2016/1387936
Review Article
Onychomycosis: Potential of Nail Lacquers in
Transungual Delivery of Antifungals
NidaAkhtar,1HemlataSharma,1andKamlaPathak2
1DepartmentofPharmaceutics,RajivAcademyforPharmacy,P.O.Chhatikara,Mathura,
UttarPradesh281001,India
2DepartmentofPharmaceutics,PharmacyCollegeSaifai,Saifai,Etawah,UttarPradesh206130,India
CorrespondenceshouldbeaddressedtoKamlaPathak;[email protected]
Received30December2015;Revised1March2016;Accepted6March2016
AcademicEditor:PaulO.Gubbins
Copyright©2016NidaAkhtaretal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Onychomycosisconstitutesthemostcommonfungalinfectionofthenail(skinbeneaththenailbed)thataffectsthefingeras
wellastoenails.Itisaninfectionthatisinitiatedbyyeasts,dermatophytes,andnondermatophytemolds.Naillacquersaretopical
solutionsintendedonlyforuseonfingernailsaswellastoenailsandhavebeenfoundtobeusefulinthetreatmentofonychomycosis.
Thus,inthepresentreviewanattempthasbeenmadetofocusonthetreatmentaspectsofonychomycosisandtheungualdelivery
ofantifungalsvianaillacquer.Severalpatentsissuedonnaillacquertilldatehavealsobeendiscussed.Penetrationefficiencywas
assessedbyseveralresearchersacrossthehumannailplatetoinvestigatethepotentialityofnaillacquerbasedformulations.Various
clinicaltrialshavealsobeenconductedinordertoevaluatethesafetyandefficacyofnaillacquersindeliveringantifungalagents.
Thus,itcanbeconcludedthatnaillacquerbasedpreparationsareefficaciousandstableformulations.Thesepossesstremendous
potentialforclinicaltopicalapplicationtothenailbedinthetreatmentofonychomycosis.
1.Introduction newer antifungal agents resulted in higher concern among
thepatientstogetcureofthediseaseandalsoamongmedical
Onychomycosis is described as a fungal infection affecting practitioners to constitute an effective therapy. However,
primarilyfingerandtoenails.Itisreportedtoinfluenceabout there has been an issue regarding the treatment which
19%ofthepopulationacrosstheworldandconstituteabout may be prescribed without prior knowledge of infection.
50% of all the nail diseases [1]. It is sometimes thought to The effectiveness of several antifungals against the fungi
be a trivial disorder acquiring only cosmetic implications is not well understood and drugs are often recommended
and might lead to embarrassment underlining self-esteem. forinappropriatetimeperiods[3].Therefore,thetreatment
If it is not treated well it can lead to discomfort and may of onychomycosis is a challenging task as the infection is
spread to the nearby tissues. It is a frequently occurring embedded within the nail. It may take a year or more to
diseaseofnailswhichisinitiatedbynondermatophytemolds getcureasnewnailgrowthmustcompletelyreplacetheold
anddermatophytes.Nondermatophytefilamentousfungiare and infected one [4]. Imidazole derivatives among several
considered as a causing agent of onychomycosis. The pre- antifungals are reported to be effective in the management
valence of this disease may vary across the globe because of onychomycosis. These are broad spectrum antifungal
of several cultural and socioeconomic factors. Prevalence agents and are active against mold, yeast, dermatophytes,
of the infection also depends upon the geographic area, and some bacteria of the actinomycetes and highly active
population,andmycologicalfeaturesordiagnosticmethods against some Gram-positive cocci and bacilli. Antifungals
employed [2]. Onychomycosis is considered as one of the havebeenavailableinmarketintheformofcream,ointment,
common dermatology based conditions. Enhanced survey lotion, powder, and solutions. These formulations require
on the prevalence of the disease as well as inventions of high concentration of active agents to be incorporated for
2 Scientifica
effective therapy because of their low efficacy. To overcome 3.TransungualDrugDelivery
thelimitationsofconventionalformulationsthereisaneed
of an effective system that can deliver the antifungals deep Transungual drug delivery is defined as system which is
intothenailbed.However,thedevelopmentofaformulation relatedtodrugtransportacrossthenailtogaintargeteddrug
for safe and effective topical delivery in the treatment of delivery in order to treat nail diseases. In the term tran-
onychomycosisisstillunderinfancy. sungual, “Trans” signifies “through” and “unguis” signifies
Topical therapy would be an attractive alternative “nails” [10]. Transungual drug transport system is thought
approachinthetreatmentofonychomycosisasitisfoundto to be quite effective to manage nail disorders because of its
becapableofovercomingmostofthelimitationsofsystemic betteradherence,localizedaction,whichprovidesminimum
administration and targeting the drug at its site of action, systemic side effects [11]. Ungual therapy provides several
with minimum interactions and adverse effects. Limited benefits over oral/systemic drug delivery such that prepa-
permeability of the drug through nail plate and blood ration is easy compared to oral dosage forms like tablets,
supply in the affected area might lead to subtherapeutic andsoforth.Druginteractionsandsystemicadverseeffects
concentration and can be overcome via topical application are absent. Less common local rash related side effects like
[5]. Further, most of the conventionally used formulations periungual erythema of the proximal nail fold gradually
arenotspecificallyadaptedtothenailsincetheyarereadily disappearafterafewminutesandusuallygetlessovertimeas
removed by rubbing or washing [6]. To overcome these thebodyadjuststothenewmedication.Systemicabsorption
barriers,ungualdeliverycanbeoptedforthedeliveryofdrug islessanditisbasedonapplicationoftopicalformulationthat
across the nail plate. Medicated nail lacquer formulations canbeeasilyremovedwhenneeded.Ungualtherapyprovides
have been developed for effective ungual drug delivery of improvedadherenceandissuitableforthosewhoareunable
antifungals to improvise the treatment of onychomycosis. totakesystemicmedication.Itispreferableincaseofelder
In one of our in-house research reports, potential of nail patientsandpatientsreceivingmultiplemedicationssoasto
lacquerhasbeeninvestigatedforthetreatmentofpsoriasis. avoid drug-drug interactions. In spite of possessing several
Asafe,stable,andefficaciousnaillacquerofisotretinoinwas benefits, it also poses various problems like composition of
formulated for effective topical clinical application. Drug the nail plate, limits penetration of drugs, and allows only
targeting has been done into the nail bed in order to treat afractionoftopicaldrugtopenetrateacrossandhencethe
nailpsoriasis[7].Sothedevelopmentandevaluationofnail desiredtherapeuticconcentrationisnotachieved[11].
lacquer for its therapeutic efficacy are a well-established
concept. Thus, in the present review an attempt has been 4.ApproachesforTransungualDrugDelivery
madetofocusonthetreatmentaspectsofonychomycosisand
thedeliveryofantifungalsvianaillacquerforungualdelivery. Nailpenetrationcanbeachievedbyvariousmethodssuchas
Severalclinicalandpatentreportshavealsobeenreviewed. mechanical,chemical(permeationenhancers),andphysical
methods(likeiontophoresis,microneedles,etc.)asdiscussed
intheprecedingtextandhighlightedinFigure1.
