Nanoparticles formed of polymer and terbinafine

Abstract

To provide a composition for use in the treatment of onychomycosis and / or tinea pedis.SOLUTION: A composition comprises a polymer capable of forming nanoparticles and terbinafine, or derivatives or salts thereof, wherein the nanoparticles are formed with and / or in the presence of terbinafine, or derivatives or salts thereof, wherein the composition is administered topically to deliver a daily dose to an infected area in the range of about 5 μg to about 50 μg of terbinafine. The invention also relates to a combination of the composition and a liquid dispensing device for dispensing a pre-defined quantity of the composition to a user's toes and / or inter-digital spaces and / or front of the foot.SELECTED DRAWING: Figure 13

Description

[Technical field]

[0001] The present invention relates to compositions (and methods of making such compositions) comprising nanoparticles formed from a polymer and terbinafine. Such compositions are particularly suitable for, but not limited to, the treatment of nail and / or skin mycoses. [Background technology]

[0002] Fungal diseases are becoming increasingly common in both humans and animals, but the treatment of such infections remains problematic due to the toxicity of antifungal compositions, the poor solubility of these compositions, and the remote locations of some infections that can prove difficult to reach using conventional pharmaceutical formulations.

[0003] Broad spectrum antifungal drugs such as amphotericin B, hamycin, filipin, and nystatin were discovered in the 1960s. However, due to toxicity, only hamycin and nystatin are used topically, while amphotericin B is used systemically. A breakthrough in antifungal therapy was the introduction of azoles, especially ketoconazole. The major classes of antifungal drugs currently used are polyenes, azoles, arylamines, lipopeptides, and pyrimidines. However, polyenes are toxic to mammalian cells. Azoles are well tolerated topically but have side effects when given systemically, and there have been some reports of resistance to azoles. Flucytosine is the most common pyrimidine used. While flucytosine has excellent tissue penetration, resistance to flucytosine can develop rapidly and produce gastrointestinal side effects. Lipopeptides exhibit low toxicity, and several clinical trials are still ongoing to test their efficacy.

[0004] The development of new antifungal drugs is limited because fungi are eukaryotic and intracellular targets, if perturbed, can also damage host cells. The increase in mycoses and the increased use of antifungal drugs have led to the development of resistance in fungi. The clinical impact of antifungal drug resistance is great, as fungal diseases cause increased morbidity and mortality in immunocompromised patients.

[0005] It is estimated that approximately 40% of newly discovered drugs fail due to inadequate delivery due to water solubility problems. For topical delivery of drugs, the barrier properties of the skin often necessitate the need for permeation enhancers to achieve the required dose of the drug.

[0006] Onychomycosis (more commonly known as fungal nail infection) causes nails to thicken, discolour, disfigure and split. If untreated, the nail can become so thick that it presses against the inside of the shoe, causing pressure, irritation and pain. There is a risk of further complications, especially in diabetics, those with peripheral vascular disease and immunocompromised patients. Fungal nail infections can cause psychological and social problems. The incidence of fungal nail infections increases with age, with a prevalence of about 30% in people over 60 years of age, with a significant incidence in Europe and even higher levels in Asia. Fungal nail infections can affect one or more toenails and / or fingernails and can completely destroy the nail if left untreated.

[0007] Current treatments for fungal infections of the nails are topical nail lacquers / paints (such as amorolfine) and / or oral antifungal drugs (such as terbinafine or itraconazole) applied once or twice a week for 6-12 months. Oral antifungal drugs can have serious side effects such as gastrointestinal upset and even liver failure. Relapses are commonly reported in 25-50% of cases, and many patients do not complete the course of treatment due to expected side effects and length of treatment time, often beginning treatment only when the disease becomes more aggressive. Current oral or topical treatments can take 6-12 months to be effective. Oral treatments require saturating the systemic circulation to reach the toes, and increased doses increase the risk of gastrointestinal and hepatic complications. Topical treatments are not effective at penetrating thickened nails and require high dosing in this regard.

[0008] Athlete's foot (otherwise known as tinea pedis, tinea pedis, or moccasin foot) is a skin mycosis commonly caused by fungi of the genus Trichophyton (most commonly T. rubrum or T. mentagrophytes). The various parasitic fungi that cause athlete's foot can also cause other skin infections such as onychomycosis and tinea cruris. Although distinct from fungal nail infections, athlete's foot also presents problems with compliance and duration of treatment.

[0009] WO 2015 / 044669 discloses a topical composition (and a method for making such a composition) for the treatment of mycoses comprising a polymer capable of forming nanoparticles and an antifungal agent. WO 2017 / 006112 discloses an antifungal composition comprising nanoparticles formed from a polymer and terbinafine, the nanoparticles comprising particles in the range of 0.5-5 nm and / or in the range of 150-250 nm. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2015 / 044669 Brochure [Patent Document 2] International Publication No. 2017 / 006112 Brochure Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to address one or more of the above problems associated with current antifungal treatments. One object of the present invention is to provide a topical antifungal treatment. Another object of the present invention is to provide a treatment that allows better penetration of the antifungal agent through the nail and / or dermis. It would be desirable if the present invention could be used as a single treatment to address both onychomycosis and tinea pedis, and be easy to apply, resulting in high treatment adherence, and having a low recurrence rate. [Means for solving the problem]

[0012] According to the present invention there is provided a composition for use in the treatment of onychomycosis and / or tinea pedis, the composition comprising a polymer capable of forming nanoparticles and terbinafine, or a derivative or salt thereof, the nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, the composition being administered topically to provide a daily dose of terbinafine in the range of about 5 μg to about 50 μg to the infected area.

[0013] According to a further alternative first aspect of the present invention there is provided a composition for use as a medicament for use in the treatment of onychomycosis and / or tinea pedis, the composition comprising a polymer capable of forming nanoparticles and terbinafine, or a derivative or salt thereof, said nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, the composition being administered topically to provide a daily dose of terbinafine in the range of about 5 μg to about 50 μg to the infected area.

[0014] According to yet a further alternative first aspect of the present invention there is provided use of a composition for the treatment of onychomycosis and / or athlete's foot, said composition comprising a polymer capable of forming nanoparticles and terbinafine, or a derivative or salt thereof, said nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, and said composition being administered topically to provide a daily dose of terbinafine in the range of about 5 μg to about 50 μg to the infected area.

[0015] According to yet another alternative first aspect of the present invention there is provided use of a composition for the manufacture of a medicament for the treatment of onychomycosis and / or tinea pedis, said composition comprising a polymer capable of forming nanoparticles and terbinafine, or a derivative or salt thereof, said nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, and said composition being administered topically to provide a daily dose of terbinafine in the range of about 5 μg to about 50 μg to the infected area.

[0016] According to yet another alternative first aspect of the present invention there is provided a method of treating onychomycosis and / or athlete's foot by topically administering to a patient in need thereof a composition comprising a polymer capable of forming nanoparticles and terbinafine, or a derivative or salt thereof, wherein the nanoparticles are formed with and / or in the presence of terbinafine, or a derivative or salt thereof, and wherein the composition is administered at a daily dose of terbinafine in the range of about 5 μg to about 50 μg to the infected area.

[0017] Preferably, the daily dose of terbinafine administered to the infected area ranges from about 10 μg to about 40 μg. More preferably, the daily dose of terbinafine administered to the infected area ranges from about 15 μg to about 35 μg. More preferably, the daily dose of terbinafine administered to the infected area ranges from about 20 μg to about 30 μg. Even more preferably, the daily dose of terbinafine administered to the infected area ranges from about 20 μg to about 30 μg. Most preferably, the daily dose of terbinafine administered to the infected area is about 25 μg.

[0018] The term "daily dose" is intended to mean an amount of terbinafine given (applied) topically to the infected areas of the digits and / or interdigital spaces and / or front of the feet, hands or limbs of an individual or animal suffering from onychomycosis and / or tinea pedis. It is envisioned that multiple areas of an individual or animal may be infected and therefore require separate doses of the composition.

[0019] The daily dose is preferably applied in the morning after the affected area has been washed or soaked and dried. Preferably, the area is not washed or soaked again for up to about 8 hours. The daily dose may be given in the morning or evening.

