GLUCONO DELTA-LACTONE FOR TREATMENT OF VAGINAL FUNGAL INFECTIONS

Abstract

The present invention relates to a pharmaceutical formulation for vaginal administration, wherein the formulation comprises a pharmaceutical acceptable excipient and glucono δ-lactone, wherein the glucono δ-lactone is present in an amount of 5 to 99 wt % of the formulation. The invention also relates to a pharmaceutical formulation according to the invention for use in the prevention or treatment of a urogenital fungal infection. Furthermore, the invention relates to glucono δ-lactone (formula (III)), for use in the in the prevention or treatment of a fungal infection.

Claims

1. A pharmaceutical formulation for vaginal administration, wherein the formulation comprises a pharmaceutical acceptable excipient and glucono δ-lactone (formula (III)), ##STR00006## wherein the glucono δ-lactone is present in an amount of 5 to 99 wt % of the formulation.

2. The pharmaceutical formulation according to claim 1, wherein the glucono δ-lactone is present in an amount of 10 to 70 wt % of the formulation, preferably 20 to 70 wt % of the formulation.

3. The pharmaceutical composition according to claim 1 or 2, wherein the composition comprises no more than 10 wt % water, preferably no more than 5 wt %.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the pharmaceutical formulation further comprises an antifungal agent selected from the group consisting of miconazole, terconazole, isoconazole, fenticonazole, fluconazole, nystatin, ketoconazole, clotrimazole, butoconazole, econazole, tioconazole, itraconazole, 5-fluoracil, and metronidazole.

5. The pharmaceutical formulation according to any one of the claims 1 to 4, wherein the pharmaceutical formulation comprises a carrier, a filler, and/or a buffering or pH-adjusting agent.

6. The pharmaceutical formulation according to any one of the claims 1 to 5, wherein the pharmaceutical formulation is formulated to release a compound according to formula (III) over an extended period of time, such as over at least 4 hours, over at least 6 hours, over at least 8 hours, or over at least 24 hours, after administration, such as intravaginal insertion.

7. The pharmaceutical formulation according to any one of the claims 1 to 6, wherein the pharmaceutical formulation is formulated as a tampon, vagitorium, vaginal aerosol, vaginal cup, vaginal gel, vaginal insert, vaginal patch, vaginal ring, vaginal sponge, vaginal suppository, vaginal cream, vaginal emulsion, vaginal foam, vaginal lotion, vaginal ointment, vaginal powder, vaginal shampoo, vaginal solution, vaginal spray, vaginal suspension, vaginal tablet, vaginal rod, vaginal disc, vaginal device, and any combination thereof, or wherein the pharmaceutical formulation is present on a sanitary article, such as a tampon, a sanitary napkin, an incontinence pad or diaper, or a panty liner.

8. The pharmaceutical formulation according to claim 7, wherein the pharmaceutical formulation is formulated as a vagitorium, vaginal insert, vaginal ring, vaginal suppository, vaginal tablet, vaginal rod, or vaginal disc.

9. The pharmaceutical formulation according to any one of the claims 1 to 8, wherein the pharmaceutical formulation has the ability to reduce or prevent biofilm formation by Candida species.

10. A pharmaceutical formulation according to any one of the claims 1 to 9 for use in the prevention or treatment of a urogenital fungal infection.

11. The pharmaceutical formulation according to any one of the claims 1 to 9 for use according to claim 10, wherein the urogenital fungal infection is vulvovaginal fungal infection.

12. The pharmaceutical formulation according to any one of the claims 1 to 9 for use according to claim 11, wherein the urogenital fungal infection is vulvovaginal candidosis.

13. The pharmaceutical formulation according to any one of the claims 1 to 9 for use according to claim 12, wherein the vulvovaginal candidosis is caused by Candida albicans, Candida glabrata, Candida krusei and/or Candida tropicalis.

14. Glucono δ-lactone (formula (III)), ##STR00007## for use in the in the prevention or treatment of a fungal infection.

15. Glucono δ-lactone (formula (III)) for use according to claim 14, wherein the fungal infection is a urogenital fungal infection.

16. Glucono δ-lactone (formula (III)) for use according to claim 15, wherein the urogenital fungal infection is vulvovaginal candidosis.

