ANTIFUNGAL COMPOUNDS AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority to U.S. Provisional Application Ser. No. 61/683,710 filed, August 15, 2012 which is hereby incorporated by reference in its entirety, including all figures.

FIELD OF THE INVENTION

[002] The present compositions and methods described herein involve topical compositions for treatment of fungal infections of the skin and treatment for using the same. The compositions comprise one or more antifungal agents and one or more penetration enhancers. In one embodiment, the composition comprises tolnaftate and dodecyl 2-(N,N-dimethylamino)-propionate or a salt thereof.

BACKGROUND OF THE INVENTION

[003] Fungal skin infections include Tinea Pedis, which is commonly known as athlete's foot or ringworm of the feet and can be difficult to cure in some affected persons. Athlete's foot is caused by fungi in the genus Trichophyton.

[004] Tinea Curis, which is commonly known as jock itch is caused by Epidermophyton floccosum, Trichophyton rubrum, or Trichophyton mentagrophytes. Tinea Curis generally occurs on the medial and upper portions of the thighs, and in the pubic area. Tinea Curis lesions have specific margins with small vessels commonly found. Acute lesions are bright red, and chronic, recurring cases tend to be hyper pigmented.

[005] Tinea Corporis is known as ringworm of the skin and is more prevalent in climates with higher humidity. Tinea Corporis usually involves infections by Trichophyton or Microsporum species. Lesions causes by Tinea corporis form on smooth and bear skin areas. These lesions begin as small circular red areas and can become scaling, raised, and pruritic areas.

[006] Tinea Versicolor is a fungal infection that generally creates cosmetic concerns. Lesions generally occur on seborrheic areas in a confetti-like configuration. This common superficial fungal infection of the stratum corneum is caused by Ptiyrosporum orbiculare.

[007] Candidiasis or moniliasis is caused primarily by Candida albicans, and usually occurs in the groin, axilla, interdigital spaces, and under the breasts. [008] There is a need to provide antifungal preparations capable of treating various fungal infections that will effectively work in medically compromised, as well as relatively healthy persons. Topical preparations are best for this situation.

SUMMARY OF THE INVENTION

[009] The disclosure provides compositions, namely topical antifungal compositions, for treatment of fungal infections of the skin and methods for using such.

[0010] In some embodiments, the compositions include an effective amount of an antifungal ingredient and one or more penetration enhancers. In some embodiments the compositions include an alcohol, a preservative, an oil, a surfactant, an emulsifying agent, a glycol compound, water; and a wax.

[0011] In some embodiments the antifungal agent is tolnaftate.

[0012] In other embodiments the compositions contain one or more penetration enhancers wherein at least one penetration enhancer is a N,N-di(Ci-Cs)alkylamino substituted, (C4-Ci ₈ )alkyl(C2-Ci ₈ )carboxylic ester or pharmaceutically acceptable acid addition salt thereof. In a related aspect, the N,N-di(Ci-Cs) alkylamino substituted, (C ₄ - Ci ₈ )alkyl(C2-Ci ₈ )carboxylic ester comprises dodecyl 2-(N,N-dimethylamino)-propionate (DDAIP) or its salts. In yet another aspect, the DDAIP is an enantiomer, such as the R or S form.

[0013] An embodiment disclosed herein is an antifungal composition wherein the penetration enhancer is dodecyl 2-(N,N-dimethylamino)-propionate (DDAIP) or its salts which includes cetyl alcohol, methylparaben, mineral oil, ceteth-10, propylene glycol, propylparaben, water, sorbitan monostearate; and stearyl alcohol.

[0014] In some embodiments, the compositions contain one or more antifungal agents, such as nystatin, clortrimazole, econazole, ketoconazole, miconazole, solconazole, oxiconizole, naftifine, terbinifine, butenafine and grisiofulvin. In a related aspect the other antifungal agent is grisiofulvin.

[0015] Also disclosed here are compositions comprising about 0.1 to 10 percent concentration of said antifungal agent, and about 0.1 to 10 percent concentration of said penetration enhancer.