[0020] The composition may be administered by spraying. For the purpose of calculating the daily dose to obtain substantially complete coverage of the fingers and / or interdigital spaces and / or front of the feet or hands or infected limbs of a patient suffering from onychomycosis and / or tinea pedis, it was estimated that approximately 50% of the spray of the composition will be applied to the patient and approximately 50% of the spray of the composition will be wasted and will cover the floor or surface on which the affected limb is placed. Therefore, although the daily dose of terbinafine is most preferably about 25 μg, when the composition is applied to the infected area, the total dose contained in the sprayed dose will be twice the desired dose, taking into account the approximately 50% loss. Therefore, preferably, when the composition is administered by spraying, the amount of terbinafine sprayed towards the infected area will be in the range of about 20 μg to about 80 μg. More preferably, when the composition is administered by spraying, the amount of terbinafine sprayed towards the infected area will be in the range of about 30 μg to about 70 μg. Even more preferably, when the composition is administered by spray, the amount of terbinafine sprayed toward the infected area ranges from about 40 μg to about 60 μg.Most preferably, when the composition is administered by spray, the amount of terbinafine sprayed toward the infected area is about 50 μg.

[0021] The composition may be administered by spray application. A daily dose may be administered using one or more spray applications. A daily dose may include up to about 10 spray applications. Alternatively, a daily dose may include up to about 8 spray applications. Preferably, a daily dose may include up to about 7 spray applications. Most preferably, a daily dose includes up to about 5 spray applications. When administered by a spray device, multiple doses have been shown to advantageously provide a larger area of ​​coverage than a single dose.

[0022] The spray needs to be effective to cover the entire infected area, fingers and / or interdigital spaces and / or front of the feet, hands or legs so that it can treat onychomycosis and / or athlete's foot.

[0023] The composition comprises: a) a ratio of terbinafine, or a derivative or salt thereof, to polymer in the range of about 1:2 to about 1:4; and b) up to about 30% (v / v) (volume / volume) alcohol may include:

[0024] The polymer may include one or more of polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB) or polyethylenehexamethylene biguanide (PEHMB).

[0025] Thus, the polymer may comprise a homogeneous or heterogeneous mixture of one or more of polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylenehexamethylene biguanide (PEHMB), polymethylene biguanide (PMB), poly(allylbiguanidino-co-allylamine), poly(N-vinyl biguanide), polyallyl biguanide.

[0026] The most preferred polymer comprises polyhexamethylene biguanide (PHMB).

[0027] The term "terbinafine, or a derivative or salt thereof" is intended to mean a pharma- ceutical active substance related to terbinafine hydrochloride, a synthetic allylamine antifungal drug originally sold under the trade name Lamisil®. The term is also intended to encompass pharmaceutical variations, derivatives, and alternative salts of terbinafine hydrochloride, such as non-toxic organic or inorganic acid or base addition salts in pharma- ceutically acceptable form.

[0028] Terbinafine, or a derivative or salt thereof, may be present in the formulation in an amount ranging from about 5 to about 1000 μg / ml (about 5 μg / ml or more and about 1000 μg / ml or less). Preferably, terbinafine, or a derivative or salt thereof, may be present in the formulation in a range of about 5 to about 600 μg / ml. More preferably, terbinafine, or a derivative or salt thereof, will be present in the formulation in a range of about 25 to about 200 μg / ml. Even more preferably, terbinafine, or a derivative or salt thereof, will be present in the formulation in a range of about 50 to about 150 μg / ml. Most preferably, terbinafine, or a derivative or salt thereof, will be present in the formulation at about 100 μg / ml.

[0029] The polymer may be present in the formulation in an amount ranging from about 15 to about 3000 μg / ml. Preferably, the polymer will be present in the formulation in the range of about 15 to about 1800 μg / ml. More preferably, the polymer will be present in the formulation in the range of about 75 to about 600 μg / ml. Even more preferably, the polymer will be present in the formulation in the range of about 150 to about 450 μg / ml. Most preferably, the polymer will be present in the formulation at about 300 μg / ml. The polymer will preferably comprise PHMB.

[0030] The alcohol may be present in the formulation in an amount ranging from about 5% to about 29% or from about 5% to about 30% (v / v). More preferably, the alcohol will be present in the formulation in an amount ranging from about 10% to about 29% or from about 10% to about 30% (v / v). Even more preferably, the alcohol will be present in the formulation in an amount ranging from about 20% to about 29% or from about 20% to about 30% (v / v). Even more preferably, the alcohol will be present in the formulation in an amount up to about 25% or 23% (v / v). Most preferably, the alcohol will be present in the formulation in an amount up to about 20% (v / v).

[0031] The alcohol will preferably comprise ethanol, although the alcohol may comprise other alcohols such as methanol or propanol (alone or in combination).

[0032] The composition may also include water. The water is preferably distilled water. The water may be present in the formulation in an amount ranging from about 70% to about 95% (v / v). Preferably, the water will be present in the formulation in an amount ranging from about 70% to about 90% (v / v). More preferably, the water will be present in the formulation in an amount ranging from about 70% to about 80% (v / v). Even more preferably, the water will be present in the formulation in an amount greater than about 77% (v / v). Most preferably, the water will be present in the formulation in an amount up to about 90% (v / v), up to about 80% (v / v), or up to about 79.6% (v / v).

[0033] Terbinafine, or a derivative or salt thereof, may be present in an amount ranging from about 0.005% w / w (weight / weight) to about 1.0% w / w. Preferably, terbinafine, or a derivative or salt thereof, may be present in a range of about 0.005% w / w to about 0.6% w / w. More preferably, terbinafine, or a derivative or salt thereof, will be present in a range of about 0.025% w / w to about 0.2% w / w. Even more preferably, terbinafine, or a derivative or salt thereof, will be present in a range of about 0.05% w / w to about 0.15% w / w. Most preferably, terbinafine, or a derivative or salt thereof, will be present at about 0.1% w / w.

[0034] The polymer may be present in an amount ranging from about 0.15% w / w to about 3% w / w. Preferably, the polymer will be present in the range of about 0.15% w / w to about 1.8% w / w. More preferably, the polymer will be present in the range of about 0.75% w / w to about 0.6% w / w. Even more preferably, the polymer will be present in the range of about 0.15% w / w to about 0.45% w / w. Most preferably, the polymer will be present at about 0.3% w / w. The polymer will preferably comprise PHMB.

[0035] The alcohol may be present in an amount ranging from about 5% w / w to about 29% w / w. Preferably, the alcohol will be present in an amount ranging from about 10% w / w to about 29% w / w. More preferably, the alcohol will be present in an amount ranging from about 20% w / w to about 29% w / w. Even more preferably, the alcohol will be present in an amount up to about 29% w / w, more preferably up to about 25%, even more preferably up to about 23% w / w, and most preferably, the alcohol is present in an amount up to about 20% w / w.

[0036] The alcohol will preferably comprise ethanol, although the alcohol may comprise other alcohols such as methanol or propanol (either alone or in combination).

[0037] The composition may also include water. The water is preferably distilled water. The water may be present in an amount ranging from about 70% w / w to about 95% w / w. Preferably, the water will be present in an amount ranging from about 70% w / w to about 90% w / w. More preferably, the water will be present in an amount ranging from about 70% w / w to about 80% w / w. Even more preferably, the water will be present in an amount up to about 70% w / w, more preferably up to about 77% w / w. Most preferably, the alcohol is present in an amount up to about 90% w / w, up to about 80% w / w or up to 79.6% w / w.

[0038] Preferably, the composition comprises: (a) terbinafine, or a derivative or salt thereof, present in an amount ranging from about 0.005% w / w to about 1% w / w; (b) terbinafine, or a derivative or salt thereof, and a polymer capable of forming nanoparticles, said nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, said polymer being present in an amount ranging from about 0.015% w / w to about 3% w / w; (c) less than about 30% w / w alcohol; and (d) up to about 90% w / w water Includes.

[0039] More preferably, the composition comprises: (a) terbinafine, or a derivative or salt thereof, present in an amount ranging from about 0.025% w / w to about 0.2% w / w; (b) a polymer present in an amount ranging from about 0.75% w / w to about 0.6% w / w; (c) an alcohol present in an amount ranging from about 20% w / w to about 29% w / w, and (d) Water, present in an amount ranging from about 70% w / w to about 90% w / w Includes.

[0040] Even more preferably, the composition comprises: (a) about 0.1% w / w of terbinafine, or a derivative or salt thereof; (b) about 0.3% w / w of a polymer; (c) about 20% w / w alcohol, and (d) up to about 79.6% w / w water Includes.

[0041] The ratio of terbinafine, or a derivative or salt thereof, to the polymer may be about 1:3±0.75. Preferably, the ratio of terbinafine, or a derivative or salt thereof, to the polymer will be about 1:3±0.5. More preferably, the ratio of terbinafine, or a derivative or salt thereof, to the polymer will be about 1:3±0.25. Even more preferably, the ratio of terbinafine, or a derivative or salt thereof, to the polymer will be about 1:3±0.1. Most preferably, the ratio of terbinafine, or a derivative or salt thereof, to the polymer will be about 1:3.

