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The invention discloses a composition comprising a sulfur-containing glycine residue combined with urea to increase the permeation of an active agent or drug through human nail tissue. The invention further provides methods of applying the composition to a nail surface.
DETAILED DESCRIPTION OF THE INVENTION
This application claims the benefit of priority from U.S. Provisional Application Ser. No. 60/300,828, filed Jun. 27, 2001, incorporated herein by reference.
Tritiated water (3H2O) (specific activity of 1 μCi/mg) was obtained from NEN™ Life Sciences Products (Boston, Mass.). Tritiated clotrimazole, [3H]-Clotrimazole, (specific activity of 8 Ci/mmol) was obtained from Moravek Biochemicals, Inc. (Brea, Calif.). Hydroxyethyl cellulose (HEC, NATROSOL®), hydroxypropyl cellulose (HPC, KLUCEL®, Aqualon Co., Wilmington, Del.), and polyethylene glycol-20-oleyl ether (PEG-20-oleyl ether, Croda, Inc., Parsippany, N.J.) were used as received. Sodium pyrithione was received from Arch Chemicals Inc. (Norwalk, Conn.). All drugs including N-acetyl-1-cysteine, N-(2-mercaptopropionyl) glycine (MPG), pyrithione, and its zinc derivative, 8-mercaptomenthone, meso-2,3-dimercapto succinic acid, clotrimazole, sodium metabisulfite, and salicylic acid were obtained from Sigma-Aldrich Chemical Co. (St. Louis, Mo.). All other laboratory chemicals (ACS grade or better), including Scintiverse I, urea, sodium lauryl sulfate, and propylene glycol were obtained from Fisher Scientific (Springfield, N.J.) and used as received. Ethanol (200 Proof) was obtained from Aaper Alcohol and Chemical Company (Shelbyville, Ky.).
Preparation of Nails
Frozen human toenails and fingernails were obtained from tissue banks based on a protocol for size, patient history, etc. The nails were thawed at room temperature for 1 hour, and the adhering skin and tissue was removed with a pair of scissors and a scalpel. The nails were cleaned by rinsing them in a mild detergent solution (1% solution of a standard liquid soap containing sodium and potassium dodecyl benzene sulfonate and ammonium laureth sulfate), followed by two rinses in deionized water (DI water). The thickness of the nails was measured with a micrometer, and the nails were immersed in 10 mL of DI water for 24 hours just prior to use to allow complete hydration and bring all the nails to the same condition for valid comparisons (Kobayashi, et al. (1998) 46(11):1797-1802; Walters, et al. (1981) 76:76-79; Mertin and Lippold (1997) 49:30-34).
Nail Permeation Cells
Franz-type diffusion cells (Crown Glass Co., Somerville, N.J.), specially designed to hold the human nail were used (Malhotra and Zatz (2000) 51:367-377). The cleaned, trimmed nails were clamped into the diffusion cells. The area of the cell available for permeation was 0.2749 cm2. A circulating water bath maintained the temperature of the receptor compartment at 37° C. Magnetic stirrer bars ensured stirring throughout an experiment.
Enhancers tested and their concentrations are shown in Table 12.
Gels were prepared containing enhancers either alone or in combination with each other, in a vehicle that was either aqueous, hydroalcoholic, or one containing dimethylsulfoxide (DMSO). The nature of the vehicle was dependent on the solubility characteristics of the enhancer. The polymer used as the gelling agent was HPC for the hydroalcoholic and DMSO containing gels. HEC was used as the gelling agent for the aqueous gels. An appropriate control gel was prepared for each of the enhancers studied. The control gel did not contain the enhancer, but had a vehicle of the same composition (ingredients in the same proportions) as the test formulation with enhancer, for example, Gel (I) was used as a control for Gels (III)-(XI). A positive control (Gel II), based on the U.S. Pat. No. 5,696,164 containing 5% N-acetyl-1-cysteine (AC) and 20% U was also studied. Representative formulations for gels containing the keratolytic agent, urea, alone (Gel IV), the mercaptan compound, MPG, alone (Gel V), a combination of MPG and urea (Gel VII), and a control (Gel I) are shown in Table 13.
