Novel antifungal compositions and methods of preparation and treatment thereof

Abstract

The present invention provides an antifungal composition comprising one or more azoles and two or more excipients, wherein the first of the two or more excipients comprise chitosan, and wherein the second of the two or more excipients comprise polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), or a combination thereof. Synergistic interaction between the azoles and the excipients allows for low amount of azole to provide therapeutic effect. The present invention also provides a method of preparation as well as method of treatment of subject suffering from fungal infection using the composition of the present invention.

Description

[0001] NOVEL ANTIFUNGAL COMPOSITIONS AND METHODS OF PREPARATION AND TREATMENT THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of antifungal compositions for treatment of dermatophytosis, candidiasis, and other fungal infections. More specifically, the present invention relates to topical antifungal medication comprising low concentration of azoles and excipients which exhibit synergistic effect.

[0004] BACKGROUND OF THE INVENTION

[0005] Dermatophytosis, colloquially known as ringworm or tinea, is a fungal infection of the skin typically causing redness, itching, and scaly, circular rashes on the skin. Such infections may further affect a subject’s hair and nails, causing hair loss in the affected area. Examples of dermatophytosis include athlete’s foot, nail fungus, scalp ringworm, etc., conditions which negatively impact the subject’s physical and mental health.Dermatophytosis can be contracted when a subject walks barefoot in public areas, share personal items, participates in contact sports, or comes in contact with animals. Individuals with obesity, excessive sweating, and / or poor immunity have a higher risk of dermal fungal infections. Examples of fungi that can cause dermatophytosis in a subject include but are not limited to: Trichophyton, Microsporum, Epidermophyton, etc., with Trichophyton being the most common pathogen to cause dermatophytosis in humans and animals.

[0006] Dermatophytosis affects 40% of the world’s population, at least 1 billion people worldwide, with up to 65% probability of recurrence. Current treatments include application of topical medication and / or systemic treatment with oral medication. Examples of topical agents used for treatment of dermatophytosis include azoles, terbinafine, allylamine, naftifin, tolnaftate, nystatin and other antifungal agents, with topical terbinafine being the best-selling drug for treatment of dermatophytosis. However, studies show that there is increased terbinafine resistance of Trichophyton clinical isolates,1and that the newly discovered1Yamada, T suyoshi et al *'T erbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene.” Antimicrobial agents and chemotherapy vol. 61,1 cOOl 15-17. 27 Jun. 2017, doi: 10.1128 / AAC.00115-17species Trichophyton indotineae exhibits in vitro resistance to terbinafine.2Azoles are a class of pharmaceutical agents known for their distinctive five-membered heterocyclic ring that contains nitrogen, along with at least one other non-carbon atom such as nitrogen, sulfur, or oxygen, and feature various side chains. These compounds exert their therapeutic effects by targeting and inhibiting sterol 14-a-demethylase, an enzyme integral to the microsomal cytochrome P450 system. By blocking this enzyme, azoles reduce the synthesis of ergosterol and cause a buildup of 14-a-demethylase sterols. This disruption interferes with the proper alignment of phospholipid acyl chains and compromises the function of certain membranebound enzyme systems. Azoles are effective against a range of fungi commonly found in patients with fungal infections, including species such as Candida, Coccidioides, Aspergillus, Cryptococcus, Trichophyton, Microsporum, and Epidermophyton etc.

[0007] In addition, azoles as antifungal drugs exhibit only fungistatic effect, rendering them less effective. As such, there is a need for a novel topical drug formulation comprising azoles for treatment of dermatophytosis capable of achieving strong fungicidal effects to treat dermatophytosis.|0008| Another common fungal infection is candidiasis, infection caused by fungi from the genus Candida. Candidiasis may affect a subject’s skin and mucosal membranes including the oral cavity and pharynx (commonly known as thrush), esophagus, and sexual organs such as the vagina and penis. One common cause of candidiasis is the opportunistic pathogenic yeast Candida albicans, which can invasively infect a subject and may form biofilms.Vulvovaginal candidiasis (VVC), Candida infection of the vulva and / or vagina, occurs at least once in a lifetime in 75% of women, with a 10% chance of recurrence. 40-50% of women relapse several times. Infections may also become invasive in rare cases, spreading systemically and resulting in candidemia. Current intravaginal medication for treatment of VVC exhibit the drawbacks of short vaginal residence time, requiring frequent application, possibly not being suitable for pregnant subjects, and having reduced or little effect on drug-resistant strains. Considering these drawbacks, there is a need for a novel intravaginal / topical drug formulation comprising azoles for treatment of candidiasis with improved fungicidal effect to treat Candida mucosal infection.2Sacheli, Rosalie, and Marie-Pierre Hayette. “Antifungal Resistance in Dermatophytes: Genetic Considerations, Clinical Presentations and Alternative Therapies.” Journal of Fungi (Basel, Switzerland), U. S. National Library of Medicine, 18 Nov. 2021, www.ncbi.nlin.nih.gov / pmc / articles / PMC8622014 / .

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides an antifungal composition comprising one or more azoles and one, two, or more excipients. In an embodiment, at least one of the one or more azoles and at least one of the one, two, or more excipients exhibit synergistic antifungal activity.

[0011] The present invention further provides a method of preparation of any antifungal composition of the present invention comprising the step of:i. Preparing a water solution, wherein the water solution comprises one or more acids, one or more co-solvents, or a combination thereof;ii. Dissolving one or more azoles and one, two or more excipients in the mixture of step i;iii. Stirring the resulting mixture of step ii until completely dissolved;iv. Adjusting the pH of the solution of step iii;v. Heating the solution of step iv in a sealed container;vi. Cooling down the solution of step v to room temperature for long-term storage.100121 The present invention additionally provides a method of treatment of fungal infection of a subject comprising the step administration of any embodiment of the antifungal composition of the present invention to the subject.

[0013] DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the antifungal effect of chitosan in combination with azoles. FIG. 1A demonstrates the antifungal effect of miconazole and chitosan on Trichophyton interdigitale, T. rubrum, T. indotineae, and Epidermophyton floccosum. Miconazole with chitosan significantly enhanced antifungal activities against Trichophyton spp. and Epidermophyton floccosum. Plates in the control group (lower panel) did not contain chitosan, while plates in the CHI group had chitosan of various concentrations in the medium as indicated. Miconazole was applied to each plate via paper disks. FIG. IB demonstrates the antifungal effect of difference azole drugs in combination with chitosan against Trichophyton. As shown by the inhibition zone diameters, itraconazole, miconazole or clotrimazole combined with chitosan showed superior antifungal activity against Trichophyton.

[0015] FIG. 2 shows the result of Checkerboard Assays performed using miconazole or fluconazole in combination with chitosan to determine antifungal effect on Trichophyton.Both miconazole (MCZ) in combination with chitosan as well as fluconazole in combination with chitosan showed synergistic anti-Trichophyton effect (FIC Index < 0.5).

[0016] FIG. 3 shows the appearances and antifungal effects of miconazole and chitosan in combination with different chemicals (excipients). Gels made with different excipients exhibit varying textures, appearances, and properties. The gel based on gelatin solidified upon storage, making it unsuitable for further experimentation. PVA, PEG, [3-glycerophosphate, and sodium alginate exhibited uniform texture and appearance, while the remaining excipients exhibited uneven texture when stirred.

