WO1999049895A2

WO1999049895A2 - Method for increasing the permeability of horny human tissue

Info

Publication number: WO1999049895A2
Application number: PCT/US1999/006743
Authority: WO
Inventors: Ying Sun; Jue-Chen Liu; Elizabeth Kimbleton; Jonas C. T. Wang
Original assignee: Johnson And Johnson Consumer Companies, Inc.
Priority date: 1998-03-31
Filing date: 1999-03-29
Publication date: 1999-10-07
Also published as: AU3212099A; AR016200A1; WO1999049895A3; CO5070608A1

Abstract

This invention relates to a novel method to increase the permeability of horny human tissue prior to the application of pharmaceutical or cosmetic agents. The horny human tissues are nails, calluses, or corns.

Description

METHOD FOR INCREASING THE PERMEABILITY OF HORNY HUMAN TISSUE

FIELD OF THE INVENTION This invention relates to a novel method to increase the permeability of horny human tissue prior to the application of pharmaceutical or cosmetic agents. The horny human tissues are nails, calluses, or corns. This method is particularly useful to increase the permeability of human nails which have

been hardened by antifungal diseases such as onychomycosis. BACKGROUND OF THE INVENTION

Onychomycosis is a fungal disease of the human nail. The symptoms of this disease are split, thickened, hardened, and rough nail plates. This is caused by any of a number of organisms and is particularly prevalent in the elderly. Typically fungal infections are treated by topical application of antifungal agents and/or oral administration of antifunal agents. Unlike other fungal infections, there is no topical treatment for onchomycosis which is approved by the United States Food and Drug Administration. It is desirable to treat this disease topically due to the potential for side effects that have been associated with some of the oral treatment regimens. One reason for the absence of a topical treatment is that in this disease, the symptomatic thickened nail plate prevents topical agents from reaching the site of the infection.

The human nail plate is comprised of hard nail keratin with a high content of disulfide-linkeages (cystine), and is approximately 0.5 mm thick for finger nails, and about 1.5 mm thick for big toe nails. A nail plate grows out from the nail matrix, a living and highly proliferative epithelial tissue under the eponychium, and overlays the nail bed, a soft and normally non-comified tissue. Although it appears visually uniform, a nail plate is made of at least two discernible macroscopic strata, i.e., the dorsal nail plate and intermediate nail plate. The dorsal nail plate is harder and thinner than the intermediate nail plate. The thick nail plate and its dense keratin nature present a formidable barrier for topical drugs to penetrate, and accounts for the failure of many topical antifungal agents to treat fungal infected nails.

Others have disclosed methods increasing nail permeability to antifungal drugs. One treatment incorporates certain mercaptans with antifungal agents to treat a diseased nail. (Morimoto, JP 8231430 A, JP 95- 41722). The incorporated mercaptans include 4-t-butylbenzyl mercaptain, thiosalicylic acid, cysteamine hydrochloride, or thioglycoiic acid and its derivatives such as ammonium thioglycolate, or butyl thiglycolate. In this disclosure, the mercaptan compound is directly incorporated in the drug formulation.

Olthoff et al. In EP 440,298, disclose the use of sulfur containing amino acid derivatives, dithiothreitol, and ammonium thioglycolate in topical preparations for the treatment of human nails. Once again this reference discloses a composition containing the aforementioned substances and pharmaceutical or cosmetic agents.

U.S. Patent 5,696,164 (Sun et al., 1997) discloses the use of sulfur containing amino acids and amino acid derivatives, such as cysteine and N- acetyl cysteine, and urea to increase drug permeability in a nail plate. The sulfhydryl (SH) compounds in the aforementioned patent disclosure are used as nail penetration enhancers to increase drug permeability in the nail. Yet again, the aforementioned substances and the pharmaceutical agents were combined in one composition.

The teachings of Morimoto, Olthoff, and Sun show a method of treating onychomycosis using a formulation which combines a penetration enhancer and an antifungal agent. However they do not teach a method of increasing the permeability of hardened nails prior to applying an antifungal drug. Such a method would be useful, for some antifungal drugs cannot be easily formulated with penetration enhancers to give stable and useful pharmaceutical compositions. Therefore it would be useful to be able to increase nail permeability prior to using antifungal medications. This way the compatibility of the nail permeability enhancer and the antifungal medication is not an issue.

In addition to diseased nails, other human horny tissues, such as ingrown nail, corns and callus, are fairly impervious to topical treatments. This can be seen in the case of a particular type of athlete's foot fungus, namely plantar tinea pedis. In that disease, the infected areas of the skin are covered with hard calluses which impede topical antifungal agents. Topical treatment for this disease is usually not effective. To date, it is generally accepted that the most effective treatment is systemic. Therefore it would be advantageous to find a topical treatment for this diseases.

