J
`
`mlr
`
`Skin Pharmacol 1991;4:89-94
`
`© 1991 S. Karger AG, Basel
`1011-0283/91/0042-008952.75t0
`
`Ciclopirox Nail Lacquer 8%: Jn vivo Penetration into and
`through Nails and in vitro Effect on Pig Skin
`
`C,G, CescMtl-Roqz~esa, It. Hb’nelb, S.M. Pruja-Bougaretc, ft. Lz¢ca, J. Vandermander%
`
`G. Michela
`
`a Laboratoire de M icrobiologie Industrielle et Virologic, Facult6 des Sciences Pharmaceutiqucs,
`
`Toulouse, France; bHoechst AG, Frankfurt am Main, BRD;
`c Centre de P.ccherehe Pierre-Fabre Mddicament, Castres, France
`
`Key Words. In vivo nail penetration ¯ Human healthy nails ¯ Ciclopirox ¯
`Microbiological assessment - Pig skin ¯ In vitro antifungal test ¯ Onychomycosis
`
`Abstract. Th is repo.q presents origi hal methods to assess the bioavailability of an antifun-
`gal drug from a varnish preparation in finger nails. For the studies with human volunteers a
`ciclopirox 8 % nail lacquer was used to determine its efficacy in the treatment of onychomy-
`coses. In vivo sludies were performed on the fingernails of healthy volunteers by determining
`the total amount of ciclopirox penetrated per milligram of nail and the partition of the drug
`in the plate of the nails (technically divided into four layers). Ciclopirox concentrations were
`evaluated by measuring the inhibition of Candida pseudotropicalis growth in vitro. The
`ciclopirox concentration after 30 days treatment was determined as 3.35 ± 0.82 gg/mg nail
`material. This is a sufficient amount to kill the fungaI pathogens. In addition, in vitro pen-
`etration experiments were carried out with excised pig skin. Lacquer formulations from 0.5
`to 8 % were used to inhibit the growth of Trichophyton mentagrophytes. Formulations from 2
`to 8 % led to a strong to total inhibition of the dermatophyte after 30 min treatment time.
`
`Introduction
`
`Fungi involved in onychomycosis can
`grow within different nail locations in the
`nail plate and/or the nail bed [1]. This phe-
`nomenon raise’.; the interest of determining
`the penetration of antifungal drugs into and
`through the nail. On the other hand, the
`ultrastructure of the nail can limit this pa-
`rameter [2]. The keratinocytes within the
`nail plate are strongly linked by numerous
`
`desmosomes [3] and surrounded by phos-
`pholipid layers [3]. The nailplate is built up
`by three different layers which could have
`different penetration characteristics, but this
`subject is discussed controversally in the lit-
`erature. The penetration characteristics in
`diseased nails are poorly investigated but it
`is rather likely that the more dehydrated dis-
`eased nail has a reduced penetration for hy-
`drophilic compounds. This report presents
`methods to test the nail penetration of an
`
`’ "i
`
`/
`
`?
`
`2
`
`ARGENTUM EX1017
`
`Page 1
`
`

`

`9O
`
`Ceschin-Roques/H’anel/Pruja-Bougaret/Luc/Vandcrmander/Michel
`
`Penetration of (
`
`antifungal drug by measuring its biological
`activity instead of using radiolabeled mole-
`cules [4]. Additionally, the evaluation of
`large numbers of lacquer formulations
`causes problems due to the limited availabil-
`ity of healthy human nails. Therefore we
`applied an additional method using excised
`pig skin [5], which is a well-established
`method for the evaluation of penetration.
`
`H~inel and Ritter [5] showed that even lac-
`quer formulations can be evaluated on the
`pig skin by applying the samples and subse-
`quent strip of the stratum corneum before
`inoculation with dermatophytes.
`
`Materials and Methods
`
`Chcm icals
`6-Cyclohexyl- l-hydroxy-4-methyl-2-( 1 H)-pyridone
`(ciclopirox free acid) and ciclopirox varnish 8%
`(Centre de Recherche Pierre-Fabre M4dicament/
`Hoechst AG, FRG; patent pending).