[0017] FIG. 4 shows the results of in vitro antifungal assay of different combinations of chitosan, azoles, and chemicals (excipients). FIG. 4A shows the antifungal effect of different formulations against Trichophyton. As shown in FIG. 4A, antifungal results of miconazole with chitosan in combination with different excipients indicate that miconazole / chitosan / PVA and miconazole / chitosan / HPC exhibited strong antifungal effect compared to the other groups. FIG. 4B shows the strong antifungal effect of miconazole / chitosan / PVA against Trichophyton interdigitale, Trichophyton indoteneae, Microsporum canis and Epidermophyton floccosum; the strong antifungal effect of miconazole / chitosan / HPC against Trichophyton interdigitale and Trichophyton indoteneae. FIG. 4C shows the antifungal effect of PVA gel containing different azole drugs. Miconazole (MCZ), itraconazole (ITZ), and sertaconazole (STZ) all showed strong anti-Trichophyton activity.

[0018] FIG. 5 shows the method of testing antifungal effect of different drug and excipient combinations using guinea pigs. Hartley guinea pigs (male, 250-300 g) were immunosuppressed with daily subcutaneous triamcinolone acetonide (dosage 20 mg per kg body weight) for 3 days prior to inoculation. Animals were anesthetized with isoflurane, and the back of each animal was depilated manually. The hairless skin was slightly abraded with sterilized sandpaper. The damaged skin (2.5 cm x 2.5 cm area) was inoculated with 25 pL Trichophyton conidia suspension (4.0 x 107conidia / mL). Following inoculation, each animal was intraperitoneally (i.p.) injected with dexamethasone (7.5 mg per kg body weight) daily for 3 days. After successful fungal infection, animals were divided randomly into groups and treated with various drugs once a day for 10 days.

[0019] FIG. 6 shows the results of different treatments on guinea pigs inoculated with Trichophyton. The efficacy of each treatment was evaluated by observing changes in skin lesions and observing fungal growth on culture plates from the inoculation site skin scrapings of inoculated animals. FIG. 6A shows photographs of the inoculation sites for clinicalevaluation, observation of changes in redness and ulcerative scaling. Treatment outcomes show that CATM-02 demonstrated superior treatment efficacy in comparison with the commercial antifungal products Lamisil and Winsolve. To determine fungal growth, a sterilized cotton swab was used to rub the inoculation site, then inoculated onto potato dextrose agar and incubated for 7 days at 25°C. The fungal growth results are shown in FIG. 6B. Altogether, CATM-02 (PVA / chitosan / miconazole) demonstrated far superior treatment outcomes due to its ability to reduce fungal survival. It should be noted that concentration of azole in CATM-02 (0.5 mg / mL) is 40 times lower than that of the commercial antifungal product Winsolve.

[0020] FIG. 7 shows the results of in vitro antifungal assay of different chemicals (excipients), chitosan, fluconazole, and combinations thereof against Candida albicans. In FIG. 7A, the label “Gel” represents the combination of chitosan and another excipient selected from PVA, sodium alginate (SA), PEG, alginic acid, and gum Arabic. As shown in FIG. 7A, PVA / chitosan + fluconazole (Gel + fluconazole; CATM-03) demonstrated significant antimicrobial effect. This result suggests that the combination of PVA and chitosan enhances antifungal effectiveness of fluconazole, leading to a more pronounced inhibition of Candida growth. FIG.7B shows the antimicrobial effect of the individual and combined components of the gel CATM-03, including PVA, chitosan, fluconazole, and combinations thereof. Group I represents application of PVA / chitosan gel with 5.4 pg of fluconazole. Group II represents PVA / chitosan gel without fluconazole. Group III represents 10% PVA without fluconazole. Group IV represents only fluconazole at 5.4 pg. Group V represents 1% chitosan with 5.4 pg of fluconazole. Group VI represents 1 % chitosan without fluconazole. The result demonstrates the superior antimicrobial effect of the combination of PVA, chitosan, and fluconazole.

[0021] FIG. 8 shows the results of antifungal assay of PVA / chitosan gel extract solutions on Candida albicans. 100 pL of 1 x 105cells / mL fibroblast cells were seeded in a 96- well plate and incubated at 37°C with 5% CO2 for 24 hrs. Culture medium was discarded from cell cultures by carefully aspirating the liquid medium. The liquid medium component of each well was replaced with 100% or 50% gel extraction solutions, then the plate was incubated at 37°C with 5% CO2 for 72 hrs. Results indicate that gel extracts No. 1 + Flu (1% chitosan, 8% PVA, 0.72 pg / mL Flu), No. 2 + Flu (1% chitosan, 10% PVA, 0.72 pg / mL Flu), No. 3 + Hu (2% chitosan, 6% PVA, 0.72 pg / mL Flu), and No. 4 + Hu (2% chitosan, 8% PVA, 0.72 pg / mL Flu) exhibited significant antifungal activity against Candida albicans. It is noted that fluconazole stock solution was dissolved in DMSO, Polysorbate 80 or / and ethanol. P < 0.05: Denoted by *. P < 0.01: Denoted by **. P < 0.001: Denoted by ***.

[0022] FIG. 9 shows the results of MTT assay of gel extracts on HDFa cells (human fibroblasts). As shown, PVA / chitosan / fluconazole gel, PVA / chitosan / miconazole gel, or PVA / chitosan / clotrimazole gel exhibit no cytotoxicity to human fibroblasts (survival rate of the human fibroblasts was greater than 70%) in comparison to the control group.

[0023] FIG. 10 shows the results of gel treatment of vaginal Candida infection in mice. The experiment includes 7 groups: PBS: physiological saline as a control; PVA / chitosan / fluconazole gel (FLU gel): gel containing PVA, chitosan, and 250 ug / mL fluconazole; PVA / chitosan / miconazole gel (MCZ gel): gel containing PVA, chitosan, and 250 pg / mL miconazole; PVA / chitosan / clotrimazole gel (CTZ gel): gel containing PVA, chitosan, and 250 pg / mL clotrimazole; PVA / chitosan gel (Gel): PVA and chitosan gel without any antifungal drugs; 5 mg / mL fluconazole solution (FLU solution): liquid solution of fluconazole; commercial antifungal cream (Commercial): a commercially available antifungal cream containing 10 mg / g clotrimazole FIG. 10A shows the counts of CFUs from the vaginal washings collected from mice after 5 days of treatment. The washings were diluted and plated to count CFUs of Candida. Results demonstrate a significant anti-Candida albicans effect of the PVA / chitosan / azole gel formulations. P < 0.05: Denoted by *. P < 0.01: Denoted by **. P < 0.001: Denoted by ***. FIG. 10B were the represented images of the vaginal appearance of mice at three different time points: Group A: Four days post Candida infection, marking the start of the treatment. Group B: Two days into the treatment period. Group C: After five days of treatment, marking the end of the experiment.

[0024] DESCRIPTION OF THE INVENTION

[0025] The compositions of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, or limitations described herein.

[0026] As used in this specification and in claims which follow, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an ingredient” includes mixtures of ingredients, reference to “an active pharmaceutical agent” includes more than one active pharmaceutical agent, and the like.

[0027] As used herein, the term “about” as a modifier to a quantity is intended to mean + 20%, ± 15%, ± 10% or ± 5% inclusive of the quantity being modified.

[0028] As used herein, the term “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0029] As used herein, the term “effective amount” or “a therapeutically effective amount” of a drug or pharmacologically active agent comprises administering an amount necessary to achieve a desired result. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the disease, the particular active agent, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a compound or pharmaceutical composition is that amount effective for inhibiting progression or reversing of any disease disclosed herein in a subject or a biological sample (e.g., in cells). In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In certain embodiments, the compound inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments of the present invention, a “therapeutically effective amount” refers to an amount of a composition sufficient to reversal of disease. In certain embodiments, the disease is reversed by about 1 %, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or any numbers and number ranges falling within these values.

