Pharmaceutical composition loaded with fluconazole having an increased antifungal activity, the formulation and the use thereof

Abstract

The object of the invention is a pharmaceutical composition containing fluconazole and a non-ionic triblock polyoxyethylene-polyoxypropylene copolymer used as a carrier, wherein the composition contains fluconazole at a concentration of 0.004% (w / v) to 0.45% (w / v) of the composition's volume, the non-ionic triblock polyoxyethylene- polyoxypropylene copolymer at a concentration of 0.08% (w / v) to 25% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume, wherein the carrier is in the form of micelles having a hydrodynamic diameter of 5.0±0.1 nm to 40±1.0 nm. The invention also encompasses a formulation containing the composition and the use thereof.

Description

[0001] Pharmaceutical composition loaded with fluconazole having an increased antifungal activity, the formulation and the use thereof

[0002] The object of the invention is a pharmaceutical composition containing fluconazole using a block polymer as a carrier for the active pharmaceutical ingredient for the use in pharmaceutical preparations for the treatment of topical and systemic fungal infections.

[0003] "Formulation & Evaluation of Fluconazole Gel for Topical Drug Delivery System" (American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2021) Volume 76, No 1, pp 124-137) discloses topically-used gel formulations with fluconazole and Carbopol 940 / NaCMC (carboxymethylcellulose sodium salt) polymers. Moreover, it describes the use of methanol as an absorption promoter. "Formulation and evaluation of fluconazole topical gel" (International Journal of Pharmacy and Pharmaceutical Sciences, Vol 4, Suppl 5, 2012) discloses fluconazole formulated as a topical gel for the use in fungal skin infections. Gel formulations were developed using various polymers, including Carbopol 940, HPMC E4M (hydroxypropyl methylcellulose), methylcellulose, pectin, and Pluronic F127 (15% and 18%). Candida albicans was used as a model strain for assaying the antifungal activity of obtained preparations. "A Novel Thermo-Sensitive Sol-Gel Reversible Buccal Adhesive Property of Fluconazole in Situ Gel For Oral Thrush" (J. Biomed. Sci. and Res., Vol 2 (2), 2010,100-109) discloses thermosensitive and bioadhesive properties of a gelling in-situ delivery system with fluconazole that may be used in the treatment of oral thrush. Bioadhesive polymers poloxamer 188 and Carbopol 934 were used as ingredients of the developed thermosensitive carrier. Furthermore, "Fluconazole Loaded Cubosomal Vesicles for Topical Delivery" (IntJ Drug Dev & Res 2015, 7:3) describes a cubosomal gel-based fluconazole formulation for the treatment of fungal infections of the skin. The aim of the work was to load large amounts of the drug for increased therapeutic effectiveness. Cubosomes were prepared using a top-down technique (fragmentation by sonication). Yet another publication, "Thermoresponsive fluconazole gels for topical delivery: rheological and mechanical properties, in vitro drug release and anti-fungal efficacy" (Pharm Dev Technol, Early Online: 1-9) presents thermosensitive poloxamer-using gels for the topical administration of fluconazole (FLZ). Eight different formulations containing 1% FLZ in poloxamer and varying concentrations of a selected co-solvent (propylene glycol (PG) orTranscutol-P) were prepared. In addition, "Smart in-situ thermo-responsive and ion activated ophthalmic sol-gel system of fluconazole" (J Res Pharm 2021; 25(2): 173-178) describes ophthalmic formulations utilizing an in-situ sol-gel process, capable of releasing fluconazole in the administration site (eye) over a prolonged time. In-situ gelling fluconazole formulations were prepared using thermosensitive Pluronic F127, sodium alginate, and a combination thereof. The optimized formulation F8 contained fluconazole (0.3% w / v), Pluronic F127 (1% w / v), sodium alginate (0.5% w / v), sodium chloride (0.9% w / v), benzalkonium chloride (0.01% w / v), and acetate buffer (pH 4, ad 100% w / v). Another publication ("Development and machine-learning optimization of mucoadhesive nanostructured lipid carriers loaded with fluconazole for treatment of oral candidiasis" DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 2021, VOL. 47, NO. 2, 246 - 258) describes a mucoadhesive nanostructured lipid carrier (NLC) with fluconazole loaded therein for a more effective treatment of oral candidiasis. NLCs were prepared via emulsification / sonication techniques. Nanoparticles contained stearic acid, oleic acid, Pluronic F127, and lecithin. Nanoparticles had a particle size of 335 ±13.5 nm, and loading efficiency was 73.1 ±4.9%. When covered with chitosan, nanoparticles adhered better to the rabbit buccal mucosa and exhibited improved activity towards Candida albicans. "Recent progress in biomedical applications of Pluronic (PF127): Pharmaceutical perspectives" (J Control Release. 2015 Jul 10;209: 120-38) discloses the use of PF127 as a polymer for the preparation of nanocarriers modified with other copolymers and / or conjugates thereof with magnetic nanoparticles. The pivotal results of these studies showed that PF127-based carriers may significantly improve the stability of loaded hydrophobic molecules, reduce in vitro cytotoxicity, and enhance cellular uptake of anticancer medicines. Providing a topical pharmaceutical composition containing fluconazole in a form of gel or a liquid, having an increased contact time with the application site, would be a technical problem. In addition, the said composition should be easy to prepare. Moreover, the composition should inhibit the growth of yeasts even at low active ingredient concentrations, be efficacious towards resistant Candida strains, whereas the qualitative content of the composition should not decrease the antifungal activity of the active pharmaceutical ingredient.