It is an object of the present invention to provide a method of increasing the permeability of active agents through horny human tissue, by pretreating the horny human tissue with the compositions of this invention before the application of the active agents. SUMMARY OF THE INVENTION This invention relates to a method of increasing the permeability of horny human tissue to active agents which comprises applying a composition comprising an effective amount of one or more sulfur containing compounds to human horny tissue for a duration of time sufficient to increase the permeability of the horny human tissue to active agents. Further, the invention contemplates a method of treating diseased or healthy human horny tissues which comprises 1 ) applying a first topical composition comprising an effective amount of one or more sulfur containing compounds to human horny tissue for a duration of time sufficient to increase the permeability of the horny human tissue to active agents and 2) applying a second topical composition containing one or more active agents to the treated horny human tissue. Still further, the invention contemplates a kit which comprises 1 ) a first topical composition which increases the permeability of horny human tissue comprising an effective amount of one or more sulfur containing compounds and 2) a second topical composition containing one or more active agents useful in the treatment of horny human tissue.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the swelling profiles of human nail clippings over 48 hours at 32°C in six solutions of different compositions.

Figure 2 is a graph showing nail re-hydration in distilled water for the nail samples which have been pretreated with chemicals as shown in Figure 1 , and have been completely air-dried.

Figure 3 is a graph showing the effect of urea concentration, and urea solution pH on nail hydration. Figure 4 is a graph showing the pH effect of a solution containing 2% calcium thioglycolate and 10% urea on nail hydration (N=2).

Figure 5 shows that the pH dependency of nail hydration at pH>9.

Figure 6 is a graph showing the pH effect of 2% thioglycerol aqueous solutions with pH's ranging pH 7-pH 10.

Figure 7 is a graph plotting the percentage weight gain of nail clippings verses the solution pH for the 24-hour treatment using thioglycerol solutions.

Figure 8 is a graph showing the effect of different concentrations of calcium, sodium, and ammonium salts of thioglycolic acid, as well as the pH of calcium thioglycolate on nail hydration.

Figure 9 is a graph showing the nail hydration of several reducing agents other than thioglycolates.

Figure 10 is a graph showing the significant enhancement of nail pretreatment on itraconazole penetration into the nail plates in vitro. Figure 11 is a graph showing that the amount of itraconazole delivered into the pretreated nail plates from another itraconazole formulation (0.45% itraconazole) progressively increased as the duration of itraconazole permeation increased from 0.25 hours to 7 days until it reached a plateau.

Figure 12 is a graph showing the result of nail pretreatment on the miconazole nail permeation in vitro (1 % calcium thioglycolate, pH 11 , 8 hours,

32°C, N=3).

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a method of increasing the permeability of horny human tissue to active agents which comprises applying a composition comprising an effective amount of one or more sulfur containing compounds to human horny tissue for a duration of time sufficient to increase the permeability of the horny human tissue to active agents.

"Horny human tissue" is defined as the keratinous, and often hyper- proliferative tissues such as diseased nails, healthy nails and hardened tissues of the skin such as corns and callus. Preferably the horny human tissues are diseased and healthy nails. Those tissues include but are not limited to the thickened diseased portions of the nail which is common in onychomycosis and psoriasis. An "effective amount" of a sulfur containing compound refers to the amount which is necessary to increase the permeability of horny human tissue. The amount ranges from about 0.1% to about 40%, preferably, from about 0.5% to about 20%, more preferably, from about 1 % to about 10%. The term "duration of time" refers to the time that is required to increase the permeability of the horny human tissue. This time period is dependent on the sulfur compound used, its concentration, the type of horny human tissue, and other factors such as composition pH. In general, if horny human tissue is a nail, the duration of time may vary from about one minute to about one day, preferably between 5 minutes and 12 hours, more preferably between 10 minutes and 30 minutes. If the horny tissue is a corn or callus, the same time periods are preferred.

According to the invention, the term "sulfur containing compounds" refers to (a) thio-compounds with one or more sulfhydryl functional groups capable of reacting with disulfide bonds of keratin, (b) thio-containing amino acids and their derivatives, and (c) certain sulfides. The thio-compounds with one or more sulfhydryl functional groups capable of reacting with disulfide bonds of keratin, include but are not limited to thioglycolic acid and its salts, such as glycolates of calcium, sodium, strontium, potassium, ammonium, lithium, magnesium, and other metal salts; thioethylene glycol, thioglycerol, thioethanol, as well as thioactic acid, thiosalicylic acid and their salts. The thio-containing amino acids and their derivatives are selected from a group consisting of L-cysteine, D-cysteine, DL-cysteine, N-acetyl-L-cysteine, DL- homocysteine, L-cysteine methyl ester, L-cysteine ethyl ester, N-carbamoyl cysteine, glutathion, and cysteamine. Certain sulfides, include but are not limited to calcium, sodium, potassium, lithium and strontium sulfides. The preferred thio-compounds with one or more sulfhydryl functional groups capable of reacting with disulfide bonds of nail keratin are the calicum and sodium salts of thioglycolic acid. The preferred thio-containing amino acids and their derivatives are L-cysteine and N-acetyl-L-cysteine. The preferred certain sulfides are sodium sulfide. Overall, the preferred sulfur containing compounds are thio-compounds with one or more sulhydryl functional groups capable of reacting with disulfide bonds of keratin.