2.OnychomycosisandItsEtiology
Onychomycosisisdefinedasafungalinfectionaffectingnails 4.1. Mechanical Methods. Mechanical methods such as nail
of finger or toes resulting in thickening, discoloration, and abrasion and avulsion have been investigated by several
separation from the nail bed. Onychomycosis affects about researchers. Nail abrasion is methodology which causes
10% of the common population but is more prevalent in thinningofthenailplate,reducingthefungaldebrisincase
adults. The risk factor of onychomycosis is 1.9-fold to 2.8- ofonychomycosis.Nailabrasiondescribessandingofthenail
foldmoreinpersonssufferingfromdiabetescomparedtothe plate in order to minimize its thickness or damage it in a
common population. However, the prevalence varies from completeform.Sandpaper(#150or180)maybeusedonthe
15 to 40% in case of patients infected with human immun- basisofintensity.Sandingshouldbecarriedoutontheedges
odeficiency virus [4]. Onychomycosis is classified into two ofnails.Ahigh-speedsandinghandpiececanbeutilizedfor
types.Thefirsttypeisdistalsubungualonychomycosisaffect- thispurpose.Further,dentist’sdrillshavealsobeenusedto
ing nail plate, nail bed, and hyponychium (infection more create small size holes in the nail plate, thus improving the
prominentlyspreadsfromtineapedisfromthesurrounding penetration[42].
skin). The diseased nails become dystrophic and thick. The On the other hand, nail avulsion either completely or
color of nails modifies from yellowish-white to brown. The partially is the removal of whole nail plate surgically or
secondtypeisproximalsubungualonychomycosisinwhich removalofinfectednailplatecarriedoutundertheactionof
the fungus usually spreads through the proximal nail folds. localanesthesia.Keratolyticsubstanceslikeureaorsalicylic
Clinically,thenailhasawhitishappearance.Aclinicaltype, acid may cause softening of the nail plate for avulsion
endonyxformofonychomycosis,affectingthenailplatewith- [12].
outonycholysisorhyperkeratosisisalsoknown.Superficial
whitetypeofonychomycosis(nailbecomeswhitewithchalky 4.2. Chemical Methods. A combination of 2-mercaptoeth-
texture)isobservedtobelessfrequent.Thefunguscausing anol and N-acetyl-l-cysteine is depicted to improvise the
thisincludesT.mentagrophytes,butnondermatophytefungi deliveryoftolnaftate,intothenails.Thepenetrationofoxi-
(Aspergillus, Fusarium, and others) can also produce these conazole via N-acetyl-l-cysteine has also been documented
changes[8,9]. [43]. Keratolytic agents like papain, urea, and salicylic acid
Scientifica 3
Approaches investigated to enhance transungual delivery
Mechanical methods Physical methods
(i) Nail abrasion Chemical methods like iontophoresis,
(ii) Nail avulsion etching, laser,
microneedles
Mercaptans/thiols Keratolytic agents Organic solvents
(i) Thioglycolic acid (i) Urea (i) Ethanol
(ii) 2-Mercaptoethanol (ii) Salicylic acid (ii) Isopropanol
Figure1:Variousapproachesforeffectivetransungualdrugdelivery.
wereusedinenhancingthepenetrationoffewantifungalslike Anemergingapproachisbasedonhydrationandocclu-
ketoconazole, miconazole, and itraconazole [44]. Further- sion.Hydrationenhancestheporesizeofnailmatrix,influ-
more, organic solvents like ethanol, isopropanol, propylene encingtheungualpenetration.Hydratednailsarefoundtobe
glycol,andpolyethyleneglycolcanbeemployedinenhancing morepermeableandelastic.Studiesbasedoniontophoresis
the drug penetration across the nail. Direct interaction of use this characteristic in order to further improvise the
organicsolventspresentinformulationwiththenailplatein penetration.ThepHofthesolutionandionicstrengthhave
caseoftransungualdrugtransportmightresultinenhanced revealed no remarkable influence on nail hydration. Water
barrierresistivityofthenail.Secondly,useoforganicsolvent anddrugsdiffusivityisenhancedwhenhumanskinbecomes
maymanipulatethehydrationstateofnail[45]. morehydrated[50].Electroporationofanadvancedphysical
technique is carried out with the use of an electric pulse of
4.3. Physical Methods. Microneedles based drug delivery approximately100–1000V/cm.Thiscausesdisturbanceinthe
systemsincludetheuseofarraysofmicroscopicsizeneedles. lipidbilayersandformationoftransientaqueouspores,thus
These open the pores present in stratum corneum. Further, allowingthesolutestopassthroughnail[46].
these also possess the benefit of being so short that they
do not stimulate the pain fibers [46]. Another approach is 5.NailLacquersfor Transungual
theuseofetchingtechnique.Etchingoccursviaexposureof DrugDelivery
surface-modifyingchemical(e.g.,phosphoricacid).Itcauses
the formation of profuse microsporocytes, can enhance the Naillacquershavebeenemployedasacosmeticinorderto
wettability along with surface area, and can decrease the provideprotectiontothenailsandtodecoratethenails.Med-
contactangle.Anoptimumsurfaceisprovidedforbonding icated nail lacquers constitute an innovative type of formu-
material.Additionallypresenceofmicroporositiesimproves lations that have been used for transungual drug delivery.
interpenetrationandbondingofapolymericdeliverysystem Table1 reviews several market formulations based on nail
andfacilitationofinterdiffusionofatherapeuticagent[47]. lacquers. These preparations consist of a solution of a film-
Iontophoresisinvolvestheapplicationofelectricfieldfor formingpolymeranddrug[12].
