\'
`\'
`
`J, Phann. Phannacol. 1997, 49: 866-872
`Received March 3, 1997
`Accepted June 2, 1997
`
`© 1997 J. Phann. Phannacol.
`
`In-vitro Permeability of the Human Nail and of a Keratin
`Membrane from Bovine Hooves: Prediction of the Penetration
`Rate of Antimycotics through the Nail Plate and their Efficacy
`
`DIRK MER TIN AND BERNHARD C. LIPPOLD
`
`Department of Pharmaceutical Technology, Heinrich-Heine-University, Universitiitsstr. l, D-40225 Diisseldoif,
`Gennany
`
`Abstract
`In contrast to the partition coefficient octanol/water the molecular size of penetrating drugs has a noticeable
`influence on the permeability of the human nail plate and a keratin membrane from bovine hooves. The
`relationship between permeability and molecular weight is founded on well-established theories. The
`correlation between the permeability of the nail plate and that of the hoof membrane allows a prediction of
`the nail penneability after determination of the drug penetration through the hoof membrane.
`The maximum flux of ten antimycotics (amoroJfine, bifonazole, ciclopirox, clotrimazole, econazole,
`griseofulvin, ketoconazole, naftifine, nystatin and tolnaftate) through the nail plate was predicted on the
`basis of their penetration rates tluough the hoof membrane and their water solubilities. An efficacy coefficient
`against onychomycoses was calculated from the maximum flux and the minimum inhibitory concentration.
`Accordingly, amorolfine, c.iclopirox, econazole and naftifine are expected to be especially effective against
`dennatophytes, whereas in the case of an infection with yeasts on1y, amorolfine and ciclopirox are promising.
`
`The influence of molecular size on permeability was investi(cid:173)
`gated in the present work, after previous studies had shown
`that drug penetration through the nail plate and a keratin
`membrane from bovine hooves is independent of the lipophi(cid:173)
`licity of the diffusing substance (Mertin & Lippold 1997).
`Since the penetration of non-electrolytes through biological
`membranes is similar to that through polymers, the diffusion
`mechanism has also been transferred (Lieb & Stein 1969).
`Although there is no consistent theory about the diffusion
`in polymers, it is assumed that the thermal movement of
`the polymer chains creates holes which are occupied by the
`diffusing molecules (Kuminis & Kwei 1968; Lieb & Stein
`1969). The penetration rate is limited by the formation fre(cid:173)
`quency and the size distribution of these free volumes. On the
`other hand, these factors are influenced by the temperature, the
`nature of the polymer and the interactions of the polymer
`chains with each other and with the diffusing molecules.
`Transferred to biological structures, free volumes can be
`fanned by separating lipid bilayers or proteins (Lieb & Stein
`1969),
`Cohen & Turnbull
`(1959) deduced an exponential
`relationship between the molecular volume VM of the diffusing
`particle and its diffusion coefficient D from statistical
`analysis of the :fluctuations of the free volume in super cooled
`liquids:
`
`(I)
`
`where D0 is the diffusion coefficient of a hypothetical molecule
`with the mole volume of 0 and p is a reciprocal value for the
`average free volume (Potts & Guy 1993). M the diffusion
`coefficient through a biological membrane is difficult to
`
`Correspondence: B. C. Lippold, Department of Pharmaceutical
`Technology, Heiruich-Heinc-University, UniversiUl.tsstr. 1, D-40225
`Dilsseldorf, Gennany.
`
`determine, it is combined with the partition coefficient bar(cid:173)
`rier/vehicle PC81v to the permeability coefficient P:
`P =Do .• -P·V" , PC,,1v
`
`(2)
`
`Talcing the logarithm leads to:
`
`p
`log P =log Do - 2-J0
`VM +log PC81v
`3
`Since both nail plate and hoof membrane are hydrophilic gel
`membranes whose PC8 /V is approximately unity (Merlin &
`Lippold 1997). equation 4 follows by combining the constant
`parameters:
`
`(3)
`
`(4)
`
`resp, log P = k - P'' , MW
`(5)
`where {J" is similar to /J' and contains a factor which reflects
`the conversion of the molecular volume into the '·molecular
`weight (MW).