[0016] Another embodiment disclosed herein are compositions comprising about 0.1 to 10 percent concentration of tolnaftate, about 0.1 to 10 percent concentration of DDAIP, about 0.001 to 1.0 percent concentration of vitamin E, about 0.5 to 10.0 percent concentration of light mineral oil; and about 0.5 to 100.0 percent concentration of white petrolatum.

[0017] Disclosed herein are methods of treating fungal infections of the skin using the compositions described herein. In some embodiments the compositions are applied to the skin of a subject in need of treatment. Such applications can be applied once, twice or more times daily. The durations of treatment can be for any period of time to achieve a satisfactory result such as curing, reducing or alleviating symptoms associated with the fungal infection. In some embodiments, the treatment may last for less than a week, one week or more, such as two weeks, three weeks or 4 weeks or longer.

[0018] The methods described herein can be used to treat fungal infections of the skin such as those caused by, without limitation, one or more of Microsporum gypseum, Microsporum canis, Microsporum audouini, Microsporum japonicum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton schoenleinii, Trichophyton tonsurans, Aspergillis niger and Epidermophytom flocossum.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1-shows the penetration of tolnaftate (1%) in skin 0%, 0.05%, and 0.5% DDAIP.

[0020] Figure 2- shows the penetration of tolnaftate (1 %) in skin at 0%, 0.05%, 0.5%, and 2.5% DDAIP.

[0021] Figure 3-shows tolnaftate (1 %) kinetics in tissue at 0%, 0.05%, and 0.5% DDAIP.

[0022] The foregoing and other features and advantages will be apparent from the following more particular description of exemplary embodiments of the disclosure, and as illustrated in the drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0023] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to fungus includes a plurality of fungi species. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[0024] The term "antifungal" or "antimicrobial" refers to an ability to kill or inhibit the growth of microorganisms (including, without limitation, viruses, bacteria, yeast, fungi, protozoa, etc.), or to attenuate the severity of a microbial infection. The antimicrobial compounds of the present invention are compounds that may be used in the treatment of disease and infection.

[0025] The term "active agent" as used herein, refers to compounds with known activity for the treatment of disease caused by microbes, and in particular agents that are effective in sublingual, intraocular, intraaural, and particularly topical, application.

[0026] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease (or infection) and/or adverse effect attributable to the disease (or infection). The terms "treatment", "treating" and the like as used herein includes: (a) preventing a microbial disease and/or infection from occurring in a subject who may be predisposed to but has not yet been diagnosed as having it; (b) inhibiting the progress or transmission of a microbial disease and/or infection, i.e., arresting its development or maintenance; or (c) relieving a bacterial disease (i.e., causing regression and/or amelioration of the disease) and/or infection.

[0027] As used herein, the term "subject" refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey); other mammals such as rodents (mice, rats), cattle, pigs, horses, sheep, goats, cats, dogs; and/or birds, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of an, agent, compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.

[0028] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise. [0029] Tolnaftate (O-2-naphthyl methyl(3-methylphenyl)thiocarbamate is used topically for the treatment of certain dermatophytoses, such as Tinea cruris, Tinea corporis, Tinea manuum, and Tinea pedis, which are caused by Trychophyton rubrum, Trychophyton tonsurans, Microsporum canis, Microsporum audouini, or Epidermophyton floccosum. The exact mode of action of tolnaftate is not known, but the drug has been demonstrated to distort the hyphae and stunt mycelial growth in susceptible fungi. In vitro, tolnaftate is fungistatic or fungicidal to Microsporum gypseum, Microsporum canis, Microsporum audouini, Microsporum japonicum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton schoenleinii, Trichophyton tonsurans, and Epidermophytom flocossum. Activity of tolnaftate against Aspergillis niger varies from one strain to another.

