European Journal of Pharmaceutical Sciences 21 (2004) 471–477
`
`In vitro permeation of several drugs through the human nail plate:
`relationship between physicochemical properties and nail
`permeability of drugs
`
`Yoichi Kobayashi a, Tsunehisa Komatsu a, Machiko Sumi a, Sachihiko Numajiri a,
`Misao Miyamoto b, Daisuke Kobayashi a, Kenji Sugibayashi a, Yasunori Morimoto a,∗
`
`a Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan
`b Nissan Chemical Co., Ltd., 3-7-1 Kanda-Nishiki-cho, Chiyoda-ku, Tokyo 101-0054, Japan
`
`Received 10 April 2003; received in revised form 10 October 2003; accepted 17 November 2003
`
`Abstract
`
`The objectives of the present study are to clarify the relationship between the physicochemical properties and the nail permeability of drugs
`through human nail plates. Homologous p-hydroxybenzoic acid esters were used to investigate the relationship between the octanol/water
`partition coefficient and the permeability coefficient of several drugs. The nail permeability was found to be independent of the lipophilicity
`of a penetrating drug. However, the nail permeability of several model drugs was found to markedly decrease as their molecular weights
`increased. The nail permeability of an ionic drug was found to be significantly lower than that of a non-ionic drug, and the nail permeability of
`these drugs markedly decreased as their molecular weights increased. The permeation of a model drug, 5-fluorouracil (5-FU), through healthy
`nail plates was also determined and compared with that through nail plates with fungal infections. The drug permeation through a nail plate
`decreased with an increase in nail plate thickness. Nail plates with fungal infections exhibited approximately the same 5-FU permeation as
`healthy nail plates. We suggest that the permeability of a drug is mainly influenced by its molecular weight and permeability through nails
`with fungal infection can be estimated from data on healthy nail permeability.
`© 2004 Elsevier B.V. All rights reserved.
`
`Keywords: Nail; Nail permeation; Fungal nail plate; Onychomycosis; Antifungal agents
`
`1. Introduction
`
`Onychomycosis has been treated mainly with oral anti-
`fungal medication (Piepponen et al., 1992; Villars and Jones,
`1992). This oral therapy, however, sometimes has severe
`systemic side effects, which interrupt treatment (Wilson and
`Plunkett, 1962). On the other hand, it is well known that
`topical treatment is not widely used in onychomycosis ther-
`apy. The anticipated low levels of nail penetration and per-
`meation during topical antifungal drug exposure are very
`significant factors in onychomycosis therapy. Only a few
`drug permeation studies have been performed on the human
`nail plate and, as a consequence, the mechanisms behind
`healthy and fungal nail permeation have yet to be confirmed.
`Walters et al. (1983) have suggested that the nail plate be-
`
`Corresponding author. Tel.: +81-49-271-7685;
`∗
`fax: +81-49-285-5863.
`E-mail address: morimoto@josai.ac.jp (Y. Morimoto).
`
`0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
`doi:10.1016/j.ejps.2003.11.008
`
`haves like a hydrophilic gel membrane and that there is an
`additional lipophilic route, as revealed by an in vitro pen-
`etration study of homologous alcohols through the human
`nail plate. Mertin and Lippold (1997) also suggested that
`the nail plate behaves like a hydrophilic gel membrane and
`that the dissociation of a penetrating drug leads to a re-
`duction in the penetration rate, as revealed by an in vitro
`penetration study of homologous nicotinic acid esters, ben-
`zoic acid and pyridine through the human nail plate and
`bovine hoof membrane. Since they used healthy nails from
`dead men and women for the in vitro permeation study, they
`were probably not able to carry out many permeation stud-
`ies using human nail plates. We have developed a modified
`side-by-side diffusion cell using nail tip pieces from healthy
`volunteers in order to investigate the nail penetration mech-
`anism and enhancing system. From the results we obtained
`using our in vitro technique, we suggest that drug diffu-
`sion in the upper layer of the human nail plate is the main
`barrier to nail permeation and that the full-thickness nail
`
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`Y. Kobayashi et al. / European Journal of Pharmaceutical Sciences 21 (2004) 471–477
`
`plate behaves like a hydrophilic gel membrane (Kobayashi
`et al., 1999). In addition, we found that N-acetyl-l-cysteine
`and 2-mercaptoethanol are able to enhance drug permeation
`through the human nail plate (Kobayashi et al., 1998). How-
`ever, it is very difficult to evaluate antifungal drug perme-
`ation, because of the very low nail permeability involved.