`On the basis of the diffusion of non-electrolytes in polymers,
`Lieb & Stein deduced an empirical equation which can also be
`transferred to biological membranes:
`
`D=Do·MW-•
`resp. log D = log D0 - z · log MW
`If the PCB/V becomes unity, it follows:
`
`logP=k-z.logMW
`
`(6)
`
`(7)
`
`(8)
`
`The parameter z is called mass selectivity coefficient which
`quantifies the sensitivity of the diffusion coefficient to altera(cid:173)
`tions of the molecular weight of the diffusing compound. It
`ranges from l·l to 3·8 in plastics, from 2·9 to 6·0 in cell
`membranes and from 0·3 to 0·5 in liquids (Lieb & Stein 1969).
`
`FlatWing Ex. 1031, p. 1
`
`

`

`(
`i
`
`PENETRATION OF ANTIMCYOTICS THROUGH NAIL PLATE AND THEffi EFFICACY
`The higher the value, the higher is the sensitivity to alterations
`of the molecular weight. The power function of Lieb & Stein
`(1969) often provides a satisfactory fit to the experimental
`data. but it is disadvantageous that the exponent z has no
`physical meaning (Potts & Guy 1993).
`However, the single consideration of the molecular volume
`or weight may lead to the wrong prediction of the diffusion
`coefficient. Investigations of the penetration of linear and
`branched paraffins through different polymers show that
`branching reduces the diffusion to a greater extent than an
`increase of the molecular volume (Flynn et al 1974). The size
`as well as the shape of the molecules is important. Due to
`taking the logarithm of the molecular weight, the equation of
`Lieb & Stein (Eqn 6) seems to be less sensitive to neglecting
`the molecular shape than the Cohen-Turnbull correlation
`(Eqn 1) (Flynn et al 1974).
`In this study, the relationship between the penneability of
`the nail plate or the hoof membrane, respectively, and the
`molecular weight of the penetrating substance has been
`investigated to enable the prediction of the nail penetration of
`potential antimycotics.
`Among the nail infections onychomycoses, i.e. infections by
`fungi, are predominant. As antimycotics, which seem to be sui(cid:173)
`table for topical application, are expected to have low fluxes due
`to their slight water solubility, only their penetration through the
`hoof membrane was studied. The prospective maximwn flux
`(Jmax) of the antimycotics amorolfine, bifonazole, ciclopirox,
`clotrlmazole, econazole, griseofulvin. ketoconazole, naftifine,
`nystatin and tolnaftate through the nail plate was calculated from
`their penetration through the hoof membrane and their water
`solubility. The efficacy of a topically applied antimycotic is not
`only influenced by the maximwn flux but also by the anti.fungal
`potency, which is quantified by the minimum inhibitory con(cid:173)
`centration (MIC). An efficacy coefficient E is calculated from
`Jmax and MIC. which predicts the topical effectiveness of an
`antimycotic against onychomycoses.
`
`867
`Belgium), naftifine hydrochloride from Sandoz (Nuremberg,
`Germany) and tolnaftate from Essex (Munich, Gennany).
`HPLC-pure acetonitrile (Acetonitril Chromasolv) and metha(cid:173)
`nol (Methanol Chromasolv) were from Riedel-de Haen
`(Seelze, Germ.any).
`
`Penetration studies
`The diffusion cells, the preparation of the nails and of the hoof
`membranes, the penetration studies, the analyses, the deter(cid:173)
`mination of the solubilities and the calculation of the perme(cid:173)
`ability coefficient P and of the maximum flux 1nwr. have
`already been described in an earlier publication (Merlin &
`Lippold 1997). The antimycotics as well as paracetamol,
`phenacetin and cWoramphenicol were presented as saturated
`solutions in their maximum thermodynamic activity. The set(cid:173)
`ting of the saturation concentrations was guaranteed by sus(cid:173)
`pending and stirring a surplus of the drug at 32°C for 48 h. Due
`to their very high water solubility, diprophylline and iopamidol
`were able to be used as non-saturated solutions (hoof mem(cid:173)
`brane: C= 1000 mg L - 1
`; nail plate: C=20000 mg L - 1
`).
`With the antimycotics, the donor compartment consisted of the
`drug suspension in ethanol 42% (v/v), pH 8·1. The penetrating
`amount per time and area therefore represented the maximum
`flux. Due to its high solubility in the medium, ciclopirox was
`an exception: it could be dissolved completely in a con(cid:173)
`
`centration of 1000 mg L - 1• Since the antimycotic with the
`least molecular size had a mole mass of 207, homologous
`nicotinic acid esters served to cover the low molecular weight
`area which ranged from 140 to 230 in a donor concentration of
`1000 mg L - 1. Ethanol 42% (v/v), pH 8·1 also served as the
`acceptor medium.