thedeliveryofacompoundacrossamembrane.Drugdiffu- Upon application to the nail plate, solvent evaporation
sionthroughthehydratedkeratinofanailmaybeenhanced takes place. The film left behind after solvent evaporation
byiontophoresis.Iontophoresissignificantlyenhanceddrug workslikeadrugdepot.Fromthisdrugstore,drugundergoes
penetration through the nail. The delivery of griseofulvin releaseandpenetrationacrossthenailforanoptimumtime
wasenhancedby8-foldwiththehelpofiontophoresis[48]. period.Highdiffusiongradientisgeneratedfordrugperme-
Another significant technique uses carbon dioxide laser. ationintothenailplate[49].Filmformationonnailplatealso
CO2 laser methodology might have produced effective but causesreductioninwaterlossfromthesurfaceofnailsurface
uncertain outcomes. Two techniques have been reported intotheatmosphere.Hyperhydrationoftheuppernailplate
up till now. One is based on the avulsion of the nail part layers takes place, further assisting in drug diffusion. The
2
accompaniedbytreatmentwithlaserat5000W/cm .Thus, active agent penetration can further be improvised via use
via this method deep tissue is directly exposed to laser of penetration enhancers like thiol compounds, hydrating
treatment.Secondmethodologyincludespenetratingacross agents, and keratolytic agents [51]. Figure2 highlights the
thenailplatebylaserbeamofCO2[49]. mechanismofdrugvianaillacquer.
4 Scientifica
Table1:Acompilationofcommerciallyavailablenaillacquers.
Therapeuticagent Brandname Companyname
Ciclopiroxamine8% Onlyac(cid:2) Dermic,Canada
Ciclopiroxamine8% Penlac(cid:2) Rochelab,Australia
Ciclopiroxamine8% Nailon(cid:2) ProtechBiosystem,India
Amorolfine5% Loceryl(cid:2) ProtechBiosystem,India
Econazole5% Econail(cid:2) MacrochemCorporation,Lexington[12]
Table2:Antifungalagentsutilizedinthetreatmentofonychomycosis.
Therapeutic
Examplesofantifungals Organisms Reference
category
T.rubrum,T.mentagrophytes,T.
Allylaminesand Terbinafine-HCl, tonsurans,E.floccosum(fungicidaleffect)
benzylamines naftifine,butenafine Candidaspp.,Scopulariopsisspp.,
Aspergillusspp.(fungi-staticactivity)
[13]
Itraconazole,fluconazole,posaconazole,voriconazole,
C.tropicalis,C.glabrata,C.krusei,
Azoles ravuconazole,isavuconazole,pramiconazole,
Zygomycetes,Cryptococcusneoformans
albaconazole,efinaconazole
6.DeliveryofAntifungalDrugs: and45%oftheapplieddrugafter24h,resp.).Itwasobserved
TreatmentofOnychomycosis that,after3h,colloidalcarrierscouldtransportthedrugto
ahigherextent.Realtimepenetrationinvestigationwasalso
Antifungaldrugshavebeenwidelyusedtotreattopicalinfec- conductedbyTeflononlinediffusioncellincombinationwith
tionsofnails.Table2listsvariousantifungalsthathavebeen FTIR-ATR spectroscopy. It was depicted that nail lacquer
usedinthetreatmentofonychomycosis.Astheoccurrence acquiredadrugcontentofabout12%becauseofthedrying
levelsofonychomycosiskeeponincreasingnowadays,need thatoccurredindonorregionoftheonlinediffusioncellafter
of topical therapy that can circumvent the threats posed by ethanol(58.6%)underwentevaporationpartially.Thoughthe
systemicdeliveryisgainingprominentdeliberations. solutionshowedmaximumpermeabilitycoefficientinitially,
Various researchers across the globe have worked in itcouldexhibithigherdrugconcentrationofonly35%inthe
the development of effective carriers for antifungal agents. acceptorcellafter24h.Ontheotherhandhydrogeldepicted
Table3highlightsthedeliveryantifungalsviaothercarriers minimumpenetrationrateandthemostmarkedlagtimeof
totreatonychomycosis. about3h.Theexperimentalresultswerefoundtobeentirely
In the preceding text various preclinical/clinical reports different in case of bovine hoof membranes as compared
arecitedthatdepictthesafetyandefficacyofnaillacquersin to the equine hoof membranes. EV-086K concentrations
deliveringantifungaldrugs forthetreatmentofonychomy- in contrast to the applied dose demonstrated conflicting
cosis. outcomes in contrast to that obtained with using equine
Kushwaha et al. had investigated the potential of AR-12 hooves. Solution was found to conquer the barrier up to a
(antifungal drug) to penetrate into and across the human greaterextent(65%in24h).Incaseofcolloidalcarrierand
nailplate.Invitropermeationstudieswereperformedacross naillacquer,35–40%oftheapplieddrugconcentrationwas
the human cadaver nail plates using Franz diffusion cells. determined in the acceptor cell. With application of hydro-
The cumulative amount of drug permeated across the nail gel, less drug concentration was detected in the acceptor
plateforoneweekwasfoundtobe0.82±0.11ng/cm2.The compartment.Colloidalcarriersandsolutionrevealedhigher
amount of drug retained in active diffusion area of the nail permeability coefficients than hydrogel and nail lacquer in
platewasobservedtobe∼0.42±0.02𝜇g/mg.TranScreen-N linearrange.Further,colloidalcarriersshowedsmalllagtime
clearlyshowedthatPEG400couldbeapotentialenhancerof ascomparedtotheotherformulationswhichwerefoundto
transungual delivery of AR-12. Further, in vitro permeation be beneficial for ungual delivery. Combining the results of
studies conducted along with 10% PEG 400 showed 6-fold the studies faster drug penetration rate was concluded via
enhancementintransungualdrugdeliveryincomparisonto colloidalcarriersthannaillacquer[53].
the control solution. The drug loaded in the nail plate was Inadditiontonovelantifungals,theestablishedantifun-
depictedtobe2-foldhigherinpresenceof10%PEG400[52]. galshavealsobeenexploredfortransungualdelivery.Vipinet
In a report by Naumann et al. four systems, namely, al.developedmedicatednaillacquerloadedwithmiconazole
naillacquers,colloidalcarriers,solution,andhydrogelofan withtheaimtoprovidesustaineddrugdelivery.Naillacquer
antifungaldrug(EV-086K),wereformulatedandcompared basedformulationswerepreparedbysimplemixing.Among
for their efficacy. Ex vivo penetration studies using human alltheformulations,naillacquercontainingdrug(2%),nitro-
nails and bovine/equine hoof membranes reported best cellulose (3%), ethyl cellulose (0.5%), salicylic acid (20%),
diffusionratebythenaillacquerandcolloidalcarriers(55% propyleneglycol(5%),andurea(5%)inhydrogenperoxide
Scientifica 5
Nail lacquer
Break
disulphide
bond
Solvent evaporation
New pores
formed
Film formation
Nail plate Better drug
penetration
Nail bed
Figure2:Mechanismofdrugpenetrationvianaillacquers.