`In order to clarify the nail permeation mechanism of drugs
`through human nail plate, we studied the relationship be-
`tween the physicochemical properties and the healthy nail
`permeability of several drugs. To observe the effect of oc-
`tanol/water partition coefficients on nail permeability coeffi-
`cients, homologous p-hydroxybenzoic acid esters were used
`as model drugs. The effects of the molecular weight and drug
`dissociation on nail permeability were also investigated in a
`nail permeation study using several model drugs. We inves-
`tigated the relationship between the flux of a model drug,
`5-fluorouracil (5-FU), and the healthy or fungal nail plate
`thickness.
`
`2. Experimental
`
`2.1. Materials
`
`Antipyrine, 5-FU, p-hydroxybenzoic acid esters (methyl
`ester, MP; ethyl ester, EP; propyl ester, PP; butyl ester,
`BP; amyl ester, AP; hexyl ester, HP) and sodium nicotinate
`were obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan).
`Aminopyrine, barbital sodium, benzoic acid, ethanol, pro-
`caine hydrochloride, pyridine and sodium benzoate were ob-
`tained from Wako Pure Chemical Industries (Osaka, Japan).
`Deuterium oxide was obtained from Merck Co. (Darmstadt,
`Germany). Isoproterenol hydrochloride, lidocaine, lidocaine
`hydrochloride and mexiletin hydrochloride were obtained
`from Sigma Chemical Co. (St. Louis, MO, USA). Crocona-
`zole hydrochloride was supplied by Shionogi & Co. (Osaka,
`Japan). Isosorbide dinitrate was supplied by Toko Pharma-
`ceutical Ind. Co. (Tokyo). All other reagents were obtained
`from commercial sources.
`
`2.2. Preparation of the nail plate
`
`Healthy nail tip pieces were obtained from the fingers and
`toes of healthy volunteers (15 males and 5 females; mean
`age 25 years, range 20–45) using nail clippers. Nail pieces,
`which had been allowed to grow for at least one month,
`were used in this permeation study. Fungal nail plates
`were supplied from Saiseikai Central Hospital (Tokyo) and
`Saitama Medical Center (Saitama Medical School, Saitama,
`Japan). The thickness of the healthy and fungal nail pieces
`was measured with a micrometer (Mitutoyo Corp., Japan)
`equipped with pointed metal attachments. Healthy nail
`plates, having a thickness of about 400 ␮m (350–450 ␮m),
`were used to evaluate the effect of the octanol/water par-
`tition coefficient, molecular weight and dissociation of the
`penetrant on the nail permeability. Both fungal and healthy
`
`nail plates were used to make a comparison between the
`different drug permeabilities. After healthy and fungal nail
`pieces were hydrated for a day, they were used to evaluate
`drug permeability.
`
`2.3. Determination of solubilities and octanol/water
`partition coefficients
`
`The drug suspensions were mixed with a magnetic stirrer
`◦
`C. After 12 and 24 h, each suspension was subjected
`at 37
`to filtration (Ekicrodisc 3; German Sciences Japan, Ltd.,
`Tokyo). The filtrate was immediately diluted with methanol
`or acetonitrile to obtain samples for analysis. No difference
`in drug solubility was observed between 12 and 24 h. The
`octanol/water partition coefficient of the drugs (Kow) was
`◦
`defined as the solubility ratio in octanol/water at 37
`C. A
`few of the values were taken from the literature (Morimoto
`et al., 1992).