`
`In vitro Pig Skin Experiments
`
`In vivo Experimet;ts
`
`The study was performed on the fingernails of 9
`
`healthy volunteers. The anaount of varnish (8% ciclo-
`
`pirox and placebo) applied daily was about 5-15
`
`rag/nail. Before each application and before sampling,
`the residual film of lacqucr was gently removed with
`
`methanol-wetted cotton wool.
`
`Sampling. Four free distal parts of nails were cut
`after 7, 14, 30 (9 subjects) and 45 {4 subjects) days of
`
`application, and aIso 7 and 14 days after the end of
`
`the treatment (4 subjects). The samples of each sub-
`
`ject were used for two different experiments. (1) For
`
`the determination of the total amount of ciclopirox
`
`per milligram of nail (two samples) direct extraction
`
`of ciclopirox and microbiological determination were
`
`performed. (2) In order to evaluate the distribution of
`
`ciclopirox in the nail (two samples) the distal parts of
`the nails were pressed fiat. Then the flattened samples
`were cut by means of a freezing microtome to obtain
`
`four layers in depth of equal thickness. Extraction was
`
`carried out with each layer separately. Repartition of
`
`ciclopirox in the four layers was determined by the
`
`microbiological method described.
`]:2xtraction. Extraction was performed in a 10%
`
`polyethylene glycol 4,000 (PEG) aqueous solution
`
`with a contact time of 48 h. For the determination of
`
`the total amount of ciclopirox per milligram of naiI,
`
`the samples were weighed and extraction was carried
`
`out with 50 I.tl PEG solution per milligram of naih To
`
`For the investigation the back skin of pigs was
`
`assess the distribution of ciclopirox in the nail depth,
`
`used as described in detail elsewhere [5]. The skin was
`shaved and kept on water agar. Lacquer formulations
`
`(5-i0 my) containing 0.5, 1, 2, 3, 4, and 8% ciclopi-
`
`rox were applied with a nail varnish brush as was the
`
`placebo lacquer. Treatment time was 30 min through-
`out the different concentrations. In previous cxpcri-
`
`ments (unpublished data) it had been ~ound that
`
`treatment time longer than 1 h leads to a complete
`
`saturation of the skin pieces with ciclopirox and
`
`therefore complete inhibition of fungal growth irre-
`spective of the concentrations used. After the treat-
`
`ment the lacquer was removed with a pair of forceps.
`
`each layer was extracted with 100 HI PEG solution.
`Extraction yield was determined under e~ vivo exper-
`
`imental conditions. A quantity Q of ciclopirox 8%
`
`nail lacquer was applied, equivalent to a total amount
`
`of Qc - 0.08 x Q of ciclopirox. Then the pieces of
`nail were put into a humid chamber during 7 days to
`
`obtain a passive diffusion of" the drug. After elimina-
`
`tion of the residual film of lacquer, ciclopirox was
`
`extracted with 50 ill PEG solution per milligram of
`nail. The extract for the microbiological determina-
`
`tion was defined as Qe (extractible amount of ciclopi-
`
`rox). Qe was identical irrespective of the elimination
`
`Subsequent adhesive tape strips exposed different
`depth of the stratum corneum of the pig skin.
`
`of the residual film, involving a total passive penetra-
`tion of ciclopirox in the nail. In all the cases, extrac-
`
`Inoculation was carried out as described [5] with
`microconidia of Trichopkyton menlagropkytes. Dur-
`ing 7 days of observation the fungal growth was
`
`recorded daily by measuring the size of the mycelium
`
`in comparison to the placebo-treated group [for more
`
`details see ref. 5].
`
`tion yield (Qe/Qc) was estimated at 0.95.
`
`Microbiological Determinatio~z. Ciclopirox con-
`
`centrations in the nail material were evaluated after
`filtrat4on of extracts at room temperature (0.2-~m
`
`pore size glass fiber) by measuring the kinetics of inhi-
`
`bition of Candida pseudotropica/is growth. Determi-
`
`Fig. 1. lnbi!
`g’owlh of 71 *neJ
`c sed pig skin a
`ment with lacc
`c3ntaining differ
`c opirox.