[0030] As used herein, the term “fungistatic” used as a noun comprises antifungal agents that inhibit fungal growth without killing the fungus. Used as an adjective herein, the term “fungistatic” refers to the effect of an antifungal agent of inhibiting fungal growth without killing the fungus. As used herein, the term “fungicidal” refers to the effect of an antifungal agent of killing fungi, or more specifically the effect of killing fungal pathogens.

[0031] As used herein, the term “co-solvent” refers to solvents used in combination with the solutes such as an API to increase the solubility thereof. In an embodiment, the co-solvent may comprise Polysorbate 80, Polysorbate 60, Polysorbate 20, methanol, ethanol, isopropanol, Dimethyl sulfoxide, propylene glycol, polyethylene glycol, glycerol, propylene glycol, other FDA approved organic solvents, or a combination thereof. In an embodiment, the co-solvent may comprise Polysorbate 80, ethanol, or a combination thereof.

[0032] The present invention provides an antifungal composition comprising one or more azoles and one, two, or more excipients. In an embodiment, at least one of the one or moreazoles and at least one of the one, two, or more excipients exhibit synergistic antifungal activity. In an embodiment, the synergistic antifungal activity allows for substantially less azole by weight in the composition than required for equal or better therapeutic effectiveness as commercially available antifungal compositions such as Lamisil and Winsolve as illustrated by the Examples below. In an embodiment, the amount of azole by weight in the antifungal composition required to achieve the equal or better therapeutic effect is less than about 0.01% to about 0.7% by weight of the amount in commercially available antifungal compositions such as Lamisil and Winsolve such as about 0.01 %, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% including any value or value ranges falling within these values.

[0033] In an embodiment, the antifungal composition of the present invention comprises an antifungal spray, an antifungal gel, or a combination thereof. In an embodiment, the antifungal composition of the present invention comprises an antifungal gel.100341 In an embodiment, the one or more azoles comprise fluconazole, itraconazole, miconazole, clotrimazole, sertaconazole, bifonazole, fenticonazole, isoconazole, omoconazole, albaconazole, isavuconazole, ravuconazole, abafungin, flutrimazole, oxiconazole, terconazole, voriconazole, butoconazole, ketoconazole, econazole, posaconazole, isavuconazonium, sulconazole, luliconazole, efinaconazole, tioconazole, levoketoconazole, oteseconazole, or a combination thereof. In an embodiment, the one or more azoles comprise a pharmaceutically acceptable salt thereof.

[0035] In an embodiment, the concentration of each of the one or more azoles of the antifungal composition of the present invention is from about 0.00005% to about 3% by weight such as about 0.00005%, about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3% including any percentages or percentage ranges falling within these values. In an embodiment, the concentration of each of the one or more azoles of the antifungal composition of the present invention is from about 0.00005% to about 0.0002%, aboutO.0002% to about0.001%, about 0.001% to about0.005%, about0.005% to about 0.02%,about 0.02% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 2%, about 2% to about 3% by weight, or any concentration falling between these ranges.

[0036] In an embodiment, the one, two or more excipients of any embodiment of the antifungal composition of the present invention comprise any pharmaceutically acceptable excipients known to those skilled in the art. In an embodiment, the one, two or more excipients comprise chitosan, polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), polyethylene glycol (PEG), beta-glycerophosphate, sodium alginate, gum Arabic, alginic acid, gelatin, or a combination thereof. In an embodiment, the one, two or more excipients consist of chitosan, PVA, HPC, or a combination thereof.

[0037] In an embodiment, the chitosan component of any embodiment of the antifungal composition of the present invention comprises oligochitosans or derivatives thereof, low molecular weight chitosans or derivatives thereof, medium molecular weight chitosans or derivatives thereof, high molecular weight chitosans or derivatives thereof, chitosan hydrochloride or derivatives thereof, or a combination thereof. The chitosan may be hydrophilic or hydrophobic. The chitosan may be about 50% to about 99% deacetylated such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% including any percentages or percentage ranges falling within these values. In an embodiment, the chitosan may be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 75% to about 80%, about 85% to about 90%, about 95% to about 99% deacetylated. In another embodiment of the present invention, the chitosan is more than about 85% deacetylated. The chitosan may be artificially synthesized, sourced from animals such as crustaceans, sourced from non-animals such as fungi or plants, or other sources. In an embodiment, the chitosan has a molecular weight of about the range from about 1 kDa to about 400 kDa such as about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 8 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 120 kDa, about 140 kDa, about 160 kDa, about 180 kDa, about 200 kDa, about 240 kDa, about 280 kDa, about 320 kDa, about 360 kDa, or about 400 kDa including any molecular weights or weight ranges falling within these values. In an embodiment, the chitosan has a molecular weight from about 20 kDa to about 35 kDa such as about 20 kDa, about 22 kDa, about 24 kDa, about 26 kDa, about 28 kDa, about 30 kDa, about 32 kDa, about 34 kDa, or about 35 kDa including any molecular weights or weight ranges falling within these values. Inan embodiment, the chitosan has a molecular weight from about 20 to about 24 kDa, from about 24 to about 28 kDa, from about 28 to about 32 kDa, from about 28 to about 35 kDa. In yet another embodiment of the present invention, the chitosan is hydrophobic.

[0038] In an embodiment, the pH of the antifungal composition of the present invention is about pH 3.5 to about pH 6.5 such as about pH 3.5, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, or about pH 6.5 including any pH values or pH ranges falling within these values. In an embodiment, the pH of the antifungal composition is about pH 3.5 to about pH 4.0, about pH 4.0 to about pH 4.5, about pH 4.5 to about pH 5.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about pH 6.0, about pH 6.0 to about pH 6.5.

[0039] In an embodiment, the concentration of each of the one, two or more excipients is from about 0.0001% to about 20% by weight of the antifungal composition such as about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% by weight, or any concentration or concentration ranges falling between these ranges.

[0040] In an embodiment, the antifungal composition of the present invention comprises chitosan at a concentration from about 0.0001% to about 10% by weight such as about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 7%, about 8%, about 9%, about 10% by weight, or any concentration or concentration ranges falling between these values.

[0041] In an embodiment, the antifungal composition of the present invention comprises PVA at a concentration from about 1% to 20% by weight such as about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% by weight, or any concentration or concentration ranges falling between these values.

[0042] In an embodiment, the antifungal composition of the present invention comprises HPC at a concentration from about 0.5% to 5% by weight such as about 0.5%, about 0.8%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.2%, about 3.5%, about 4%, about 5% by weight, or any concentration or concentration ranges falling between these values.

[0043] In an embodiment, the antifungal composition of the present invention further comprises one or more acids, wherein the acid comprises acetic acid or other acceptable acids as known in the art. In an embodiment, the acid concentration is about 0.1% to 10% by weight such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, or any concentration or concentration ranges falling within these values. In an embodiment, the acid concentration of the antifungal composition of the present invention can be adjusted by a skilled person in the art to achieve a desired pH of the antifungal composition of the present invention.

[0044] In an embodiment, the antifungal composition of the present invention further comprises one or more co-solvents, wherein the one or more co-solvents comprise Polysorbate 80, Polysorbate 60, Polysorbate 20, methanol, ethanol, isopropanol, Dimethyl sulfoxide, propylene glycol, polyethylene glycol, glycerol, propylene glycol, other FDA approved organic solvents, or a combination thereof. In an embodiment, the one or more co-solvents consist of polysorbate 80 and ethanol. In another embodiment, the one or more co-solvents consist of dimethyl sulfoxide.