[0004] The first object of the invention is a pharmaceutical composition containing fluconazole and a carrier being a non-ionic triblock polyoxyethylene-polyoxypropylene copolymer, wherein the composition contains fluconazole at a concentration of 0.004% (w / v) to 0.45% (w / v) of the composition's volume, the non-ionic triblock polyoxyethylene-polyoxypropylene copolymer at a concentration of 0.08% (w / v) to 25% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume, wherein the carrier is in the form of micelles having a hydrodynamic diameter of 5.0±0.1 nm to 40±1.0 nm.

[0005] In a preferred embodiment, the composition contains fluconazole at a concentration of 0.004% (w / v) to 0.013% (w / v) of the composition's volume, the non-ionic triblock polyoxyethylene-polyoxypropylene copolymer at a concentration of 0.08% (w / v) to 5% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume.

[0006] In another preferred embodiment, the composition contains fluconazole at a concentration of 0.13% (w / v) to 0.45% (w / v) of the composition's volume, the non-ionic triblock polyoxyethylene-polyoxypropylene copolymer at a concentration of 10% (w / v) to 25% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume. The second object of the invention is a pharmaceutical formulation, wherein the formulation is a micellar aqueous solution containing the pharmaceutical composition according to the first object of the invention.

[0007] In a preferred embodiment of the invention, in the concentration range of the non-ionic triblock polyoxyethylene-polyoxypropylene copolymer of 0.08% (w / v) to 15% (w / v) of the composition's volume, the formulation is in a liquid form.

[0008] In another preferred embodiment of the invention, in the concentration range of the nonionic triblock polyoxyethylene-polyoxypropylene copolymer of 20% (w / v) to 25% (w / v) of the composition's volume, the composition is gelling.

[0009] In yet another preferred embodiment of the invention, the pharmaceutical formulation is stable over an 84-day period.

[0010] Another object of the invention is the use of the pharmaceutical composition according to the first object of the invention, or the pharmaceutical formulation according to the second object of the invention, for the treatment of topical and systemic fungal infections.

[0011] In a preferred embodiment of the invention, the use comprises topical administration or administration by injection.

[0012] Yet another object of the invention is the use of the pharmaceutical composition according to the first object of the invention, or the pharmaceutical formulation according to the second object of the invention, in the preparation of medicines for use in the treatment of topical and systemic fungal infections.

[0013] The carrier used in the formulation has surface-active properties. When the critical micelle concentration (CMC) is exceeded, it forms polymeric micelles in a solution. As the concentration of the block polymer increases in the aqueous solution, a hydrogel is formed.