[0030] Exemplary penetration enhancer compounds include dodecyl 2- (dimethylamino) propanoate 4-methylbenzenesulfonate, dodecyl 2-(dimethylamino) propanoate sulfate, dodecyl 2-(dimethylamino) propanoate 2-hydroxypropane- 1,2,3- tricarboxylate, dodecyl 2-(dimethylamino) propanoate phosphate, dodecyl 2- (dimethylamino) propanoate benzenesulfonate, dodecyl 2-(dimethylamino) propanoate maleate, dodecyl 2-(dimethylamino) propanoate methanesulfonate, dodecyl 2- (dimethylamino) propanoate ethanesulfonate, dodecyl 2-(dimethylamino) propanoate heminaphthalene-l,5-disulfonate, dodecyl 2-(dimethylamino) propanoate 2- hydroxyethanesulfonate, dodecyl 2-(dimethylamino)-3-hydroxybutanoate hydrochloride, dodecyl 2-(dimethylamino) acetate hydrochloride, dodecyl 2-(dimethylamino)-3- methylbutanoate hydrochloride, dodecyl 2-(dimethylamino)-3-phenylpropanoate hydrochloride, dodecyl 2-(dimethylamino)-4-methylpentanoate hydrochloride, D-dodecyl 2-(dimethylamino) propanoate hydrochloride, L-dodecyl 2-(dimethylamino) propanoate hydrochloride, dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride, dodecyl 2-(methylamino) propanoate hydrochloride, dodecyl 2-(isopropylamino) propanoate hydrochloride, dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride, dodecyl 2- ((2-(diethylamino)ethyl)amino) propanoate dihydrochloride, tridecan-2-yl 2- (dimethylamino) propanoate hydrochloride, 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride, tetradecyl 2-(dimethylamino) propanoate hydrochloride, undecyl 2-(dimethylamino) propanoate hydrochloride, decyl 2-(dimethylamino) propanoate hydrochloride, tridecyl 2-(dimethylamino) propanoate hydrochloride, octyl 2- (dimethylamino) propanoate hydrochloride, and tridecan-2-yl 2-(dimethylamino)-2- methylpropanoate. [0031] In some embodiments, the penetration enhancer is an N,N-di(d- Cs)alkylamino substituted, (C4-Ci ₈ )alkyl(C2-Ci ₈ )carboxylic ester or pharmaceutically acceptable acid addition salt thereof.

[0032] In certain embodiments, the penetration enhancer is selected from the group consisting of dodecyl 2-(methylamino) propanoate hydrochloride, dodecyl 2- (isopropylamino) propanoate hydrochloride, dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride, and dodecyl 2-((2-(diethylamino)ethyl)amino) propanoate dihydrochloride.

[0033] In addition to the active ingredient and one or more penetration enhancers, the composition may also include: an alcohol, a preservative, oil, a surfactant, an emulsifying agent, a glycol compound, water, a wax, and/or a fragrance compound.

[0034] In a set of embodiments, in addition to the active ingredient and one or more penetration enhancers, the composition may also include one or more of the following: cetyl alcohol, methylparaben, mineral oil, ceteth-10, propylene glycol, propylparaben, water, sorbitan monostearate, and/or stearyl alcohol.

[0035] Cetyl alcohol, also called 1 -hexadecanol (CH ₃ (CH ₂ )i ₅ 0H, is a solid organic compound. Dodecyl-2-N,N-Dimethylaminopropionate (DDAIP) is a pharmaceutical ingredient added to topical products to increase penetration through the skin. Chemically, DDAIP is an ester of N,N-dimethylalanine and dodecanol. DDAIP is sometimes formulated as its hydrochloride salt (DDAIP.HC1). In some embodiments, DDAIP is used as the free base while in other embodiments, DDAIP is used as a salt. The salt is generally obtained by reacting the free base with an acid. In certain embodiments, the salt is a pharmaceutically acceptable salt.

[0036] Methylparaben (Methyl paraben; Methyl p-hydroxybenzoate; Methyl arahydroxybenzoate; Nipagin M) also methyl paraben, one of the parabens, is a preservative with the chemical formula CH ₃ (C ₆ H ₄ (OH)COO). It is the methyl ester of p- hydroxybenzoic acid.