[0042] The nanoparticles may comprise particles formed as two different diameter species, a first species in the range of 0.5-5 nm and a second species in the range of 50-350 nm.

[0043] The relative amounts of the first species to the second species may be approximately equal to each other, or one species may be the more abundant species in the composition.

[0044] Preferably, the particles of the first species are in the range of 0.5-3 nm. More preferably, the particles of the first species are in the range of 0.5-2.5 nm. Most preferably, the particles of the first species are in the range of 0.5-2 nm. Preferably, the particles of the second species are in the range of 75-325 nm. More preferably, the particles of the second species are in the range of 100-300 nm. Most preferably, the particles of the second species are in the range of 150-200 nm or 215 nm.

[0045] Preferably, the average diameter of the particles in said first species will be in the range of 0.5-1.5 nm. More preferably, the average diameter of the particles in said first species will be in the range of 0.6-1.4 nm. Even more preferably, the average diameter of the particles in said first species will be in the range of 0.7-1.2 nm. Most preferably, the average diameter of the particles in said first species is in the region of about 0.9 nm.

[0046] Preferably, the average diameter of the second type of particles will be in the range of 50 to 350 nm. More preferably, the average diameter of the second type of particles will be in the range of 100 to 300 nm. Even more preferably, the average diameter of the second type of particles will be in the range of 150 to 200 nm. Most preferably, the average diameter of the second type of particles will be in the region of about 160 to about 176 nm.

[0047] Preferably, the mean mode diameter of the second type of particles will be in the range of 150 to 225 nm. More preferably, the mean mode diameter of the second type of particles will be in the range of 155 to 220 nm. Even more preferably, the mean mode diameter of the second type of particles will be in the range of 160 to 215 nm. Most preferably, the mean mode diameter of the second type of particles will be in the region of about 164 to about 211 nm.

[0048] It will be apparent to one of skill in the art that the composition may further comprise one or more of the following ingredients: buffers, excipients, binders, oils, solvents, water, emulsifiers, glycerin, antioxidants, preservatives and fragrances, or any other ingredients typically found in pharmaceuticals, and especially topical creams and ointments. Furthermore, the composition could be in several forms, such as a paste or suspension. Alternatively, and more preferably, the composition is substantially free of other ingredients.

[0049] Several techniques such as centrifugation, electrophoresis, chromatography or filtration may be used to further process the mixture to select nanoparticles in the required size range. The measurement of the size / diameter of the nanoparticles is preferably performed using dynamic light scattering analysis.

[0050] In a further aspect of the present invention there is provided a spray device for the topical delivery of the compositions described herein above.

[0051] In a further aspect of the present invention, there is provided a method of topically administering a composition as hereinbefore described by using a spray device such as a pump dispenser. The composition may be sprayed from the pump dispenser at a distance of about 0-30 cm from the target area. Preferably, the composition may be sprayed from the pump dispenser at a distance of about 5-25 cm from the target area. Most preferably, the composition may be sprayed from the pump dispenser at a distance of about 5-15 cm from the target area. Advantageously, a spray distance of 5-15 cm has been shown to result in complete coverage of the target area and less than 50% loss of the sprayed dose to the surrounding area.

[0052] According to another aspect of the present invention there is provided a combination of a composition as hereinbefore described with a liquid dispensing device for dispensing a predefined amount of said composition onto an infected area (such as the toes and / or fingers of the hand or infected limb, and / or interdigital spaces and / or front of the limb), said device comprising a container for containing the composition, a nozzle for expelling (discharging) the composition from the container, and a user operable pump action mechanism for drawing a predefined amount of the composition from the container and expelling it through the nozzle, thereby atomizing the composition towards the infected area.

[0053] The device may further comprise a metered valve through which a predefined amount of the composition is dispensed. Preferably, the predefined amount of the composition comprises up to about 200 μl. More preferably, the predefined amount of the composition comprises up to about 150 μl. Most preferably, the predefined amount of the composition comprises up to about 100 μl or about 100 μl.

[0054] The device may further include and / or be associated with a distance indicating or measuring device to indicate the correct distance at which a user should place the nozzle of the device relative to the infected area (such as the user's fingers and / or interdigital spaces and / or front of the foot or hand or limb).

[0055] In use, when the nozzle of the device is placed within about 5-15 cm of the toes and / or interdigital spaces and / or front of the foot, the nozzle of the device can dispense the entire required dose in up to about 5 draws. Preferably, in use, when the nozzle of the device is placed within about 10 cm of the infected area (such as the fingers and / or interdigital spaces and / or front of the foot or hand or limb), the nozzle of the device can dispense the entire required dose in about 5 draws.

[0056] Embodiments of the invention will now be described, by way of example only, with reference to the following experiments and the accompanying drawings. [Brief description of the drawings]