[0045] In an embodiment, the concentration of each of the one or more co-solvents is about 0.1% to about 90% by weight of any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% by weight, or any concentration or concentration ranges falling within these values. In an embodiment, the concentration of each of the one or more co-solvents is about 0.1% to about 20% by weight of any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about2%, about 3%, about 4%, about 5%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% by weight, or any concentration or concentration ranges falling within these values. In an embodiment, the concentration of each of the one or more cosolvents is about 0.1% to about 10% by weight of any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% by weight, or any concentrations or concentration ranges falling within these values.

[0046] Some exemplary embodiments of the antifungal composition comprising antifungal spray of the present invention are illustrated by the following examples (Tables 1-2):

[0047] Table 1.0048] Table 2

[0049] Some exemplary embodiments of the antifungal composition comprising antifungal gel of the present invention are illustrated by the following Examples (Tables 3-10):

[0050] Table 3.

[0051] Table 4.0052] Table 5.053] Table 6.054] Table 7.

[0055] Table 8.0056] Table 9.0057] Table 10.

[0058] The present invention further provides a method of preparation of any antifungal composition of the present invention comprising the step of:vii. Preparing a water solution, wherein the water solution comprises one or more acids, one or more co-solvents, or a combination thereof;viii. Dissolving one or more azoles and one, two or more excipients in the mixture of step i;ix. Stirring the resulting mixture of step ii until completely dissolved;x. Adjusting the pH of the solution of step iii;xi. Heating the solution of step iv in a sealed container;xii. Cooling down the solution of step v to room temperature for long-term storage.

[0059] In an embodiment, the one or more azoles comprise fluconazole, itraconazole, miconazole, clotrimazole, sertaconazole, bifonazole, fenticonazole, isoconazole, omoconazole, albaconazole, isavuconazole, ravuconazole, abafungin, flutrimazole, oxiconazole, terconazole, voriconazole, butoconazole, ketoconazole, econazole, posaconazole, isavuconazonium, sulconazole, luliconazole, efinaconazole, tioconazole, levoketoconazole, oteseconazole, or a combination thereof. In an embodiment, the one or more azoles may comprise a pharmaceutically acceptable salt thereof.

[0060] In an embodiment, the concentration of each of the one or more azoles of the antifungal composition of the present invention is about 0.00005% to about 3% by weight such as about 0.00005%, about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3% including any percentages or percentage ranges falling within these values. In an embodiment, the concentration of each of the one or more azoles of the antifungal composition of the present invention is from about 0.00005% to about 0.0002%, about 0.0002% to about 0.001%, about 0.001% to about 0.005%, about 0.005% to about 0.02%, about 0.02% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 2%, about 2% to about 3% by weight, or any concentration falling between these ranges.

[0061] In an embodiment, the one, two or more excipients of any embodiment of the antifungal composition of the present invention comprises any pharmaceutically acceptable excipients known to those skilled in the art. In an embodiment, the one, two or more excipients comprise chitosan, PVA, HPC, PEG, beta-glycerophosphate, sodium alginate, gum Arabic, alginic acid, gelatin, or a combination thereof. In an embodiment, the one, two or more excipients consist of chitosan, PVA, HPC, or a combination thereof.

[0062] In an embodiment, the concentration of each of the one, two or more excipients is from about 0.0001 % to about 20% by weight of the antifungal composition such as about 0.0001 %, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% by weight, or any concentration or concentration ranges falling between these values.

[0063] In an embodiment, the antifungal composition of the present invention comprises PVA at a concentration from about 1% to about 20% by weight such as about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% by weight, or any concentrations or concentration ranges falling within these values.

[0064] In an embodiment, the antifungal composition of the present invention comprises HPC at a concentration from about 0.5% to about 5% by weight such as about 0.5%, about 0.8%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.2%, about 3.5%, about 4%, about 5% by weight, or any concentrations or concentration ranges falling within these values.

[0065] In an embodiment, the chitosan component of any embodiment of the antifungal composition of the present invention comprises oligochitosans or derivatives thereof, low molecular weight chitosans or derivatives thereof, medium molecular weight chitosans or derivatives thereof, high molecular weight chitosans or derivatives thereof, chitosan hydrochloride or derivatives thereof, or a combination thereof. The chitosan may behydrophilic or hydrophobic. The chitosan may be about 50% to about 99% deacetylated such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% including any percentages or percentage ranges falling within these values. In an embodiment, the chitosan may be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 75% to about 80%, about 85% to about 90%, about 95% to about 99% deacetylated. In another embodiment of the present invention, the chitosan is more than about 85% deacetylated. The chitosan may be artificially synthesized, sourced from animals such as crustaceans, sourced from non-animals such as fungi or plants, or other sources. In an embodiment, the chitosan has a molecular weight of about the range from about 1 kDa to about 400 kDa such as about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 8 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 120 kDa, about 140 kDa, about 160 kDa, about 180 kDa, about 200 kDa, about 240 kDa, about 280 kDa, about 320 kDa, about 360 kDa, or about 400 kDa including any molecular weights or weight ranges falling within these values. In an embodiment, the chitosan has a molecular weight of about the range from about 20 kDa to about 35 kDa such as about 20 kDa, about 22 kDa, about 24 kDa, about 26 kDa, about 28 kDa, about 30 kDa, about 32 kDa, about 34 kDa, or about 35 kDa including any molecular weights or weight ranges falling within these values. In an embodiment, the chitosan has a molecular weight of about the range from about 20 to about 24 kDa, from about 24 to about 28 kDa, from about 28 to about 32 kDa, from about 28 to about 35 kDa. In yet another embodiment of the present invention, the chitosan is hydrophobic.

[0066] In an embodiment, the antifungal composition of the present invention comprises chitosan at a concentration from about 0.0001% to about 10% by weight such as about 0.0001 %, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 7%, about 8%, about 9%, about 10% by weight, or any concentration or concentration ranges falling within these values.

[0067] In an embodiment, the pH of the solution of step iii of the present method of preparation is adjusted to about pH 3.5 to about pH 6.5 such as about pH 3.5, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, or about pH 6.5, including any pH values or pH ranges falling within these values. In an embodiment, the pH of the solution of step iii of the present method of preparation is adjusted to about pH 3.5 to about pH 4.0, about pH 4.0 to about pH 4.5, about pH 4.5 to about pH 5.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about pH 6.0, about pH 6.0 to about pH 6.5.

[0068] In an embodiment, the one or more acids comprises acetic acid or other acceptable acids as known in the art. In an embodiment, the acid concentration is about 0.1% to about 10% by weight of the any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, or any concentrations or concentration ranges falling within these values. In an embodiment, the acid concentration of the antifungal composition of the present invention can be adjusted by a skilled person in the art to achieve a desired pH of the antifungal composition of the present invention.

[0069] In an embodiment, the one or more co- solvents comprise Polysorbate 80, Polysorbate 60, Polysorbate 20, methanol, ethanol, isopropanol, Dimethyl sulfoxide, propylene glycol, polyethylene glycol, glycerol, propylene glycol, other FDA approved organic solvents, or a combination thereof. In an embodiment, the one or more cosolvents consist of polysorbate 80 and ethanol. In an embodiment, the one or more cosolvents consist of dimethyl sulfoxide.