[0037] Mineral Oil is a clear, colorless, oily liquid that is a by-product of the distillation of petroleum. Mineral oil is used in medicine as a laxative and as an emollient. Given orally, it coats the bowel and softens the stool mass, thus, easing the latter' s passage. Mineral oil is completely indigestible and is not absorbed by the intestine. Its prolonged use may cause vitamin deficiencies, however, because it carries fat-soluble vitamins out of the digestive system, and, thus, prevents their absorption. Other oils, such as fish oils, such as mandarin oil, shark liver extract, salmon oil, and purified Omega-3 fatty acid fish oils, may be used

[0038] Ceteth-10 is a surfactant and emulsifying agent, and a polyethylene glycol ether of Cetyl Alcohol.

[0039] Propylene glycol, also called 1,2-propanediol or propane- 1 ,2-diol, is an organic compound (a diol or double alcohol) with formula C ₃ H ₈ O2 or HO-CH2-CHOH- CH ₃ . It is a colorless, nearly odorless, clear, viscous liquid with a faintly sweet taste, hygroscopic and miscible with water, acetone, and chloroform.

[0040] Propylparaben (propyl 4-hydroxybenzoate), the n-propyl ester of p- hydroxybenzoic acid, occurs as a natural substance found in many plants and some insects, although it is manufactured synthetically for use in cosmetics, pharmaceuticals and foods. It is a preservative typically found in many water-based cosmetics, such as creams, lotions, shampoos and bath products. As a food additive, it has the E number E216.

[0041] Sodium propyl p-hydroxybenzoate, the sodium salt of propylparaben, a compound with formula Na(C ₃ H ₇ (C ₆ H ₄ COO)0), is also used similarly as a food additive and as an anti-fungal preservation agent.

[0042] Sorbitan monostearate (Octadecanoic acid [2-[(2/?,35',4/?)-3,4-dihydroxy- 2-tetrahydrofuranyl]-2-hydroxyethyl] ester) is an ester of sorbitan (a sorbitol derivative) and stearic acid and is sometimes referred to as a synthetic wax It is primarily used as an emulsifier to keep water and oils mixed. Sorbitan monostearate is used in the manufacture of food and healthcare products, and is a non- ionic surfactant with emulsifying, dispersing, and wetting properties. Sorbitans are also known as "Spans". It is also employed to create synthetic fibers, metal machining fluid, brighteners in the leather industry, as an emulsifier in coatings, in pesticides, and in various applications in the plastics, food and cosmetics industries.

[0043] Stearyl Alcohol (Octadecan-l-ol) (also known as octadecyl alcohol or 1- octadecanol) is a substance prepared from stearic acid by the process of catalytic hydrogenation. It is a fatty alcohol. It takes the form of white solid granules or flakes, which are insoluble in water, with a melting point of 60 °C and boiling point of 210 °C (at 15 mmHg or 2.0 kPa). It has a wide range of uses as an ingredient in lubricants, resins, perfumes and cosmetics. It is used as an emollient, emulsifier, and thickener in ointments of various sorts, and is widely used as a hair coating in shampoos and hair conditioners. Stearyl alcohol is even used as a liquid solar blanket in swimming pools by forming a molecule thick layer on the surface of the water and slowing the evaporation rate of the pool water.

[0044] Suitable formulations of the composition include, for example, creams, ointments, lotions, gels, semi-solids, spray-aerosols, oils, aqueous solutions, oil-in-water or water-in-oil emulsions, ointments, pastes, and solutions or suspensions. The specific compounds of a composition to be used, as will be appreciated by those skilled in the art, is one that provides for optimum drug delivery and effectiveness of the active ingredient or ingredients.

[0045] The disclosed compositions are used for methods of treatment to relieve itching, burning and cracking, and with daily use prevents athlete's foot.

[0046] In some embodiments, the area to be treated is cleaned with, for example, without limitation, soap and water and dried thoroughly. Compositions described herein are applied, for example, as a thin layer of cream. In a related embodiment, the compositions are applied twice daily (for example, morning and night) to the affected area(s) for full treatment period of about 4 weeks or until the infection is cured, reduced and/or alleviated.

[0047] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent ( ), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0048] In some embodiments, the agent or compound prevents the condition or can be used at prophylactically effective amounts.

[0049] As used herein, unless otherwise specified, a "prophylactically effective amount" of an agent, compound and/or drug, when administered alone or in combination, prevent the condition, or one or more symptoms associated with the condition, or prevent its recurrence. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. The prophylactic amount need not result in a complete prevention of the condition; partial prevention or reduction of the fungal condition is encompassed by this term.