[0057] [Figure 1] Figure 1 is a graph showing the results of a nail soak experiment with BB2603 (terbinafine and PHMB nanoparticles) and terbinafine alone. In this experiment, 3 mm human nail discs from healthy nail segments were suspended in equivalent concentrations of the active ingredients (0.1, 1 and 10 mg / ml) of BB2603 and terbinafine solutions. Washed and dried nails were dissolved in 5M NaOH and terbinafine levels were determined by quantitative LC-MS / MS. [Figure 2A] FIG. 2A is a photograph of frozen sectioned histology samples of nail slices from healthy human volunteers that were immersed in solutions of 0.25 mg / ml PHMB, 0.05 mg / ml FITC-labeled PHMB (1:5 addition of fluorescently labeled PHMB), and 0.1 mg / ml terbinafine at 32° C. for 24 hours (scale bar is approximately 100 μm). [Figure 2B] Figure 2B is a photograph of a frozen sectioned histology specimen of a nail section from a healthy human volunteer, immersed as in Figure 2A. Two images are presented: the entire nail section (scale bar on the left hand side is approximately 100 μm) and the central region of the nail only (scale bar on the right hand side is approximately 20 μm). Staining is clearly seen around the edge of the nail that penetrates at least 20 μm into the nail structure itself. [Diagram 3]FIG. 3 is a scatter plot showing the penetration of terbinafine into healthy human nail samples treated with BB2603 (nanoparticles of terbinafine and PHMB) or terbinafine solution. The concentration of terbinafine was determined by LC-MS / MS in ethanol washes from healthy human nails treated with BB2603 or 0.1 mg / ml terbinafine solution. Individual samples are plotted. (Diamond markers) All terbinafine samples (n=4) were below the limit of detection (<0.1 ng / ml). The two sample sets were compared using an unpaired independent two-group parametric Student's T-test, assuming that the concentration of terbinafine passing through the nails treated with terbinafine solution is 0.1 ng / ml (the limit of LC-MS / MS detection). The p-value for this test was 0.04. [Figure 4] FIG. 4 is a scatter plot showing the levels of terbinafine associated with nails treated with BB2603 (nanoparticles of terbinafine and PHMB) or terbinafine solution. The concentration of terbinafine was determined by LC-MS in lysed nail samples from healthy human nails treated with BB2603 or 0.1 mg / ml terbinafine solution. Individual samples are plotted. The two sample sets were compared using an unpaired independent two-group parametric Student's T-test. The p-value for this test was 0.02. [Diagram 5] Figure 5A is a scatter plot summarizing Franz cell data from multiple doses of BB2603 (terbinafine and PHMB nanoparticles) added to human nails, where the concentration of terbinafine (determined by LC-MS / MS) in ethanol washes from healthy human nails treated with multiple small doses of BB2603 was examined. Figure 5B is a scatter plot summarizing Franz cell data from multiple doses of BB2603 (terbinafine and PHMB nanoparticles) added to human nails, where the concentration of terbinafine (determined by LC-MS / MS) in lysed nail samples from healthy human nails treated with multiple small doses of BB2603 was examined. [Figure 6]Figures 6A-E are photographic images of yeast extract peptone dextrose (YEPD) agar plates with Trichophyton mentagrophytes after 4 days of incubation at 30°C. Each plate had a 10 mm sterile paper disk placed in the center of a Trichophyton mentagrophytes lawn. 40 μl of double distilled water or terbinafine solutions of various concentrations were spotted onto each paper disk. The concentrations of terbinafine solutions used were 0 μg / ml (control, Figure 6A), 0.06 μg / ml (Figure 6B), 0.6 μg / ml (Figure 6C), 6.00 μg / ml (Figure 6D), and 60.0 μg / ml (Figure 6E). [Figure 7A] Figures 7A and 7B are photographic images of YEPD plates evaluating the efficacy of BB2603 (nanoparticles of terbinafine and PHMB) and terbinafine samples passing through healthy human nails against T. mentagrophytes. A lawn of T. mentagrophytes was spread on a YEPD agar plate supplemented with 50 μg / ml chloramphenicol. Aqueous samples from Franz cell collection chambers after 7 days of treatment of healthy human nails with either 0.1 mg / ml terbinafine (Figure 7A) or BB2603 (Figure 7B) were spotted onto a 10 mm paper disk. The disk was placed in the center of a T. mentagrophyte plate, which was then incubated at 30°C for 5 days to allow fungal growth. The antifungal activity of terbinafine from the BB2603-treated nails is seen as a zone of clearance around the disk. [Figure 7B]Figures 7A and 7B are photographic images of YEPD plates evaluating the efficacy of BB2603 (nanoparticles of terbinafine and PHMB) and terbinafine samples passing through healthy human nails against T. mentagrophytes. A lawn of T. mentagrophytes was spread on a YEPD agar plate supplemented with 50 μg / ml chloramphenicol. Aqueous samples from Franz cell collection chambers after 7 days of treatment of healthy human nails with either 0.1 mg / ml terbinafine (Figure 7A) or BB2603 (Figure 7B) were spotted onto a 10 mm paper disk. The disk was placed in the center of a T. mentagrophyte plate, which was then incubated at 30°C for 5 days to allow fungal growth. The antifungal activity of terbinafine from the BB2603-treated nails is seen as a zone of clearance around the disk. [Figure 8] 8 is a scatter plot showing terbinafine concentrations in ethanol washings from nails treated with multiple doses of BB2603 (terbinafine and PHMB nanoparticles). The concentration of terbinafine (determined by LC-MS / MS) in ethanol washings from healthy human nails treated with multiple small doses of BB2603 was either 20% (v / v) ethanol (left hand side) or 30% (v / v) ethanol (right hand side). [Figure 9] 9 is a scatter plot showing terbinafine concentrations in lysed nails from nails treated with multiple doses of BB2603 (terbinafine and PHMB nanoparticles). The concentration of terbinafine (determined by LC-MS / MS) in lysed nails from healthy human nails treated with multiple small doses of BB2603, either in 20% (v / v) ethanol (left hand side) or 30% (v / v) ethanol (right hand side). [Figure 10A-10B] Figures 10A-10E are images of blue food dye spots from spot spray tests, where spraying was done at distances of 5 cm (Figure 10A), 10 cm (Figure 10B), 15 cm (Figure 10C), 20 cm (Figure 10D) and 25 cm (Figure 10E) from the sheet of paper. Images are all shown to the same scale. Scale bar is 1 cm. [Fig. 10C-10E] Figures 10A-10E are images of blue food dye spots from spot spray tests, where spraying was done at distances of 5 cm (Figure 10A), 10 cm (Figure 10B), 15 cm (Figure 10C), 20 cm (Figure 10D) and 25 cm (Figure 10E) from the sheet of paper. Images are all shown to the same scale. Scale bar is 1 cm. [Figure 11] FIG. 11 is a line graph showing the diameter of the total area of ​​visible dye coverage (n=2) from the spot spray test and the diameter of the area showing 100% dye coverage, plotted against the distance of the spray from the sheet of paper. [Figure 12] Figure 12 is an image of a spot from a spot spray test with blue food dye, performed by spraying at a distance of 10 cm from a sheet of paper and performing 5 repeated spray wickings. This image is not to scale. The total visible coverage and the diameter of 100% coverage were measured and are shown on the image. [Figure 13] Figures 13A and 13B are images of a paw template (Figure 13A) and underlying paper (Figure 17B) that were treated with five dye sprays from a distance of 10 cm. Areas of 100% dye coverage are indicated by lines on both images. The area of ​​the paper corresponding to 100% dye coverage was cut out and the paper was weighed as a way to assess the relative area of ​​coverage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] The objective of the following study was to investigate the feasibility, efficacy and dosing of a formulation containing terbinafine and the cationic polymer polyhexamethylene biguanide (PHMB) for use in treating onychomycosis and / or tinea pedis.

[0059] Nanoparticle formation using terbinafine and PHMB Experiments were first performed to form nanoparticles of terbinafine and PHMB, which were designated BB2603 throughout the initial experiments.

[0060] BB2603 nanoparticles were initially formed by combining terbinafine.HCl and PHMB in 30% (v / v) ethanol to final terbinafine concentrations equivalent to 0.1 mg / ml, 1 mg / ml, or 10 mg / ml. Nanoparticle formation was routinely verified on a Nanosight LM10 Zetasizer instrument (Malvern Instruments). Control terbinafine solutions were made by dissolving terbinafine.HCL in 30% (v / v) ethanol to final concentrations of 0.1 mg / ml, 1 mg / ml, or 10 mg / ml.

[0061] Initial formulation of terbinafine with PHMB in 30% ethanol was shown to significantly increase the number of nanoparticles formed, resulting in the formation of more monodisperse nanoparticles than those formed with terbinafine alone in 30% ethanol. The results showed that PHMB can be used to form monodisperse nanoparticles with antifungal drugs that can be used in the preparation of topical medicaments for the subsequent treatment of a range of potential fungal diseases.

[0062] Nanoparticle Analysis A solution of BB2603 at the equivalent concentration of 0.1 mg / ml terbinafine would produce a large amount (typically 5-10x10 8 Monodisperse particles were observed with diameters ranging from 170 to 210 nm (nanoparticles / ml). Higher concentrations of BB2603 (equivalent concentrations of terbinafine at 1 mg / ml and 10 mg / ml, respectively) were also prepared for use in initial nail immersion experiments (as described below), but these showed a loss of monodispersity, which was attributed to the higher polymer concentration allowing the formation of larger nanoparticle aggregates (data not shown).

[0063] Finally, the long-term stability of a solution of BB2603 in 30% (v / v) ethanol was evaluated by measuring the nanoparticles in solution over a period of 170 days. The analysis was performed using a Nanosight LM10, therefore only considering the larger diameter population of BB2603 nanoparticles. This analysis demonstrated that despite some initial reduction in the number of particles in solution and some variability in the mode diameter of the particles, the BB2603 nanoparticles are substantially stable for at least 5 months at room temperature under ambient light conditions.

[0064] Nail immersion experiment Healthy human nail samples were pre-incubated in ddH2O for 2 hours at 30°C. 3 mm disks were then cut from the sections using a 3 mm biopsy punch. The nail disks were placed in 250 μl of test solution in a 1.5 ml tube and incubated at 32°C in a humidified incubator with 0.5% (v / v) CO2 for 24 hours. The nail samples were removed and washed in a large volume of ddH2O to remove any drug solution on the nail. The nails were dried using clean tissue and then weighed. The weighed nails were dissolved in 200 μl of 5 M NaOH for 1 hour at 37°C. After dissolution, 200 μl of methanol was added to the sample to ensure that any terbinafine in the sample remained in solution. The amount of terbinafine in the dissolved nail sample solutions was analyzed using quantitative LC-MS / MS mass spectrometry.

[0065] Quantitative mass spectrometry (MS) was used to detect and quantitate terbinafine in the samples. Sample identifiers were masked before submission for analysis. The analysis used high performance liquid chromatography with tandem mass spectrometry (LC-MS / MS) using a Waters Acquity I-Class UPLC chromatography system coupled with a Waters Xevo TQ-S Mass Spectrometer. Terbinafine levels were quantified against a standard curve of drug standards from 0.1 to 10 ng / ml terbinafine. Samples were appropriately diluted to ensure they fell within the range of the standard curve. Samples below 0.1 ng / ml terbinafine were below the detection limit for this analysis. The concentration of terbinafine in the nail samples was normalized to the total volume of the nail and expressed as "ng terbinafine / mg of nail."

[0066] Early studies focused on using simple nail "soak" experiments in which 3 mm disks of human nails were incubated in different formulations and test solutions. These experiments could only detect whether terbinafine was associated with the nail, but did not provide direct evidence of nail penetration. However, they were technically simple to perform, relatively high throughput, and allowed a range of different formulations to be evaluated.