[0070] In an embodiment, the concentration of each of the one or more co-solvents is about 0.1 % to about 90% by weight of any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% by weight, or any concentration or concentration ranges falling within these values. In an embodiment, the concentration of each of the one or more co-solvents is about 0.1% to about 20% by weight of any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% by weight, or any concentration or concentration rangesfalling within these values. In an embodiment, the concentration of each of the one or more cosolvents is about 0.1% to about 10% by weight of any embodiment of the antifungal composition of the present invention such as about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, or any concentrations or concentration ranges falling within these values.

[0071] In an embodiment, step v of the present method of preparation comprises heating the solution of step iv to about 30°C to 70°C such as about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, including any temperatures falling within these ranges.

[0072] In an embodiment, step v of the present method of preparation comprises heating the solution of step iv for about 10 min to about 3 h such as about 10 min, about 20 min, about 30 min, about 40 min, about 50 min, about 1 h, about 1.4 h, about 1.6 h, about 1.8 h, about 2.2 h, about 2.6 h, about 3 h, including any time duration falling within these ranges.

[0073] The present invention additionally provides a method of treatment of fungal infection of a subject comprising the step of administration of any embodiment of the antifungal composition of the present invention to the subject. In an embodiment, the fungal infection comprises Trichophyton infections, Candida infections, Microsporum infections, Epidermophyton infections, or a combination thereof of a subject. In an embodiment, the fungal infection comprises dermatophytosis of a subject. In an embodiment, dermatophytosis comprises dermatophytosis in general. In an embodiment, the fungal infection comprises candidiasis of a subject. The fungal infection comprises an infection of the skin, nails, scalp, mucosal membranes including the oral cavity, pharynx, esophagus, vulva, vagina, penis, and / or other parts of a subject. In an embodiment, the subject comprises murines, simians, humans, farm animals, sport animals, pets, etc. In an embodiment, the subject is human. In an embodiment, the antifungal composition of the present invention is applied topically, intravaginally, within the oral cavity, within the pharynx, and / or on the affected parts and / or infected area of a subject. In an embodiment, the affected parts and / or infected area comprise the skin of a subject. In another embodiment, the affected parts and / or infected area comprise the mucosal membranes, scalp, and / or other parts of a subject. In an embodiment, the antifungal composition of the present invention is administered to the subject for several days. In an embodiment, the antifungal composition of the present invention is administered to the subject for several consecutive days.

[0074] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, intramuscular injection, intrathecal, intraventricular, intracerebral, or intracerebroventricular administration. The route and site of administration may be chosen to enhance delivery or targeting of the disrupting agent comprising a sitespecific targeting moiety to a particular location.

[0075] Typically, in therapeutic applications, the treatment would be for the duration of the infection, disease state or condition. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the infection, disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Such optimum conditions can also be determined using conventional techniques.

[0076] In many instances, it will be desirable to have several or multiple administrations of a pharmaceutical composition described herein. For example, they may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations may be from about one to about twelve week intervals, and in certain embodiments from about one to about four week intervals. Periodic re-administration may be desirable in the case of recurrent exposure to a particular pathogen targeted by a pharmaceutical composition described herein.

[0077] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.

[0078] Examples

[0079] Example 1: CATM-01

[0080] To prepare the CATM-01 spray solution, chitosan and azole (miconazole, clotrimazole or itraconazole) were dissolved in a mixture of acetic acid, Polysorbate 80 and ethanol in water under stirring. The mixture was then adjusted pH to 4.5 or pH 5.5 and heated to 50°C in asealed container for 1 hour and cooled to room temperature to produce CATM-01. The formulation details of CATM-01 were shown in Table 1 and Table 2.

[0081] In vitro antifungal assays

[0082] PDA medium was prepared aseptically under sterile conditions and autoclaved. The molten PDA medium was supplemented with different dilutions of chitosan such as 0.023% as final concentrations. Then the PDA medium was poured into sterilized plates and allowed to solidify.

[0083] Dermatophytes were subcultured on potato dextrose agar (PDA) at 25°C for 4 to 15 days. Following growth, conidia were harvested in sterile water, and the conidial suspension was adjusted to 1.0 x 106conidia / mL using a hemocytometer. PDA plates or PDA agar with chitosan were inoculated by dipping a sterile cotton swab into the inoculum suspensions and streaking the swab in three directions over the entire agar surface. The lids were left ajar for 3 minutes in a laminar flow cabinet to allow any excess surface moisture to be absorbed into the agar before disks were applied.

[0084] Paper disks (6 mm in diameter) were loaded with 10 pL of the prepared miconazole solutions, then placed onto the agar plates. The plates were then inverted and incubated at 30°C for 4 to 7 days to allow for fungal growth. Inhibition zone diameters were measured in millimeters. The results are shown in FIG. 1 A.

[0085] FIG. 1 A demonstrates the antifungal effect of miconazole and chitosan on Trichophyton interdigitale, T. rubrum, T. indotineae, and Epidermophyton floccosum. Miconazole with chitosan significantly enhanced antifungal activities against Trichophyton spp. and Epidermophyton floccosum. Plates in the control group (lower panel) did not contain chitosan, while plates in the chitosan (CHI) group (upper panel) had chitosan of various concentrations in the medium as indicated. Miconazole was applied to each plate via paper disks.

[0086] FIG. IB demonstrates the antifungal effect of different azole drugs in combination with chitosan against Trichophyton. As shown by the inhibition zone diameters, itraconazole combined with chitosan showed superior antifungal activity against Trichophyton.

[0087] Determination of the minimal inhibition concentration and synergistic effect

[0088] To evaluate antifungal activity of different combinations of chitosan and azoles, Trichophyton was cultured at 25°C on PDA for 2 weeks to produce an adequate amount of conidia. A mixed suspension of conidia and hyphae fragments was obtained by covering the fungal colonies with sterile water and gently rubbing the colonies with an inoculation loop.The suspension was then filtered through sterile qualitative filter paper to remove the hyphae. These conidia were then suspended in RPMI 1640 medium. The conidia concentration was adjusted with the medium to 0.5 x 104to 5 x 104CFU / mL using hemocytometer counts. The conidia suspension was seeded into 96-well plates that contained a serial two-fold dilution of azole drug (miconazole or fluconazole) and chitosan starting from the concentrations of 2 pg / mL and 0.125%, respectively. 100 pL fungal suspension and 100 pL samples at various concentrations were added to a 96-well culture plate. Each treatment was run in triplicates. After 7 days of incubation in a plate shaker at 28, the optical density at 520 nm (OD) of each well was measured using a microplate reader and the fungal growth inhibition was determined using the equation below:Growth Inhibition % — - x 100Ac

[0089] Ac represents the average OD value of the controls, and At represents the OD value of the treatment wells. The minimal inhibitory concentration (MIC) is defined as the antifungal concentration at the point when detected absorbance is 50% decreased from the control.

[0090] Based on the MIC obtained from the aforementioned experiment, two drugs (miconazole or fluconazole, in combination with chitosan) each at a volume of 50 pL with different concentrations were added to a 96-well plate along with 100 pL fungal suspension at a concentration of 0.5 x 104to 5 x 104CFU / mL. The combined effect of the two drugs was evaluated using the fractional inhibitory concentration index (FICI). The FICI of the two drugs was calculated using the following formula:T? T T > MIC., (joint). MIC, (joint)T It i —M1Ca,lc)~THC, (a]onc)If FICI < 0.5, the effect is synergistic; if FICI is between 0.5 and 4, the effect is indifference; if FICI > 4, the effect is antagonism.

[0091] FIG. 2 shows results of the Checkerboard Assays. Both miconazole (MCZ) in combination with chitosan as well as fluconazole in combination with chitosan showed synergistic anti-Trichophyton effect (FICI < 0.5).