`
`Materials and Methods
`
`Chemicals
`Phosphate buffered saline pH 7.4 (Ph. Eur.) and, in the case of
`the antimycotics, a mixture of phosphate buffer pH 7·4 of a
`higher buffer capacity with ethanol (resulting ethanol con(cid:173)
`centration 42% v /v) were used as media. Since the ethanol
`restrains the dissociation of phosphate, the pH value of the
`mixture is 8· 1. The selection of the model compounds was
`reduced to water-soluble substances with the exception of the
`antimycotics.
`Paracetamol was obtained from Boehringer lnge1heim
`(lngelheim, Germany), phenacetin and bifonazole from Bayer
`(Leverkusen, Germany), diprophylline from Knoll (Ludwig(cid:173)
`shafen, Germany), chloramphenico1 and clotrimazole from
`Caesar & Lorentz (Hilden, Germany), iopamidol from Byk
`Gulden (Konstanz), methyl, ethyl, butyl and hexyl nicotinate
`were obtained from Aldrich-Chemie (Steinheim, Germany),
`octyl nicotinate from the Department of Phannaceutical
`Chemistry of the University of Dilsseldorf, Germany, amor(cid:173)
`olfine from Hoffmann-La Roche (Basel, Switzerland), ciclo(cid:173)
`pirox olamine and griseofulvin
`from Cassella-Riedel
`(Frankfurt, Germany), econazole nitrate from Cilag (Schaff(cid:173)
`hausen, Switzerland), ketoconazole from Janssen (Beerse,
`
`Determination of the dissociation constants
`For the determination of the acid constants of the antimycotics,
`the potentiometric method of Albert & Serjeant (1984) was
`performed. Solutions (0·02-0·10 mol) of the antimycotics
`were used due to their slight solubility. The pH values were
`recorded with two decimal places after each addition of the
`titrant at 32± 1°C and the pKa value was detennined according
`to the Henderson-Hasselbalch equation. Since the titrations
`were carried out in ethanol 42% (v/v), the pH-meter (Digital(cid:173)
`pH-Meter 644, Knick, Berlin) with glass electrode (U
`402/165, Ingold, Frankfurt) was calibrated with ethanol 42%
`(v /v) containing 0·001 mol benzoate, salicylate and ammo(cid:173)
`nium buffer solutions. The corresponding pKa values in etha(cid:173)
`nol 42% (v/v) are 5·24 (benzoic acid), 3·62 (salicylic acid)
`(Grunwald & Berkowitz 1951) and 8·78 (ammonium chloride)
`(Gutbezahl & Grunwald 1953).
`
`Results and Discussion
`
`Penneability and molecular weight
`Table 1 shows the molecular weights (MW) and the perme(cid:173)
`ability coefficients of the drugs, calculated from the con(cid:173)
`centration increase in the acceptor through the nail plate (PN)
`and the hoof membrane (P8 ). The corresponding data of the
`homologous nicotinic acid esters (Mertin & Lippold 1997)
`were included in the analysis. Since the antimycotics were only
`investigated in ethanol 42% (v/v), pH 8·1, these results were
`analysed separately from those of the other substances. Figs 1
`and 2 show the correlation between the penneability coeffi-
`
`FlatWing Ex. 1031, p. 2
`
`

`

`868
`
`DIRK MERTIN AND BERNHARD C. LIPPOLD
`
`Table 1. Molecular weights (MW) of antimycotics and other drugs and their permeability coefficients
`through nail plate PN and hoof membrane Pa.
`
`MW
`
`Medium: aqueous phosphate buffer pH 7·4
`Paracetamol
`151·2
`179·2
`Phenacetin
`Diprophylline
`254·3
`Chloramphenicol
`323·1
`Iopamidol
`777·1
`
`1·78±0·32
`1·40±0·47
`0· 142±o-055
`0·182±0·047
`O·OlO±o-002
`
`Medium: ethanol-containing phosphate buffer pH 8· 1
`Amorolfine
`317·5
`Bifonazole
`310·4
`207·3
`Ciclopirox
`Clotrimazole
`344·8
`Econazole
`381·7
`Griseofulvin
`352·8
`Ketoconazole
`531-4
`Nafilline
`287 ·4
`Nystatin
`926·1
`Tolnaftate
`307.4
`
`n.d.
`n.d.
`n.d.
`n.d.
`n.d.
`n.d.
`n.d.
`n.d.
`n.d.
`n.d.
`
`Results are presented as means ±s.d., n=4. n.d., not determined.