17. Glucono δ-lactone (formula (III)) for use according to claim 16, wherein the vulvovaginal candidosis is caused by Candida albicans, Candida glabrata, Candida krusei and/or Candida tropicalis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 show changes in optical rotation in the hydrolysis of GDA in distilled water (unfilled circles), pH 4 buffer (filled squares), pH 5 buffer (unfilled squares), and pH 7 buffer (filled circles).

[0072] FIG. 2 shows normalized biofilm formation of Candida albicans in the minimal media at pH 2.6-6.6 with phosphate buffer (unfilled circles, dotted line) or GDA (filled squares, solid line). The biofilm was measured after 24 h and the staining was performed with crystal violet.

[0073] FIG. 3a shows normalized biofilm formation of Candida albicans treated with GDA. A pellet of GDA was added to a buffer solution of pH 3.71 (10 mL) at 37° C. Samples (4 mL) were taken after 1, 2, 3, 4, 5, 6 and 24 hours and new buffer solution (4 mL) was added. The samples were diluted 50 times with biofilm medium and the amount of biofilm formation was measured after 24 h.

[0074] FIG. 3b shows normalized biofilm formation of Candida glabrata treated with GDA. A pellet of GDA was added to a buffer solution of pH 3.71 (10 mL) at 37° C. Samples (4 mL) were taken after 1, 2, 3, 4, 5, 6 and 24 hours and new buffer solution (4 mL) was added. The samples were diluted 50 times with biofilm medium and the amount of biofilm formation was measured after 24 h.

[0075] FIG. 4a shows the viability of biofilms of C. albicans and C. glabrata after treatment with GDA at different concentrations for 24 h. The biofilm staining was performed with XTT. Optical density measured at 485 nm. Diagonal stripes indicate data for C. albicans. Filled black columns indicate data for C. glabrata.

[0076] FIG. 4b shows the viability of biofilms of C. albicans and C. glabrata after treatment with GDA at different concentrations for 48 h. The biofilm staining was performed with XTT. Optical density measured at 485 nm. Diagonal stripes indicate data for C. albicans. Filled black columns indicate data for C. glabrata.

[0077] FIG. 5 shows the effect of GDA on mature biofilm of C. albicans and C. glabrata. Mature biofilm (grown for 48 h) was incubated with GDA for 5 h at 37° C. and then cells at serial dilution were plated on YPD plate to estimate cell survival.

[0078] FIG. 6a shows microfluidics study of biofilm development of untreated C. albicans in minimal medium pH 7.0. The untreated cells mainly form hyphae.

[0079] FIG. 6b shows microfluidics study of biofilm development of C. albicans treated in minimal medium with a hydrolysate of GDA at ×50 final concentration pH 3.8. The addition of GDA caused C. albicans to grow predominantly as yeast form, but not as hyphae.

MATERIALS AND METHODS

Glucono-δ-Lactone

[0080] Solid glucono-δ-lactone (GDA, CAS 90-8-2) was obtained from commercial suppliers.

Biofilm Formation Assay

[0081] Yeast strains were grown at 37° C. in complete medium YPD (0.5% (weight/volume) yeast extract, 1% (weight/volume) peptone, 2% (weight/volume) glucose) or minimal medium consisting of YNB (yeast nitrogen base without amino acids and ammonium sulphate, FORMEDIUM™, CYN0505) supplemented with 0.5% (weight/volume) ammonium sulphate, 0.2% (weight/volume) glucose and 100 mM L-proline. If needed 2% (weight/volume) agar was used to solidify media. The liquid minimal medium (YNB (yeast nitrogen base without amino acids and ammonium sulphate, FORMEDIUM™, CYN0505) supplemented with 0.5% ammonium sulphate, 0.2% (weight/volume) glucose and 100 mM L-proline) was used for biofilm assay (biofilm medium).

[0082] In the experiments on the impact of pH on biofilm (Example 2 below) the pH values (from 2.6 to 6.6) were obtained using either different potassium phosphate buffers at the final concentration 0.25 M, or by the addition of GDA to the biofilm medium.

Yeast Strains

[0083] The strains used in the biofilm formation experiments are described in Table 1.