[0092] Example 2: CATM-02

[0093] Preparation of the gel

[0094] To prepare the CATM-02 gel, several chemicals (excipients) were tested. Each of the chemicals (excipients) except for chitosan was sterilized by autoclaving. Chitosan and azole(miconazole, itraconazole, or sertaconazole) and tested chemicals were dissolved in a mixture of acetic acid, Polysorbate 80 and ethanol in water under stirring. The mixture was then adjusted pH to 4.5 or pH 5.5 and heated to 50°C in a sealed container for 1 hour and cooled to room temperature to produce CATM-02. The tested chemicals were listed below and the formulation details of CATM-02 were shown in Tables 3-6. Chemicals (excipients) used: 10% polyvinyl alcohol (PVA), 10% PEG, 8% P-glycerophosphate, 5% sodium alginate, 1% gum Arabic, 10% alginic acid, 2% hydroxypropylcellulose (HPC), 10% gelatin, or 0.04% chitosan.

[0095] As shown in FIG. 3, gels made with different excipients exhibit varying textures, appearances, and properties. The gel based on gelatin solidified upon storage, making it unsuitable for further experimentation. PVA, PEG, P-glycerophosphate, and sodium alginate exhibited uniform texture and appearance, while the remaining excipients exhibited uneven texture when stirred.

[0096] In vitro antifungal assay

[0097] Trichophyton rubrum was subcultured on potato dextrose agar (PDA) at 25°C for 4 to 15 days. After growth, conidia were harvested in sterile distilled water, and the conidial suspension was adjusted to 1.0 x 106conidia / mL using a hemocytometer. Dermatophytes were inoculated by dipping a sterile cotton swab into the inoculum suspensions and streaking the swab in three directions over the entire agar surface. Plate lids were left slightly open for 3 minutes in a laminar flow cabinet to allow excess surface moisture to absorb into the agar.

[0098] 20 pL of various substances, including different chemicals (excipients), chitosan, miconazole, and their respective combinations, were applied onto agar plates. The inhibition zone diameters were measured in millimeters after 4 to 7 days of incubation at 25°C. FIG. 4 shows the results of in vitro antifungal assay of different combinations of chitosan, azoles, and excipients. FIG. 4A and FIG. 4B show the antifungal effect of different formulations against Trichophyton, Microsporum, or Epidermophyton. As shown in FIG. 4A and FIG. 4B, antifungal results of miconazole with chitosan in combination with different excipients indicate that miconazole / chitosan / PVA and miconazole / chitosan / HPC exhibited strong antifungal effect compared to the other groups. FIG. 4C shows the antifungal effect of PVA gel containing different azole drugs. Miconazole (MCZ), itraconazole (ITZ), and sertaconazole (STZ) all showed strong anti-Trichophyton activity.

[0099] Animal model, fungal inoculation, and drug application

[0100] FIG. 5 shows the method of testing antifungal effects of different drug and excipient combinations using guinea pigs. Hartley guinea pigs (male, 250-300 g) were immunosuppressed with daily subcutaneous triamcinolone acetonide (dosage 20 mg per kg body weight) for 3 days prior to inoculation. Animals were anesthetized with isoflurane, and the back of each animal was depilated manually. The hairless skin was slightly abraded with sterilized sandpaper. The damaged skin (2.5 cm x 2.5 cm area) was inoculated with 20 pL Trichophyton conidia suspension (4.0 x 107conidia / mL). Following inoculation, each animal was intraperitoneally (i.p.) injected with dexamethasone (7.5 mg per kg body weight) daily for 3 days. After successful fungal infection, animals were divided randomly into groups and treated with various drugs once a day for 10 days.

[0101] The efficacy of each treatment was evaluated by observing changes in skin lesions, histopathological evaluation of skin tissue to determine the clinical lesion core, and observing fungal growth on culture plates from the inoculation site skin scrapings of inoculated animals.

[0102] FIG. 6A shows photographs of the skin lesions and inoculation sites for clinical evaluation, observation of changes in redness and ulcerative scaling. Treatment outcomes show that CATM-02 demonstrated superior treatment efficacy in comparison with the commercial antifungal products Lamisil and Winsolve.

[0103] To determine fungal growth, a sterilized cotton swab was used to rub the inoculation site, then inoculated onto potato dextrose agar and incubated for 7 days at 25°C. The fungal growth results are shown in FIG. 6B. Altogether, CATM-02 (PVA / chitosan / miconazole) demonstrated far superior treatment outcomes due to its ability to reduce fungal survival. It should be noted that concentration of azole in CATM-02 is 40 times lower than that of the commercial antifungal product Winsolve.

[0104] Example 3: CATM-03

[0105] Preparation of the gel

[0106] Chitosan and azole (miconazole, fluconazole or clotrimazole) and tested chemicals were dissolved in a mixture of acetic acid, Polysorbate 80 and ethanol in water under stirring. The mixture was then adjusted pH to 4.5 or pH 5.5 and heated to 50°C in a sealed container for 1 hour and cooled to room temperature to produce CATM-03. The tested chemicals were listed below and the formulation details of CATM-03 were shown in Tables 7-10. Chemicals(excipients) used: 10% polyvinyl alcohol (PVA), 10% PEG, 8% P-glycerophosphate, 5% sodium alginate, 1% gum Arabic, 10% alginic acid, 2% hydroxypropylcellulose (HPC), 10% gelatin, or 1% chitosan

[0107] Gels made with different chemicals (excipients) exhibited varying textures, appearances, and properties, but only azole / chitosan / PVA (CATM-03) demonstrated strong antifungal activity.

[0108] In vitro antifungal assay

[0109] Candida albicans SC5314 was subcultured on yeast extract peptone dextrose (YPD) at 30°C overnight. After growth, cells were harvested in sterile distilled water, and the cell suspension was adjusted to 2.0 x 106CFU / mL. The C. albicans cells were inoculated by dipping a sterile cotton swab into the inoculum suspensions and streaking the swab in three directions over the entire agar surface. Lids were left slightly open for 3 minutes in a laminar flow cabinet to allow excess surface moisture to absorb into the agar. 300 pL of various substances, including different chemicals (excipients), chitosan, azole, and their respective combinations, were applied onto the agar plates. The inhibition zone diameter was measured in mm after 24 hours of incubation at 30°C. The results are shown in FIG. 7A, wherein the label “Gel” represents the combination of chitosan and another excipient selected from PVA, sodium alginate (SA), PEG, alginic acid, and gum Arabic. As shown in FIG. 7A, PVA / chitosan + fluconazole demonstrated significant antimicrobial effect. This result suggests that the combination of PVA and chitosan enhances antifungal effectiveness of fluconazole, leading to a more pronounced inhibition of Candida growth.

[0110] FIG. 7B shows the antimicrobial effect of the individual and combined components of the gel CATM-03, including PVA, chitosan, fluconazole, and combinations thereof. Group I represents application of PVA / chitosan gel with 5.4 pg of fluconazole. Group II represents PVA / chitosan gel without fluconazole. Group III represents 10% PVA without fluconazole. Group IV represents only fluconazole at 5.4 pg. Group V represents 1% chitosan with 5.4 pg of fluconazole. Group VI represents 1 % chitosan without fluconazole. The result demonstrates the superior antimicrobial effect of the combination of PVA, chitosan, and fluconazole.