[0014] According to the invention, an active pharmaceutical ingredient loaded in micelles is fluconazole. Fluconazole belongs to azole antifungals and is used topically and systemically. The authors do not exclude loading other azole drugs to which microbes may have developed resistance. Fluconazole is used in the treatment of candidiases of mucosal membranes in the oral cavity, throat, esophagus, and vagina; skin candidiases; generalized candidiases involving, for example, the respiratory tract, urinary tract, meninges, serosa, and endocardium. In Poland, registered fluconazole preparations include solid forms, namely tablets and capsules at a dose of50 mg, 100 mg, 150 mg, and 200 mg. Among liquid formulations, a 2 mg / mL solution for infusion and 5 mg / mL syrup are registered. In compounding, 2 mg / mL fluconazole eye drops are prepared using the said solution for infusion.

[0015] Due to an increasing number of systemic and topical fungal infections, it is desired to prepare a formulation enhancing the efficacy of the antifungal treatment. The costs of fungal infection treatment are increasing globally due to the developing multidrug resistance. Resistance particularly concerns azole derivatives. Among yeasts, Candida albicans is the most frequent cause of severe infections, while Candida glabrata is particularly prone to multiple drug resistance, possibly due to its haploid genome. Cellular drug efflux via pump membrane transporters is one of the main resistance mechanisms in yeasts. Gene mutations lead to the overexpression of proteins regulating azole transport into the cell, which results in the increased efflux of the drug via the fungal cell membrane. Another resistance mechanism consists of variations in an enzyme, namely sterol 14a-demethylase encoded by the ERG11 gene, targeted by azoles. This gene is involved in ergosterol synthesis, which is a crucial structural component of the fungal cell membrane. A point mutation in this gene leads to the overexpression of a protein with less affinity to the binding site.

[0016] The use of Pluronic® F-127 in the inventive formulation is particularly preferred, as this is a non-toxic and biocompatible polymer. It exhibits gelling and mucoadhesive properties, and depending on the concentration and temperature can be in the form of a solution or gel suitable for topical skin, mucosa, or eye applications, which allows for a prolonged in situ drug release. Particles (micelles) in the analyzed micellar system are characterized by small diameter, not exceeding 10 nm, and— when Pluronic® F-127 content in the formulation is >1% w / v— 40 nm, which allows for their potential use in IV administration. The active pharmaceutical ingredient in the described system is solubilized by the carrier, which slightly improves its solubility. This allows for the development of a formulation with increased fluconazole content when compared to the marketed solution for infusion, and eye drops (2 mg / mL) obtained therefrom. Physical and chemical properties of Pluronic® and the fact that fluconazole addition to Pluronic® F-127-containing preparation are responsible for statistically significant inhibition of growth in the assayed yeasts, and the increased growth inhibition zone in microbiological in vitro assays. It can therefore be concluded that the used block polymer, to some extent, affects the structure of the yeast cell wall / membrane and the aforementioned pump function, which leads to the observed higher efficacy of a drug delivered to the cell.

[0017] The embodiments of the invention are shown in the drawings, wherein:

[0018] - Fig. 1 is a multivariate analysis of variance (MANOVA)— multidimensional analysis of absorbance values using a microdilution method. Effect: F(2, 1520)=23.511; p<0.00001, average ± 95% Cl;

[0019] - Fig. 2 is a multivariate analysis of variance (MANOVA)— multidimensional analysis of absorbance values using a microdilution method. Effect: F(61, 1520)=9.2569; p<0.00001, average ± 95% Cl;

[0020] - Fig. 3 is a multivariate analysis of variance (MANOVA)— multidimensional analysis of growth inhibition zone diameters in a disk diffusion test. Effect: F(3, 1001)=709.20; p<0.00001, average ± 95% Cl;

[0021] - Fig. 4 is a multivariate analysis of variance (MANOVA)— multidimensional analysis of growth inhibition zone diameters in a disk diffusion test. Effect: F(9, 1001)=39.355; p<0.00001, average ± 95% Cl;