The first topical compositions of the invention include one or more sulfur containing compounds. Aside from those compounds, other substances, such as additional chemical additives, cosmetic additives, and pharmaceutical excipients may be included in these compositions. The chemical additives include but are not limited to substances which aid the composition of the invention in increasing the permeability of the horny human tissue. Examples of such additives include but are not limited to urea and salicylic acid. The choice of which additive to use is dependent upon the identity of the sulfur containing compound. For basic sulfur containing compounds urea, is the additional chemical additive of choice. For acidic sulfur containing compounds, salicylic acid and urea are the additional chemical additives of choice. If additional chemical additives, such as urea are used, the concentration of urea to be used ranges from about 1 % to about 50%, preferably from about 5% to 40%. If salicylic acid is used as an additional chemical additive, the concentration of salicylic acid to be used ranges from about 0.1% to about 40%, preferably from about 1 % to about 20%. The cosmetic additives may include but are not limited to those agents which prevent potential skin irritation, such as emollients, vitamins (e.g., vitamin E) and herbal extracts (e.g., aloe vera).

The pharmaceutical excipients include but are not limited to pH modifying agents such as pH-modifying agents (e.g., HCI, sodium hydroxide, and calcium hydroxide), organic solvents (e.g., propylene glycol, glycerol, etc.), cetyl alcohol, kaolin, talc, zinc oxide, titanium oxide, cornstarch, sodium gluconate, oils (e.g., mineral oil), ceteareth-20, ceteth-2, surfactants and emulsifiers, thickener (e.g., cellulose derivatives and gums), perfume, anti- oxidants, preservatives, and water. The choice of which pharmaceutical excipient to use is often controlled or affected by the type of sulfur containing compounds used in the composition. For example, when the sulfur containing compounds are thio-containing amino acids and their derivatives, preferably the pH of the composition is preferably below pH 7, and more preferably, below pH 5. Alternately, if the sulfur containing compounds are thio-compounds and sulfides, the pH of the composition is preferably above pH 6, and more preferably, above pH 9.

The term "applying" refers to any method of physically transferring the compositions of the invention to the nail and the skin. Such methods include but are not limited to painting the composition on the nail; spraying the composition using a spray pump, and combining the composition with a propellant so that it sprayed on the skin as an aerosol. The term "aerosol" refers to systems consisting of "pressurized packages" with either compressed gases or liquefied gases as propellants. Examples of compressed gases are compressed nitrogen and air. Examples of liquefied gas propellants are propane, isobutane, n-butane, dimethyl ether, and mixtures thereof. The preferred propellants are dimethyl ether, and mixture of dimethyl ether and one or more hydrocarbon propellants. The preferred weight ratio of dimethyl ether to the hydrocarbon propellant(s) ranges from greater than or equal to about 3:2 (> 3.2) respectively. The sulfur containing composition of the present invention may be

prepared in a number of forms which can be applied to horny human tissue. For example, the composition may be applied in a gel, cream, ointment, liquid, spray liquid, paint-/brush-on preparation, plaster, hydrogel-device. In addition, the composition may be applied with a bandage-like device. This device can be used to affix the composition to the horny human tissue and to provide desirable occlusion and protection from accidental wiping off. A device similar to that described by Sun et al in US Patent 5,696,164 may be used, and is hereby incorporated by reference. This device is constructed in such a way that during the treatment, only the horny human tissue is in direct contact with the composition of the invention. The contact between the composition and the surrounding skin tissues is minimized to prevent any skin irritation. Still further, the composition of the invention may be incorporated in an adhesive layer, which is coated on a pliable polymeric backing sheet. This device resembles an adhesive tape, only with the composition embedded in the adhesive layer. As another alternative, the compositions of the invention are incorporated in a hydrogel device. An aqueous solution or suspension of the softening composition are immobilized in a hydrogel layer, which is coated on a pliable polymeric backing sheet. During the application, the hydrogel layer will be situated over the horny human tissue. The hydrogel device is secured to its position by the adhesion between the hydrogel layer and the horny human tissue and surrounding skin tissue. Alternatively, the hydrogel device is secured by an adhesive layer coated on the polymeric backing sheet at the edges of the hydrogel device in a configuration commonly known in the art as an "island-type bandage". Further, the invention contemplates a method of treating diseased or healthy human horny tissues comprises 1) applying a first topical composition comprising an effective amount of one or more sulfur containing compounds to human horny tissue for a duration of time sufficient to increase the

permeability of the horny human tissue to active agents and 2) applying a second topical composition containing one or more active agents to the treated horny human tissue.

Still further, the invention contemplates a kit which comprises 1 ) a first topical composition which increases the permeability of horny human tissue comprising an effective amount of one or more sulfur containing compounds and 2) a second topical composition containing one or more active agents useful in the treatment of horny human tissue.

The terms "horny human tissue," "effective amount," "sulfur containing compounds," "duration of time," and "applying," are as described in the