`
`nations were pc:
`parison with a
`determination. I
`solution were ca
`JJl). After additio
`g:ucose broth (D?
`plates were inoet
`t,’opicalis ( 10~ yc
`t:on was carric’d
`(650 nm) v, as re~
`the MIC tb.e t~:st>
`detection limit. "~
`been estimated t,
`data) due to the
`the extraction >,
`ciclopirox in ihe
`in the tour layers
`biological mctiv~
`r,)x in the [ac-luc
`
`In vitro !;,-,
`In lhc test u
`of ciclopirox i~
`campared (fig.
`of fungal grow,
`(10 strips) :
`
`Page 2
`
`

`

`-mander!Michcl
`
`Penetration of Ciclopimx Nail Varnish
`
`tingcrnails of 9
`¯ r~isi~ (8 % ciclo-
`as about 5-15
`c fore sampling,
`. removed with
`
`" nails ,;,ere cut
`Jbjccts) days of
`f~er lhe end of
`-s of each sub-
`mcnts. (I) For
`t cA cich~pirox
`rcct exm~ction
`ruination were
`distribtaion of
`distal paris of
`t c n,,:d samples
`om.c to obtain
`!!x traction was
`Rcparlition of
`mi:,cd by the
`
`~cd ira a 10%
`21~US solution
`::rmim~tion of
`[gram of nail,
`n was carried
`tan of nail. To
`1,.: nail depth,
`
`’E(} solution.
`v, vivo cxper-
`iclopirox 8%
`toted }llllO unt
`the pieces of
`J*,g 7 day’s to
`.i’tcr elimina-
`CiOl:’irox ;’,.as
`milligram of
`
`] determina-
`nt oi"ciciopi-
`
`elimination
`dye pcnctra-
`ascs, exlrac-
`
`oplrox con-
`t] UZltc,;t after
`
`Ire (0.2-11 n1
`
`.:tics f~lillhi-
`
`h, l)ctclnli-
`
`91
`
`[~ 10 strips
`
`6 strips
`
`[Z~ 2 strips
`
`toc
`
`90
`
`80
`
`7o-
`
`60-
`
`50--
`
`t
`
`Fig. 1. Inhibition of fungal
`
`growth of T mentagrophyte.s’ on ex-
`cised pig skin after 30-rain treat-
`ment with lacquer formulations
`
`containing different amounts of ei-
`clopirox.
`
`8 4 3 2 1 0.5
`Concentration of cic!opirox in lacquer, %
`
`nations were performed on microtiter plates in com-
`parison with a ciclopirox solution to evaluate the
`determination. Dilutions of samples and ciclopirox
`solution were carried out with a PEG solution (100
`uI). After addition of the same volume of Neopeptone
`glucose broth (DIFCO, Mich.) to each well, microtiter
`plates were inoculated with suspensions of C. pseudo-
`tropicalis (104 yeasts/ml final concentration). Incuba-
`tion was carried out at 28.5 °C and optical density
`(650 nm) was read at 18 h. At concentrations close to
`the MIC the tests were run for 24 or 48 h to lower the
`detection limit. The limit of ciclopirox detection had
`been estimated to be 0.04 lag/mg of nail (unpublished
`data) due to the sensitivity of C. pseudotropiealis and
`the extraction yield. To assess the distribution of
`ciclopirox in the nail matrix, the relative percentage
`in the four layers was determined by the same micro-
`biological method. The limit of solubility of ciclopi-
`rox in the lacquer is around 12%.
`
`Results
`
`In vitro Experiments
`In the test using pig skin six concentrations
`of ciclopirox in the lacquer formulation were
`compared (fig. 1): 0.5 % led to a low inhibition
`of fungal growth close to the stratum tucidum
`(10 strips) : 63.4%, whereas in the upper
`
`layers 84.1% (6 strips) and 88.11% (2 strips)
`inhibition of fungal growth was recorded. 1%
`lacquer led to comparative results. The 2%
`formulation was only tested down to the 6-
`strip layer and showed 100% inhibition at
`this depth. 3 % lacquer was also tested at the
`stratum lucidum (10 strips) and led to
`99.72% inhibition just like the 4% lacquer.