`
`2.4. Permeation studies
`
`A piece of nail plate was sandwiched between two
`adapters made of polypropylene with an O-shaped ring
`(effective diffusion area 0.049 cm2) and mounted in a
`side-by-side diffusion cell (1.5–2.5 ml) with a water jacket
`◦
`connected to a water bath at 37
`C (Kobayashi et al., 1998).
`The dorsal nail plate side was filled with a drug suspension
`or solution (almost all drugs were applied as suspensions,
`but for drugs with a high solubility, the dorsal nail plate side
`was filled with drug solution. The maximum flux was eval-
`uated from the flux obtained with the applied concentration
`and drug solubility. In both cases, the drug concentration in
`the dorsal nail plate side was almost constant throughout the
`nail permeation studies) and the ventral nail plate side was
`filled with distilled water. No preservative was added be-
`cause the receiver solution was clear even at the end of the
`experiment. Drug permeation was measured by sampling
`the solution on the ventral nail plate side at pre-determined
`times. The experimental period was 5–17 days, because of
`the low degree of nail permeability of the drugs used.
`
`2.5. Analytical methods
`
`Deuterium oxide was quantified from the intensity of
`−1 with
`the O–D stretching vibrational band at 2512 cm
`an infrared spectrophotometer (260-30, Hitachi, Tokyo)
`(Hatanaka et al., 1993). Ethanol was measured by GC
`as described previously (Kobayashi et al., 1997). Other
`drugs were determined by HPLC. Sample solutions were
`injected into an HPLC consisting of a pump system
`(LC-10A, Shimadzu Seisakusho, Kyoto, Japan), an UV
`detector (SPD-10A, Shimadzu), a chromatopack (C-R5A,
`Shimadzu), a system controller (SCL-10A, Shimadzu), an
`auto injector (SIL-10A, Shimadzu), and a reverse-phase
`column (Inertsil ODS 250 mm × 4.6 mm i.d., GL Sci-
`ences Inc., Tokyo). The mobile phase, which consisted
`
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`Y. Kobayashi et al. / European Journal of Pharmaceutical Sciences 21 (2004) 471–477
`
`473
`
`of methanol/0.1% phosphoric acid or acetonitrile/0.1%
`phosphoric acid mixtures, with or without ion-pair chro-
`matography reagents (sodium 1-hexanesulfonate or sodium
`dodecylsulfate), was pumped at flow rate ranging from 1 to
`1.5 ml/min. No other peak apart from the drug and inter-
`nal standard was observed on the HPLC recording. It was
`clear that the permeating drugs were stable and no material
`leached from the nail plates during the permeation studies.
`
`3. Results and discussion
`
`3.1. Influence of the octanol/water partition coefficient on
`the permeability coefficient
`
`through the
`The steady-state permeation of drugs
`solution–diffusion membrane is characterized by Fick’s law:
`J = DmKmCV
`h
`
`(1)
`
`in which J is the steady-state flux, Km is the membrane/donor
`vehicle partition coefficient of the drug, Dm is the diffusion
`coefficient of the drug in the membrane, CV is the concen-
`tration in the donor solvent and h is the membrane thick-
`ness. The permeability coefficient (P) can be characterized
`as follows:
`P = DmKm
`h
`
`(2)
`
`.
`
`To predict the skin permeability of drugs, Potts and Guy
`(1992) carried out their analysis using a mathematical model
`based on permeant size (molecular volume (MV) or molec-
`ular weight (MW)) and octanol/water partition coefficient
`(Kow). The functional dependence of Dm on MW is expo-
`nential and it can be characterized by:
`Dm = D0 exp(−β MW)
`
`(3)
`
`(5)
`
`MW
`
`where D0 represents the diffusivity of a hypothetical
`molecule having zero molecular weight and β is a constant.