[0022] - Fig. 5 depicts a hydrodynamic micelle diameterforformulations with the increasing Pluronic® F-127 and fluconazole content; and

[0023] - Fig. 6 shows a relationship between the dynamic viscosity and the formulation temperature for the formulations with 20.0% and 25.0% w / v content of Pluronic® F-127. Example 1. Pharmaceutical composition

[0024] The developed preparations of fluconazole (CAS 86386-73-4) loaded into aqueous Pluronic® F-127 (non-ionic triblock polyoxyethylene-polyoxypropylene copolymer, CAS 9003-11-6) resulted in the formulations having an increased antifungal activity towards resistant Candida strains. Table 1 lists inventive formulations.

[0025] Table 1. Inventive micellar formulations containing fluconazole. Critical micelle concentration (CMC), namely the concentration above which micelle formation is seen in a polymer solution, was determined using spectrofluorimetry and surface tension measurements. It was 0.060% w / v and 0.062% w / v, respectively.

[0026] 1 FORMULATIONS WERE OBTAINED VIA A DIRECT DISSOLUTION METHOD INVOLVING DISSOLVING POLYMER IN AN AQUEOUS SOLVENT AND ADDING AN ACTIVE PHARMACEUTICAL INGREDIENT. N EXT, THE SPONTANEOUS AGGREGATION OF POLYMER MOLECULES, WITH MICELLE FORMATION, AND THE LOADING OF THE ACTIVE PHARMACEUTICAL INGREDIENT ARE OBSERVED. API, DEPENDING ON THE REFERENCE, IS IN THE FORM OF A PRE-PREPARED SOLUTION, OR IS ADDED DIRECTLY, AS A POWDER, TO THE POLYMER SOLUTION, AS DESCRIBED IN "NOVEL NANOMICELLAR FORMULATION APPROACHES FOR ANTERIOR AND POSTERIOR SEGMENT OCULAR DRUG DELIVERY" (RECENT PAT NANOMED. 2012; 2(2): 82-95).

[0027] Formulations containing up to 5.0% w / v Pluronic® F-127 were obtained by dissolving the polymer in sterile purified water at 2-8°C. Next, the polymer solution was transferred to the fluconazole solution and stirred until the dissolution of fluconazole.

[0028] Formulations containing more than 5.0% w / v Pluronic® F-127 were obtained by transferring the solution of fluconazole in sterile purified water to the polymer solution and storing at 2- 8°C until polymer dissolution.

[0029] Hydrodynamic diameter of micelles in the developed formulations was determined using dynamic light scattering (DLS) and transmission electron microscopy (TEM) methods. It ranged from 5.0±0.1-8.4±0.6 nm to 38±1.0-40±1.0 nm, depending on the fluconazole and polymer concentrations (Fig. 5). Micelles aggregate at higher (>3% w / v) concentrations, therefore a population of particles with a higher determined diameter (38±1.0-40±1.0 nm) is observed, which does not undergo changes when the polymer concentration increases to 5% w / v. For carrier concentrations from 0.08% w / v to 20% w / v, the system is in a liquid form at room temperatures. Above 20% w / v of the carrier, a phase transition to sol-gel is observed at room temperatures. Given the fact that across the entire range of the tested concentrations (up to 5% w / v), the micelle diameter was constant, it can be concluded that, with increasing polymer concentration, the size of the formed aggregates remains unchanged, while their concentration and packing density increase. As a consequence, due to their specific spatial organization, a hydrogel is formed. Fig. 5 also shows that the diameters are higher for lower, near-CMC (up to 0.08% w / v) concentrations. This is due to the instability of the system at low concentrations, which stabilizes when CMC is exceeded. Addition of fluconazole at a wide concentration range from 1.6 to 16.3 mM did not affect the hydrodynamic micelle diameter when compared to micelles without fluconazole. It can therefore be concluded that formulations of increased antifungal activity, containing 0.013-14.7 mM fluconazole, were characterized by a constant micelle diameter.