`The 8% lacquer however resulted in total
`inhibition (100%) in all layers investigated.
`
`In vivo Experiments
`Kinetics of Cic/opirox Penetration. After
`correction by the extraction yield, individual
`ciclopirox concentrations and means and
`standard errors were calculated and are
`given in micrograms per milligram of nail in
`table 1. Figure 2 shows the increase of the
`ciclopirox concentration in the nail during
`the first 30 days and the persistence of a
`steady state between the 30th and the 45th
`day of the daily application. The repartition
`of ciclopirox in the nail plate is indicated in
`table2 by relative percentage values ob-
`served in the four layers. After 7 days of
`daily application, the greater amount of ci-
`
`Page 3
`
`

`

`i °
`
`92tz"~ t
`
`Ceschin-RoqueslH~inel/Pruja-Bougaret/Luc/Vanderma n der/Michel
`
`Penelrali
`
`clopirox was obtained in the first layer
`(51.5 %) and a progressive decrease occurred
`in the deeper layers. Ciclopirox levels were
`not detectable in layer 3 for 2 subjects and in
`layer 4 for 3 subjects, in al! cases, penetra-
`
`tion through tile nail down to the lburth
`
`layer was relatively low. A more homoge-
`neous partition of ciclopirox appeared after
`
`14 days of application, always with a higher
`
`level in the first layer (32%). At this time,
`
`penetration occurred down to the fourth
`
`layer (23%). The same phenomenon could
`
`be observed after 30 days of application.
`Ciclopirox Concentration afler Treat-
`
`mont. These studies were performed on 4
`
`Fir{. 2
`clop, irox
`mulatien
`jects. Tre
`30 or to ,
`
`/
`
`,/
`
`Table 1. Concentrations of cicIopirox in nails
`(pg/mg nail)
`
`Subject
`
`Duration of treatment
`
`7
`days
`
`0, I
`
`1.1
`
`2.6
`
`1.3
`
`0,18
`
`0.6
`
`0.45
`
`14 30
`days
`days
`
`0.3
`
`1.5
`
`3,3
`
`3,8
`
`0.5
`
`1.6
`
`0.95
`
`0.95
`
`1.7
`
`3.2
`
`6
`
`1.3
`
`2,3
`
`1.2
`
`45
`days
`
`ND
`
`1.55
`
`3.3
`
`ND
`
`ND
`
`1.94
`1.26
`
`ND
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`0.7
`1
`
`1.3
`2.8
`
`6.5
`7
`
`ND
`
`Mean 0,892 1,783 3.35 2.01
`SE
`0,413
`0.252
`0.822
`0.45
`
`ND = Not determined; SE = standard error.
`
`healthy volunteers, 7 and 14 days after the
`
`end of the treatment. Results are presented
`
`in table 3 and indicate a progressive decrease
`
`of the active drug concentrations, which felt
`
`below the limit of detection 14 days after the
`
`end of ciclopirox treatment.
`
`Tnble 2. Relative percentages of ciclopirox in the four nail layers (1-4)
`
`Subject 7-day sample
`
`14-day sample
`
`30-day sample
`
`1
`
`32
`
`52
`
`2
`
`28
`
`41
`
`4
`
`11
`
`3
`
`29
`
`7.
`
`1
`
`2
`
`1
`
`34
`
`30
`
`2
`
`25
`
`22
`
`3
`
`21
`
`22
`
`4
`
`20
`
`26
`
`1
`
`33
`
`40
`
`2
`
`45
`
`21
`
`3
`
`12
`
`19
`
`4
`
`10
`
`treat or
`
`Among
`tare [7],
`zole [9]
`since 1
`expcrir.,-
`were p,
`
`on.veho~
`
`ketocon
`prior ~
`
`3
`
`4
`
`5
`
`6
`
`7
`
`60
`
`45
`
`56
`
`41
`
`48
`
`26
`
`34
`
`44
`
`33
`
`43
`
`9
`
`17
`
`-
`
`18
`
`5
`
`4
`
`8
`
`30
`
`32
`
`36
`
`34
`
`20
`
`28
`
`29
`
`28
`
`24
`
`18
`
`!7
`
`13
`
`26
`
`22
`
`i8
`
`25
`
`15
`
`38
`
`41
`
`36
`
`38
`
`20
`
`26
`
`24
`
`20
`
`22
`
`19
`
`16
`
`21
`
`20
`
`20
`
`14
`
`24
`
`21
`
`"Far, k:
`nai!) al t!