`The relationship between Km and Kow is better expressed as:
`Km = [Kow]f
`(4)
`where the coefficient, f, accounts for the difference between
`the partitioning domain presented by octanol and that pre-
`sented by the membrane lipids. A combination of Eqs. (2),
`(3) and (4) yields Eq. (5):
`log P = log(D0/ h) + f log Kow − β(cid:8)
`where β(cid:8) = β/2.303.
`The effect of the octanol/water partition coefficient on
`the permeability coefficient of each drug was investigated.
`Fig. 1 shows a typical permeation profile of one of the
`model drugs, p-hydroxybenzoic acid methyl ester, through
`the healthy human nail plate. Steady-state fluxes of homol-
`ogous p-hydroxybenzoic acid esters can be observed from
`4.5 to 6.5 days allowing calculation of the nail permeability
`coefficients.
`Table 1 shows the physicochemical parameters and nail
`permeability coefficients of homologous p-hydroxybenzoic
`acid esters used as model drugs in this investigation. The
`molecular weights of homologous p-hydroxybenzoic acid
`esters covered a narrow range from 152.12 to 222.28. The
`logarithm values of the octanol/water partition coefficients
`varied widely from 1.53 to 4.25.
`Fig. 2 shows the relationship between the octanol/water
`partition coefficient
`and the permeability coefficient
`of p-hydroxybenzoic acid esters. The permeability of
`p-hydroxybenzoic acid esters did not increase with an in-
`crease in lipophilicity. If a nail plate behaves as a lipid par-
`tition membrane, the slope (f) should be clearly greater than
`0; but it was nearly zero (f = −0.160). Multiple regres-
`sion analysis was carried out to clarify which parameters
`(molecular weight or octanol/water partition coefficient)
`contribute to the permeability of p-hydroxybenzoic acid
`
`Flux
`
`180
`
`150
`
`120
`
`90
`
`60
`
`30
`
`MPflux (ug/cm2.day)
`
`Cumulative amount
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`0
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`900
`
`800
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`MP permeated (ug/cm2)
`
`Time (day)
`Time (day)
`Fig. 1. Typical permeation profile of p-hydroxybenzoic acid methyl esters through the healthy human nail plate. Each value represents the mean ± S.E.
`(n = 4).
`
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`Y. Kobayashi et al. / European Journal of Pharmaceutical Sciences 21 (2004) 471–477
`
`in the human nail plate has been found to be much lower than
`in the stratum corneum of skin (Walters and Flynn, 1983).
`Thus, there does not appear to be an additional lipophilic
`route in the human nail plate.
`
`3.2. Influence of the molecular weight and dissociation on
`the permeability coefficient
`
`Nail permeability was found to be independent of the
`lipophilicity of the drug used. From the findings in this early
`study, the coefficient, f, inEq. (5) can be assumed to equal
`zero. Thus, substitution of f = 0 in Eq. (5) gives a simple
`(cid:1)
`(cid:2)
`model as follows:
`log P = log
`D0
`h
`
`− β(cid:8)
`
`MW.
`
`(6)
`
`We investigated the nail permeability of several model
`drugs having various molecular weights. Because the influ-
`ence of drug dissociation on the nail permeability is not
`clearly understood, the nail permeability of non-ionic and
`ionic drugs was evaluated separately.
`Table 2 summarizes the pKa, pH of the donor solution,
`molecular weight and permeability coefficient of the model
`drugs used in this investigation. All the model drugs were
`in the non-ionic form in the donor solution. The molecular
`weight of the model drugs ranged from 20 to 240. The nail
`permeability of drugs having a molecular weight of above
`240 could not be determined, because of the low nail perme-
`ability. The nail permeation data of p-hydroxybenzoic acid
`esters (pKa = 8.4, donor pH = 3.8–5.8) were added to
`this investigation because they exist in non-ionic form in the
`donor solution.