All gels were prepared using the same general procedure. Briefly, the enhancers were dissolved in the appropriate vehicle with vortex agitation. All the gels were spiked with 3H2O so as to obtain a “hot”: “cold” ratio of 1:1000, with the exception of PTO and ZnPTO gels (Gels XIV-XVII), which had a spiking ratio of 1:400. HEC/HPC was added to the mixture, and gel formation was allowed by agitating on a mechanical shaker bath at 37° C. overnight. The apparent pH of the gels was measured. Prior to use, three 10 μL aliquots of the gel were analyzed by Liquid Scintillation Counting (LSC) to validate uniformity of spiking.
Enhancer Screening Studies
Each gel formulation was screened using three replicates. Excised human toenails were used to screen all the gels, except for the gel containing the surfactant (XI), where thumbnails were used due to the limited availability of toenails. Before the start of the study, a baseline normalized water flux, J*, defined as the product of flux (J) and nail thickness, was established for all the nails. This was accomplished by monitoring the permeation of H2O through the nails from aqueous gels, followed by a washout procedure. The nails were assigned to different treatment groups, comprising three nails each, such that the mean normalized water flux for each group was approximately equal.
Hydrated, trimmed nails were clamped in the diffusion cells and the receptor compartment was filled with the receptor fluid which was water containing 0.5% PEG-20-oleyl ether, a wetting agent that helped to remove entrapped air bubbles and was nondestructive to nails (Bronaugh and Stewart (1984) 73:1255-1258). A temperature of 37° C. and constant stirring was maintained throughout each study. The cells were left uncovered for 1 hour prior to application of the donor formulation to allow the top surface of the nails to dry out. The diffusion cells were then dosed with 200 μL of the gel formulations under investigation and covered with PARAFILM™. Samples were drawn every 8 hours over a period of 6-14 days. Initial studies were run for 2 weeks to establish when steady state conditions were reached, and based on these earlier studies, the overall duration was later reduced to 6 days. At each sampling time, the entire contents of the receptor compartment were removed and replenished with an equal volume of fresh receptor fluid. Additionally, at each sampling time, the donor gel adhering to the nail from a previous application was wiped off completely using tissues dipped in 65% alcohol. The cells were redosed with 200 μL of the donor gel, and were again covered. Due to solubility constraints, the combination of 10% PTO and 20% U was screened by alternately dosing with two separate gels, XVIa and XVIb, containing 10% PTO and 20% urea, respectively.
At the end of the study, the nails were removed from the diffusion set up and rinsed repeatedly in 65% alcohol (DMSO for the PTO and ZnPTO formulations, XIV-XVII), followed by DI water, for radioactivity and residual enhancer extraction. The extractions were carried out by shaking on a mechanical shaker bath at 25° C. for successive 24-hour periods, until the radioactive counts from the extractions approached blank values.
Donor Formulations for Antifungal Studies
Infinite Dose Studies. To study the effect of various enhancers on antifungal permeation, gels were prepared containing 5% clotrimazole and the different chemical modifiers (Table 14).
Since MPG and the keratolytic agents were soluble in a hydroalcoholic medium, all the gels (A-H) were prepared in a vehicle comprising ethanol, propylene glycol, and water. The polymer used as the gelling agent was HPC. Gels A-H had similar compositions to the gels used in enhancer screening studies with water as a probe, however, in this case the gels each contained 5% clotrimazole. Representative formulations for gels containing urea alone (Gel C), MPG alone (Gel D), a combination of MPG and urea (Gel F), and a control (Gel A) are shown in Table 15.
The gels were prepared using the same general procedure as that used for the enhancer screening studies. Briefly, clotrimazole and the enhancers were dissolved in the hydroalcoholic vehicle with vortex agitation. Butylated hydroxy toluene (BHT) was added as an antioxidant to prevent oxidation of MPG. Disodium EDTA was also added as a chelating agent. All gels were spiked with [3H]-Clotrimazole to obtain a ratio of “hot”:“cold” drug of approximately 1:60,000. This very dilute spiking ratio was sufficient due to the high specific activity of [3H]-Clotrimazole (8 Ci/mmol). HPC was added to the mixture, and gel formation was allowed by agitating on a mechanical shaker bath at 37° C. overnight. The apparent pH of the gels was measured. Prior to use, three 10 μL aliquots of the gels were analyzed by LSC to validate uniformity of spiking.
To study the effect of concentration of clotrimazole on nail permeation of gels containing enhancers, additional formulations were prepared containing 0.5% and 2% clotrimazole in conjunction with the effective enhancer combination of 10% MPG and 20% urea. These were studied along with the gel containing 5% clotrimazole and the same enhancers (Gel F, Table 14).