`later
`
`8
`
`9
`
`!0
`
`47
`
`55
`
`60
`
`36
`
`21
`
`40
`
`9
`
`11
`
`16
`
`-
`
`6
`
`8
`
`-
`
`33
`
`23
`
`40
`
`28
`
`15
`
`28
`
`22
`
`24
`
`22
`
`18
`
`23
`
`37
`
`27
`
`20
`
`25
`
`28
`
`34
`
`28
`
`31
`
`25
`
`25
`
`26
`
`32
`
`23
`
`20
`
`26
`
`I9
`
`24
`
`21
`
`20
`
`18
`
`Mean 49.6 34.6 11.6 4.2 32 ~4.1 21.2 22.7 34.7 26.4 19.7 19.2
`
`SE
`
`2.8 2.4 2.8 1.3
`
`1.5 1,4 1.1 2.1
`
`1.5 2.4 1.2 1.4
`
`Subject
`
`2
`
`3
`
`6
`
`7
`
`Page 4
`
`

`

`ander/Michel
`
`t]rst layer
`e occurred
`evcls were
`ects and in
`s, pcnetra-
`tim fourth
`: homoge-
`:arcd after
`h a higher
`this time,
`hc fBurth
`~on could
`cation.
`:’r Treal-
`ned on 4
`after the
`presented
`: decrease
`uhJch fell
`; alier the
`
`l 0
`
`2O
`
`2O
`
`14
`
`24
`
`21
`
`24
`
`2t
`
`2(;
`
`18
`
`Penetration of Ciclopirox Nail Varnish
`
`93
`
`1E+02
`
`~’ IK+01
`K
`
`! I E+00
`
`,E-o t I[.y
`
`i IE-02
`
`i
`0 ~0 20 30 40
`Days af~er star1 of treatment
`
`i
`50
`
`1
`60
`
`Fig. 2. Ungual penetration ofci-
`clopirox from an 8% lacquer for-
`mulation in the fingernails of 9 sub-
`jects. Treatment from day 0 to day
`30 or to day 45.
`
`Discussion
`
`Numerous attempts have been made to
`treat onychomycoses by topical antifungals.
`Among these were clotrimazole [6], tolnaf-
`tate [7], miconazole [8], and recently bifona-
`zole [9]. Ciclopirox has proved to be effective
`since 1979 [10]; also cow horn penetration
`experiments with ciclopiroxolamine cream
`were performed. The systemic therapy of
`onychomycoses with azole-compounds like
`ketoconazole has to be considered carefully
`prior to treatment due to the possibility
`
`Table 3. Residual amounts of ciclopirox (,ug/mg
`nail) at the end of treatment (D45) and 7 and 14 days
`later
`
`Subject
`
`2
`
`3
`
`6
`
`7
`
`D45
`
`1.55
`
`3,3
`
`1.94
`
`1.26
`
`7 days I4 days
`
`005
`
`0.32
`
`0.13
`
`0.(/4
`
`0.04
`
`0.04
`
`0.04
`
`0.04
`
`of serious side effects [11]. From its composi-
`tion the nail plate is more like hair than like
`stratum corneum [12]; therefore the penetra-
`tion characteristics arc remarkably different
`from the skin. From the in vivo results (fig. 3)
`it is obvious that under treatment a steady
`state is reached which enables the inhibition
`and killing of fungi like Trichophyton spp.
`Also the MICs for fungi like Hendersonula
`spp. and other causative agents ofonychomy-
`coses are more or less within the range of
`concentrations calculated.
`Among the group of nails investigated
`there were some (e.g. subjects 1, 5 and 7)
`which did not accumulate high concentra-
`tions of the compound. The variation of the
`nail structure in the individua! onychomyco-
`sis patient is also of clinical importance. In
`all clinical trials a considerable part of ’non-
`responders’ cannot be explained by a lack of
`compliance since it is often due to hydration
`problems and other mo~lghological differ-
`ences in the nails.