[0067] Terbinafine in a simple solution of terbinafine was shown to associate with human nail samples. The amount of terbinafine associated with the nail was concentration dependent between 0.1 mg and 1 mg / ml, but did not show any further increase at the higher concentration of 10 mg / ml. This would indicate that above 1 mg / ml, the nail disc reached an upper limit for the amount of terbinafine that could associate with the nail disc.

[0068] No significant differences were observed between BB2603 and terbinafine solutions at equivalent concentrations up to 0.1 mg / ml terbinafine, both of which resulted in comparable concentrations of drug in the dissolved nail samples. As observed for terbinafine, there was also an increase in drug association between BB2603 at terbinafine concentrations equivalent to 0.1 mg / ml and 1 mg / ml, but no further increase at 10 mg / ml. Again, above 1 mg / ml, BB2603 suggests that it has reached the limit of the amount of drug that can be absorbed into the nail disc in 24 hours. However, the maximum amount of drug that can be associated with BB2603-treated nails was much higher (1.3-2.5-fold) compared to terbinafine-treated nails. This increase was not attributable to differences in accessible nail surface or overall nail material, as all tests were performed on 3 mm nail discs with essentially the same surface area and with weights that differed by less than 10% between samples. Therefore, these experiments suggest that BB2603 increases the maximum amount of terbinafine that can associate with human nail, indicating that the formulation enhances drug delivery to tissues.

[0069] Although this nail immersion experiment suggested that BB2603 enhanced drug delivery to the nail, it was not possible to distinguish between increased drug penetration into the nail and increased drug binding to the nail. Therefore, it was decided to proceed with the 0.1 mg / ml BB2603 formulation for histological studies to try to obtain a direct evidence of nanoparticle penetration into tissues. This concentration was chosen because it produced the most robust and consistent nanoparticle formulations and, as previously discussed, higher concentrations of BB2603 were much more variable in nanoparticle formation.

[0070] histological study A formulation of BB2603 was made at an equivalent concentration of 0.1 mg / ml terbinafine with 1% (w / w) "loaded" Nanocin™ (a nanoparticle-based delivery platform consisting of PHMB, commercially available from Tecrea Ltd, The London Bioscience Innovation Center, 2 Royal College Street, London, NW1 0NH, UK) conjugated to FITC. This labeled BB2603 was used in the nail immersion experiments described above. The washed and dried nails were then sent for histological analysis. Histological examination and fluorescence microscopy were performed on frozen sections of the nails.

[0071] Exemplary images from a histological study using FITC-labeled BB2603 are shown in Figures 6A and 6B. Strong fluorescence was observed around the edge of the nail, consistent with BB2603 binding to the surface of the nail. In addition, we observed that staining penetrated from the surface into the nail. Although the level of staining varied, we were able to detect fluorescence deep within the nail structure itself (particularly as shown in Figure 6A).

[0072] Although this data is highly suggestive of BB2603 nanoparticles penetrating into human nails, we need to exclude the possibility that the observed staining was only due to free FITC-Nanocin™. Therefore, we decided to proceed from these histological experiments to use Franz cells to directly measure drug passage across human nail samples.

[0073] Franz Cell Nail Penetration Study The nail sections were immersed in water at 30°C overnight and quickly dried. A disk biopsy of the nail section was taken using a 3mm diameter punch. Each nail disk was added to a Franz cell and the upper chamber of the cell was attached. 40μl of the following formulation was added to the upper chamber: 0.3mg / ml PHMB + 0.1mg / ml terbinafine; or 10mg / ml terbinafine. The lower collection chamber of the Franz cell was filled with water (approximately 600μl) and the hole in the bottom of the sample chamber was also filled with ddH2O to prevent air bubbles from forming under the nail. The upper sample chamber was carefully placed into the collection chamber, making sure not to introduce air bubbles. Excess liquid from the collection chamber was drained at this point, leaving a final volume of 500μl of liquid in the lower chamber. Parafilm® was used to wrap the joint between the upper and lower chambers to prevent evaporation of the liquid.

[0074] For single-dose (continuous exposure) experiments, 40 μl of the relevant test sample (BB2603 or terbinafine control) was added to the upper sample chamber using a small pipette tip, ensuring that no air bubbles were introduced at the nail / liquid interface. The upper chamber was sealed to limit evaporation. For multiple-dose experiments, 5 μl of sample was added to the upper sample chamber directly onto the nail using a small pipette tip each day for 7 days, ensuring that no air bubbles were introduced at the nail / liquid interface. The chamber was left open to allow the sample to evaporate. The franzsae were incubated at 32° C. in a humidified incubator with 0.5% (v / v) CO2.

[0075] After this Franz cell incubation, the sample chamber and collar assembly were carefully removed and all of the liquid was removed from the lower collection chamber and the holes in the bottom of the collar. The sample chamber and collar assembly was inverted and then the underside of the nail was gently washed with 5 x 20 μl of ethanol to remove any drug associated with the underside of the nail. The combined ethanol washes were retained for analysis (100 μl total). This wash was intended to capture any terbinafine that may have passed through the nail. Terbinafine found in either the lower collection chamber or the ethanol washes under the nail represented drug that had passed through the nail.

[0076] The nail discs from the Franz cells were also analyzed for the presence of terbinafine as follows: the remaining test sample was removed from the upper sample chamber and discarded, and the sample chamber was washed five times with 100 μl of ddH2O to remove any residual test solution remaining in the sample chamber, discarding each wash. The sample chamber and collar were then disassembled and the nail sample removed. The nail was washed by immersion in a large amount of ddH2O, dried using clean tissue, and weighed. The weighed nail was then dissolved in 200 μl of 5M NaOH at 37° C. for 1 hour. After dissolution, 200 μl of methanol was added to the sample to ensure that any terbinafine in the sample remained in solution.

[0077] Figures 3-5B summarize data from Franz cell analysis of drug passage across human nail samples. Only data present from lysed nail samples and undernail ethanol washes are provided as these observations appeared to be the most robust across samples. However, it was always possible to detect terbinafine in the underchamber of BB2603-treated nails, sometimes at very high levels (>0.6 μg / ml). This analysis is believed to represent a conservative view of the amount of terbinafine passing through the nail in BB2603-treated samples.

[0078] Single dose (constant exposure) experiment 40 μl of BB2603 solution (equivalent to 0.1 mg / ml terbinafine) or terbinafine (0.1 mg / ml) in 30% (v / v) ethanol was added to the sample chamber of a Franz cell containing healthy human nail samples. The cell was then incubated at 32° C. for 7 days. The sample remained in contact with the upper nail surface for the duration of each experiment. After 7 days, samples from the underside of the nail (ethanol washes) were collected and analyzed by LC-MS / MS. Day 7 nail samples were washed and dissolved using 5 M NaOH as previously described. All collected samples were analyzed for the presence of terbinafine using high performance liquid chromatography with tandem mass spectrometry (LC-MS / MS) on a Waters Acquity I-Class UPLC chromatography system coupled with a Waters Xevo TQ-S Mass Spectrometer. Terbinafine levels were quantified against drug standards. The detection limit in these analyses was 0.1 ng / ml.

[0079] As shown in Figures 3 and 4, samples treated with BB2603 consistently demonstrated penetration of terbinafine into healthy human nail samples. Terbinafine could be detected in both the collection chamber solution and in ethanol washes from the underside of the nail as early as one day of incubation. Analysis of ethanol washes from the underside of nails treated with BB2603 for 7 days revealed robust drug delivery through the nail by BB2603 (as shown in Figure 3). The amount delivered varied between samples, likely due to natural variability in nail samples, but in all cases the amount delivered was predicted to be greater than that required to achieve a mycologically killing dose.

[0080] In contrast, terbinafine solutions did not penetrate the nail, and in all experiments using an equivalent drug concentration to BB2603 (0.1 mg / ml), the amount of terbinafine passing through the nail was below the detection limit in LC-MS / MS (<0.1 ng / ml) (as illustrated in Figure 3). By assuming that terbinafine penetrated to a concentration of 0.1 ng / ml, a statistical test could be applied to the data above to reveal that the results of BB2603 were significantly different from those of plain terbinafine solution. The p-value of 0.04 calculated for this analysis is an underestimate of significance, since it assumed the highest possible concentration of terbinafine in the plain terbinafine-treated samples.

[0081] The amount of dissolved nail sample terbinafine from the Franz cells was also measured on day 7 (as shown in FIG. 4). This represents the amount of drug bound to the top surface of the nail (not washed off prior to dissolution with 5M NaOH) along with any drug in the nail itself, i.e., drug that penetrated the nail but did not reach the other side. We found significantly greater amounts of terbinafine associated with nails treated with BB2603 compared to terbinafine alone (approximately 2-fold median difference, p=0.02). This is again consistent with the idea that BB2603 increases the delivery of terbinafine into (through) the nail.