[0050] In some embodiments, the composition is provided in a tube or other container, such as, without limitation, for example, a 20-40g tube.

[0051] In some embodiments, the pH of the composition is about pH 5.5, 6.5 or

7.5. [0052] In some embodiments, the composition includes a penetration enhancer comprising DDAIP.HC1 enantiomers, for example, a racemic mixture or separate R and S forms.

[0053] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLES

[0054] The following non-limiting examples further illustrate the various embodiments described herein.

Example 1: 1% Tolnaftate formulations:

[0055] Following in Table 1 are two different embodiments of Tolnaftate formulations.

Table 1 FORMULA 1 FORMULA 2

COMPONENT w/w (%) w/w (%)

Tolnaftate, USP 1.00 1.00

DDAIP (Base) 0.50 1.00

Vitamin E 0.002 0.002

Light Mineral Oil 3.00 3.00

White Petrolatum Q.S. Q.S.

Example 2: Process for Making Tolnaftate 1 % Formulations:

[0056] Steps for making 1 % Tolnaftate in cream form. pH=5.37: 1 % Tolnaftate, 0.5% DDAIP: 5076mg of tolnaftate+25.4mg DDAIP base. pH=5.78; 1% Tolnaftate, 0.05% DDAIP: 5016mg of tolnaftate+2.5mg DDAIP base. pH=5.55; the creams were mixed with a handheld stirrer.

[0057] The franz cells to be dosed with the 0.5 and 0.05% formulations of DDAIP were pre-treated with \0μL· neat DDAIP base for 2 hours prior to dosing. Receiving buffer: Phosphate buffered saline, pH=7.4 containing 0.1% gentamycin sulphate and 0.05% thiourea. [0058] Another embodiment comprises: Tolnaftate (1%), butylated hydroxytulene, cetanol, liquid paraffin, methylparaben, polyoxyethylene cetylether, propylene glycol, propylparaben, purified water, sorbitan monostearaie, stearyl alcohol, DDAIP (0.5, 1.0 or 2.5%).

3-Epidermal Penetration Assessment:

OBJECTIVE/RATIONALE

[0059] The objective of this study is to assess epidermal penetration of a topical product using human skin cadaver.

Materials and Methods

Test System

Species/strain: cadaver skin

Physiological state: normal

Age/weight range at start Should be adult cadaver, average 40 years old

Cadaver Supplier: Skin Banks (US Tissue & Cells)

Number/sex of cadaver: 18 of 1 inch diameter pieces of skin in round shape/group, 3 groups, all same type of skin, and all male or all female

Identification: Back, Shoulder, Thigh, Arm depend on availability

Randomization: None

Justification: 6 pieces of skins per group to achieve statistical significance Replacement: Skins will not be replaced during the course of the study.

a. Test Article

Dosage form: Tolnaftate (1%) in three formulations:

• With 0.0 % DDAIP.HC1

• With 0.5 % DDAIP.HC1

• With 0.05 % DDAIP.HC1

Will dose 1 mg tolnaftate/ 100 μΙ7 well

Dosage preparation/storage: Prepared stored at 2-8 °C

Stability/expiration date: Prepare one day before dosing [0060] Experimental Design

I. MATERIALS AND METHODS:

1. Skin Preparation:

[0061] Human cadaver skin from UCSD Skin Bank stored in a deep freezer -80 ⁵ C will be thawed by immersing in 0.9% sodium chloride in water for 30 minutes prior to the experiment. These skin sections are mounted onto Franz diffusion cells (Permegear, Model # VC9).

2. Equipment Set-up

[0062] The receptor compartment is filled to capacity with a solution of phosphate buffered isotonic saline (PBS), pH 7.4 ± 0.1 containing 0.1% gentamicin sulfate and 0.05% thiourea. The donor compartment is clamped onto the top of the epidermis allowing for a tight fit between the dosing and receiver compartment. The receptor solution temperature is maintained at 32 ± 0.5 ⁵ C. The epidermis is allowed to equilibrate to temperature for 30 minutes prior to dosing.