Table3:Topicaldrugdeliveryviaseveralothercarriersforthetreatmentofonychomycosis.
Drug System Concludingremarks Reference
Ciclopirox Gel Enhancedpermeationintothenail [13]
Terbinafine Transfersomes Significantantifungalactivity [14]
Microemulsionbasedgelexhibitedbetter
Itraconazole Microemulsiongel retention,penetration,andantifungal [15]
activity
Betterantifungalactivityagainst
Fluconazole Microemulsiongel Aspergillusnigerascomparedtothe [16]
commercialgel
Betterretentionabilitywasobservedfor
Ciclopirox Nanoemulsiongel [17]
nanoemulsionbasedgel
Effectiveformulationsforungual
delivery,suggestingthatliposomebased
Terbinafine-HCl Liposomes/ethosomes [18]
poloxamergelformulationcouldbea
potentialcarrierforungualdrugdelivery
Formulationbasedonlipid Effectiveinthetopicaltreatmentof
Ciclopirox [19]
diffusionenhancers onychomycosis
Effectiveantifungalactivityagainst
Clotrimazole Nanoemulsiongel [20]
Candidaalbicans
Poloxamer PermeationcoefficientfromP407-based
Ciclopirox 407-based formulationswashigherincomparison [21]
formulations tothenaillacquer
Effectivetransungualdeliveryof
Efinaconazole Solution [22]
efinaconazole
Liposomalfilmbasedformulation
Terbinafine-HCl Liposomalfilm showedbetterantifungalactivityon [23]
fungalinfectednails
1.4-foldhigherhydrationanddrug
Nailpenetrationenhancer uptakeenhancementintonailclippings
Sertaconazole containingnanovesicles showedsignificantlyhigherzoneof [24]
(nPEVs) inhibitionforTrichophytonrubrumthan
themarketedcream
showedbettergloss,nonvolatilecontent,smoothnesstoflow, and the findings depicted extended release of 94.25% up
drugcontent,drugrelease,andantifungalactivity.Diffusion to 36h. Further, sustained drug release was obtained by
studies conducted across artificial membranes using Franz increasing the concentration of ethyl cellulose up to 0.5%,
diffusion cell showed drug release of about 96.19% in 20h. whichcansustainthedrugreleaseupto48h.Acumulative
The permeation characteristics and hydration by propylene drug release of about 96.03% (48h) was depicted. Further,
glycol improvised the drug permeation. To sustain drug in vitro ungual permeation studies were carried out using
release ethyl cellulose was taken at concentration of 0.25% bovine hoof membrane. Optimized formulation exhibited
6 Scientifica
optimum rate of diffusion across the membrane and hence and miconazole, respectively. Differences were observed
wasselectedforinvitroungualpermeationstudies.Thefind- becauseoftheirdifferencesinmolecularweight.Thus,itwas
ings demonstrated superimposable diffusion profiles. This concludedbytheauthorsthataninverserelationshipexisted
confirmed that artificial cellophane membrane can imper- between logarithm of permeability and molecular weight
sonate the features of ex vivo bovine hoof membrane. The [56].
authors concluded that miconazole nail lacquer possessed The use of hydroxypropyl-𝛽-cyclodextrin (HP-𝛽-CD)
effectiveness in inhibiting the growth of the nail fungi, wasstudiedasaneffectivetransungualpermeationenhancer
Candidaalbicans.Therefore,naillacquerbasedformulation forterbinafinehydrochloridedeliverybyChouhanandSaini.
loaded with miconazole can be employed for transungual Anaillacquerbasedformulationwasdevelopedwithanaim
drugdeliveryformanagementofonychomycosis[54]. to transport the drug across the nail. In vitro transungual
OnsimilarlinesDessaietal.formulatednaillacquerfor permeability studies were carried out using nail clippings
preungual delivery of fluconazole for the management of placed in Franz diffusion cell. From the findings, it was
onychomycosis.Diffusionstudiesacrossartificialmembrane observed that the formulations prepared with HP-𝛽-CD
showedthattheformulationcontainingminimumpolymeric exhibited greater flux than the control formulation. The
concentration and maximum concentration of penetration formulationhavingHP-𝛽-CD(10%w/v)depictedmaximum
enhancers showed highest cumulative percent drug release flux of 4.586 ± 0.08𝜇g/mL/cm in comparison to the con-
(95.55%). Conversely, the formulation prepared with maxi- trol formulation having flux 0.868 ± 0.06𝜇g/mL/cm. This
mumpolymericconcentrationandminimumconcentration investigation suggested the use of HP-𝛽-CD as an effective
ofpenetrationenhancersexhibitedthemostsustaineddrug permeationenhancerfortransungualdeliveryofterbinafine
release (85.90%, 10h). In vitro transungual permeability hydrochloride[57].