foregoing paragraphs. As used herein, the term "active agents" refers drugs for treating diseased nails, nutrients or nail conditioners which may be used to improve damaged nails or maintain healthy nails, and nail growth promoters which may be used on damaged or healthy nails. Typically these agent include but are not limited to antifungal agents, antibiotic agents, antibacterial agents, antipsoriatic agents, and anti-inflammatory agents. Most of these agents are known and may be used at concentrations and for durations of time which have proved effective against their respective disease states. All of the aforementioned types of active agents may be used to treat the tissue surrounding the nail, and skin on other areas of the human body whether that tissue is healthy or diseased. When the active agents are antifungal drugs, they may be used to treat onychomycosis and other fungal diseases. When the active agents are antibiotics (or antiseptics) they may be used to treat athlete's foot, bacterial infection of nails, bacterial infections of the tissue surrounding the nails and other human tissues, When the active agents are antipsoriatic drugs they may be used to treat psoriatic nail and skin conditions. Examples of antifungal drugs include but are not limited to miconazole, econazole, ketoconazole, itraconazole, fluconazole, bifoconazole, terconazole, butoconazole, tioconazole, oxiconazole, sulconazole, saperconazole, clotrimazole, undecylenic acid, haloprogin, butenafine, tolnaftate, nystatin, ciclopirox olamine, terbinafine, amorolfine, naftifine, elubiol, griseofulvin, and their pharmaceutically acceptable salts. The preferred antifungal drugs are an azole, an allylamine, or a mixture thereof. Preferred azoles are selected from the group consisting of itraconazole, ketoconazole, miconazole, econazole, fluconazole, voriconazole, clotrimazole, butenafine, undecylenic acid, clioqinol, and their pharmaceutically acceptable salts. Preferred allylamines are selected from the group consisting of terbinafine, naftifine and mixtures thereof.

Examples of antibiotics (or antiseptics) include but are not limited to mupirocin, neomycin sulfate bacitracin, polymyxin B, /-ofloxacin, tetracyclines (chlortetracycline hydrochloride, oxytetracycline hydrochloride and tetrachcycline hydrochoride), clindamycin phsphate, gentamicin sulfate, benzalkonium chloride, benzethonium chloride, hexylresorcinol, methylbenzethonium chloride, phenol, quaternary ammonium compounds, triclocarbon, triclosan, tea tree oil, and their pharmaceutically acceptable salts. Preferred antibiotics and antiseptics include mupirocin, neomycin sulfate, bacitracin, polymyxin B, /-ofloxacin, tetracyclines, benzalkonium chloride, benzethonium chloride, triclocarbon, and triclosan.

Examples of antipsoriatic drugs include but are not limited to corticosteroids (e.g., betamethasone dipropionate, betamethasone valerate, clobetasol propionate, diflorasone diacetate, halobetasol propionate, triamcinonide, dexamethasone, fluocinonide, fluocinolone acetonide, halcinonide, triamcinolone acetate, hydrocortisone, hydrocortisone verlerate, hydrocortisone butyrate, aclometasone dipropionte, flurandrenolide, mometasone furoate, methylprednisolone acetate), methotrexate, cyclosporine, calcipotriene and anthraline. Preferred antipsoriatic drugs include betamethasone dipropionate, betamethasone valerate, and clobetasol propionate.

When the active agents are nail growth promoters, such agents include but are not limited to minoxidil, minoxidil sulfate, retinoids, cysteine and acetyl cysteine, methionine, glutathione, biotin, finasteride and ethocyn, as well as pharmaceutically acceptable salts of these compounds. The preferred growth promoters are minoxidil, minoxidil sulfate, retinoids, cysteine and acetyl cysteine. The particularly preferred nail growth promoters are 2% minoxidil, 2% minoxidil sulfate, and 0.1 % retinol. When the active agents include nutrients, they include to vitamins, amino acids, and their derivatives. Examples of such agent include but are not limited to vitamin B complex: thiamine, nicotinic acid, biotin, pantothenic acid, choline riboflavin, vitamin B₆, vitamin B₁₂, pyridoxine, inositol, camitine; ascorbic acid, ascorbyl palmitate, vitamin A, vitamin K, vitamin E, vitamin D, cysteine and N-acetyl cysteine, herbal extracts, and their derivatives.

When the active agents include nail conditioners they include but are not limited to mineral-containing compounds, flavonoids and retinoids. These nail conditioners improve general nail conditions, such as strengthening the nails to prevent nail chipping and cracking, and to beautify the nails. Examples of such agents include but are not limited to calcium pantothenate, calcium carbonate, and calcium gluconate. Examples of retinoids include but not limited to retinol (Vitamin A alcohol), retinal (Vitamin A aldehyde), retinyl acetate, etinyl palmitate, retinoic cid, 9-cis-retinoic acid and 13-cis-retinoic acid. When retinoids are the active agents, the concentration of retinoids is from about 0.01 % to about 0.5%, preferably, from about 0.05 to about 0.1 %. Examples of flavonoids include but not limited to naringenin, quercetin, catechins (e.g., epigallocatechin gallate), theaflavins, robustaflavone, hinokiflavone, amentoflavone, agathisflavone, volkensiflavone, morelloflavone, rhusflavanone, and succedangeafiavanone. The active agents may also be cosmetic agents, nutrients and minerals to improve the appearance or condition of the tissue underlying the horny human tissue. Such tissue includes but is not limited to the nail matrix, the nail bed or lower nail plate underneath a hardened nail plate as well as the skin below a callus or a corn. Cosmetic agents include but are not limited to pigments, dye, and other additives (e.g., silica, talk, zinc oxide, titanium oxide, clay powders). The formulation containing active agents may be in the form of a conventional topical formulation, e.g., as solution, gel, cream, lotion, ointment, rinse-off formulation, spray liquid, spray powder, aerosol, paint-on formulation such as lacquer/varnish and tincture. The formulation may be applied directly to the pretreated tissue, or applied in a bandage-like device, or in a hydrogel device similar to the methods described in the previous sections for pretreating horny human tissue. Optionally, a protective means, in the form of a polymeric coating, an adhesive-coated pliable tape, or a bandage is applied to the treated tissue to prevent the actives from leaching out and being washed off.