[0030] Solubility and stability of formulated fluconazole were determined using the high- performance liquid chromatography (HPLC) method. Pluronic® F-127 improved the maximal fluconazole solubility in water at a polymer concentration of at least 3.0% w / v. Based on the results, it was concluded that fluconazole was stable in the polymer solution over a 84-day period for all tested concentrations. Drug content in the preparations was 100±2.8%, depending on the formulation.

[0031] Viscosity of the obtained gels was measured using a rotational rheometer. The relationship between the dynamic viscosity and the formulation temperature shows that formulations with 20.0% and 25.0% w / v of Pluronic® F-127 (Fig. 6) are thermosensitive. The phase transition temperature decreased with an increase of polymer concentration (25.8±0.2°C and 21.3±0.0°C, respectively). Fluconazole addition had no effect on the sol-gel transition. The phase transition temperature was below human body temperature (36.6°C) and human skin surface temperature (34.0°C), which indicates the possibility of gelling at the application site. Formulations with lower Pluronic® F-127 concentrations (10.0% w / v and 15.0% w / v; F127- 10_FLU-7, F127-10_FLU-8, F127-10_FLU-9, F127-10_FLU-10, F127-10, and F127-15_FLU-7, F127-15_FLU-8, F127-15_FLU-9, F127-15_FLU-10, and F127-15, respectively) did not show phase transition. Therefore, these formulations can be useful as liquids.

[0032] Example 2. Antifungal activity Antifungal activity of the obtained formulations was assayed using yeasts (Candida spp.) exhibiting fluconazole resistance (C. albicans, C. glabrata, and C. tropicalis) and Candida krusei as a control using microdilution and disk diffusion methods. Strains were maintained in a TSB (tryptic soy broth, CAS 1132-61-2, catalogue #: PS 23, Biomaxima) medium at -80°C. Before each assay, yeasts were inoculated onto Sabouraud medium (Sabouraud agar with dextrose and chloramphenicol, catalogue #: 620203, Liofilchem) and incubated at 35°C for 24 hrs. Next, using a densitometer, a cell suspension characterized by a density of 0.5 McFarland in RPMI 1640 medium with L-glutamine and without sodium bicarbonate (medium, catalogue #: R6504, Sigma-Aldrich) containing MOPS (3-(N-morpholino)propanesulfonic acid, CAS 1132- 61-2) was prepared.

[0033] Antifungal activity assays of the described formulations were carried out using the microdilution method and 22 fungal strains:

[0034] - C. krusei fCTCC (American Type Culture Collection) 6258 fluconazole-resistant reference strain (a control strain, naturally resistant to fluconazole);

[0035] - C. albicans fCTCC MYA-574 fluconazole-resistant reference strain;

[0036] - C. albicans fCTCC 64124 fluconazole-resistant reference strain;

[0037] - C. albicans 1444 fluconazole-resistant clinical strain;

[0038] - C. albicans 3057 fluconazole-resistant clinical strain;

[0039] - C. albicans 3089 fluconazole-resistant clinical strain;

[0040] - C. tropicalis 3151 fluconazole-resistant clinical strain;

[0041] - C. glabrata 2586 fluconazole-resistant clinical strain;

[0042] - C. glabrata 2738 fluconazole-resistant clinical strain;

[0043] - C. glabrata 140 fluconazole-resistant clinical strain;

[0044] - C. glabrata 769 fluconazole-resistant clinical strain; - C. glabrata 773 fluconazole-resistant clinical strain;

[0045] - C. glabrata 1941 fluconazole-resistant clinical strain;

[0046] - C. glabrata 1973 fluconazole-resistant clinical strain;

[0047] - C. glabrata 2342 fluconazole-resistant clinical strain; - C. glabrata 3154 fluconazole-resistant clinical strain;

[0048] - C. glabrata 1467 fluconazole-resistant clinical strain;

[0049] - C. glabrata 2853 fluconazole-resistant clinical strain;

[0050] - C. glabrata 3010 fluconazole-resistant clinical strain;

[0051] - C. glabrata 137 fluconazole-resistant clinical strain; - C. glabrata 2124 fluconazole-resistant clinical strain; and

[0052] - C. glabrata 3081 fluconazole-resistant clinical strain.