investigations using cattle hooves obtained depicted drug Hafeez et al. formulated ketoconazole loaded nail lac-
releaseofabout99.52%(in22h).Frombothinvitrodiffusion quer for transungual delivery. In this investigation, the
and in vitro permeation investigations, it was depicted that efficacyofnaillacquerinimprovisingpenetrationof[14C]-
thioglycolic acid could work as an effective penetration ketoconazole was evaluated by comparing nail distribution,
enhancer in contrast to dimethyl sulfoxide. This enhancing absorption, and penetration. Penetration efficacy was then
actionofthioglycolicacidwasattributedtoitssmallmolec- comparedwithketoconazolecream.Invitroinvestigationwas
ular weight and its damaging effect on keratin structure. carriedoutbyselectingfinitedosemodel.Tothehumannail
Reducedlipidcontentpresentinthenaillayer(dorsal)caused plates, all formulations were applied up to seven days. The
by thioglycolic acid loosened the nail structure, thus facil- absorptionofdrugwasdeterminedbyobservingappearance
itating fluconazole to undergo penetration easily. Further, rate in each nail layer and in the supporting bed. After
antifungalactivitywasevaluatedbycupplatemethodagainst completion of the study, cumulative amount of drug in the
Candida albicans. Inhibition zone in case of pure drug was deeper portion of nail layer and nail bed was found to be
observed to be 26mm and for optimized formulation it notably higher than marketed form of ketoconazole (𝑝 <
was 25.4mm. Thus, it was concluded by the authors that 0.05). These findings suggested that the delivery of keto-
optimizedformulationwasasefficientasthepuredrug[55]. conazolevianaillacquerpossessedtremendouspotentialto
Theeffectofpermeationenhancerandmolecularweight treatonychomycosis[58].
on the permeability of several antifungal drugs was also AnotherreportonketoconazolebyShireeshetal.claims
establishedbyMironetal.Formulationscontaininggeraniol, ungual delivery of ketoconazole via nail lacquer for treat-
penetration enhancer, antioxidant, and ethanol were pre- ment of onychomycosis. In vitro permeability studies were
pared. Lipo-lacquer was then prepared by taking lacquer conducted across the human nail plates employing Franz
base, geraniol, and ethanol. Permeation studies were con- diffusion cells which reported cumulative drug permeation
ducted across bovine hoof membranes for all the formula- of 92.10 ± 0.08 and 84 ± 0.04 in case of thioglycolic acid
tions. Further, formulations containing geraniol, nerol, flu- andureahydrogenperoxide,respectively,attheendof24h.
conazole,miconazole,fluconazolebutenafinehydrochloride, Significantly greater permeation was observed in case of
and terbinafine hydrochloride were prepared and subjected thioglycolicacidincomparisontotheureaperoxide.These
to permeation studies. From the results, it was observed findings claim thioglycolic acid to be a better permeation
that ascorbic acid (used as an antioxidant) can significantly enhancer than urea hydrogen peroxide in improving the
improve the geraniol permeability across the membrane permeability.Thus,itwasconcludedbytheauthorsthatnail
in comparison to the control formulation. Ascorbic acid lacquerpreparedbytakingthioglycolicacidcanprovetobe
acts by modifying the structure of keratin, thus improving remarkably better in treating onychomycosis by delivering
the permeability. Further, presence of acetylcysteine in the ketoconazoledeepintothenailbed[59].
formulations improvised the geraniol permeability. Rela- Traynoretal.demonstratedtheinfluenceofpermeation
tion between permeability and molecular weight of ionized enhancers on two marketed nail lacquers and terbinafine
drugs (miconazole, fluconazole, terbinafine, and butenafine transportacrossthehumannailsamplesinvitrousingmod-
hydrochloride) and unionized drugs (geraniol, nerol) was ifiedFranzcell.Itwasobservedthatpermeationenhancing
alsoestablished.Theresultsconcludedthatmolecularweight system enhanced the permeation of both drugs present in
ofdrugsignificantlyaffectsdrugpermeabilityascomparedto naillacquersandterbinafinethroughhumannails.Further,
thedegreeofdissociation.Permeabilitycoefficientsofgeran- ATPassayassuredthatthesystemalsoimprovisedtheper-
iol and nerol were 30- and 175-fold higher than terbinafine meation of terbinafine across infected cadaver nail, thus
Scientifica 7
causing decreased levels of ATP equivalent to those of topicalantifungaldrugsinonychomycosishasbeenimpeded
uninfected negative control samples. It was concluded that bylessnailpermeability.Toexerteffectiveactionantifungals
theadditionofpermeationenhancersmodifiesthechemical mustacquiretherapeuticefficacyandcanefficientlyundergo
structure of nail. These enhancers not only improvised the permeation across the nail plate [27]. Various clinical trials
therapeuticefficacyoftheexistingtopicalproductsbutalso havebeenconductedonantifungalsandarestillinongoing
initiatetransungualtransportandefficacyofterbinafine[60]. process to evaluate the potential of antifungals in treating
Gunt and Kasting evaluated the influence of hydration on onychomycosis. Some newer antifungals like efinaconazole
ketoconazole penetration across the excised human nails. and oxaboroles like tavaborole and AN2690 have also been
Nailsprocessedwith[3H]-ketoconazolesolventkeptonthe investigatedfortheireffectivenessintreatingonychomycosis.
∘
dorsalsurface,maintainedat32 Cinincubatorsandexposing Oxaboroles were designed with properties believed to be
themsequentiallytorelativehumidityof15,40,80,and100% required to allow for easier transit through the nail plate.
for a period of about 40 days. The ventral side was kept Hui et al. had reported the nail penetration results of four
in a phosphate buffer, pH 7.4. Descending and ascending oxaborolesthatledtotheselectionofAN2690,theresultsof
humidity regimens were evaluated. Flux was depicted to thenailpenetrationofAN2690 fromfourvehicles, and the
2
be increased from 0.175g/cm /h (15% relative humidity) to nail penetration of AN2690 in its chosen vehicle compared
0.527g/cm2/h(100%relativehumidity).Asuddenincrement to a commercial control, ciclopirox. It was reported by the
influx(twofold)wasobservedinthezoneof80–100%relative authorsthatAN2690hassuperiorpenetrationcomparedto
humidity. This was the humidity zone in which the nail ciclopiroxandachieveslevelswithinandunderthenailplate
water content enhanced most frequently. Increased relative thatsuggestthatithasthepotentialtobeaneffectivetopical
humidityfrom15to100%improvisedthepermeationofradi- treatment for onychomycosis [63]. Table4 lists the clinical
olabelrelatedto[3H]-ketoconazolethreetimes.Therefore,it statusofantifungaldrugsusedinonychomycosis.
wasconcludedthatformulationswhichcauseanincreasein
nailhydrationpossessedtremendouspotentialtoimprovise 8.PatentReportsonNailLacquer
topicaltherapyforthemanagementofonychomycosis[61].