`
`PH, PH.E10H resp.
`10-8 cm2 s- 1
`
`20-97±5·15
`20·78±5·05
`6·14±2·01
`9·01±2·61
`1·44±0·34
`
`2·03±o2S
`3·05±o37
`2·13±0-65
`2·3o±o·65
`3·37±1·20
`1·00±0·26
`0-84±0·18
`4·08±0·98
`0·10±().02
`3·44±1·03
`
`-6
`
`-7
`
`.. -8
`"' 3
`
`-9
`
`-10
`
`-11
`100 200
`
`-6
`
`-7
`
`.. -8
`O>
`
`3 -9
`
`-10
`
`300 400 500 600
`MW
`
`700
`
`800
`
`-11+-~~~~~~~~~~~~~~~~
`2·1
`2·2 2•3 2·4 2·6 2·6
`2·7 2·8 2·9
`Log MW
`
`FIG. I. Relationship between the logarithm of the penneability
`coefficient P for the nail plate (.6., .6.) or the hoof membrane (0, e)
`and the molecular weight (n = 3--8, means, s.d. see Fig. 3). Medium:
`phosphate buffer pH 7·4. Pis expressed in cm2 s- 1 0, A oicotinic
`acid esters; e, .& remaining substances. Plot according to Cohen &
`Turnbull (1959) (Eqn 5): log PN= -7·296-0.()()3708 "MW, r=0·933
`(nail plate) or log PH= -6·284 -0-002071 MW, r=0·920 (hoof
`membrane).
`
`FIG. 2. Relationship between the logarithm of the flC!Illeability
`coefficient P for the nail plate (A, .&) or hoof membrane (0, e) and
`the logarithm of the molecular weight MW (n = 3-8, means, s.d. see
`Fig. 3). Medium: phosphate buffer pH 7·4, Pis expressed in cm2 s- 1•
`0, A nicotinic acid esters; e, .& remaining substances. Plot according
`to Lieb & Stein (1969) (Eqn 8): log PN= -0-427 -3·341 log "MW,
`r=0·981 (nail plate) or log PH= -2·635 -1·782 log MW, r=0·924
`(hoof membrane).
`
`cient and the molecular weight according to the theory of
`Cohen & Turnbull (log P vs MW) and Lieb & Stein (log P vs
`log MW), respectively, in the aqueous milieu pH 7·4, where
`the investigated substances were nearly undissociated (Table 1,
`upper part).
`There was a linear relationship with a negative slope
`between the penneability coefficient and the molecular weight
`for both the nail plate (generally lower P-values) and the hoof
`membrane. Although giving of the correlation coefficient r is
`only permitted for regressions of the second kind (x and y as
`random variables) (Documenta Geigy 1975), it was never(cid:173)
`theless considered. as it simplifies a judgement of the rela(cid:173)
`tionship. The correlation coefficients show that the plot
`according to Lieb & Stein (Fig. 2) was either equal or slightly
`superior to the Cohen-Turnbull plot (Fig. 1). The slopes of the
`nail-plate data and the hoof-membrane data differed in both
`
`conelations by a factor of 1·8 to 1·9. This meant that the
`penneability of the nail plate was about twice as sensitive to a
`change of the molecular size as that of the hoof membrane.
`Both results (the lower permeability, but higher slope in the
`case of the nail plate) could be explained by the denser net(cid:173)
`work of the nail keratin matrix. Th.is demanded that the
`molecules bad to diffuse a longer way due to the greater pore
`tortuosity and the penetration rate was therefore reduced in
`general. On the other band, the penetration rate in the pores
`was reduced by the increased friction between the diffusing
`molecules and the gel network, which meant that the radius of
`the solvated molecule (rs) became closer to the pore radius of
`the network (rp) (Flynn et al 1974). The close-meshed keratin
`network of the nail plate contains few pores in the order of
`magnitude of the larger diffusing molecules, which are hin(cid:173)
`dered to a stronger extent than smaller ones. The higher dif-
`
`FlatWing Ex. 1031, p. 3
`
`

`

`PENETRATION OP ANTIMCYOTICS THROUGH NAD.. PLATE AND TIIEffi EFFICACY
`
`869
`
`I ,
`
`.- 10-s
`~
`"'e
`~ 10-9
`
`10-10.l!:::!!:::!..~~~~~~~~~~~~~~
`10-e
`10-8
`
`FIG. 3. Relationship between the permeability coefficient of the nail
`plate PN and the permeability coefficient of the hoof membrane PH at
`32°C (n = 3-8, means ± s.d.). 0 nicotinic acid esters; e remaining
`substances. Log PN=3·723 + 1·751 log PH, r=0·971.