`
`Nevertheless, ciclopiroxolamine proved
`to be effective even as a 1% cream-solution
`
`Page 5
`
`

`

`o
`
`94
`
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`
`Ceschin-Roques/H~nel/Pruja-Bougaret/[.uc/Vandcrnlandcr/Michel
`
`7 days
`
`14 days
`
`30 days
`
`combination therapy for !2.7 _+ 5.6 weeks.
`Among 150 patients 57% showed complete
`clinical and mycological cure [13]. We con-
`sider ciclopirox as one of the few antimy-
`cotic compounds with the proven ability to
`penetrate through the human nail plate.
`
`References
`
`1 Baran R, Dawber RPR: Guide mfidico-chirurgical
`des onychopathies. Paris, Arnette, 1990.
`2 Waiters KA: Penetration of chemicals into, and
`through the nail plate. Pharm Int April 1985:86-
`89.
`3 Bereiter-Hahn J, Malolsy AG, Richard KS: Biol-
`ogy of the Integument. Berlin, Springer, 1986, vol
`2.
`4 Tuttgen G, Bauer E: Bioavailability, skin and nail
`penetration of topically applied antimycotics. My-
`kosen 1982;25:74-80.
`5 Hfinel H, Ritter W: Formulations; in Ryley JF
`(ed): Chemotherapy of fungal diseases. Handbook
`Exp Pharmacol. Berlin, Springer, 1990, vol 97, pp
`251-278.
`6 Schubert E: Klinische Erfahrungen bei der Bc-
`handlung yon Nagelmykosen mit dem neuen An-
`timykotikum BAY b 5097 (Clotrimazol). Z
`Hautkr 1973;48:887-891.
`7 Ishii M, Hamada T, Asai Y: Treatment ofonycho-
`mycosis by ODT therapy with 20% urea ointment
`
`Fig. 3. Relative percentages of
`ciclopirox in four different layers of
`human fingernails after 7, 14, and
`30 days of treatment with an 8%
`ciclopirox lacquer formulation. Ab-
`solute values are given in table 2.
`
`and 2 % tolnaflate ointment. Dermatologica 1983;
`167:273-279.
`8 Rollman O: Treatment ofonychomycosis by par-
`tial nail avulsion and topical miconazole. Derma-
`tologica 1982;165:54-61.
`9 Nohing S: Non-traumatic removal of the nail and
`simultaneous treatment of onychomycosis. Der-
`matologica 1984;169(suppl 1):117-120.
`I0 Dittmar W, Grau W: Ciclopirox: Substanz mit
`Aspekten ffir Mykologic und Kosmetik. )~rztl
`Kosmetol 1979;9:209-214.
`I 1 Zaias N, Drachman D: A method for the determi-
`nation of drug effectiveness in onychomycosis. J
`Am Acad Dermatol ! 983;9:912-919.
`12 Walters KA, Flynn GL: Permeability characteris-
`tics of the human nail plate. Int J Cosmet Sei
`1983;5:231-246.
`13 Quadripur SA, Horn G, Hochler T: Zur Lokalwirk-
`samkeit von Ciclopiroxolamin bei Nagelmyko-
`sen. Arzneimittelforsch ung 1981 ;31:1369-1372.
`
`Received: blarch 30, 1990
`Accepted: October 18, 1990
`
`C.G. Ceschin-Roques
`Laboratoire de Microbiologie Industrielle et
`Virologie
`Faculte des Sciences Pharmaceutiques
`31, all~es Jules-Guesde
`F-31000 Toulouse (France)
`
`Skin
`
`G. ,l
`
`Lab,
`Touh
`Cen
`
`Ciclor;i
`
`Ab~t
`cream. (cid:128)
`stratum
`pigs inc
`inhibiti
`stratum
`in four l
`lion fro
`which a
`
`lntro
`
`Ciclc
`fungal h
`
`clinical
`3]. Cuta
`tophyte.~
`
`lar a ft] ~
`
`Therefo:
`drugs in
`the first
`
`value. T
`
`pig skin
`
`Page 6
`
`