[0082] Multiple dose experiments In the single dose experiment, the test solution remained in constant contact with the upper surface of the nail throughout the incubation period. This does not necessarily reflect the reality of patient application, where the drug would be applied daily to the infected nail and then left to dry. Therefore, in an attempt to replicate this situation, an experiment was performed in which 5 μl of BB2603 was added daily to the nail in a Franz cell. This small amount was sufficient to cover the nail disc surface, but volatilized before the next addition, which more closely mimics a patient applying BB2603 as a daily topical treatment. Samples from the ethanol washes of the underside of the nail and the nail itself were collected and analyzed by LC-MS / MS for the presence of terbinafine as described above.

[0083] In this multiple dose experiment, significant amounts of terbinafine were detected associated with the nail itself (as illustrated in Figures 5A and 5B). This was at levels not significantly different from those observed in the single dose (constant exposure) experiment with BB2603 (as shown in Figure 4), and again higher than the terbinafine control from the single dose experiment. Significant amounts of terbinafine were also detected in the ethanol wash from the underside of the nail, indicating that the drug also passed through the nail in this experiment. Compared to the single dose (constant exposure) experiment, the levels of drug delivered through the nail were much lower in the multiple dose experiment. This is consistent with the idea that longer treatment with BB2603 results in greater delivery of terbinafine through the nail.

[0084] Trichophyton mentagrophytes antifungal assay Although previous experiments clearly demonstrated that BB2603 delivers terbinafine through the nail, it was necessary to confirm that the passage of the drug through the nail did not cause chemical denaturation that would lead to a loss of efficacy. Therefore, an antifungal assay using a lawn of Trichophyton mentagrophytes was performed.

[0085] Trichophyton mentagrophytes is a laboratory fungal species related to the major pathogens associated with onychomycosis (see, e.g., Wade Foster et al., J. American Acad. Dermatology. 2004.50(5):748-752), and therefore we expected that efficacy against this species would translate to efficacy against pathogenic Trichophyton species such as T. rubum (Table 1 below).

[0086] [Table 1]

[0087] A single colony of T. mentagrophytes was picked from a stock plate and grown in 5 ml of YEPD (yeast extract, peptone, dextrose) medium at 30°C for 48 h. A sterile swab was dipped into the resulting culture, which was then used to spread a lawn of T. mentagrophytes on a YEPD agar plate supplemented with chloramphenicol (50 μg / ml). Chloramphenicol was included because samples from Franz cells were not sterile and showed bacterial growth on regular YEPD plates. A 10 mm sterile paper disk was dipped into the test solution, excess liquid was removed, and the disk was placed on top of the lawn of T. mentagrophytes. The plate was inverted and incubated at 30°C for 5 days.

[0088] The first experiment performed was to establish the approximate MIC (minimum inhibitor concentration) for terbinafine against Trichophyton mentagrophytes in a paper disc assay. To do this, a 1:10 dilution series of terbinafine.HCl in ddH2O was generated from 60 μg / ml to 0.06 μg / ml. 10 mm sterile paper discs were then dipped into the various dilutions and these were placed on top of a lawn of Trichophyton mentagrophytes. After 5 days of incubation, zones of clearance (no fungal growth) were observed around the discs with concentrations of terbinafine that had antifungal activity against this species (as shown in Figure 6). The MIC for terbinafine in this assay was 0.6 μg / ml, and no clear zones of clearance were observed below this concentration range. We noted that the MIC in this assay was >100-fold higher than the MIC (6 ng / ml) previously reported for terbinafine against Trichophyton mentagrophytes (see Table 1 above). The reported figures were certainly derived from liquid MIC assays, which are known to be more sensitive, and thus the paper disc assay represented a substantially more stringent test of drug efficacy.

[0089] This assay was also used to address the question of whether terbinafine passing through the nail in samples treated with BB2603 still retained its antifungal efficacy. To do this, a Trichophyton mentagrophytes flora assay was performed using a sample of the aqueous phase from one of the Franz cell experiments that quantitative LC-MS / MS analysis demonstrated to contain >0.6 μg / ml terbinafine (Figure 7A-B). Consistent with the MIC experiment and quantitative MS results for this sample, a clear zone of clearance could be seen for BB2603, but no effect for the terbinafine control sample. Thus, terbinafine passing through healthy human nails treated with BB2603 retained its efficacy and was still able to kill Trichophyton mentagrophytes.

[0090] Potential efficacy of BB2603 in onychomycosis The objective with BB2603 was to compare the performance of oral terbinafine with topical formulations of terbinafine that were not expected to have the safety issues associated with systemic drug exposure. Compared to terbinafine solution, BB2603 was shown to significantly increase the delivery of drug through healthy human nails. An important question is whether the dose achieved by BB2603 dosing is predicted to be effective in treating onychomycosis. To address this question, the terbinafine concentrations observed in the Franz cell experiments described above were compared to those reported in the nails of patients treated with oral terbinafine (Leyden, J. Am. Acad. Dermatol. 1998.38:S42-7).

[0091] Following oral dosing, terbinafine reaches a nail concentration of 0.1 μg / g after 7 days of treatment, rising to approximately 0.25 μg / g after 3 weeks and 0.55 μg / g after 18 months (Leyden, 1998). All of these levels are higher than the MICs for a range of important fungal species associated with onychomycosis (Table 1), thus illustrating the drug's efficacy in treating fungal infections of the nail in these patients.

[0092] BB2603 appears to greatly exceed this level in dissolved nails (FIGS. 4 and 5A-5B), achieving a median concentration equivalent to approximately 1 mg / g drug in the nail after 7 days (10,000-fold higher than oral dosing). However, although lower, terbinafine alone also demonstrated significant levels of drug associated with dissolved nails (median concentration of approximately 0.5 mg / g), clinical trials with topical terbinafine (at doses much higher than those used in these experiments) failed to demonstrate efficacy in treating onychomycosis (Elewski et al., Journal of the European Academy of Dermatology and Venereology. 2013,27(3),pp. 287-294).

[0093] Although a significant amount of drug was found associated with the nails treated with the terbinafine solution, no significant amount of terbinafine was measured passing through the nail in any of our samples (FIG. 7), and for these samples we concluded that a significant amount of the drug was either bound to the upper surface of the nail or had not penetrated very deeply into the tissue.

[0094] In contrast to terbinafine treated samples, terbinafine was always detected on the underside of BB2603 treated nails indicating that the drug must have entered and passed through the nail. Thus, measurements of terbinafine in dissolved nails from BB2603 treated samples represent not only drug associated with the upper surface but also drug present throughout the entire depth of the tissue.

[0095] It is highly likely that the BB2603 treated nail confirms an asymmetric distribution of the drug, with higher concentrations at the upper (treated) surface and the lowest concentrations of drug found towards the bottom of the nail. For this reason, we estimated the concentration of terbinafine in the lower part of the nail, as this was believed to be the lowest concentration of drug in our samples. To do this, we assumed that the level of drug found on the underside of the nail (in the ethanol wash) was equivalent to the concentration in the nail just above the underside of the nail in the nail disc. Although a 3 mm diameter disc of nail was used in the Franz cell experiment, only a 1.5 mm diameter circle of the nail was in contact with the solution in the upper and lower chambers (the rest of the nail forms a seal with the chamber itself). This suggests that terbinafine in the ethanol wash at the bottom of the nail was distributed over approximately 1.8 mm of the nail. 2 This means that the concentration of the nail disc is derived from the surface area of ​​the nail. To calculate the approximate concentration under the nail, it was assumed that this part of the nail has a depth of 0.1 mm. In total, the nail is about 0.5 mm thick, so this 0.1 mm represents about one fifth of the total volume of the nail disc. Therefore, the volume under the nail disc is 0.18 mm. 3which is equal to 0.18 μl. To calculate the concentration of terbinafine under the nail, it was assumed that this volume of the nail contained an amount of terbinafine equivalent to that found in the ethanol wash.

[0096] The median concentration of terbinafine found on the underside of the nail in the multiple-dose experiment was 0.4 ng / ml (Figures 5A-5B), which is equivalent to 0.04 ng of total terbinafine in the sample. From this, we estimated that the concentration of terbinafine at the bottom of the nail was therefore 220 ng / ml (0.04 ng / 0.18 μl). Finally, the density of healthy human nail is 1.34 g / ml (Baraldi et al. 2015, Pharm. Res. 32(5), 1626-33), and therefore the concentration of terbinafine at the bottom of the nail is approximately equal to 0.165 μg / g (0.22 μg / ml / 1.34 g / ml).