`According to Eq. (6), Fig. 3 shows the relationship
`between the permeability coefficient and the molecular
`weight of each non-ionic drug. The logarithm of the nail
`permeability coefficient decreased as the molecular weight
`of the penetrating drug increased. A linear relationship
`(r = −0.860, P <0 .01) existed between the permeability
`coefficient and the molecular weight of the model drug,
`the slope (β(cid:8)
`) and the intercept (log(D0/h)) being 0.00856
`
`Table 2
`Physicochemical parameters and nail permeability coefficients (h =
`400 ␮m) of non-ionic model drugs
`
`Drug
`
`pKa
`
`pHa
`
`MW
`
`b
`
`Pnon
`45.52 ± 4.30
`Deuterium oxide
`–
`7.81
`20.0
`19.81 ± 2.21
`Ethanol
`–
`7.43
`46.1
`6.36 ± 0.40
`Pyridine
`5.19
`7.35
`79.1
`12.84 ± 0.05
`Benzoic acid
`4.19
`3.21
`122.1
`2.08 ± 0.13
`5-Fluorouracil
`8.0, 13.0
`4.65
`130.1
`0.53 ± 0.07
`Antipyrine
`1.50
`6.37
`188.2
`0.09 ± 0.02
`Aminopyrine
`5.00
`8.44
`232.3
`0.39 ± 0.14
`Lidocaine
`7.92
`10.21
`234.3
`1.51 ± 0.29
`Isosorbide dinitrate
`–
`5.72
`236.1
`Each value represents mean± S.E. (5-FU, n = 30; other drugs, n = 3–4).
`a The pH in the donor solution.
`b Pnon: permeability coefficient (×107 cm/s) of non-ionic drugs.
`
`Table 1
`Physicochemical parameters and nail permeability coefficients (h =
`400 ␮m) of p-hydroxybenzoic acid esters
`
`Drug
`
`MW
`
`log Kow
`
`a
`
`152.15
`
`166.18
`
`180.20
`
`194.23
`
`208.25
`
`p-Hydroxybenzoic acid
`methyl ester (MP)
`p-Hydroxybenzoic acid
`ethyl ester (EP)
`p-Hydroxybenzoic acid
`propyl ester (PP)
`p-Hydroxybenzoic acid
`butyl ester (BP)
`p-Hydroxybenzoic acid
`amyl ester (AP)
`p-Hydroxybenzoic acid
`222.28
`hexyl ester (HP)
`Each value represents mean ± S.E. (n = 4).
`a Kow: octanol/water partition coefficient.
`b P: permeability coefficient (×107 cm/s).
`
`1.53
`
`2.23
`
`2.75
`
`3.13
`
`3.65
`
`4.25
`
`Pb
`3.68 ± 0.08
`2.43 ± 0.48
`2.01 ± 0.35
`2.38 ± 0.32
`2.24 ± 0.39
`1.24 ± 0.32
`
`esters. It was suggested that the molecular weight makes
`a greater contribution to the permeability coefficient than
`the octanol/water partition coefficient. The F-values of the
`molecular weight or octanol/water partition coefficient were
`1.9399 or 0.1058, respectively.
`From our findings, it was evident that nail plates behave
`as a hydrophilic gel membrane rather than a lipophilic parti-
`tion membrane. Although this suggestion agreed with the in-
`vestigations of Walters et al. (1983) and Mertin and Lippold
`(1997), an additional lipophilic route suggested by Walters
`et al. (1985) could not be found in the human nail plate. It
`has been reported that lipids are present in the dorsal and
`ventral plates, but only at very low levels in the intermediate
`plate which forms the main nail body (Jarrett and Spearman,
`1966; Kobayashi et al., 1999). In addition, the lipid content
`
`MP
`
`EP PP BP AP
`
`HP
`
`2
`
`3
`Log Kow
`
`4
`
`5
`
`-4
`
`-5
`
`-6
`
`-7
`
`-8
`
`-9
`
`1
`
`Log P (cm /s)
`
`Fig. 2. Relationship between the permeability coefficient (P) and the
`octanol/water partition coefficient (Kow) ofp-hydroxybenzoic acid esters.
`Each value represents the mean±S.E. (n = 4). The abbreviations attached
`to the closed circle (䊉) represent the model drugs in Table 1.