[0053] Table 2. Control formulations used for the comparison of the antimicrobial activity by microdilution method.

[0054] For the microdilution method, 50 pL of the micellar system (formulations with 0.16% w / v and 10.0% w / v Pluronic® F-127) containing 0.008-0.256 mg / mL fluconazole was transferred into a well of a 96-well microplate. 50 pL of fungal suspension was transferred into each well. Each solution was diluted twice after the addition of the fungal suspension (i.e., the final concentrations of the tested formulations were 0.004-0.128 mg / mL; 0.0004-0.0128% w / v; 0.013-0.42 mM, Table 1; F127-0.08_FLU, F127-0.08_FLU-l, F127-0.08_FLU-2, F127-0.08_FLU- 3, F127-0.08_FLU-4, F127-0.08_FLU-5, F127-0.08_FLU-6, F127-5_FLU: F127-5_FLU-1, F127- 5_FLU-2, F127-5_FLU-3, F127-5_FLU-4, F127-5_FLU-5, and F127-5_FLU-6 formulations). The final fungal concentration per well was approximately 1.5xl05CFU / mL. The plate was incubated at 35°C for 24 hrs. The minimum inhibitory concentration (MIC), i.e., the lowest drug concentration that inhibits fungal cell growth by at least 50% versus control, was derived from the absorbance values measured at 530 nm. The assay conformed to the EUCAST (European Committee on Antimicrobial Susceptibility Testing) standard. As a control, 0.008- 0.256 mg / mL fluconazole solution without polymer (Table 2; FLU-1, FLU-2, FLU-3, FLU-4, FLU- 5, and FLU-6 formulations) and 0.16% w / v and 10.0% w / v micellar solution without loaded fluconazole were used, therefore the final concentrations of the tested formulations were 0.08% w / v and 5% w / v, respectively; Table 2; F127-0.08, and F127-5 formulations. 2-4 wells per each tested strain were used as a growth control (without the addition of the tested system). 2-4 plate wells were used as a sterility control (without fungal suspension). Verapamil (as a hydrochloride, CAS 152-11-4), a yeast cell efflux pump inhibitor belonging to the ABC group inhibiting yeast growth, was used as a control for the assay (Table 2; WER, WER_FLU-1, WER_FLU-2, WER_FLU-3, WER_FLU-4, WER_FLU-5, and WER_FLU-6 formulations).

[0055] Statistical analysis showed that 5.0% w / v Pluronic® F-127 formulation with fluconazole caused a statistically significant reduction of the absorbance as a measure of yeast growth (Fig. 1; Table 3; F127-5_FLU-l-6 vs. FLU-1-6, p <0.000001). There was no statistically significant difference in absorbance after fluconazole addition to the lowest concentration, i.e., slightly exceeding CMC, polymer solution (Fig. 1; Table 3; FLU-1-6 vs. F127-0.08_FLU-l-6, p = 0.138034). Considering the individual strains, in case of C. albicans TCC MYA-574, C. albicans 1444, C. glabrata 2586, 2738, 2853, 1973, 1467, and 2124 the highest differences in absorbance between Pluronic® F-127 formulation with fluconazole and fluconazole solution without polymer were observed (Fig. 2; Table 3; F127-5_FLU-l-6 vs. FLU-1-6, p <0.000001; p <0.000001; p = 0.000036; p <0.000001; p <0.000001; p = 0.000001; p = 0.000010; and p = 0.000011, respectively).

[0056] Table 3. One-dimensional significance tests of absorbance values (FLU-1-6, F127-0.08_FLU-l- 6, F127-5_FLU-l-6, and WER_FLU-l-6 formulations), sigma-restricted, effective hypotheses decomposition.