[0111] Antifungal assay of gel extract solutions

[0112] Candida albicans SC5314 was subcultured on yeast extract peptone dextrose (YPD) at 30°C overnight. After growth, cells were harvested in sterile distilled water, and the cell suspension was adjusted to 2.0 x 105CFU / mL. To prepare the gel extract solution, 200 pL ofPVA / chitosan gel was added into 5 mL of RPMI-1640 (pH 7.0) liquid medium and mixed thoroughly until dissolved. 2 mL of the gel extract solution was added into a 12-well plate. 20 pL of the cell suspension (OD = 0.01) adjusted to achieve a final concentration of 2 x 103CFU / mL was added into each well of the 12-well plate and incubated at 30°C for 24 hrs. Then 100 pL of each test sample was plated onto YPD agar plates and incubated at 30°C. Cell colonies were counted after 48 hrs. In the assays, 200 pL and 100 pL of gel dissolved in 5 mL of RPMI-1640 liquid medium is defined as 100% and 50% gel extract solutions, respectively. The experiment includes four groups: PVA / chitosan gel extract, PVA / chitosan gel extract with 0.72 pg / mL fluconazole, 0.72 pg / mL fluconazole, and a control group with only the microbial suspension.

[0113] FIG. 8 shows the results of antifungal assay of PVA / chitosan gel extract solutions on Candida albicans. Results indicate that gel extracts No. 1 + Flu (1% chitosan, 8% PVA, 0.72 pg / mL Flu), No. 2 + Flu (1% chitosan, 10% PVA, 0.72 pg / mL Flu), No. 3 + Flu (2% chitosan, 6% PVA, 0.72 pg / mL Flu), and No. 4 + Flu (2% chitosan, 8% PVA, 0.72 pg / mL Flu) exhibited significant antifungal activity against Candida albicans. P < 0.05: Denoted by *. P < 0.01: Denoted by **. P < 0.001: Denoted by ***.

[0114] MTT assays

[0115] Human fibroblasts were used for cytotoxicity testing. The cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1 % antibiotics (penicillin / streptomycin solution) in an incubator at 37°C with 5% CO2. This study assesses cytotoxicity using PVA / chitosan / azole gel extract solutions and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to measure the cell viability of human fibroblasts exposed to diluted gel extract solutions (100% and 50%). The experiment is divided into two main parts: co-culturing the gel extract solutions with cells, and then performing the MTT assay to determine cell viability.

[0116] 100 pL of 1 x 105cells / mL fibroblast cells were seeded in a 96- well plate and incubated at 37°C with 5% CO2 for 24 hrs. Culture medium was discarded from cell cultures by carefully aspirating the liquid medium. The liquid medium component of each well was replaced with 100% or 50% gel extraction solutions, then the plate was incubated at 37°C with 5% CO2 for 72 hrs.

[0117] After 72 hrs, gel extract solution was removed from the wells. Each well was washed gently with 200 pL of PBS. 200 pL of 0.5 mg / mL MTT solution was added to each well, thenthe plate was incubated at 37°C with 5% CO2 for 4 hrs. Supernatant was removed, then the purple-blue crystals left over were dissolved by adding 200 pL of DMSO to each well. The mixtures were mixed thoroughly, and the absorbance at 520 nm measured using a spectrophotometer. The absorbance values were compared to those of the control group to calculate cell viability.

[0118] Cell viability calculation

[0119] Cell viability was calculated using the formula:OD (experimental group) -0D(blank)OD (control group) -OD (blank) xl00%,wherein OD(experimental group) is the optical density of the wells treated with the gel extract solutions; OD(control group) is the optical density of the wells with only fresh culture medium; and OD(blank) is the optical density of wells without cells (blank control).

[0120] FIG. 9 shows the results of MTT assay of gel extracts on HDFa cells (human fibroblasts). As shown, PVA-chitosan-azole gel No. 1 (1% chitosan, 8% PVA) + 250 pg / mL of either FLU, MCZ, or CTZ, No. 2 (1% chitosan, 10% PVA) + 250 pg / mL of either FLU, MCZ, or CTZ, No. 3 (2% chitosan, 6% PVA) + 250 pg / mL of either FLU, MCZ, or CTZ, and No. 4 (2% chitosan, 8% PVA) + 250 pg / mL of either FLU, MCZ, or CTZ exhibit no cytotoxicity to human fibroblasts (survival rate of the human fibroblasts was greater than 70%) in comparison to the control group.

[0121] Animal trials: mouse infection and treatment

[0122] C57BL / 6 mice were used to evaluate the therapeutic efficacy of chitosan / PVA / azole gels in treating Candida vaginal infections and to assess their antifungal effectiveness against Candida albicans from the vaginal cavity of the mice. The experiment is divided into two parts: establishing a mouse model of Candida vaginal infection and treating the infected mice with the gels.

[0123] Establishing the mouse Candida vaginal infection model

[0124] Mouse selection and preparation

[0125] Female C57BL / 6 mice aged 5 to 6 weeks were selected. 0.5 mg of 0-estradiol was dissolved in 100 pL of sesame oil and administered subcutaneously to the mice 48 hrs prior to infection, in order to induce a pseudo-estrus state in the mice.

[0126] Infection Procedure

[0127] On the day of infection, Candida albicans suspension was adjusted to 5 x 107CFU / mL.20 LIL of the prepared Candida suspension was dropped into each mouse’s vagina to establish the Candida vaginal infection. Day 0 of the experiment is defined as 4 days after infection. Starting from Day 0, the mice were treated with the gels for 5 consecutive days.

[0128] Gel treatment for vaginal Candida infection

[0129] Experimental groupsEach group consisted of 3 mice. The groups are listed below:-Control group (no Candida infection)-PVA / chitosan / fluconazole gel (250 pg / mL fluconazole; 1% chitosan; 10% PVA) -PVA / chitosan / miconazole gel (250 pg / mL miconazole; 1% chitosan; 10% PVA) -PVA / chitosan / clotrimazole gel (250 pg / mL clotrimazole; 1% chitosan; 10% PVA) -PVA / chitosan gel without any drug (1% chitosan; 10% PVA)-5 mg / mL fluconazole solution-Commercial antifungal cream (10 mg / g clotrimazole)-Physiological saline (PBS)

[0130] Treatment procedureStarting from Day 0, respective treatments were applied daily for 5 consecutive days.

[0131] Evaluation

[0132] On Day 0 and Day 5, mice were euthanized using CO2. Each mouse’s vagina was washed with 50 pL of PBS, and the washings were collected and diluted with sterile doublefiltered water. Serial dilutions of 10-1, 10-2, and 10-3were performed on the washings, then the solutions were plated on agar in order for colony-forming units (CFUs) to be counted the next day. This approach allows for the assessment of the gel’s effectiveness in treating Candida vaginal infections and provides insight into how different formulations impact fungal clearance.

[0133] FIG. 10 shows the results of gel treatment of vaginal Candida infection in mice. The experiment includes 7 groups: PBS: physiological saline as a control; PVA / chitosan / fluconazole gel (FLU gel): gel containing PVA, chitosan, and fluconazole; PVA / chitosan / miconazole gel (MCZ gel): gel containing PVA, chitosan, and miconazole; PVA / chitosan / clotrimazole gel (CTZ gel): gel containing PVA, chitosan, and clotrimazole;PVA / chitosan gel (Gel): PVA and chitosan gel without any antifungal drugs; 5 mg / mL fluconazole solution (FLU solution): liquid solution of fluconazole; commercial antifungal cream (Commercial): a commercially available antifungal cream containing 10 mg / g clotrimazole; control group (no Candida infection): mice not infected with Candida, serving as a baseline for comparison. FIG. 10A shows the counts of CFUs from the vaginal washings collected from mice after 5 days of treatment. The washings were diluted and plated to count CFUs of Candida. Results demonstrate a significant anti-Candida albicans effect of the PVA / chitosan / azole gel formulations. P < 0.05: Denoted by *. P < 0.01: Denoted by **. P < 0.001: Denoted by ***.