3. Experimental Run

Finite Dose Study:

[0063] The experiments include 18 Franz cells with a dosing surface area of 0.64 cm ² for permeation evaluation. Each group of formulations is applied to 6 permeation cells. An exact volume of 100 μΕ of tolnaftate (1%) in cream formulation with 0, 0.5 or 0.05% DDAIP.HCl, is applied to each of the 6 Franz cells directly onto the surface of the epidermal membranes using a positive displacement pipette.

4. Sampling Plan

[0064] Samples will be stored at approximately 2-8 ⁵ C until ready for delivery for analysis.

Dose Study:

[0065] A 200 μΕ of sample in syringe is collected from each receptor chamber at 0 hr, 1 hr, 2 hrs, 4 hrs, 8 hrs and 24 hrs time points following dosing. Fresh receptor media replaced the amount removed at each collection time. An aliquot of 200 μΕ of each sample is processed for assay of tolnaftate by LCMSMS. 5. Cadaver Skin Discarded

[0066] The cadaver skin after the experiment will be discarded in accordance with the California Medical Waste Management Act.

6. Assay Method

[0067] The samples will be sent out to HSP for analysis with LCMSMS.

7. Data Evaluation

a. Values <LLQ will be assigned as zeros

b. Values > mean + 3SD will be assigned as outliers

c. The amount of G-CSF permeated Q is calculated from the following equation

Q = C _t V +∑o ^x 0.2 (C _t _ _x )

[0068] Where C _t is the concentration μg/mL of the sample at time point t and V is the volume of the cell (5.2 mL) and t-x represent all previous sampling time points. ∑o ^x 0.2 (C _t -x) is the correction for all of the sampling amounts at previous time points.

d. The flux is calculated by dividing Q _t by the area of the Franz Cell, which is 0.64 cm ² at each time point.

8. Statistical Evaluation

[0069] Differences between test formulations are evaluated using the Student's t- test or other ANOVA methods. FORMULATION

1. Group 1 : 1% tolnaftate 0% DDAIP.HC1

2. Group 2: 1% tolnaftate 0.5% DDAIP.HC1

3. Group 3: 1% tolnaftate 0.05% DDAIP.HC1

[0070] For Groups 2 and 3, the cadaver skin will be pre-treated with 10 neat DDAIP.base for two hours, after mounting on the Franz Cell apparatus

Example 4: Assessment of Epidermal Penetration:

OBJECTIVE/RATIONALE

[0071] The objective of this study is to assess epidermal penetration of a topical product using human skin cadaver. Materials and Methods

Test System

Species/strain: cadaver skin

Physiological state: Normal

Age/weight range at start Should be adult cadaver, average 40 years old

Cadaver Supplier: Skin Banks (US Tissue & Cells)

Number/sex of cadaver: 36 of 1 inch diameter pieces of skin in round shape/group, 4 groups, all same type of skin, and all male or all female

Identification: Back, Shoulder, Thigh, Arm depend on availability

Randomization: None

Justification: 9 pieces of skins per group to achieve statistical significance Replacement: Skins will not be replaced during the course of the study.

Test Article

Dosage form: Tolnaftate in four formulations:

• Tolnaftate (1 %) with 0 % DDAIP.base

• Tolnaftate (1%) with 0.05 % DDAIP.base

• Tolnaftate (1 %) with 0.5 % DDAIP.base

• Tolnaftate (1 %) with 2.5 % DDAIP.base

1 mg tolnaftate/ 100 μΙ7 well

Dosage preparation/storage: Prepared stored at 2-8 °C

Stability/expiration date: Prepare one day before dosing

Experimental Design

MATERIALS AND METHODS:

1. Skin Preparation:

[0072] Human cadaver skin from UCSD Skin Bank stored in a deep freezer -80 ⁵ C will be thawed by immersing in 0.9% sodium chloride in water for 30 minutes prior to the experiment. These skin sections are mounted onto Franz diffusion cells (Permegear, Model # VC9).