`
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`
`475
`
`Fig. 3. Relationship between the permeability coefficient (P) and the
`molecular weight (MW) of non-ionic and ionic drugs. Each value repre-
`sents the mean ± S.E. (5-FU, n = 30; other drugs, n = 3–6). The dotted
`line represents the 95% confidence interval of the regression line. The
`closed circle (䊉) represents the non-ionic drugs in Table 2. The open
`square (䊐) represents the ionic drugs in Table 3.
`
`and −5.260, respectively. This finding suggests that nail
`permeability depends on the molecular weight of the pen-
`etrating drug, i.e. the drug diffusivity in the nail plate. The
`nail permeability of non-ionic drugs (Pnon) can be pre-
`dicted approximately from the linear regression (log Pnon =
`−0.00856 MW − 5.260) and molecular weight of each
`drug.
`Table 3 summarizes the pKa, pH of the donor solution,
`molecular weight and permeability coefficient of the other
`model drugs. The model drugs existed in ionic form in the
`donor solution. The molecular weights of the ionic drugs
`used in this investigation ranged from 120 to 312.
`Fig. 3 shows the relationship between the permeability
`coefficient and the molecular weight of the ionic drug. The
`logarithm of the nail permeability coefficient decreased as
`
`Table 3
`Physicochemical parameters and nail permeability coefficients (h =
`400 ␮m) of ionic model drugs
`
`the molecular weight of the penetrating ionic drug increased.
`A linear relationship (r = −0.966, P <0 .01) also existed
`between the permeability and the molecular weight of the
`ionic drug; the slope (β(cid:8)
`) and intercept (log(D0/h)) were
`0.01030 and −5.907, respectively. The nail permeability of
`ionic drugs (Pion) can also be predicted from the linear re-
`gression (log Pion = −0.01030MW − 5.907) and molecular
`weight of the ionic form. Multiple regression analysis was
`carried out to clarify which parameters (molecular weight
`or degree of dissociation) contribute to the permeability of
`model drugs. The degree of dissociation of the model drugs
`was calculated from the pKa and pH in the donor solution.
`It was found that the molecular weight makes a greater con-
`tribution to the permeability coefficient of model drugs than
`the degree of dissociation. The F-values of the molecular
`weight or degree of dissociation were 10.9254 or 0.8599,
`respectively.
`The permeability coefficient (12.84 × 10
`−7 cm/s) of the
`non-ionic form of benzoic acid, which is an acidic drug, was
`about 10 times higher than that (0.91 × 10
`−7 cm/s) of its
`ionic form. The permeability coefficient (39.31×10
`−9 cm/s)
`of the non-ionic form of lidocaine, which is a basic drug,
`was also about 10 times higher than that (3.10× 10
`−9 cm/s)
`of its ionic form. In addition, the regression lines, which
`show the relationship between the permeability coefficient
`and the molecular weight of the non-ionic and ionic drugs,
`were parallel to each other. Furthermore, the regression line
`of the non-ionic form was about 10 times higher in position
`than that of the ionic form, as shown in Fig. 3. These results
`make it clear that dissociation leads to a reduction in nail
`permeability, irrespective of the charge on the drug. It is
`thought that the decrease in the permeability of ionic drugs
`is caused by a small increase (about 100) in the apparent
`molecular weight due to ion hydration.