Overall, the preferred active agents are miconazole, itraconazole, econazole, ketoconzaole, clotrimazole, minoxidil, terbinafine, and their pharmaceutically acceptable.

As discussed for said first topical composition, the second topical composition may include other substances, which include but are not limited to additional chemical additives, cosmetic additives, and pharmaceutical excipients. Those agents are discussed in greater detail in the paragraphs describing the first topical composition.

For example if the horny human tissue is a thick hardened nail plate

(due to onychomycosis, psoriasis, or other diseases), this nail plate may be treated with a composition of the invention, containing calcium thioglycolate as the sulfur containing compound for about 15 minutes. One such useful composition is the commercial depilatory, Nair® (Carter-Wallace, Inc., New

York, listed ingredients include sodium thioglycolate, calcium thioglycolate, calcium hydroxide, water, mineral oil, ceteareth-20, fragrance and iron oxide). Generic commercial depilatories typically contains 5 to 7.5% calcium thioglycolate ("Cosmetic and Toiletry Formulations", 2nd Ed. Vol.2, pages 243 & 255, by E.W. Flick, Noyes Publicaitons, New Jersey, 1991 ). Therefore commercial depilatories containing the sulfur containing compounds of the invention may be used in the method of this invention.

Immediately after treating the nail plate with the sulfur containing compositon, the remaining composition is rinsed off (typically with water) and a composition containing miconazole nitrate as the active agent is applied to the nail. In this instance, the miconazole nitrate was in a lacquer formulation. Examples of the preparation of this lacquer formulation are disclosed in a

US. Patent application (Sun et al., U.S. Serial No. 60/080,116 which is hereby incorporated by reference. Aside from the antifungal agent, this lacquer may also contains cosmetic ingredients, which include but are not limited to, pigments, dye, and other additives (e.g., silica, talk, zinc oxide, titanium oxide, clay powders) for improved mechanical properties and non-shiny appearance. In addition to or instead of the nail lacquer, other formulation forms such as antifungal containing, liquids, creams, ointments, liquid sprays and aerosols may also be used.

In addition to onychomycosis, fungal skin diseases such as plantar tinea pedis may be treated using the methods and compositions of this invention.

Typically the treatment regimen for nail or skin fungal infections is once or twice per day, preferably once per day, with a duration from less than a week to four weeks, preferably equal or less than two weeks. The topical treatment of the invention may be employed in combination with systemic treatment. For example, an antifungal drug, such as, itraconazole, terbinafine, griseofulvin or other antifungal drugs, can be given orally over a period of time. This time period may be concurrently during the entire topical treatment regimen, or concurrently during a portion (usually the latter phase) of the topical treatment regimen, or following the topical treatment.

Even if the horny human tissue is not infected with a disease, this invention may be used prophylactically, to prevent infections. Typically under prophylactic use the method sulfur containing compositions of the invention may be applied once or twice per month. Typically the prophylactic treatment regimen for skin and fungal infections, can vary from once or twice per week to once or twice per month, with the interval between treatments shorter for the skin and longer for the nail.

In order to illustrate the invention the following examples are included. These examples do not limit the invention. They are meant only to suggest a method of practicing the invention. Those knowledgeable in the treatment of horny human tissue as well as other specialties may find other methods of practicing the invention. However, those methods are deemed to be within the scope of this invention. EXAMPLES

Example 1. Nail Swelling and Drug Uptake by the Nail The effect of nail pretreatment using S-compounds on nail swelling and drug uptake by the nail was studied by in vitro experiments. The experimental procedure is briefly described as the following. Clean human finger nail clippings were equilibrated to a constant weight by placing them in a desiccator over saturated CaCI₂.6H₂0 aqueous solution (29% relative humidity at room temperature) for at least 48 hours before use. Approximately 30 mg of nail clippings were weighed in a glass vial, the exact initial weight recorded. Three milliliters of a test vehicle, (pre-warmed to 32°C) were added to the vial and maintained at 32°C under stirring in a heating/Stirring Module (Reacti-Therm III, Pierce). The changes in nail weight were monitored at predetermined intervals over a period of time. To study whether the effect of nail pretreatment is a reversible phenomena for the nail, namely, whether the pretreatment has altered the absorption property of the nail in a permanent way, the nail samples used in the pretreatment experiment were air-dried in a fume-hood for 72 hours to remove completely the moisture content. Another nail swelling study was then conducted with these nail samples using distilled water as the medium with the procedure identical to that described above. All the percentage values hereafter are by weight unless otherwise specified. To study the extent of drug uptake by nail, a drug partitioning experiment was conducted following the nail swelling experiment. The nail sample was immersed in one milliliter of a drug formulation at 32°C under stirring for a predetermined period of time, the nail sample was thoroughly washed with alcohol to remove surface-bound drug. The nail sample was then digested, and drug content in the nail was determined by High Pressure Liquid Chromotography (HPLC).