InareportbyHuietal.,penetrationenhancementeffect While a number of research reports can be found in litera-
of2-n-nonyl-1,3-dioxolaneoneconazoledeepintothehuman ture,variouspatentshavealsobeenreportedspecifyingthe
nail was studied. Aliquots (10microL) of Econail lacquer commercial potential of nail lacquers for transungual drug
formulation containing 0.45mg of [(14)C]-econazole with delivery. Table5 highlights several patents on transungual
18% 2-n-nonyl-1,3-dioxolane (test group) or without 2-n- drugdeliveryvianaillacquers.
nonyl-1,3-dioxolane(controlgroup)wereappliedtwicedaily
for14daystohumannailsthathadbeenwashedwithethanol 9.EmergingTopicalTherapiesfor
beforeeachmorning’sapplication.Thehydrationofthenail Onychomycosis: FuturePerspective
samplewaswellcontrolledtosimulatenormalphysiological
conditions.After14daysofdosing,theinnerventralsection Topicaltherapyintreatingonychomycosishasbeenfoundto
ofthenailplatewasassayedforabsorbeddrugcontent,using benotaseffectiveasminimumamountofdrugpenetrating
a micrometer-controlled drilling and nails powder removal intothenailplate.Nailmorphologyspecificallyitsthickness
system.Themassbalancevaluesof[(14)C]-econazoleinthis andcompactconstructionmakesitabarriertotheentryof
study were 90.8 and 96.4% for the test and control groups, topically applied agents. Concentration of the drug across
respectively. The weight-normalized econazole content in the nail falls about 1000 times from outer to the inner
the ventral/intermediate nail plate center in the test group surface.Asanoutcome,thedrugconcentrationpresumably
was 6 times more than the control (𝑝 = 0.008). The total hadnotreachedatherapeuticallyeffectivelevelintheinner
econazole absorbed into the supporting bed cotton ball in ventrallayer [8]. The limited success rate of topical therapy
thetestgroupwasnearly200timeshigherthanthecontrol in nail disorders is mainly because of the low permeability
group (𝑝 = 0.008) over the 14-day period. The surface nail of keratinized nail plates [64]. The existing clinical reports
contained more econazole, that is, unabsorbed drug, where suggestedthatakeytosuccessfultreatmentofonychomycosis
2-n-nonyl-1,3-dioxolanewasnotpartofthedosingsolution. by a topical antifungal product lies in overcoming the nail
However, the concentration in the deep nail layer in the barrier. Recently, available topical treatments via lacquers
test group is 14,000-fold higher than minimum inhibitory havelimitedeffectiveness,possiblybecausetheycannotsuf-
concentration believed necessary to inhibit the growth of ficientlypenetratethenailplatetotransportatherapeutically
infectingfungi.Inasubsequentstudy,[(14)C]-dioxolanedid sufficient quantity of antifungal drug to the target sites and
not penetrate the nail well. Therefore, the mechanism of eradicate the infection. To achieve an effective chemical
penetration enhancement of econazole was found to be at concentrationinto/throughthehumannailplate,penetration
formulation/nailinterface[62]. enhancersarerequiredtosupplementthetherapythattend
topromotediffusionthroughtheskin’shornylayer[8].This
7.ClinicalStatusofAntifungals mightbethereasonbehindthelimitedeffectivenessofnail
lacquers. Preparation of nail lacquers also requires the use
Clinical trials of transungual delivery are initiated with the of penetration enhancers to deliver the drug effectively in
finding that drugs administered topically could enter deep the nail bed. As reported by Joshi et al. [7], permeation
into the nails. Safe and efficacious transungual transport of enhancerthioglycolicacidwasincorporatedtoenhancethe
8 Scientifica
Table4:Clinicalstatusofantifungaldrugsusedinonychomycosis.
Therapeuticagent Clinicalstatus Reference
Amorolfine/RV4104A/Ciclopiroxolamine Unknown [25]
Econail(cid:3)(econazole) PhaseII(completed) [26]
Phytonail/Loceryl Unknown [25]
Terbinafinehydrochloride/amorolfine Completed [25]
Terbinafinehydrochloride Completed [25]
Ciclopirox Completed [25]
Amorolfine/terbinafine Completed [25]
MycoVa(cid:3) PhaseIII(completed) [26]
Er:YAGlaser(device)withamorolfine Unknown [25]
Luliconazole(solution) PhaseIIb/III(ongoing) [25]
Efinaconazole(10%topicalsolution) PhaseIII(completed) [27]
TDT067(liquidspray) PhaseIII(ongoing) [26]
P-3058 PhaseII(ongoing) [26]
Isavuconazole PhaseIII(completed) [28]
Tavaborole PhaseIII(completed) [29]
Albaconazole PhaseII(completed) [30]
Posaconazole PhaseII(completed) [30]
VT-1161 PhaseII(ongoing) [31]
penetration.Further,viscositymodificationisanotherissue twice above the minimum inhibitory concentration. Thus
that has to be looked after. Too low viscosity could pose a iontophoresisimprovedterbinafinedeliveryintoandacross
threat in its handling, that is, why adjustment of viscosity the nail plate [66]. Laser therapy that results in formation
isnecessarysothatitcan remainonthelacquerbrushand of partial microsize holes in the nail plate facilitated better
caneasilyflowtocoverthenail.Furthermore,toimprovise terbinafine penetration via application of lacquer solution.
thepenetration,ahumectantcanalsobedeliveredwhichcan ThistreatmentapproachisbeinginvestigatedinEuropefor
modify the structural integrity of nail barrier and provides its possible commercialization [67]. Laser therapy is con-
better results. Panthenol, the alcohol form of pantothenic sideredasanonpharmacologicalapproachfortreatingony-
acid (vitamin B5), is believed to act as a humectant and chomycosis. Laser systems are endorsed by FDA on the
improvetheflexibilityandstrengthofnailsassuggestedby basis of resemblance to established devices and are recom-
Huietal.Aliquidnailtreatmentformulatedwithpanthenol mendedtotemporarilyfacilitateclearnailinonychomycosis.