`
`fusional resistance of the nail keratin cannot only be explained
`by the lower swelling in the aqueous milieu compared with
`that of the hoof membrane. With approximately 27% it was
`only slightly below the corresponding value of the hoof
`membrane (36%) (Mertin 1995). A distinct difference
`regarding the structure of the two barriers must be the reason.
`The permeabilities of both the nail plate and the hoof
`membrane derive from the molecular size of the drugs and can
`be therefore calculated. It is not necessary to have infonnation
`about partition parameters as in the case of the stratum cor(cid:173)
`neum; correlations using the molar volume instead of the
`molecular weight showed that this parameter has no advantage
`(Mertin 1995).
`
`Prediction of the nail permeability
`Since nail plates are only available to a certain extent for the
`preclinical development of topical drugs, it is of interest to
`calculate the expected permeability coefficient of the nail from
`a determined value using the hoof membrane mode I. Although
`the permeability coefficients of the nail plate and the hoof
`membrane differ from each other, it has been shown that the
`bovine hoof membrane may serve as an appropriate model for
`the nail, because both are hydrophilic gel membranes (Mertin
`& Lippold 1997). As the logarithm of the permeability coef(cid:173)
`ficient represents the correlating parameter, the nail plate
`penneability of a drug can be derived directly from a plot of
`log P of the nail plate (log PN) vs log P of the hoof membrane
`(log PH) after experimental determination of PH (Fig. 3). The
`drug permeability of the nail plate evaluated by this procedure
`should better correspond to the real value than the direct cal(cid:173)
`culation using molecular weight, according to equations 5 and
`8, or the parameters in Figs 1 and 2, respectively, since the
`experimental. determination of the penetration through the hoof
`membrane considers the characteristics of a substance (e.g.
`interactions with keratin) to a larger extent.
`The hoof membrane is therefore a suitable in-vitro model
`regarding the prediction of the penneability of the nail plate.
`The result is the following equation (Fig 3):
`log PN = 3.723 + J.751 log P8
`
`(IO)
`
`Penetration of the antimycotics and its prediction
`The permeability coefficients of the antimycotics through the
`hoof membrane in the ethanol-containing medium range from
`
`-7·0 00
`
`a."
`CD -8·0
`.s
`
`-9·0
`
`200
`
`400
`
`600
`MW
`
`800
`
`1000
`
`Flo. 4. Relationship between the logarithm of the penneability
`coefficient of the anlimycotics through the hoof membrane Pa and
`the molecular weight "MW (n =4, means). Medium: ethanol 42% (v/v)
`pH 8·1. Pa is expressed in cm2 s- 1• 0 nicotinic acid esters;
`• antimycotics. Plot according to Cohen & Turnbull (1959) (Eqn 5):
`log P1tEt0e= -6·795 -0·002427 MW, r=0·931.
`
`-7·0
`
`o..x.-8·0
`_g
`
`-9·0
`
`•
`
`nystatin
`
`2·0
`
`2·2
`
`2·4
`2·6
`Log MW
`
`2·8
`
`3·0
`
`FIG. 5, Relationship between the logarithm of the permeability
`coefficient of the antimycotics through the hoof membrane Pa and
`the logarithm of the molecular weight MW (n=4. means). Medium:
`ethanol 42% (v/v) pH 8·1. Pa is expressed in cm2 s- 1• 0 nicotinic
`acid esters; • antimycotics. Plot according to Lieb & Stein (1969)
`(Eqn 8): log Pa.acm= -2·224-2·181 log MW, r=0·915.
`
`O·lOx 10-8 to4·08x10-8 cm2 s- 1 (Table1, lower part and
`Figs 4 and 5, respectively). The distinct decrease in the
`penetration rate to one-fourth or one-fifth compared with the
`pure aqueous milieu was a result of the de-swelling effect of
`the ethanol. The swelling of the keratin membrane decreased
`from 36% (m/m)- to 27% (m/m) due to replacing water by
`ethanol 42% (v/v) (Mertin 1995). The correlation according to
`Cohen & Turnbull (Fig. 4) as well as according to Lieb & Stein
`(Fig. 5) again showed that there was a clear relationshlp
`between the permeability coefficient and the molecular weight.