[0134] FIG. 10B shows the vaginal appearance of mice at three different points of time: Day 0 (A Group): 4 days post Candida infection, marking the start of the treatment. Day 2 (B Group): 2 days into the treatment period. Day 5 (C Group): After 5 days of treatment, marking the end of the experiment.

[0135] It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

What is claimed is:

1. An antifungal composition comprising one or more azoles and two or more excipients, wherein the first of the two or more excipients comprise chitosan, and wherein the second of the two or more excipients comprise polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), or a combination thereof.

2. The antifungal composition of claim 1, wherein the antifungal composition comprises an antifungal gel.

3. The antifungal composition of claim 1, wherein the one or more azoles comprise fluconazole, itraconazole, miconazole, clotrimazole, sertaconazole, bifonazole, fenticonazole, isoconazole, omoconazole, albaconazole, isavuconazole, ravuconazole, abafungin, flutrimazole, oxiconazole, terconazole, voriconazole, butoconazole, ketoconazole, econazole, posaconazole, isavuconazonium, sulconazole, luliconazole, efinaconazole, tioconazole, levoketoconazole, oteseconazole, pharmaceutical acceptable salt form thereof, or a combination thereof.

4. The antifungal composition of claim 1, wherein the concentration of the one or more azoles is from about 0.00005% to about 3% by weight of the antifungal composition.

5. The antifungal composition of claim 1, wherein the concentration of each of the two or more excipients is from about 0.0001% to about 20% by weight of the antifungal composition.

6. The antifungal composition of claim 1, wherein the chitosan has a molecular weight from about 20 kDa to about 35 kDa.

7. The antifungal composition of claim 1, wherein the chitosan is more than about 85% deacetylated.

8. The antifungal composition of claim 1, wherein the two or more excipients comprise chitosan and PVA.

9. The antifungal composition of claim 8, wherein the concentration of chitosan is from about 0.0001% to about 10% by weight of the antifungal composition.

10. The antifungal composition of claim 8, wherein the concentration of PVA is from about 1% to about 20% by weight of the antifungal composition.

11. The antifungal composition of claim 1, wherein the two or more excipients comprise chitosan and HPC.

12. The antifungal composition of claim 11, wherein the concentration of chitosan is from about 0.0001% to about 10% by weight of the antifungal composition.

13. The antifungal composition of claim 11, wherein the concentration of HPC is from about 0.5% to about 5% by weight of the antifungal composition.

14. The antifungal composition of claim 1, wherein the pH of the composition is from about pH 3.5 to about pH 6.5.

15. The antifungal composition of claim 1, further comprises one or more co-solvents.

16. The antifungal composition of claim 15, wherein the one or more co-solvents comprise polysorbate 80, ethanol, dimethyl sulfoxide, or a combination thereof.

17. The antifungal composition of claim 15, wherein the concentration of each of the one or more co-solvents is from about 0.1% to about 20% by weight of the antifungal composition.

18. The antifungal composition of claim 1, further comprises one or more acids, 19. The antifungal composition of claim 18, wherein the one or more acids comprise acetic acid.

20. The antifungal composition of claim 18, wherein the concentration of each of the one or more acids is from about 0.1 % to about 10% by weight of the antifungal composition.

21. A method of preparation of the antifungal composition of claim 1, comprising the steps of:i. preparing a solution, wherein the solution comprises water, one or more cosolvents, one or more acids, or a combination thereof;ii. dissolving one or more azoles and two or more excipients in the solution of step i; iii. stirring the resulting solution of step ii until completely dissolved;iv. adjusting the pH of the solution of step iii;v. heating the solution of step iv in a sealed container;vi. cooling down the solution of step v to room temperature for long-term storage.

22. The method of claim 21, wherein the one or more azoles comprise fluconazole, itraconazole, miconazole, clotrimazole, sertaconazole, bifonazole, fenticonazole, isoconazole, omoconazole, albaconazole, isavuconazole, ravuconazole, abafungin, flutrimazole, oxiconazole, terconazole, voriconazole, butoconazole, ketoconazole, econazole, posaconazole, isavuconazonium, sulconazole, luliconazole, efinaconazole, tioconazole, levoketoconazole, oteseconazole, and pharmaceutical acceptable salt form thereof, or a combination thereof.

23. The method of claim 21, wherein the concentration of the one or more azoles is from about 0.00005% to about 3% by weight of the antifungal composition.

24. The method of claim 21, wherein the concentration of each of the two or more excipients is from about 0.0001% to about 20% by weight of the antifungal composition.

25. The method of claim 21, wherein the two or more excipients comprise chitosan and PVA.

26. The method of claim 25, wherein the concentration of chitosan is from about 0.0001% to about 10% by weight of the antifungal composition.

27. The method of claim 25, wherein the concentration of PVA is from about 1% to about 20% by weight of the antifungal composition.

28. The method of claim 25, wherein the chitosan has a molecular weight from about 20 kDa to about 35 kDa.

29. The method of claim 25, wherein the chitosan is more than about 85% deacetylated.

30. The method of claim 21, wherein the two or more excipients comprise chitosan and HPC.

31. The method of claim 30, wherein the concentration of chitosan is from about 0.0001% to about 10% by weight of the antifungal composition.

32. The method of claim 30, wherein the concentration of HPC is from about 0.5% to about 5% by weight of the antifungal composition.

33. The method of claim 30, wherein the chitosan has a molecular weight from about 20 kDa to about 35 kDa.

34. The method of claim 30, wherein the chitosan is more than about 85% deacetylated.

35. The method of claim 21, wherein in step iv the pH of the composition is adjusted to between about pH 3.5 to about pH 6.5.

36. The method of claim 21, further comprises one or more co-solvents.

37. The method of claim 36, wherein the one or more co-solvents comprise polysorbate 80, ethanol, dimethyl sulfoxide, or a combination thereof.

38. The method of claim 36, wherein the concentration of each of the one or more cosolvents is from about 0.1% to about 20% by weight of the antifungal composition.

39. The method of claim 21, further comprises one or more acids,40. The method of claim 39, wherein the one or more acids comprise acetic acid.

41. The method of claim 39, wherein the concentration of each of the one or more acids is from about 0.1% to about 10% by weight of the antifungal composition.

42. The method of claim 21, wherein step v comprises the step of heating the solution of step iv to about 30°C to about 70°C.

43. The method of claim 21, wherein step v comprises the step of heating the solution of step iv for about 10 min to about 3 h.

44. A method of treatment of fungal infection of a subject comprising the step of administering the antifungal composition of claim 1 to the subject.

45. The method of 44, wherein the fungal infection comprises Trichophyton infections, Candida infections, Microsporum infections, Epidermophyton infections, or a combination thereof.

46. The method of 44, wherein the fungal infection comprises dermatophytosis.

47. The method of 44, wherein the fungal infection comprises candidiasis.

48. The method of 44, wherein the fungal infection comprises an infection of the skin, nails, scalp, mucosal membranes including the oral cavity, pharynx, esophagus, vulva, vagina, penis, and / or other parts of a subject.

49. The method of 44, wherein the subject comprises murines, simians, humans, farm animals, sport animals, or pets.

50. The method of 44, wherein the antifungal composition is applied topically, intravaginally, within the oral cavity, within the pharynx, and / or on the affected parts and / or infected area of a subject.

51. The method of 44, wherein the antifungal composition is administered to the subject for several days or several consecutive days.

Images