(2%) was compared to a solution of panthenol (2%) in Neodymium-yttrium garnet lasers classified as short pulse
water. Fingernail specimens were dosed daily for 7 days andlongpulselasersandq-switchedlasersystemshavebeen
with either the nail treatment (nonlacquer film forming) investigatedtoincreasethemycologicalcurerates[59].
formulation or aqueous solution with sampling performed Mechanicalremovalofinfectednailmightberapidlyand
every 24h. Panthenol concentrations were determined in easilycarriedoutbyanailclipper.Chemicalavulsionofthe
the dorsal surface, interior (by drilling and removal), and nailiscarriedoutemploying40%urea,whichisappliedon
the supporting bed under the human nail. Panthenol levels the infected nail under occlusion (7 to 14 days). After nail
in the dorsal nail, nail interior, and nail supporting bed removal,anantimycoticagentlikebifonazoleisthenapplied
showedasignificantlinearincreasewitheachdayofdosing. topicallyupto4weeks[68].Anewsubstituteintheeffective
Significantlymorepanthenolwasdeliveredintotheinterior management of onychomycosis might be a photodynamic
nailandsupportingbedbyanailtreatmentformulationthan therapy,whichwassuggestedinpatientshavingacontraindi-
fromanaqueoussolution.Thefilmnotonlyactsasareservoir cation for systemic treatment. After the nail removal, 5-
ofpanthenolbutalsoactstoincreasethehydrationofthenail aminolevulinic acid methyl ester (20% solution) was used,
andthethermodynamicactivityofpanthenolaswell,thereby along with treatment by radiation of the nail with excimer
2
enhancingdiffusion[65]. laser630nmat100J/cm (sixtoseventreatments)untilcom-
However,risingtreatmentmodalitiesrelyprominentlyon pletelycurableresultswereobtained[9].Photodynamicther-
the application of chemicals as well as physical approaches apywasalsousedeffectivelyinanotherpatientsufferingwith
that initiate the persistence and penetration of antifungal onychomycosis caused by T. rubrum that was found to be
agentsininfectednail.Physicalenhancingmethodbasedon unresponsivetothepreviouslygiventreatment.Thenailplate
iontophoresiscouldprovetobemorefruitfulthanchemical wasthensoftenedby40%urea(upto7days)underocclusive
methods.Thismethodologyhasbeenemployedinorderto conditions. The keratotic debris was then removed, and 5-
improviseterbinafinedeliveryintothenails.Thecumulative aminolevulinic acid was applied up to 3h under occlusive
amountofreleaseddruginvitrowasfoundtobemorethan conditions. Broadband red light (630nm) was applied at
Scientifica 9
Table5:Patentreportsonnaillacquerfortransungualdrugdelivery.
Patent/applicationnumber
Originalassignee/applicant Comment Reference
(yearofissue/publication)
GyurikRJ,Bentley
Pharmaceuticals,Inc., Depictinganinnovationbasedona
Exeter,NewHampshire, pharmaceuticalcompositioncontaininga
CA2400178A1(2001) USA;Cpex permeationenhancerthatenhancesthe [32]
Pharmaceuticals,Inc., rateofpassageofapharmaceutical
Wilmington,Delaware, compoundacrossthenailormembrane
USA
E.I.DuPontDeNemours Inventingacrylicbasednailpolishandits
US6254878B1(2001) &Company,Wilmington, compositioncontainingbindersand [33]
Delaware,USA dispersantsorboth
Disclosingatherapyanditsapplicationin
thetreatmentofnailfungalinfection
Astan,Inc.,Birmingham,
US6391879B1(2001) (onychomycosis)composedofan [34]
Alabama,USA
antifungaldrugwithDMSOin
polyglycolicsolution
Describingnaillacquerforfungal
infectionscontainingdrugslike
MacrochemCorporation,
US6495124B1(2002) econazole/ciclopiroxinapolymericfilm [35]
Wellesley,Massachusetts
ofpentadecalactone/lactoneandasolvent
(volatile)
ImxLabs,Inc.,
Disclosinganailpolishcolor.Itsmethods
US6622064B2(2003) Birmingham,Oakland, [36]
anddispensinghavebeendescribed
Michigan
Revealinganaturallydevelopednail
US8771655B2(2014) DebraMarino polishpreparedwithfruits/vegetables [37]
containingacrylicpolymer
Disclosingacoloredandtranslucentnail
US20120178056A1(2012) CindyNelson [38]
polishpreparedwithdifferentcolors
Providinglongresidingandeasilydried
MichaelMitsuoHomma,
US20130149266A1(2013) nailpolishcomprisingalacquerand [39]
VictorMasaruHomma
pigmentcomposition
Inventingagelbasedpolishcomprising
PakalyEnterpriseCo.,Ltd., (meth)acrylatemonomerlikeethyl
US20150190331A1(2015) [40]
Chung-Ho,Taipei,Taiwan methacrylateand
hydroxyethylmethacrylate
Disclosinganailpolishpreparation
BennyBorsakian,Janel
WO2003007675A2(2003) containingabase,colorant,anda [41]
Faraci
photochromicpowder
2
37J/cm for 7min 24sec for removal of dermatophytes, upto84%clinicalandmycologicalcuringrate.Thisfinding
which was then revealed by direct mycologic investigation reachedupto94%,whenitraconazolecontinuedtobeused
and culture. Repeated treatment (twice) with an interval of for 12 weeks along with nail lacquer (amorolfine) for fur-
2weekswasfixed.Nosideeffectswerementioned.However, ther six months. When itraconazole was used all alone, the
completeremovalofhyperkeratoticmaterialandinfectednail mycological and clinical curable rate was found to be 69%
beforeusingphotodynamictherapywasvitaltogetoptimum [70]. Another example of combination therapy involves the
outcomes[69]. useoffluconazole(150mg)onceinaweekalongwithamorol-
In order to treat and manage onychomycosis, topical fine (5%) nail lacquer once in a week. This combination
therapy is supplemented with the systemic therapy to gain showed the curing rate in between 75% and 86% [71]. In
more effective results and to cure the disease efficiently. To another investigation conducted on 157 patients treatment
designoptimumdosageregimenvarioustopicalproductsare wasgivenbyapplyingamorolfinenaillacqueronceinaweek
beingusedincombinationwithoralantifungalsdrugs.Some for12monthsalongwithanoralantifungaldrug(terbinafine)
ofthesearediscussedintheprecedingtext.Itraconazoleina once in a day up to 3 months. Further, itraconazole pulse
dailydoseof200mgdailyuptosixweekscanbeusedincom- therapy for 3 months, and fluconazole once in a week for
bination with amorolfine (5%) nail lacquer used once in a 6 months, depicted same curable rate (71% to 73%) in the
week up to six months. This combination therapy showed threegroups[72].Combinedtherapyusingitraconazoleand
10 Scientifica
terbinafinetogetherwascomparedwithterbinafinealonein were kept in a Teflon one-chamber diffusion cell in order
aninvestigationon190patientsupto72weeks.Itraconazole tomaintainthephysiologicalconditionsoftemperatureand
pulse used for 1 week in a dose of 400mg each month humidity.Thissystemwascomprisedofnailsurfacethatwas
for 2 months, followed by one or two additional pulses of openintheairandinnersurfacewhichwasincontactwith
terbinafine (500mg daily for 1 week per month), was then thecottonballandplacedonthechamberbeneaththenail
compared with three to four pulses of terbinafine alone. plate. Degree of hydration was observed and determined.