`The correlation coefficients were similar to those determined
`in the pure aqueous milieu, but the Cohen-Turnbull plot
`seemed to have a slight superiority. For both correlations the
`regression coefficients of the respective straight lines of the
`data determined in ethanol 42% (v /v) were higher than in the
`case of the aqueous solutions: 0·002427 vs 0·002071 (Cohen &
`Turnbull) and z.181vs1·782 (Lieb & Stein). This was also the
`consequence of the decrease of the membrane swelling in
`ethanol with a higher sensitivity of the permeability towards an
`
`FlatWing Ex. 1031, p. 4
`
`

`

`\ ~
`
`870
`
`DIRK MERTIN AND BERNHARD C. LIPPOLD
`
`Table 2. Physicochemical and antimicrobial properties of the antimycotics.
`
`pK,.,EIOH
`
`Pat0H
`
`Amorolfi.ne
`Bifonazole
`Ciclopirox
`Clotnmazole
`Econazole
`Griseofulvin
`Kctoconazole
`Naftifine
`Nyscatin
`
`6·6 (water)"'
`5·11±0·04
`8-07±0·05
`4·74±0·04
`5·38±0·04
`no acidic or basic groups
`5·20±0·10
`6-80±0·03
`pK.1: about 4·0
`pK.,: 7-73±0-03
`
`Tolnaftatc
`
`no acidic or basic groups
`
`0·0316 (water)
`0·0010
`0·517
`0·0004
`0-0019
`
`0·0013
`0-0477
`
`1·00
`0·299 (zwitter ion)
`0·701 (negative)
`
`c ..
`
`9995
`0-35
`8590
`3·0
`1020
`10·4
`10·6
`8650
`
`18°6
`
`Ca1-4
`
`8·8
`0-13
`1020
`2·1
`11-5
`10·1
`7.9
`2·9
`
`18·5
`
`0-07
`
`0-11
`
`MlC0
`
`0·01
`0.1
`2·0
`2·3
`0-35
`3·1
`2·23
`0-55
`
`4.5
`
`O·SS
`
`Ml Cy
`
`o.ss
`0·89
`2-0
`35
`100·0
`
`25
`so
`3
`
`PK..Bt0H: dissociation constant in ethanol 42% (v /v) (n = 6--9, means :l: max. deviation). Pa oH: degree of dissociation in ethanol 42% (v /v) at
`pH 8· l. C1.,.: water solubility at 32°C (means, n = 2) expressed in mg L - i. C.74:solubility in phosphate buffer pH 7·4 at 32°C (means, n = 2)
`expressed in mg L _,. MICo. MICy: MIC against dennatophytes or yeasts, respectively, calculated as the geometrical mean of the limits of the
`highest range given in the literature (Plempel & Stetter 1987; Wilson & Ryley 1990; McEvoy & Litvak 1993) expressed in mg L - 1. •Hofmann·La
`Roche AG (1992). ED, EY: efficacy coefficients against dermatophytes and yeasts, respectively, taking into account the calculated maximum fluxes
`from water, expressed in cm s- 1
`•
`
`alteration of the molecule size due to the denser structure of the
`keratin filaments.
`Antimycotics, which differ to a larger extent from the
`regression line, are labelled in the diagrams. Ciclopirox,
`deviating in both plots, was dissociated at pH 8-1 to about SOo/o
`(Table 2) and was inhibited as an anion in its penetration
`through the negatively charged keratin membrane due to the
`Donnan equilibrium (Mertin & Lippold 1997). A similar
`argwnent can be applied to nystatin, which was present as an
`anion to 70%. Although griseofulvin had a high affinity
`towards keratin (ICl·Pharma 1981), its rather low penneability
`coefficient was probably not due to the sorption phenomenon.
`It rather represented, as did the deviation of econazole, a
`normal experimental error.
`Since the Coben·Tumbull correlation led to a better adap·
`tation of the permeability coefficients and was theoretically
`better sustained than the Lieb--Stein plot, it was used in the
`following calculations to predict the penetration of the anti·
`mycotics through the nail plate. A direct calculation of the naiJ
`plate permeability according to Fig. 3 was not possible due to
`the different substances and media used. Combining the
`regression equations concerning the permeability of the nail
`plate in water (Eqn 11) and also the hoof membrane in ethanol
`42% (Eqn 12) resulted in equation 13 after transformation:
`
`log PN = -7·296-0·003708 MW
`
`log Pa.E<OH = -6·795 -0·002427 MW
`
`(11)
`
`(12)
`
`log PN = 1·528 x log Pa.&<lH + 3·085
`According to equation 13, the permeability coefficients of the
`antimycotics through the nail plate in an aqueous medium
`could be derived from the experimental data in ethanol 42%
`(v/v). Taking the water solubility of the drug C,w (Table 2)
`into account, the maximum flux through the nail plate was
`calculated according to equation 14:
`
`(13)
`
`PN
`Jmax = he . Csw
`
`(14)
`
`The values were standardized to a barrier thickness of
`he= 1000 µm (Jmax(lOOO µm)). As information about the pH
`value in the nail or its buffer capacity was not available, the
`water solubility C,w instead of the solubility in phosphate
`buffer pH 7.4 was used.