`In the investigation of Walters et al. (1983), they con-
`cluded that there was little or no dependence of human nail
`permeability on the dissociation of miconazole. However,
`Mertin and Lippold (1997) contradicted this conclusion be-
`cause they found that the dissociation of benzoic acid and
`pyridine leads to a reduction in their penetration rate through
`bovine hoof membrane. They concluded that the decreased
`permeation caused by dissociation, is due to the Donnan
`effect or electrostatic repulsion between the keratin mem-
`brane and the diffusing molecule. The conclusions that we
`drew from our investigation using human nail plate agree
`with those of the latter researchers. The former researchers
`used citrate/phosphate buffer solution to evaluate the ef-
`fect of miconazole dissociation on nail permeability. Their
`buffer solution has a high ionic strength and is composed of
`compounds exhibiting different ionic forms (mono-, di- and
`tri-ionic forms) with changing pH, compared with that (con-
`stant ionic strength, µ = 0.158) used in the investigation
`of the latter group. The diffusion coefficient of the tri-ionic
`forms for citrate or phosphate is lower than that of the di-,
`mono-, and non-ionic forms (Southard et al., 1991). It seems
`that the decrease in permeability due to dissociation could
`
`Drug
`
`pKa
`
`pHa
`
`MWb
`
`Sodium benzoate
`4.19
`8.12
`Sodium nicotinate
`4.85
`7.21
`Barbital sodium
`7.91
`10.29
`Mexiletin hydrochloride
`9.0
`4.87
`Isoproterenol hydrochloride
`8.57
`4.06
`Lidocaine hydrochloride
`7.86
`4.33
`Procaine hydrochloride
`8.8
`5.27
`Croconazole hydrochloride
`6.0
`1.96
`Each value represents mean ± S.E. (n = 4–6).
`a The pH in the donor solution.
`b The molecular weight with an ionic form of the drug.
`c Pion: permeability coefficient (×107 cm/s) of ionic drugs.
`
`121.1
`122.1
`183.2
`179.3
`211.2
`235.3
`237.3
`311.8
`
`c
`
`Pion
`0.910 ± 0.136
`0.606 ± 0.204
`0.135 ± 0.016
`0.202 ± 0.057
`0.084 ± 0.013
`0.031 ± 0.003
`0.110 ± 0.016
`0.017 ± 0.009
`
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`476
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`Y. Kobayashi et al. / European Journal of Pharmaceutical Sciences 21 (2004) 471–477
`
`not be confirmed due to the interaction of various ions in
`the buffer solution. We did not use a buffer solution because
`it would have complicated the assessment of drug diffusiv-
`ity in the nail plate. The diffusivity of ω-dicarboxylic acids
`decreased about 5% following complete ionization (Albery
`et al., 1967). In a comparison of the diffusivity of zwitteri-
`onic glycine and neutral glycolamide, which have the same
`molecular formula, the diffusivity of the ionic compound is
`nearly 10% less than that of the neutral compound (Flynn
`et al., 1974). We suggest that the decrease in permeability
`is caused by a decrease in diffusivity due to ion hydration
`rather than the Donnan effect or electrostatic repulsion be-
`tween nail keratin and the penetrating drug.
`
`3.3. Comparison between healthy and fungal nail plate
`permeation of the drugs used
`
`Nail plates have different thicknesses in the fingers and
`toes of the human body. To evaluate drug permeation through
`healthy and fungal nail plates, 5-FU was selected as a model
`drug. 5-FU was used because nail permeation can easily be
`determined and because it is comparatively soluble in water
`(17.1 mg/ml). In this permeation study, 5-FU was suspended
`in all the donor vehicles.
`Fig. 4 shows the relationship between the 5-FU flux and
`the healthy nail plate thickness. 5-FU penetrates healthy nail
`plates of thickness ranging from 225 to 1050 ␮m at aflux
`of 1–28 ␮g/cm2/h. The 5-FU flux increased as the nail plate
`thickness decreased.
`The 5-FU permeation through fungal nail plates from
`eight patients was investigated (Table 4) and compared
`with that through nail plates from healthy volunteers. The
`5-FU flux through the fungal nail plate ranged from 0.5 to
`15.5 ␮g/cm2/h and tended to increase with a decrease in nail
`
`Table 4
`5-FU flux (J) through fungal nail plates from eight patients
`
`Patient number
`
`Age/gender
`
`1
`2
`3
`4
`5
`6
`7
`8
`
`–
`52/female
`–
`38/male
`42/female
`–
`48/male
`30/male
`
`ha
`
`502
`570
`626
`665
`820
`930
`955
`1158
`
`Jb
`
`6.25
`13.07
`15.41
`9.67
`5.93
`4.26
`3.23
`0.51
`
`a h: nail thickness (␮m).