2. Equipment Set-up

[0073] The receptor compartment is filled to capacity with a solution of phosphate buffered isotonic saline (PBS), pH 7.4 ± 0.1 containing 20% ethanol 0.1% gentamicin sulfate and 0.05% thiourea. The donor compartment is clamped onto the top of the epidermis allowing for a tight fit between the dosing and receiver compartment. The receptor solution temperature is maintained at 32 + 0.5 ⁵ C. The epidermis is allowed to equilibrate to temperature for 30 minutes prior to dosing.

3. Experimental Run

Finite Dose Study:

[0074] The experiments include 36 Franz cells with a dosing surface area of 0.64 cm ² for permeation evaluation. Each group of formulations is applied to 9 permeation cells. An exact volume of 100 μΕ of tolnaftate in cream formulation with 0, 0.05, 0.05 or 2.5% DDAIP.base, is applied to each of the Franz cells directly onto the surface of the epidermal membranes using a positive displacement pipette.

4. Sampling Plan

Samples will be stored at approximately 2-8 ⁵ C until ready for delivery for analysis. Dose Study:

[0075] A 200 μΕ of sample in syringe is collected from each receptor chamber at 8 hrs following dosing. Fresh receptor media replaced the amount removed at each collection time. An aliquot of 200 μΕ of each sample is processed for assay of Tolnaftate by LCMSMS.

5. Cadaver Skin Discarded

[0076] The cadaver skin after the experiment will be discarded in accordance with the California Medical Waste Management Act.

6. Assay Method

[0077] The samples will be analyzed by LCMSMS.

7. Data Evaluation

a. Values <LLQ will be assigned as zeros

b. Values > mean + 3SD will be assigned as outliers

c. The amount of G-CSF permeated Q is calculated from the following equation

[0078] Where C _t is the concentration μg/mL of the sample at time point t and V is the volume of the cell (5.2 mL) and t-x represent all previous sampling time points. ∑o ^x 0.2 (C _t -x) is the correction for all of the sampling amounts at previous time points.

d. The flux is calculated by dividing Q _t by the area of the Franz Cell, which is 0.64 cm ² at each time point. 8. Statistical Evaluation

[0079] Differences between test formulations are evaluated using the Student's t- test or other ANOVA methods.

II. FORMULATION

1. Group 1 : 1% tolnaftate 0% DDAIP.base

2. Group 2: 1% tolnaftate 0.5% DDAIP.base

3. Group 3: 1 % tolnaftate 0.05% DDAIP.base

All groups will be heated to 37°C prior to dosing.

Example 5: NON-GLP Studies to Compare the in vitro Permeation of Tolnaftate (1 %) in the Presence or Absence of DDAIP.HCL Through Human Cadaver Epidermal Membrane Using Franz Cells:

I. FORMULATION

1. Group 1 : 1% tolnaftate 0% DDAIP.HC1

2. Group 2: 1% tolnaftate 0.5% DDAIP.HC1

3. Group 3: 1% tolnaftate 0.05% DDAIP.HC1

[0080] Results of permeation studies are shown in Table 2

Table 2-Permeation of tolnaftate with and without DDAIP

[0081] The results shown in Table 2 are also depicted in Fig. 1. Fig. 3-shows tolnaftate (1%) kinetics in tissue at 0%, 0.05%, and 0.5% DDAIP

Example 6: NON-GLP Studies to Compare the in vitro Permeation of Tolnaftate (1%) in the Presence or Absence of DDAIP.HCL Through Human Cadaver Epidermal Membrane During The Course Of a Franz Cell Permeation Study:

I. FORMULATION

Dosage form: Tolnaftate (1%) with 0 % DDAIP.base

Tolnaftate (1%) with 0.05 % DDAIP.base

Tolnaftate (1%) with 0.5 % DDAIP.base

Tolnaftate (1%) with 2.5 % DDAIP.base

Table 3-Permeation of tolnaftate

[0082] The results shown in Table 3 are depicted in Fig. 2.

[0083] Although the principles, preferred embodiments and preferred operation of the present invention have been described in detail herein, the disclosure herein is not limited to the particular illustrative forms disclosed. Persons skilled in the art will recognize that various modifications of the disclosed embodiments can be made without departing from the spirit or scope of the invention as defined by the appended claims.