TABLE-US-00001 TABLE 1 Yeast strains used in biofilm experiments. Laboratory Original strain name designation Description Reference Candida Y775 Wild-type, virulent in a [A. M. Gillum, et albicans mouse model of systemic al. Mol. Gen. SC5314 infection, sequenced strain Genet. 1984, 198, 179-182] Candida Y1092 Wild-type, type strain [B. Dujon, et al. glabrata isolated from human feces, Nature, 2004, 430, CBS138 sequenced strain 35-44]

Measurement of Biofilm

[0084] Biofilm was measured in liquid culture as described [K. Scherz et al., G3 (Bethesda), 2014, 4, 1671-1680. I. Serrano-Fujarte et al. Biomed Res Int. 2015; 2015:783639] with some modifications. Prior the biofilm assay, yeast cultures were grown in liquid YPD medium for 24 hours until stationary phase cells were then pelleted by centrifugation (1699×g), washed with sterile water and the cells were further inoculated into test biofilm medium (YNB (yeast nitrogen base without amino acids and ammonium sulphate) supplemented with 0.5% ammonium sulphate, 0.2% glucose and 100 mM L-proline pH7.0) at final concentration of 0.2 OD.sub.600/ml and incubated in 96-well flat-bottom polystyrene microtiter plates (Sigma Aldrich, Corning® Costar® culture plates, CLS3596-50EA) for 72 hours at 37° C. thermostat. At defined time points crystal violet (HT901-8FOZ; Sigma Aldrich) was added to the media at the final concentration 0.05% In addition, total biomass was measured. After 24 hours of cells staining, plate wells were washed four times with 200 μl of water to remove planktonic cells (cells which are not part of a biofilm), biofilms were then dried and dissolved in 200 μl of 96% ethanol. Total biomass and crystal violet biofilm staining measurements were performed at OD.sub.560 with FLUOstar OPTIMA plate reader, BMG LABTECH. Crystal violet biofilm measurements were normalized to the total biomass (OD.sub.560Biofilm/OD.sub.560 total biomass).

EXAMPLES

Example 1—Hydrolysis of Glucono-δ-Lactone (GDA)

[0085] In water solution glucono-δ-lactone (GDA) is in equilibrium with gluconic acid (GA, CAS 526-95-4). GDA (200 mg) was added to distilled H.sub.2O (20 mL), pH 4 buffer, pH 5 buffer, or pH 7 buffer at 37° C. The optical rotation and pH were measured over time. Optical rotation, measured at 37° C., sodium D line, C=10 mg/mL, path length=10 cm. The optical rotation of GDA is approximately 66°. The optical rotation of gluconic acid is approximately 5° [D. T. Sawyer, J. B. Bagger, J. Am. Chem. Soc., 1959, 81, 5302-5306]. This experiment shows that GDA is slowly hydrolyzed to a mixture of GDA and GA (FIG. 1). The equilibrium is pH-dependent and relevant concentrations of GDA are present at all buffered conditions.

Example 2—Biofilm Formation of Candida albicans at Different pH

[0086] As can be seen from FIG. 2, GDA shows strong effects on the biofilm formation of C. albicans, while the effects from phosphate buffer are much less pronounced. Further, GDA shows strong effect also at pH values up to around at least 6, whereas the effect from buffer are diminished already at pH 5.

Example 3—Biofilm Formation in a Model of In Vivo Conditions Using GDA

[0087] Pellets of GDA (2.5 g, duplicate samples) were added to buffer solution of pH 3.71 (0.5 M KH.sub.2PO.sub.4/ortho phosphoric acid, 10 mL) at 37° C. Samples (4 mL) were taken at fixed time points (1, 2, 3, 4, 5, 6 and 24 h) and new buffer solution (4 mL) was added. The samples were diluted 50 times with biofilm medium (vide supra) and the amount of biofilm formation was measured after 24 h as described above. As seen from FIG. 3a, the released GDA significantly reduces the amount of biofilm formation in C. albicans. Further, the hydrolysis of the pellet is seemingly slow enough to provide a preventive effect for at least up to 24 hours, likely far more. The effect is less pronounced with C. glabrata (FIG. 3b).

TABLE-US-00002 TABLE 2 Biofilm formation of C. albicans and C. glabrata treated with GDA in a model of in vivo conditions. Samples were taken after 1 h, diluted 50 times with biofilm assay medium and the amount of biofilm formation was measured after 24 h. Normalized biofilm (% of control), 1 h Candida albicans 8.3 Candida glabrata 71

[0088] The results show that biofilm formation of both C. albicans and C. glabrata was reduced in the presence of GDA. In addition to diminished biofilm formation, GDA may affect the viability of mature biofilm of C. albicans and C. glabrata.