[0057] When MIC values considered, a decrease was observed for 5 tested strains (C. krusei ATCC 6258, C. albicans 3057, C, glabrata 2586, C. glabrata 2738, and C. glabrata 2853) after Pluronic® F-127 block polymer was added to the fluconazole solution (Table 4). Table 4. Antifungal activity of fluconazole (FLU-1-6 formulations) and fluconazole loaded into Pluronic® F-127 at 0.08% w / v (F127-0.08_FLU-l-6 formulations) and 5.0% (F127-5_FLU-l-6 formulations). Based on the results presented, it was observed that polymer aggregation above CMC and fluconazole loading into micelles did not decrease the antifungal activity of fluconazole. Block polymer without fluconazole was not inhibitory towards yeast growth (except for C. albicans 3089 and C. tropicalis 3151 in 5.0% w / v Pluronic® F-127 formulation, which were excluded from the MIC determination and statistical analysis; denoted with (*) in Table 4). Therefore, the micelle solution without the loaded drug substance was non-toxic for yeast cells.

[0058] Antifungal activity of the described formulations was measured using the disk diffusion method and 15 fungal strains:

[0059] - C. krusei TCC 6258 fluconazole-resistant reference strain;

[0060] - C. albicans 3057 fluconazole-resistant clinical strain;

[0061] - C. glabrata 2586 fluconazole-resistant clinical strain;

[0062] - C. glabrata 2738 fluconazole-resistant clinical strain;

[0063] - C. glabrata 2853 fluconazole-resistant clinical strain;

[0064] - C. glabrata 3010 fluconazole-resistant clinical strain;

[0065] - C. glabrata 1467 fluconazole-resistant clinical strain;

[0066] - C. glabrata 2124 fluconazole-resistant clinical strain;

[0067] - C. glabrata 137 fluconazole-resistant clinical strain;

[0068] - C. glabrata 140 fluconazole-resistant clinical strain;

[0069] - C. glabrata 1941 fluconazole-resistant clinical strain;

[0070] - C. glabrata 1973 fluconazole-resistant clinical strain;

[0071] - C. glabrata 2342 fluconazole-resistant clinical strain;

[0072] - C. glabrata 3154 fluconazole-resistant clinical strain; and

[0073] - C. glabrata 773 fluconazole-resistant clinical strain. Table 5. Control formulations used for the comparison of the antimicrobial activity by disk diffusion method.

[0074] Candida cells density inoculated onto Mueller-Hinton agar with glucose and methylene blue was 0.5 McFarland. Wells having 7 mm in diameter were cut in the agar using a sterile cork borer. 20 pL of fluconazole solution (Table 5; FLU-7-10 formulation), or 10.0% w / v, 15.0% w / v, and 20.0% w / v Pluronic® F-127 solution containing fluconazole (Table 1; F127-10_FLU-7-10, F127-15_FLU-7-10, F127-20_FLU-7-10, and F127-25_FLU-7-10 formulations) was transferred into each well in the agar plate. Final fluconazole content per well was 25 pg, 50 pg, 75 pg, and 90 pg. To ensure that the addition of the polymer does not inhibit growth in yeasts, further control wells, filled with Pluronic® F-127 solutions without fluconazole (Table 5; F127-10, F127-15, F127-20, and F127-25) were prepared. After inoculation, plates were incubated at 35°C for 24 hrs, and the growth inhibition zone was measured (Tables 6a and 6b).

[0075] Based on the results, the addition of even high concentrations of the block polymer did not inhibit fluconazole antifungal activity, similarly as observed for the microdilution method. The polymer addition did not inhibit yeast growth in wells filled with Pluronic® F-127 solution without fluconazole, regardless of the polymer concentration. For 10 out of 15 tested strains, the growth inhibition zone increased, and the highest increase versus fluconazole solutions was seen for 10.0% w / v Pluronic® F-127 solution (Table 1; F127-10_FLU-7-10 formulations). Tables 6a and 6b. Antifungal activity of fluconazole solution (FLU-10-7 formulations) and fluconazole loaded into Pluronic® F-127 at 10.0% w / v (F127-10_FLU-7-10), 15.0% (F127- 15_FLU-7-10), and 20% w / v (F127-20_FLU-7-10).