The results in the Figure 1 shows the nail swelling profiles in six solutions of different compositions (Table 1 ) over a 48-hour period. The rank order of nail hydration enhancement capability of the tested chemicals is: calcium thioglycolate > urea > salicylic acid > ethanol. It can be seen that 10% lactic acid and 50% ethanol had little effect on nail hydration (26% and 29%, respectively), as compared to the nail swelling in pure water (23%). The solutions containing 7% salicylic acid and 10% urea produced a slightly higher hydration (31% and 35%, respectively). Apparently, keratolytic agents, such as salicylic acid and urea, have a rather limited effect on nail hydration enhancement under the test conditions. In contrast, a significant increase in the nail hydration (up to 185%) was achieved with 1 % calcium thioglycolate solution at pH 10. The nail clippings treated with calcium thioglycolate solution became swollen and more elastic. Thioglycolates can reduce the disulfide bonds of nail keratin. Because the nail contains a high level of structural cystine in the form of disulfide bonds, the reduction of the structural disulfides would result in loosening the nail keratin structure, leading to an increased liquid uptake. The resulting softer nail plate also suggested enhanced nail permeability. Figure 2 shows result of the second nail swelling study (rehydration in distilled water) conducted with the pretreated nail samples, after being completely dried for three days. It can been seen that the nail samples previously treated with calcium thioglycolate retain a significantly elevated water uptake capability (over 60%), whereas all the other chemicals tested show the water uptake value comparable to the intrinsic capability of nail- water uptake. This phenomenon, i.e., that a thioglycolate-pretreated nail retains its enhanced ability to absorb liquid after being dried, has never been reported before. This finding suggests that thioglycolates and other thio- compounds with sulfhydryl functional groups can be used to pretreat the nail to increase its absorption ability to topically applied active agents for a prolonged period of time. The improved elasticity and softness associated with the pretreated, swollen nail provide much needed enhancement in nail permeability for the active agents to penetrate into or through the nail plate. Our results in Figure 3 suggest that the nail's ability for enhanced nail absorption of active agents will remain to a desirable degree, even after the pretreated swollen nail is dried as under a practical situation.

Forty percent urea in a petrolatum base has been reported to be used by dermatologists to soften fungal infected nails for the purpose of non- surgical nail avulsion [Farber, E.M. and South, D.A. CUTIS, 22 (1978) 689- 692; South, D.A. and Farber, E.M. CUTIS, 25 (1980) 609-612; Buselmeier, T.J. CUTIS, 25 (1980) 397-405]. The effect of urea at comparable concentrations on nail hydration was therefore studied (Figure 3). At pH 4.5, as the urea concentration increased from 20% to 40%, nail hydration increased from 30% to 50%. The increased nail swelling appeared to be pH- dependent: lowering urea solution pH from pH 4.5 to pH 2 increased the nail hydration from 50% to 70% at 40% urea level. Further acidification of urea solution to pH 1 reduced nail hydration. However, the effect of urea on nail swelling is rather weak when compared to calcium thioglycolate (Figure 1 ). For this reason, there is only a limited value using urea alone for nail pretreatment to enhance nail absorption of active agents.

The pH effect of thio-compounds on nail softening and nail hydration was studied. The results were shown in Figures 4 and 5. For the experiment shown in Figure 4, all the testing solutions contained 2% calcium thioglycolate and 10% urea. Urea was used here to assist thioglycolate to break the disulfide bonds of nail protein. From pH 7 to pH 9, only about 150% nail hydration could be achieved. At the higher pH's, i.e., with more alkaline solutions, the nail hydration increased to 229% at pH 10 and to 258% at pH 11. The pH dependency of nail hydration at pH>9 can be seen more clearly when the results were plotted with the percentage weight gain of nail clippings verses the solution pH (Figure 5). There is a linear increase in nail hydration, as a result of the treatment with 2% calcium thioglycolate and 10% urea aqueous solutions from pH 9 to pH 11. This finding indicates that the preferred pH range to enhance nail absorption is equal to and above pH10. The pH effect of a thio-compound other than thioglycolate was investigated using 2% thioglycerol aqueous solutions with pH's ranging pH 7 - pH 10 (Figure 6). There was a slight increase in nail hydration as the solution pH increased from pH 7 to pH 9. Similar to the nail hydration enhanced by calcium thioglycolate in Figures 4 & 5, a steep increase in nail hydration occurred at pH>9 (Figure 7). Figure 8 compares the nail hydration effect of calcium, sodium and ammonium salts of thioglycolic acid of various concentrations at pH 10. Nail hydration in water was included as a control. The result for 1 % calcium thioglycolate at pH 11 was also included. It can be seen that at the same thioglycolic acid concentration (e.g., 2%) and pH condition, sodium salt and ammonium salt appears to have a greater nail hydration capability than calcium salt. In general, nail hydration increased as the thioglycolate concentration increased, and as the pH of calcium thioglycolate solution (1 %) increased (from pH 10 to pH 11 ). Figure 9 shows the nail hydration results from several non-thioglycolate reducing agents tested. Among them, thioethylene glycol, thiolactic acid and thioglycerol of 2% at pH 10 were found to be able to produce significant nail hydration. The rank order of nail hydration enhancement capability of the tested chemicals at 2% concentration and pH 10 is: thioethylene glycol > thiolactic acid > thioglycerol > sodium sulfide > thiosalicylic acid > sodium sulfite > sodium bisulfite > sodium thiosulfate = thiourea. Sodium thiosulfate and thiourea, which do not have any sulfhydryl functional group in their structure, were included in the nail hydration experiment to demonstrate the importance of sulfhydryl groups for the purpose of nail penetration enhancement. As can be seen from the data, the absence of sulfhydryl groups in sodium thiosulfate and thiourea made them completely ineffective in cleaving keratin disulfide bonds, as indicated by the poor nail hydration. For the same reason, ten percent ascorbic acid did not increase nail hydration as compared to water. Example 2. Nail Pretreatment Followed by Nail Penetration of Active Agents To test the nail treatment regimen, itraconazole permeation experiments were conducted using human cadaver nails with and without calcium thioglycolate pretreatment. Briefly, a nail plate was mounted on a modified Franz diffusion cell. In the nail pretreatment experiment, 1% calcium thioglycolate solution (pH 11 ) was placed in the donor chamber for 8 hours, then removed and washed thoroughly with distilled water. The donor chamber was then charged again with an itraconazole formulation of concentration 0.96%. The receptor solution was 20% HP-β-cyclodextrin at pH 4. The experiments were run under occlusive conditions at 32°C. At the end of the 225-hour permeation experiment, the itraconazole content in the nail plate was also determined by HPLC after an extraction procedure. As shown in Figure 10, nail pretreatment with 1 % calcium thioglycolate for 8 hours increased the amount of itraconazole penetrated into the nail plate by 55 fold, from 36 μg/cm³

