`

`(
`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-
`
`MYLAN - Ex. 1031, p. 2
`
`

`

`

`

`

`

`\ ~
`
`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
`
`MYLAN - 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
`Albert, A., Serjeant, E. P. (1984) The Detennination of Ionization
`Constants. Chapman and Hall, New York, pp 14-38
`Cohen, M. H., Turnbull, D. (1959) Molecular transport in liquids and
`glasses. J, Chem. Phys. 31: 1164-1169
`Documenta Geigy (1975) Wissenscbaftliche Tabellen. G. Thieme
`Verlag, Stuttgart
`Flynn, G. L., Yalkowsky, S. H., Roseman, T. J. (1974) Mass transport
`phenomena and models: theoretical concepts. J, Phann. Sci. 63:
`479-510
`Galgiani, J. N. (1987) Antifungal susceptibility tests. Antimicrob.
`Agents Chemother. 31: 1867-1870
`Grunwald. E., Berkowitz., B. J. (1951) The measurement and correla(cid:173)
`tion of acid dissociation constants for carboxylic acids in the system
`ethanol-water. Activity coefficients and empirical activity functions.
`J, Am. Chem. Soc. 73: 4839-4944
`Gutbezahl, B., Grunwald, E. (1953) The effect of solvent on equili(cid:173)
`brium and rate constants. II. The measurement and correlation of
`acid dissociation constants of anilinium and ammonium salts in the
`system ethanol-water. J. Am. Chem. Soc. 75: 559-574
`Hoffmann-La Roche AO (1992) Loccryl - Standard information flir
`Krankenhausapotheker., Grenzach-Wyhlen
`ICI-Phanna (1981) Fulcin S 500, Fulcin S., Plankstadt
`Kuminis, C. A., Kwei, T. K. (1968) Free volume and other theories. In:
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`London, pp 107-139
`Lieb, W.R., Stein, W. D. (1969) Biological membranes behave as non(cid:173)
`porous polymeric sheets with respect to the diffusion on non·
`electrolytes. Nature 224; 240-243
`
`MYLAN - Ex. 1031, p. 6
`
`

`

`872
`DIRK MERTIN AND BERNHARD C. LIPPOLD
`McEvoy, G. K., Litvak, K. (1993) AHFS Drug Wormation 93.
`Potts, R. 0., Guy, R. H. (1993) The prediction of percutaneous
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`penetration: a mechanistic model. In: Gumy, R., Teubner, A.
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`Herkunft. Verlag Mainz, Aachen pp 114-118
`pp 153-160
`Rex, J. H., Pfaller, M. A., Rinaldi, M. G., Polak, A., Galgiani, J. N.
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`Wilson, R. G., Ryley,J. F. (1990) Screening and evaluation in vitro. In:
`their permeability and ma.ximwn flux. J. Phann. Phannacol. 49: 30-34
`Pleropel, M., Stelter, J, (1987) Antimykotika. In: Kleemann, A.,
`Ryley, J.P. (ed.) Chemotherapy of Fungal Diseases (Handbook of
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`Experimental Pharmacology, Vol. 96). Springer-Verlag, Berlin, pp
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`115-128
`
`MYLAN - Ex. 1031, p. 7
`
`

`

`' I
`‘5.
`
`•
`
`•
`
`\
`
`MYLAN - Ex. 1031, p. 8
`
`MYLAN - Ex. 1031, p. 8
`
`