[0097] From this calculation, it can be seen that in the multiple dose experiment, BB2603 delivered a greater amount of terbinafine to the bottom of the nail than the concentration of terbinafine achieved after 7 days by oral dosing (0.165 μg / g compared to 0.1 μg / g). This level of drug is 2-3 times higher than the level required to kill the least sensitive fungal species associated with onychomycosis (>0.06 μg / ml, see Table 1). For parts of the nail closer to the treatment surface, we predicted the concentration would be much higher. These figures are based on the most conservative data from the multiple dose experiment described above. For the single dose experiment, the median concentration of terbinafine found in the ethanol washes was 185 ng / ml (Figure 3), which would mean a predicted equivalent nail concentration of drug at the bottom would be 8 μg / ml, significantly exceeding the concentration achieved by oral dosing and required for antifungal efficacy.

[0098] In summary, topical application of BB2603 for 7 days promotes much greater association of terbinafine with healthy human nails than simple terbinafine solution. Moreover, BB2603 allows terbinafine to penetrate widely through the nail, indicating that this increase in nail drug levels is due, at least in part, to increased drug penetration into the tissue. Even the most distal part of the nail from BB2603 application is predicted to achieve drug concentrations that exceed those provided by equivalent oral dosing. This level is higher than the MIC of the fungal species involved, and is therefore likely to be effective in treating onychomycosis.

[0099] Oral terbinafine is currently the gold standard for the treatment of onychomycosis, with the highest cure rates (>80% cure after 3-6 months of medication) with the shortest treatment times. However, its use in the treatment of this disease is limited by its safety profile and by the fact that terbinafine has significant drug-drug interactions. A great many of these problems are almost certainly due to oral medication (e.g., hepatotoxicity, CNS effects) and the subsequent high systemic drug exposure. Other topical onychomycosis treatments require long treatment regimens (up to 18 months of treatment), have low cure rates (20-40%), and exhibit a high rate (>50%) of disease recurrence (Halmy, KJ Am. Acad. Dermatol, 2005. 52(3):126-126; Scher et al. J Am Ac Dermatol. 2007;56(6):939-944). Producing an effective topical formulation of terbinafine is a very attractive approach to the treatment of onychomycosis, as such a topical formulation would employ the drug with the most proven clinical efficacy and eliminate the safety issues associated with systemic exposure. This has proven difficult to achieve, and many previous attempts with topical terbinafine solutions have failed to demonstrate any significant efficacy in the treatment of onychomycosis.

[0100] As noted above, the amount of terbinafine present in BB2603 for topical application is much less than would be required for current oral doses. Current systemic treatments would typically use a 250 mg dose of oral terbinafine daily for 7 days. After daily topical application of small amounts of BB2603 to nail samples for 7 days (mimicking daily patient application), levels of terbinafine in the nail were achieved that were higher than those reported for the oral dose. The drug levels seen in the nail are much higher than would be required to demonstrate efficacy against all relevant fungal species associated with onychomycosis (Table 1). One idea from these experiments is that 1.75 g terbinafine for oral treatment would be 1.5 g of terbinafine for an average nail (100 mm 2 The dose of BB2603 that would have been required to treat 100% HBV was approximately 200 μg per week, i.e., an 8750-fold lower dose.

[0101] Healthy human nails are a much more stringent test of drug penetration. A recent publication by Baraldi et al. (Baraldi et al., 2015) revealed that although the nail is thicker in onychomycosis, the nail loses significant integrity, which meant that the nail is much more permeable (3-4 times more) to aqueous solutions. Therefore, we expected that BB2603 would show even better drug penetration properties in diseased tissues.

[0102] Comparison of BB2603 in 20% (v / v) ethanol vs. 30% (v / v) ethanol All the above experiments with BB2603 were performed in a solution of 30% (v / v) ethanol. Initial formulation studies revealed that 30% (v / v) ethanol produced the highest number of BB2603 nanoparticles, while experiments with solutions of 10% (v / v) ethanol or less showed a significant drop in particle number. Although 30% (v / v) ethanol is an acceptable solution for use in treating topical fungal infections, different % (v / v) ethanol were evaluated to see if lower ethanol contents still maintained efficacy. Therefore, it was decided to focus on the formulation of BB2603 in 20% (v / v) ethanol.

[0103] Formulations of BB2603 were made as described above, but with 20% (v / v) ethanol instead of 30% (v / v). Analysis on a NanoSight LM10 showed no detectable differences in either particle number or particle distribution of BB2603 in the 20% (v / v) formulation compared to 30% (v / v) ethanol. Therefore, several multi-dose Franz cell experiments were performed with BB2603 formulations in 20% (v / v) ethanol, as these most closely mimic the type of daily topical medications that patients would use and therefore were most meaningful in modeling the efficacy of drug treatments in onychomycosis.

[0104] The amount of terbinafine found in ethanol washes from undersides of nails treated for one week with daily addition of 5 μl of BB2603 in 20% (v / v) ethanol was analyzed by LC-MS / MS as described in the text (as illustrated in FIG. 8). These revealed consistent levels of terbinafine passing through the nails with a mean value of 0.5 ng / ml in the washes. These data showed a slightly higher trend in the amount of terbinafine passing through nails treated with BB2603 in 20% (v / v) ethanol, suggesting that BB2603 in 20% (v / v) is more effective at delivering the drug through the nail. Consistent with this, the amount of terbinafine in lysed nails treated with BB2603 in 20% (v / v) ethanol was 3-fold higher than in lysed nails treated with BB2603 in 30% (v / v) ethanol (as illustrated in FIG. 9).

[0105] Taken together, these results demonstrate that using a formulation of BB2603 in 20% (v / v) ethanol further increases the delivery of terbinafine into and through human nails in Franz cell multiple dose (daily addition) experiments. A substantially higher amount of drug was found to be associated with the nail, and the amount of terbinafine that passes through the nail is also higher. Calculations indicate that the median amount of drug in the underside of the nail treated with this formulation of BB2603 would be 0.21 μg / g, which is twice the value achieved in the nail on day 7 after oral dosing and well above the value required to kill the fungal species involved in onychomycosis. This result is consistent with the observations of Baraldi et al. (Baraldi et al., 2015) that compounds in aqueous solutions have higher levels of penetration in both healthy and diseased nails compared to compounds in 50% (v / v) ethanol solutions.

[0106] In summary, reducing the ethanol concentration in a solution of BB2603 from 30% to 20% (v / v) has no detectable effect on nanoparticle formation, but interestingly, formulation of BB2603 in 20% (v / v) ethanol revealed improved terbinafine delivery properties into and through healthy human nails in Franz cell experiments mimicking daily application of the drug in the treatment of onychomycosis.

[0107] Onychomycosis medicinal composition In light of the above experiments, it is envisioned that the following formulations would be effective as topical medications for onychomycosis.

[0108] [Table 2]

[0109] Other formulations may also provide effective topical medication.

[0110] [Table 3]

[0111] [Table 4]

[0112] [Table 5]

[0113] [Table 6]

[0114] A formulation according to the present invention was prepared according to formulation F below and designated BB2603.

[0115] [Table 7]

[0116] The formulation of Formula F was placed in a spray bottle. This formulation was then tested by periodically spraying it onto the toes of patients suffering from onychomycosis (and in some cases, tinea pedis) for a period of 1-2 weeks. This treatment proved successful, successfully and quickly treating patients suffering from onychomycosis (and tinea pedis) with no subsequent recurrence.

[0117] Usage / Dosage Experiments were conducted to identify the optimum spray distance from the foot to provide complete coverage of the toes and webs.A formulation according to the present invention was prepared according to Formulation F, as described above (BB2603).

[0118] The total topical dose that a patient would receive after spray application of BB2603 was estimated. It was assumed that patients would use 5 draws of the spray per treatment. Each draw delivers 100 μl of BB2603, so the total applied volume from the 5 draws is 500 μl.

[0119] The basic spray characteristics of the pump were evaluated using a solution of blue food dye. 400 μl of blue food dye was diluted in 20 ml of 20% v / v ethanol and transferred to a clean spray bottle. The spray was prepared by pumping the spray five times to remove any air from the spray nozzle. The dye spray was then sprayed once onto a sheet of white paper to form a dye spot. Spraying was performed at distances of 5 cm, 10 cm, 15 cm, 20 cm and 25 cm from the paper, resulting in a range of spots with different diameters (Figures 10A-E).