`While the expected permeability coefficients of the various
`antimycotics through the nail only differed by a factor of
`the maximum
`fluxes
`to
`100,
`from 10-s
`ranged
`10-3 mg cm- 2 s- 1 (Table 3) due to the influence of the
`
`Table 3. Permeability coefficients PN and maximum flux Imax(lOOO µm) of the antimycotics through the nail
`plate and their predicted efficacy against dennatophytes En and yeasts Ey, calculated from the experimental
`data (Paetruil according to equations 13, 14 and 15.
`1_(1000 µml
`(mg cm- 2 s- )
`
`PN
`(cm2 s- 1)
`
`E,,
`(cm s-1)
`
`Ev
`(cm s- 1)
`
`Amorolfine
`Bifonazole
`Ciclopirox
`Clotrimazole
`Econazole
`Griseofulvin
`Ketoconazole
`Naftifine
`Nystatin
`Toloaftate
`
`2·15x 10- 9
`3·98x 10-9
`2·30x 10-9
`2.59 x 10-9
`4·66x 10-9
`7·27x 10- 10
`5·52x 10- 10
`6-23 x 10-9
`2·16x 10- 11
`4·80x 10- 9
`
`2·15 x 10-4
`1·39 x 10-8
`1·98 x 10-4
`7.77 x 10-s
`4·74X 10-S
`1·56 x 10-s
`5·85 x 10-s
`5-38 x 10-4
`4·02X 10-9
`3·36 x 10-9
`
`2·15 x 10- 2
`1·39x10-7
`9·87 x 10-s
`3·38 x 10- 8
`1·35 x 10-4
`2·44X 10- 8
`2·62x io- 8
`9·78x 10-4
`8·93X10-IO
`6·11x10-9
`
`3.91x10-4
`1·56x 10-8
`9·87 x 10-s
`2·22x 10-9
`4·74x 10-7
`
`2·34x 10-9
`1·08 x 10-s
`1·34x 10-9
`
`FlatWing Ex. 1031, p. 5
`
`

`

`PENETRATION OF ANTIMCYOTICS TIIROUGH NAIL PLATE AND THEIR EFFICACY
`
`solubiiity (Table 2). The maximum flux of the investigated
`compounds was influenced to a larger extent by their saturation
`concentrations than by their permeability coefficients. The
`high solubility of the antimycotic salts (amorolfine hydro(cid:173)
`chloride, ciclopirox olamine, econazole nitrate and naftifine
`hydrochloride) in water, in contrast to buffer pH 7·4 (Table 2).
`resulted as expected in a high maximum flux. So the pene(cid:173)
`tration inhibition, which the protonated molecules were sub(cid:173)
`jected to in a keratin membrane, can be over-compensated by
`the solubility improvement (Mertin & Lippold 1997). The
`slightly-water-soluble bases amorolfine, econazole and nafti(cid:173)
`fine profited from this, while ciclopirox already had a high
`basal solubility in water (about 500 mg L - 1). The calculated
`maximum fluxes of about 10- 7 mg cm-2 s- 1 (clotrimazole,
`griseofulvin, ketoconazole) were of a medium size, whereas
`only small fluxes could be expected for bifOnazole and tol(cid:173)
`naftate due to their slight solubility, and for nystatin due to its
`high molecular weight. Assuming a buffered milieu in the nail
`(pH 7-4), the maximum fluxes of amorolfi.ne, econazole and
`naftifine strongly decreased, as they now were only slightly(cid:173)
`water-soluble bases (Mertin 1995).