[0076] Table 6a.

[0077] Table 6b.

[0078] C. k. - C. krusei; C. a. - C. albicans; C. g. - C. glabrata. If no growth inhibition zone was observed, the table lists the well diameter. Results presented as mean ± standard deviation. C. glabrata 1941, 1973, 2342, 3154, and 773 strains were omitted, as no inhibition zone was seen for these around plate wells for both fluconazole and fluconazole loaded into Pluronic® F-127 formulations. Statistical analysis confirmed that the growth inhibition zone was statistically significantly larger when fluconazole was combined with block polymer, regardless of the polymer concentration (Fig. 3 and Table 7; FLU-7-10 vs F127-10_FLU-7-10; p <0.000001; FLU vs F127- 15_FLU-7-10; p <0.000001; and FLU vs F127-20_FLU-7-10; p <0.000001). This effect was also seen for individual fluconazole content per well (25 pg, 50 pg, 75 pg, and 90 pg; FLU-7, FLU-8, FLU-9, FLU-10, respectively; Table 1) upon loading to the polymer- containing formulation (Fig. 4; Table 7), whereas Table 7 lists statistical analysis results for the comparison of FLU-7-10, F127-10_FLU-7-10, F127-15_FLU-7-10, and F127-20_FLU-7-10 formulations from Tables 1 and 5, namely4 distinct types of formulations, with 4 samples with different fluconazole concentration per type, amounting to 16 formulations in total.

[0079] Table 7. One-dimensional significance tests of absorbance values (FLU-7-10, F127-10_FLU-7- 10, F127-15_FLU-7-10, and F127-20_FLU-7-10 formulations), sigma-restricted, effective hypotheses decomposition.

Claims

Claims1. A pharmaceutical composition containing fluconazole and a non-ionic triblock polyoxyethylene-polyoxypropylene copolymer as a carrier, characterized in that the composition contains fluconazole at a concentration of 0.004% (w / v) to 0.45% (w / v) of the composition's volume, the non-ionic triblock polyoxyethylenepolyoxypropylene copolymer at a concentration of 0.08% (w / v) to 25% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume, wherein the carrier is in the form of micelles having a hydrodynamic diameter of 5.0±0.1 nm to 40±1.0 nm.

2. The composition of claim 1, wherein the composition contains fluconazole at a concentration of 0.004% (w / v) to 0.013% (w / v) of the composition's volume, the non- ionic triblock polyoxyethylene-polyoxypropylene copolymer at a concentration of 0.08% (w / v) to 5% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume.

3. The composition of claim 1 or 2, wherein the composition contains fluconazole at a concentration of 0.13% (w / v) to 0.45% (w / v) of the composition's volume, the non- ionic triblock polyoxyethylene-polyoxypropylene copolymer at a concentration of 10% (w / v) to 25% (w / v) of the composition's volume, and purified water up to 100% (w / v) of the composition's volume.

4. A pharmaceutical formulation, wherein the formulation has the form of an aqueous solution containing the pharmaceutical composition of claim 1.

5. The pharmaceutical formulation of claim 4, wherein in the concentration range of the non-ionic triblock polyoxyethylene-polyoxypropylene copolymer of 0.08% (w / v) to 20% (w / v) of the composition's volume, the formulation is in a liquid form.

6. The pharmaceutical formulation of claim 4, wherein in the concentration range of the non-ionic triblock polyoxyethylene-polyoxypropylene copolymer of 20% (w / v) to 25% (w / v) of the composition's volume, the formulationis gelling.

7. The pharmaceutical formulation of any of the claims 4 to 6, wherein it is stable over an 84-day period.

8. Use of the pharmaceutical composition of claim 1 or the pharmaceutical formulation of claim 4 in the treatment of topical and systemic fungal infections.

9. The use of claim 8, wherein the use comprises topical administration or administration by injection.

10. Use of the pharmaceutical composition of claim 1 or the pharmaceutical formulation of claim 4 in the preparation of medicines for use in the treatment of topical and systemic fungal infections.

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