obtained from the control (i.e., no pretreatment) to 2000 μg/cm³. Figure 11

shows that the amount of itraconazole delivered into the pretreated nail plates from another itraconazole formulation progressively increased as the itraconazole permeation prolonged. Similar experiments were also conducted for miconazole nail permeation

(Figure 12). With 2% miconazole base in a tincture formulation, a 3.6 fold increase in the amount of miconazole penetrated into the nail was achieved, from 590 μg/cm³ in the control nails to 2114 μg/cm³ in the pretreated nails. Example 3 Skin Irritation Test of Nail Penetration Enhancers Because a formulation containing a thio-compound and urea may unintentionally be in contact with the adjacent skin during the nail pretreatment procedure, the skin irritability of such a preparation was tested with human volunteers as follows. Two male volunteers participated in the test. The test formulation contained 5% calcium thioglycolate, 20% urea, and 75% distilled water. The pH of the test formulation was measured to be pH 11.5. Approximately 0.3 ml of the test formulation was placed in a standard Hill Top Chamber (2.5 cm diameter with an absorbant pad; The Hill Top Companies, Cincinnati, OH). The Hill Top Chamber with the test solution was affixed to the skin at the ventral side of the upper arm by a bandage, with the solution-soaked absorbant pad situated directly on the skin. A total of four Hill Top Chambers were tested, and two Hill Top Chambers were placed on each volunteer. Three Hill Top Chambers were removed 10 hours later. The test skin sites were evaluated. There were no signs of any skin irritation: i.e., no erythema, edema, or itchiness at all three test sites. The other remaining Hill Top Chamber was removed 24 hours after the initiation of the test, and again, there were no signs of skin irritation. These results were very surprising, since the test solution was so alkaline (pH 11.5), and calcium thioglycolate in the test solution could react with the skin keratin in the stratum corneum as well. This finding indicates that the approach disclosed in this invention can be used to treat the nail without adverse effects such as skin irritation. Table 1.

Example 4 Incompatiblity of Sulfhydryl Penetration Enhancers in Formulations Sulfhydryl compounds are highly reactive. For this reason, some of them (e.g., N-acetyl cysteine and cysteine) are used as anitoxidants in liquid and semisolid preparations to remove dissolved oxygen through an oxidation- reduction reaction. This reaction can also be catalyzed by the other formulation components. In this process, the sulfhydryl compound is oxidized, and is no longer useful as penetration enhancer. This phenomenon was observed with a topical antifungal formulation containing 1 % itraconazole, 5% N-acetyl cysteine, 10% urea, 5% salicylic acid and 0.1 % butylated hydroxytoluene in organic solvents. A stability test of this formulation revealed a rapid degradation of N-acetyl cysteine, failing the stability requirement of a two-year shelf life. Similar observations were made with other drugs as well. In an experiment to examine the compatibility of thio-compounds with azole antifungal formulations, calcium thioglycolate (5% by weight) was added to a gel formulation containing 1% itraconazole in organic solvents gelled with hydroxypropyl celullose. Upon mixing there was a strong odor of hydrogen sulfide released upon mixing, indicating the degradation of calcium thioglycolate as a result of undesirable interactions with the formulation components.

Claims

What is claimed is:

1. A method of increasing the permeability of horny human tissue to active agents which comprises applying a composition comprising an effective amount of one or more sulfur containing compounds to human horny tissue for a duration of time sufficient to increase the permeability of the horny human tissue to active agents.

2. The method of claim 1 wherein the horny human tissue is a diseased nail, corns or calluses.

3. The method of claim 1 wherein the horny human tissue is a diseased nail.

4. The method of claim 1 wherein said one or more sulfur containing compounds are thio-compounds with one or more sulfhydryl function groups capable of reacting with disulfide bonds of keratin.