[0120] As can be seen in Figures 10A-10E, each spot essentially has three zones: in the center there is a zone with high dye load, surrounding this is a zone of 100% dye coverage (no white paper visible), and on the outside there is a more diffuse zone of dye coverage.

[0121] Ideally, topical treatment with BB2603 should achieve 100% coverage of the infected area and surrounding tissues (toes and interdigital spaces) to ensure that any unseen mycoses, either of the toenails or adjacent skin, are treated with the drug. For this reason, the main consideration when optimizing the spray distance for treatment is the diameter of the zone of 100% coverage. To analyze each spot, the diameter of the zone of 100% coverage was measured along with the diameter of the entire spot (visible dye edge). These were plotted on a graph against the distance of the spray from the paper, as shown in Figure 15. This revealed an approximately linear relationship between the width of the entire spot and the spray distance (Figure 11). However, the area of ​​100% dye coverage decreased when the spray distance was greater than 15 cm.

[0122] Based on this spot test, spray distances between 5 and 15 cm resulted in the largest diameter of the zone of 100% coverage. As this distance increased beyond 15 cm, the area of ​​100% coverage from a single application decreased, although the total area of ​​visible dye increased (Figure 11).

[0123] The effect of multiple applications on the area of ​​dye coverage was evaluated by spraying the paper five times from a distance of 10 cm (Figure 12). This was achieved by spraying the paper five times from a distance of 10 cm with a diameter of 10 cm and a surface area of ​​approximately 78 cm. 2 Assuming a 10 cm foot width (spanning the toes), this should be enough to cover an area of ​​over 7 cm from the tips of the toes towards the ankle, which is more than enough to cover the toes and interdigital spaces.

[0124] Thus, a spray distance of 5-15 cm appears to be optimal for patient treatment with BB2603 from this spray device.

[0125] Foot template experiment Spray testing showed that the optimal distance for spray application of BB2603 from the spray bottle is 5-15cm. Multiple application testing (Figure 12) showed that this should be sufficient to provide 100% drug coverage over the patient's toes, interdigital spaces and forefoot based on spot area. To confirm this and to calculate the approximate total dose, the patient was subjected to a series of experiments using a foot template which were performed.

[0126] A foot template was constructed from paper by drawing a line around the participant's foot (male, UK size 10). The paper template was cut and then placed on a sheet of blank paper. The dye was sprayed 5 times from a distance of 10 cm above and in front of the paper "foot" to ensure that all "toes" were treated. The dye was visible on both the foot template and the underlying paper (Figures 13A and 13B). After the dye had dried, a line was drawn around the area of ​​100% dye coverage from both the foot and the underlying paper, cut and weighed. The percentage of drug dose on the "foot" was calculated as follows:

number

[0127] Apparently, it was clear that 5 sprays from a distance of 10 cm were sufficient to give 100% coverage over the entire paw template. By weighing the area of ​​100% dye coverage, it was possible to compare the approximate area of ​​coverage and estimate the percentage of the dose received locally on the paw. The data are summarized in Table 2.

[0128] [Table 8]

[0129] Based on this experiment, we estimate that patients spraying BB2603 on their feet from a spray distance of 10 cm will receive approximately 50% of the total applied dose locally.

[0130] In summary, these experiments show that a spray application of BB2603 using five "pick-ups" from a spray bottle at a distance of 5-15 cm should be sufficient to provide 100% coverage of the toes, interdigital spaces, and front of the foot. From the foot template experiments, it is estimated that such an application would result in the patient receiving a topical dose of approximately 50% of the sprayed volume. For five "pick-ups", this would be equivalent to a topical dose of 250 μl or a total dose of 25 μg of terbinafine.

[0131] The above embodiments are not intended to limit the scope of protection afforded by the claims, but rather to illustrate examples of how the invention may be practiced.

Claims

1. 1. A composition for use in the treatment of onychomycosis and / or athlete's foot, the composition comprising a polymer capable of forming nanoparticles and terbinafine, or a derivative or salt thereof, the nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, the composition being administered topically to provide a daily dose of terbinafine in the range of about 5 μg to about 50 μg to the infected area.

2. 2. The composition of claim 1, wherein the daily dose of terbinafine to the infected area ranges from about 10 μg to about 40 μg.

3. 3. The composition of claim 2, wherein the daily dose of terbinafine to the infected area ranges from about 15 μg to about 35 μg.

4. 4. The composition of claim 3, wherein the daily dose of terbinafine to the infected area ranges from about 20 μg to about 30 μg.

5. 5. The composition of claim 4, wherein the daily dose of terbinafine to the infected area is about 25 μg.

6. 6. The composition of any one of claims 1 to 5, which is administered by spray application.

7. The composition of claim 6, wherein the daily dose is administered using one or more spray applications.

8. 8. The composition of claim 7, wherein said daily dose comprises up to eight spray applications.

9. 8. The composition of claim 7, wherein said daily dose comprises up to five spray applications.

10. 10. A composition according to any one of claims 1 to 9, wherein the spray is effective to cover the fingers and / or interdigital spaces and / or front of the feet or hands.

11. (a) terbinafine, or a derivative or salt thereof, present in an amount ranging from about 0.005% w / w to about 1% w / w; (b) a polymer and terbinafine, or a derivative or salt thereof, capable of forming nanoparticles, said nanoparticles being formed with and / or in the presence of terbinafine, or a derivative or salt thereof, said polymer being present in an amount ranging from about 0.015% w / w to about 3% w / w; (c) less than about 30% alcohol w / w; and (d) up to about 90% w / w water The composition of any one of claims 1 to 10, comprising:

12. (a) terbinafine, or a derivative or salt thereof, present in an amount ranging from about 0.025% w / w to about 0.2% w / w; (b) a polymer present in an amount ranging from about 0.75% w / w to about 0.6% w / w; (c) an alcohol present in an amount ranging from about 20% w / w to about 29% w / w; and (d) water, present in an amount ranging from about 70% w / w to about 90% w / w The composition of claim 11 comprising:

13. (a) about 0.1% w / w of terbinafine, or a derivative or salt thereof; (b) about 0.3% w / w of a polymer; (c) about 20% alcohol w / w, and (d) up to about 79.6% w / w water The composition of claim 12 comprising:

14. 14. The composition of any one of claims 1 to 13, wherein the polymer comprises a linear and / or branched or cyclic polymonoguanide / polyguanidine, polybiguanide, analogues or derivatives thereof.

15. 15. The composition of claim 14, wherein the polymer comprises polyhexamethylene biguanide (PHMB).

16. 16. The composition of any one of claims 1 to 15, wherein the alcohol comprises ethanol.

17. 17. The composition of any one of claims 1 to 16, further comprising one or more of the following: buffers, excipients, binders, oils, water, emulsifiers, glycerin, antioxidants, preservatives, flavorings, and urea.

18. 17. The composition of any one of claims 1 to 16, which is substantially free of other ingredients.

19. 19. A composition according to any one of claims 1 to 6 and claims 10 to 18 in combination with a liquid dispensing device for dispensing a predefined amount of said composition onto the toes and / or interdigital spaces and / or front of a user's feet, said device comprising a container for containing the composition, a nozzle for expelling the composition from the container, and a user operable pump action mechanism for drawing a predefined amount of the composition from the container and expelling it through the nozzle, thereby atomising the composition towards the toes and / or interdigital spaces and / or front of a user's feet.

20. 20. The device of claim 19, wherein the device further comprises a metered valve through which a predefined amount of the composition is dispensed.

21. 21. The device of claim 19 or claim 20, wherein the predefined amount of the composition comprises up to about 200 μl.

22. 22. The device of claim 21, wherein the predefined amount of the composition comprises up to about 150 μl.

23. 23. The device of claim 22, wherein the predefined amount of the composition comprises up to about 100 μl.

24. 23. The device of claim 22, wherein the predefined amount of the composition comprises about 100 μl.

25. 25. A device according to any one of claims 19 to 24, wherein the device further comprises and / or is associated with a distance indicator for indicating the correct distance at which the user should place the nozzle of the device relative to the infected area.

26. 25. A device according to any one of claims 19 to 24, wherein in use, the nozzle of the device is capable of dispensing all of the required dose in up to about 5 draws when the nozzle of the device is placed within about 5 cm to 15 cm of the infected area.

27. 25. A device according to any one of claims 19 to 24, wherein in use, when the nozzle of the device is placed about 10 cm from the infected area, the nozzle of the device is capable of dispensing all of the required dose in about 5 draws.

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