Pulsed sequential therapy provided clinical (56.0% versus In an experiment by Murthy and Maibach, it was observed
38.9%) and mycologic (72.0% versus 48.9%) curable rates that average hydration of the wet cotton balls resembled
superior to those obtained by the use of terbinafine alone the hydrationof the human nail bed obtainedfrom human
[73]. These findings suggest the effectiveness of combina- cadaver.Themainbenefitofthissystemwasthatitprovided
tionaltherapyemployingtopicalaswellassystemictherapy nonocclusive and hydration controlled environment where
altogether. normal physiological conditions can be achieved. However,
toanalyzethedrugconcentrationdrillingsystemwasdevel-
10.DevelopmentofPenetrationAssaysand oped which is comprised of a nail sampling stage and a
TheirComparativeAnalysis drill, which provided the possibility to collect the sample
from inner region of the nail [76]. Thus, development of
Inordertoanalyzethepenetrationofdrugdeepintothenail thesemethodologiesprovidesaneasyaccesstoevaluatethe
bed, several penetration assays have been developed. These penetrabilityacrossthenailbed.
assaysweredesignedtoinvestigateamountofdrugabsorbed
inthedeeperlayersaftertheapplicationoftopicaldose.Hui 11.ConcludingRemarks
etal.developedanassayproceduretoperformcomparative
analysisofnaildrugpenetrationusingpenetratingenhancing Limited permeability of antifungals across the nail poses a
formulation (test formulation). Test formulation and saline threat in transungual delivery in order to treat deep seated
formulationsweretestedusingradiolabeledurea,ketocona- nail fungal infections. Nail lacquers have been suggested to
zole, and salicylic acid. Inner portions of nail plate were possessefficientpermeabilityacrossthenailplate.Adiverse
assayed,aftertwicedosingdailyapplicationfor7daystofind range of antifungals like azoles and allylamines have been
out the absorbed drug content via unique drilling/removal investigatedforthemanagementofonychomycosis.Delivery
system.Thefindingssuggestedthattheradioactivitycontents ofthesedrugsvianaillacquersisfoundtobesafeandeffective
ofthreechemicalsintheinnerintermediatenailplatecenter inthetherapyforonychomycosis.
inthetestformulationweretwotimeshigherthanthesaline
CompetingInterests
formulation. Further, in saline formulation, salicylic acid
depictedmaximumbindingtotheouternail,thusmakingit
Theauthorsdeclarethattheyhavenocompetinginterests.
lessbioavailablefortheinnernailarea.Thiswasnotobserved
incaseofcarrierformulation.Thus,itwasconcludedbythe
References
authorsthattopicaltreatmentincaseofonychomycosisisnot
yetprovedtobethatmucheffective,asminimumamountof [1] I.Vejnovic,L.Simmler,andG.Betz,“Investigationofdifferent
drug penetrates into the inner nail plate where the disease formulationsfordrugdeliverythroughthenailplate,”Interna-
actuallyresides.Theassaysystemdevelopedhastheunique tionalJournalofPharmaceutics,vol.386,no.1-2,pp.185–194,
featureofassayingtheinnerpartofthenailwherethedisease 2010.
isperpetuated[74].Chemicalenhancershavebeenemployed [2] B.Ahmadi,S.J.Hashemi,F.Zainietal.,“Acaseofonychomy-
to improvise the drug transport across the nail plate. How- cosis caused by Aspergillus candidus,” Medical Mycology Case
ever, selecting an effective chemical enhancer for the given Reports,vol.1,no.1,pp.45–48,2012.
drug and formulation is highly essential in determining [3] D.T.Roberts,W.D.Taylor,andJ.Boyle,“Guidelinesfortreat-
the efficacy of topical therapy of nail diseases. Keeping this ment of onychomycosis,” British Journal of Dermatology, vol.
objective,Murthyetal.havedevelopedTranScreen-N,ahigh 148,no.3,pp.402–410,2003.
throughputmethodofoptimizingtransungualdrugperme- [4] D. P. Westerberg and M. J. Voyack, “Onychomycosis: current
trendsindiagnosisandtreatment,”AmericanFamilyPhysician,
ationenhancers.Itisarapidmicrowellplatebasedmethod-
vol.88,no.11,pp.762–770,2013.
ologycomprisingtwodifferenttreatmentaspects:thesimul-
[5] H. N. Shivakumar, A. Juluri, B. G. Desai, and S. N. Murthy,
taneous exposure treatment and the sequential exposure
“Ungualandtransungualdrugdelivery,”DrugDevelopmentand
treatment.VariousenhancerswerescreenedbyTranScreen-
IndustrialPharmacy,vol.38,no.8,pp.901–911,2012.
N and by diffusion studies employing Franz diffusion cell.
[6] S.Firoz,M.N.Sirisha,andR.Rajalakshmi,“Transungualdrug
In TranScreen-N technique, the enhancers can be grouped
delivery: a review,” International Journal of Innovative Drug
accordingtowhethertheyneedtobeappliedbeforeorsimul-
Discovery,vol.1,no.1,pp.9–14,2011.
taneously with drugs (or by either procedure) to enhance
[7] M. Joshi, V. Sharma, and K. Pathak, “Matrix based system
the drug delivery across the nail plate. This technique can
of isotretinoin as nail lacquer to enhance transungal delivery
remarkably minimize the cost and duration required to acrosshumannailplate,”InternationalJournalofPharmaceu-
screentheenhancers[75]. tics,vol.478,no.1,pp.268–277,2015.
A hydration controlled nail incubation system was also [8] X.Hui,R.C.Wester,andH.I.Maibach,“Nailpenetration,”in
devised in order to monitor the penetration. Nail samples TextbookofCosmeticDermatology,R.BaranandH.Maibach,