Finite Dose Studies. Formulations similar to gels A (control) and F (10% MPG+20% Urea), shown in Table 14 and Table 15, were prepared for finite dose studies. Clotrimazole was present in a concentration of 5%, as for infinite dose studies. However, in this case a higher spiking ratio of 1:40,000 was used.
Receptor Fluid Studies for Antifungal Studies
The choice of receptor fluid depends on both physiologic considerations, and solubility requirements to achieve sink conditions. Clotrimazole has a limited solubility in water (<0.01 mg/mL), but is very soluble in alcohol (95 mg/mL) and some other organic solvents, at 25° C. (Hoogerheide and Wyka (1982) In: Analytical profiles of drug substances, Vol. 11, K. Florey (ed.), Academic Press, New York, pp. 225-255). For the present invention differing concentrations of the nonionic surfactant PEG-20-oleyl ether (HLB=16) were used. This surfactant at a concentration of 6% was reported to be a receptor fluid of choice for in vitro skin permeation studies with highly hydrophobic molecules (Bronaugh and Stewart (1984) 73:1255-1258). Moreover, when this surfactant solution was used as the receptor fluid, the barrier integrity of the skin was retained as assessed by monitoring the permeation of control substances such as cortisone, urea, and water.
Aqueous solutions of PEG-20-oleyl ether in concentrations of 0.5%, 1%, 2%, and 5% were prepared. Excess clotrimazole was added to vials containing 10 mL of surfactant solution. For each surfactant concentration, the solubility determination was done in triplicate. The vials were agitated in a mechanical shaker bath (Precision Scientific, Chicago, Ill.) maintained at 37° C. for 24 hours. The vials were then centrifuged (Fisher Scientific, Springfield, N.J.) at 4600 rpm for 30 minutes at 25° C. The supernatant was analyzed for drug concentration by High Performance Liquid Chromatography (HPLC).
The HPLC conditions were adapted from well known methods, for example Hoogerheide, et al. ((1981) 64:864-869) and Rifai, et al. ((1995) 41:387-391). The HPLC analysis was performed using a Waters 600E System Controller Pump, Waters 717 Autoinjector, Waters 486 Tunable Absorbance Detector, and Waters 700 Data Module Integrator. A reverse-phase C-18 column (Waters Novapak, 3.9×150 mm, 4μ) was used. The mobile phase consisted of a mixture of 0.025M dibasic potassium phosphate (pH adjusted to approximately 6.3 with o-phosphoric acid) and HPLC grade methanol in the ratio 1:3. A flow rate of 1 mL/minute was maintained and the detector wavelength was set at 254 nm.
Standard solutions of clotrimazole in mobile phase were prepared in the concentration range of 5-200 μg/mL. The injection volume of standard and sample solutions was 20 μL. The run time was 7.0 minutes and clotrimazole had a retention time of 5.74 minutes.
The chromatography conditions yielded a linear standard curve (R2=0.9995). From the standard curve, the solubility of clotrimazole in different concentrations of PEG-20-oleyl ether was quantitatively determined. The solubility results are summarized in Table 16.
Clotrimazole solubility increased with increasing surfactant concentration. The relationship between drug solubility and PEG-20-oleyl ether concentration was found to be linear (R2=0.9995). Thus, clotrimazole solubility was highest in the 5% surfactant solution. The solubility of clotrimazole in a 0.5% PEG-20-oleyl ether solution was 75.16 μg/mL. This was about 75-fold higher than the expected clotrimazole concentration of 1 μg/mL. Thus, the use of a 0.5% PEG-20-oleyl ether solution as the receptor phase would adequately ensure that sink conditions would be maintained throughout the experiment. Also, the lowest possible surfactant concentration was desirable so as to maintain as much physiological relevance as possible.
Antifungal Permeation Studies
Infinite Dose Studies. Each antifungal gel formulation was studied using 3 replicates. Excised, human fingernails (thumbnails and other fingernails), which had been standardized as described above, were used. Prior to the start of the study, baseline water permeation parameters were established for all nails as described above. The nails were assigned to different treatment groups, comprising 3 nails each, such that the mean, normalized water flux (J*) for each group was approximately equal. Permeation studies were conducted as described for enhancer screening studies.