Example 4—Viability of Mature Biofilms of C. albicans and C. glabrata Treated with GDA

[0089] Viability of biofilms of C. albicans and C. glabrata after treatment with GDA at different concentration and different time periods was evaluated by staining the cells with XTT. XXT is a colorimetric assay for quantification of cellular viability, and cytotoxicity. The assay is based on the cleavage of the tetrazolium salt XTT, a conversion that only occurs in viable cells. The mature biofilm was exposed to GDA for 24 h. Then the cells were washed 2 times with PBS, after which the XTT reaction mixture was added. After 30 min the optical density measured at 485 nm.

[0090] The XTT assay showed a strong decrease in viability for C. glabrata already after 24 h of incubation (FIG. 4a). The effect was less pronounced for C. albicans but clearly seen after 48 h (FIG. 4b).

[0091] Furthermore, mature biofilm (grown for 48 h in YNB, 0.2% glucose, 100 mM proline) of C. albicans and C. glabrata was incubated with GDA of different concentrations (0.05-0.5 g/ml) at 37° C. For this purpose biofilm medium (YNB, 0.2% glucose, 100 mM proline) was removed and GDA was added, which was dissolved either in water at concentration 0.05, 0.1, 0.2 and 0.5 g/ml. After incubation with GDA for 5 h or 73 h, 5 μl of cells were plated at serial dilution (1:10 to 1:1000) on the agar medium YPD to estimate cell survival. The plated cells were incubated for 24 h at 37° C. and visually analyzed. The cells from the mature biofilm treated with water were used as a control. It was found that the GDA decreases cell viability of C. albicans and C. glabrata, particularly at high concentrations. At the concentrations of 0.2 and 0.5 g/ml after 5 h of incubation the cell viability was decreased by about 100 times for both C. albicans and C. glabrata. After 73 h of incubation with 0.5 g/ml of GDA the cell viability of C. albicans was decreased by about 1000 times (data not shown). C. glabrata proved to be more sensitive to GDA (FIG. 5).

Example 5—Microfluidics Study of Biofilm Development

[0092] To monitor the C. albicans cell morphology we studied the biofilm development also using microscopy and microfluidics. After the yeast cells were inoculated, hyphae started to form within first hour of incubation in the biofilm medium (YNB supplemented with 100 mM proline and 0.2% glucose, pH7.0). FIG. 6a shows untreated cells after 5 h. A pellet of GDA (2.5 g) was added to buffer solution of pH 3.71 (0.5 M KH.sub.2PO.sub.4/ortho phosphoric acid, 10 mL) at 37° C. A sample was taken after 1 h and diluted 50 times with biofilm medium and added to C. albicans. After 5 h most treated cells were planctonic (FIG. 6b).

Example 6—Viability of Different Candida Species in the Presence of GDA

[0093] Other Candida sp. studied were also sensitive (i.e. cell viability measure using the XTT assay, cf Example 4) to GDA. However, they displayed different levels of sensitivity. Candida albicans SC5314 displayed the lowest susceptibility and Candida krusei silicone isolate A4-1 displayed the highest susceptibility. The GDA-toxicity is mediated through cell wall damage as the cells exposed to GDA had lower viability on the medium with calcofluor white compared to that supplemented with osmotic stabilizer (0.5M sucrose) and compared to the untreated cells on these media. Table 3 summarizes qualitative effects shown by GDA.

TABLE-US-00003 TABLE 3 Sensitivity of different Candida species to GDA Strain Sensitivity to GDA, 24 h exposure, plates C. albicans SC5314 + C. glabrata CBS138 ++++++ C. tropicalis silicone isolate U3-3 ++++++++ C. krusei silicone isolate U3-5 ++ C. tropicalis silicone isolate A6-1 +++ C. krusei silicone isolate U2-12 ++++++++ C. krusei silicone isolate A5-2 ++ C. krusei silicone isolate A4-1 +++++++++

[0094] To conclude, (i) GDA can break mature biofilm formed by C. albicans and C. glabrata, (ii) upon texposure to GDA, C. albicans transforms into yeast form, while the viability of C. glabrata decreases, (iii) the effect is clear even on other strains, i.e. C. tropicalis and C. krusei.