`
`Prediction of the topical efficacy of the antimycotics
`Not only the flux of the drug through the nail plate and
`therefore the obtained concentration therein is of importance
`for the clinical success of a topical therapy against onycho(cid:173)
`mycoses, but also the sensitivity of the fungi towards the
`antimycotic. Numerous investigations show that the antifungaJ
`in-vitro activity, whose measure is the minimum inhibitory
`concentration (MIC), does not always coincide with the inhi(cid:173)
`bition in-vivo (Galgiani 1987; Wilson & Ryley 1990; Rex et al
`1993) as the determination of the MIC reacts in a very sensi(cid:173)
`tive way to the experimental conditions.
`Due to the lack of other parameters, characterizing the
`activity of the antimycotics, the MIC should still lead to an
`estimation of the therapeutic efficacy. It is proportional to the
`drug concentration available at the site of action, which
`depends on the maximum flux, and to the reciprocal of the
`MlC. An efficacy coefficient E is therefore introduced, which
`should be maximum for high therapeutic effectiveness:
`
`E
`
`(15)
`
`J~(IOOOµm)
`MIC
`In the literature (Plempel & Stelter 1987; Wilson & Ryley
`1990; McEvoy & Litvak 1993), the MIC is usually given as a
`range and differs for varying strains. To obtain just one char(cid:173)
`acteristic value, the geometrical mean was calculated from the
`lowest and highest value of the referenced range. As the MIC
`vaJues for the different dennatophyte types on the one hand
`and the yeast types on the other hand do not differ considerably
`from each other, a unified value for dennatophytes and yeasts
`respectively was taken in order to simplify the calculation. In
`the case of contradictory values in the literature, the greatest,
`that is for the therapeutic success most adverse MIC, was
`chosen (Table 2). Dennatophytes are usually more sensitive
`towards antimycotics than yeasts; griseofulvin and toJnaftate
`do not posses any activity against yeasts. Moulds were not
`taken into consideration, because they are only of secondary
`importance in the case of onychomycoses.
`The efficacy coefficient E (Table 2), which may estimate the
`therapeutic potency of an antimycotic against onychomycoses
`
`871
`caused by dermatophytes or yeasts, respectively, was calcu(cid:173)
`lated on the basis of the maximum flux from water (Table 3). A
`high maximum flux through the nail plate, expected for sub(cid:173)
`stances which possess a high water solubility (amorolfine
`hydrochloride, ciclopirox olamine, econazole nitrate and naf(cid:173)
`tifine hydrochloride) resulted also in an elevated E value
`against dennatophytes oo-4-10- 2 cm' s- 1
`). The high in(cid:173)
`vitro activity of bifonazole and tolnaftate (Table 2) compen(cid:173)
`sated for their low flux only to a certain extent. With the
`exception of ciclopirox and nystatin the efficacy coefficients
`towards yeasts decreased due to the lower activity of the
`antimycotics against these micro-organisms. However, eco(cid:173)
`nazole nitrate, naftifine hydrochloride and especially amor(cid:173)
`olfine hydrochloride and ciclopirox olamine could be judged
`favourably relative to the other drugs. If the maximum flux was
`determined by the drug solubility at pH 7.4, only amorolfine
`and ciclopirox showed a high activity against both classes of
`fungi, the former due to its low MIC and the latter due to the
`high solubility of the undissociated form. On the contrary,
`econazole and naftifine are only indicated for infections by
`dermatophytes. The remaining antimycotics did not seem to be
`suitable for the topical treatment of onychomycoses compared
`with those previously mentioned. Thus, the investigations
`support the fact that among the studied drugs, only amorolfine
`and ciclopirox are present in the market as topical preparations
`against onychomycoses. However, econazole and naftifine
`hydrochloride are also potential candidates, if one sue<:eeds in
`excluding the probable buffer effect of the nail interior and is
`therefore able to capitalize on their high solubility in water.
`Although the results should be interpreted with caution they
`suggest that by determining the penetration rate of an anti(cid:173)
`mycotic through hoof membrane in conjunction with calcula(cid:173)
`tion of the efficacy coefficient E, prediction of the
`effectiveness of an antifungal drug in the topical therapy of
`onychomycoses appears promising.
`
`References
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`Constants. Chapman and Hall, New York, pp 14-38
`Cohen, M. H., Turnbull, D. (1959) Molecular transport in liquids and
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`Documenta Geigy (1975) Wissenscbaftliche Tabellen. G. Thieme
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`phenomena and models: theoretical concepts. J, Phann. Sci. 63:
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`Galgiani, J. N. (1987) Antifungal susceptibility tests. Antimicrob.
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`Hoffmann-La Roche AO (1992) Loccryl - Standard information flir
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`ICI-Phanna (1981) Fulcin S 500, Fulcin S., Plankstadt
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