`b J: flux (␮g/cm2/h).
`plate thickness. A linear relationship (r = 0.796, slope =
`0.642 ␮g/cm/h, intercept = −3.38 ␮g/cm2/h, P <0 .01) ex-
`isted between the 5-FU flux (J) through the healthy nail plate
`and the reciprocal of the nail plate thickness (1/h) according
`to Eq. (1) (Fig. 4). In thick nail plates (800–1200 ␮m), the
`5-FU flux through fungal nail plates was very similar to that
`through healthy nail plates. However, the 5-FU flux through
`thin fungal nail plates (500–700 ␮m) tended to be a little
`higher than that through healthy nail plates. No significant
`difference (P = 0.05, Fisher’s pairing t-test) was observed
`between the permeability–thickness products (P × h) of
`the healthy and fungal nail plates, calculated using Eq. (2).
`The mean thickness of the fungal nail plates used in this
`study was a little thicker than that of the healthy nail plates
`used. A fungal nail plate, particularly the deepest layer
`in the nail plate (ventral nail plate), generally becomes
`thicker than a healthy nail plate (Sagher, 1948; Jillson and
`Piper, 1957). The 5-FU flux through the fungal nail plate
`can be estimated from a change in nail plate thickness in
`Fig. 5.
`
`30
`
`20
`
`10
`
`5-FU flux (µg/cm2/hr)
`
`30
`
`20
`
`10
`
`5-FU flux (µg/cm2/hr)
`
`0
`
`0
`
`200
`
`400
`
`600
`800
`h (µm)
`
`1000
`
`1200
`
`Fig. 4. Relationship between the 5-FU flux and healthy nail plate thickness
`(␮m) (n = 78).
`
`0
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`1/h (cm-1)
`
`Fig. 5. Comparison between the healthy and the fungal nail plate fluxes of
`5-FU. The closed circle (䊉) represents fungal nail plate (n = 8), the open
`square (䊐) represents healthy nail plate (n = 78); h: nail thickness (cm).
`
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`

`Y. Kobayashi et al. / European Journal of Pharmaceutical Sciences 21 (2004) 471–477
`
`477
`
`In this investigation, very heavy fungal nail plates could
`not be used for two reasons. Firstly, the thickness of the nail
`plate cannot be determined because it is very uneven. Sec-
`ondly, the uneven nail plate collapses in water. The flux of
`drug through a very heavy fungal nail plate may be higher
`than that through a healthy nail plate because of nail de-
`struction by fungi.
`We found that the healthy nail permeability depends on
`the diffusivity of penetrant. Since an increase in nail thick-
`ness leads to a decrease in healthy or fungal nail flux, the
`fungal nail permeability may also depend on the diffusivity
`of penetrant. As a result, we suggest that the permeability
`through healthy and fungal nail plates is not significantly
`different and the fungal nail permeability can be estimated
`from healthy nail permeability data.
`
`4. Conclusion
`
`In the present study, we investigated the in vitro nail
`permeation of several model drugs. Nail permeability was
`analyzed using a simple model based on the octanol/water
`partition coefficient and the molecular weight of the drug.
`Although nail permeability was independent of the oc-
`tanol/water partition coefficient of the penetrating drug, it
`markedly decreased with increasing molecular weight. The
`dissociation of the drug led to a decrease in nail permeabil-
`ity. It appears that the decrease in the permeability of ionic
`drugs is caused by a small increase (about 100) in the ap-
`parent molecular weight due to ion hydration. It may be that
`the permeability of the fungal nail plate is approximately
`the same as the permeability of the healthy nail plate.
`
`Acknowledgements
`
`The authors wish to thank the volunteers at Josai Univer-
`sity for supplying the healthy nail samples and the Saitama
`Medical Center (Saitama Medical School) and the Saiseikai
`Central Hospital for supplying the fungal nail samples.
`
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