5. The method of claim 1 wherein said one or more sulfur containing compounds are selected from the group consisting of thioglycolic acid, calcium thioglycolate, sodium thioglycolate, strontium thioglycolate, potassium thioglycolate, ammonium thioglycolate, lithium thioglycolate, magnesium thioglycolate, thioethylene glycol, thioglycerol, thioethanol, thioacetic acid, and thiosalicylic acid.

6. The method of claim 1 wherein said one or more sulfur containing compounds are selected from the group consisting of selected from the group consisting of thioglycolic acid, calcium thioglycolate, sodium thioglycolate, strontium thioglycolate, potassium thioglycolate, ammonium thioglycolate, lithium thioglycolate, and magnesium thioglycolate.

7. The method of claim 1 wherein said one or more sulfur containing compounds are thio-containing amino acids and their derivatives.

8. The method of claim 1 wherein said one or more sulfur containing compounds are selected from the group consisting of L-cysteine, D- cysteine, DL-cysteine, N-acetyl-L-cysteine, DL-homocysteine, L-cysteine methyl ester, L-cysteine ethyl ester, N-carbamoyl cysteine, glytathion, and cysteamine.

9. The method of claim 1 wherein said one or more sulfur containing compounds are selected from the group consisting of L-cysteine and N-acetyl-L-cysteine.

10. The method of claim 1 wherein said one or more sulfur containing compounds are certain sulfides.

11. The method of claim 1 wherein said one or more sulfur containing compounds are selected from the group consisting of potassium sulfide, calcium sulfide, sodium sulfide, lithium sulfide and strontium sulfide.

12 The method of claim 1 wherein said effective amount of one or more sulfur containing compounds range from about 0.1 % to about 40 %.

13. The method of claims 6, 8, or 11 wherein said effective amount of said one or more sulfur containing compounds range from about 0.5% to about 20%.

14. The method of claims 6, 8, or 11 wherein said effective amount of said one or more sulfur containing compounds range from about 1 % to about 10 %.

15. The method of claim 1 wherein said duration of time is from about 1 minute to about 1 day.

16. The method of claim 1 wherein said duration of time is from about 5 minutes and 12 hours.

17. The method of claim 1 wherein said duration of time is from about 10 minutes to about 30 minutes.

18. The method of claim 1 wherein said one or more sulfur containing compound is selected from the group consisting of thioglycolic acid, calcium thioglycolate, sodium thioglycolate, strontium thioglycolate, potassium thioglycolate, ammonium thioglycolate, lithium thioglycolate, and magnesium thioglycolate, said effective amount is about 1 % to about 10%, and said duration of time is from about 10 minutes to about 30 minutes.

19. A method of treating diseased or healthy human horny tissues which comprises 1 ) applying a first topical composition comprising an effective amount of one or more sulfur containing compounds to human horny tissue for a duration of time sufficient to increase the permeability of the horny human tissue to active agents and 2) applying a second topical composition containing one or more active agents to the treated horny human tissue.

20. The method of claim 19 wherein said one or more active agents are selected from the group consisting of miconazole, itraconazole, econazole, ketoconazaole, clotrimazole, butenafine, terbinafine and pharmaceutically acceptable salts thereof.

21. The method of claim 19 wherein said one or more active agents are selected from the group consisting of mupirocin, neomycin sulfate, bacitracin, polymyxin B, /-ofloxacin, tetracyclines, benzalkonium chloride, benzethonium chloride, triclocarbon, and triclosan.

22. The method of claim 19 wherein said one or more active agents are selected form the group consisting of betamethasone dipropionate, betamethasone valerate, clobetasol propionate, minoxidil, minoxidil sulfate, retinoids, cysteine and acetyl cysteine.

23. The method of claim 19 wherein said one or more sulfur containing compounds are selected from the group consisting of N-acetyl cysteine, thioglycolic acid, calcium thioglycolate, sodium thioglycolate, strontium thioglycolate, potassium thioglycolate, ammonium thioglycolate, lithium thioglycolate, and magnesium thioglycolate and said duration of time is from about 10 minutes to about 30 minutes.

24. A kit which comprises 1 ) a first topical composition which increases the permeability of horny human tissue comprising an effective amount of one or more sulfur containing compounds and 2) a second topical composition containing one or more active agents useful in the treatment of horny human tissue.

25. The kit of claim 24 wherein said horny human tissue is a diseased nail.

26. The kit of claim 24 wherein said one or more sulfur containing compounds are selected from the group consisting of N-acetyl cysteine, thioglycolic acid, calcium thioglycolate, sodium thioglycolate, strontium thioglycolate, potassium thioglycolate, ammonium thioglycolate, lithium thioglycolate, and magnesium thioglycolate; said one or more active agents are selected from the group consisting of miconazole, itraconazole, econazole, ketoconzaole, clotrimazole, butenafine, terbinafine and pharmaceutically acceptable salts thereof; and said duration of time is from about 10 minutes to about 30 minutes.

27. The kit of claim 24 wherein said effective amount is about 1 % to about 10%, and said duration of time is from about 10 minutes to about 30 minutes.

28. The kit of claim 24 wherein said one or more sulfur containing compounds are selected from the group consisting of calcium thioglycolate or ammonium thioglycolate and said one or more active agents are selected from the group consisting of miconazole, itraconazole, econazole, and ketoconazaole.