Finite Dose Studies. The control gel and best test formulation, containing 10% MPG and 20% urea, were also studied using finite dosing to mimic the most convenient method of consumer application. The procedure was the same as that described for enhancer screening studies, however, the volume of each application was smaller. In the finite dose studies, the cells were dosed with 5 μL of the donor gels, the gels were spread evenly on the nail surface with a thin, plastic rod, and the diffusion cells were left uncovered. The exact amount of clotrimazole applied was calculated by subtracting the amount adhering to the rod from the total amount present in 5 μL volume. The rest of the procedure was as described above.
Analysis of Samples
Each of the samples (receptor solution and extractions) obtained during the permeation study was mixed with 10 mL of Scintiverse I (scintillation cocktail) and analyzed by Liquid Scintillation Counting (LSC, Beckman model LS 5000T, Beckman Instruments, Somerset, N.J.). Each sample was counted for at least 5 minutes and the counts in disintegrations per minute (dpm) were converted to amount of active, using the conversion factor 1 μCi =2.2×106 dpm and the specific activity of 3H2O or [3H]-Clotrimazole.
Data Analysis of Enhancer Screen
The flux (J, mg cm−2h−1) was computed from the slope of the steady-state portion of the plot of cumulative amount of water permeated per unit area as a function of time. The normalized flux (J*, mg cm−1h−1) was calculated by multiplying the raw parameter by nail thickness. The normalized flux values (J*) from test formulations with enhancers were compared with the J* value of the appropriate control formulation having the same vehicle composition, but no enhancer using a t-test at the 0.05 level of significance. For effective enhancers, a statistically significant increase in normalized water flux was expected relative to the control.
A relative enhancement factor for water, EFwater, defined by Equation 1, was computed for each enhancer.
where J*=normalized flux of water (mg cm−1h−1).
The enhancers that resulted in the highest EFwater values were considered to be the most effective.
Data Analysis of Antifungal Permeation Studies
The flux (J, μg cm−2day−1) was determined from the steady-state portion of the plot of cumulative amount of clotrimazole permeated per unit area as a function of time. The permeability coefficient (P, cm day−1) was calculated by dividing the flux by the donor concentration of clotrimazole. The lag time (tlag, days) was calculated from the x-intercept of the steady-state region of the permeation profile. The diffusion coefficient (D, cm2day−1) was computed from Equation 2.
The normalized clotrimazole flux (J*, μg cm−1day−1) and normalized permeability coefficient (p*, cm2day−1) were calculated by multiplying the raw parameter by nail thickness, as shown in Equation 3 and Equation 4, respectively:
where J*=normalized flux (mg cm−1h−1) and h=nail thickness (cm), and;
where P*=normalized permeability coefficient (cm 2h−1) The normalized flux values (J*) from the antifungal test formulations with enhancers were compared with the J* value of the control formulation having the same vehicle composition but no enhancer, using a t-test at the 0.05 level of significance.
A relative enhancement factor for clotrimazole, EFclotrimazole, defined by Equation 5, was computed for each enhancer.
where J*=normalized flux of clotrimazole (μg cm−1day−1).
The EF value obtained for clotrimazole with a given enhancer was compared with the corresponding value obtained from screening studies with water. This comparison elucidated the degree to which a given enhancer increased the permeation of lipophilic vs hydrophilic molecules.
The amount of drug permeating into the nail was calculated from the sum of all extractions except the first extraction which was thought to contribute to drug at the nail surface. The amount of drug in the nail, expressed in μg/g, was compared for formulations with enhancers vs. the control using a t-test at the 0.05 level of significance.
Test for Barrier Integrity of Nails
Water permeation parameters were obtained after treatment with enhancers and antifungal agents and washout, and were compared with baseline values that had been established before screening had commenced. For the enhancer studies, the ratio of J* posttreatment to J* of the untreated nail was a measure of the barrier integrity. For the antifungal permeation studies, the relative increase in water flux due to a given enhancer, indicating a compromised barrier, was compared for lipophilic molecules (antifungals) and hydrophilic molecules (water) Thus, the degree to which the nail permeation barrier was compromised due to treatment with a given enhancer was compared for compounds of differing size and polarity.
1. A composition comprising sulfur-containing glycine residues and urea which increases the permeation of an active agent through nail tissue.
2. A method of treating a nail disease comprising topically applying the composition of claim 1 in an amount sufficient to increase the permeation of an active agent through nail tissue.
3. A method for enhancing the permeation of an active agent through nail tissue comprising administering an effective amount of the composition of claim 1.
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