Treatment of a bacterial vaginal infection

Abstract

The present invention relates to furazidin for vaginal use in the treatment of a bacterial vaginal infection. Preferably, the bacterial vaginal infection is caused by Gardnerella vaginalis and/or Atopobium vaginae bacteria.

Claims

1. A method of treating a vaginal bacterial infection in an infected woman comprising vaginally administering 10 mg to 300 mg per day of furazidin to the infected woman to thereby treat the vaginal bacterial infection, wherein the vaginal bacterial infection is caused by Gardnerella vaginalis or Atopobium vaginae or a combination of the two.

2. The method according to claim 1, wherein the vaginal bacterial infection is caused by Gardnerella vaginalis.

3. The method according to claim 1, wherein the vaginal bacterial infection is caused by Atopobium vaginae.

4. The method according to claim 1, wherein the vaginal bacterial infection is a mixed type vaginal bacterial infection.

5. The method according to claim 4, wherein the mixed type vaginal bacterial infection is caused by Gardnerella vaginalis, and Atopobium vaginae.

6. The method according to claim 1, wherein the vaginal bacterial infection is a recurrent vaginal bacterial infection.

7. The method according to claim 1, wherein the pharmaceutical composition is in the form of tablets, capsules, cream, gel, lotion, foam, pessary, or globules.

8. The method according to claim 1, wherein the pharmaceutical composition is in the form of tablets, capsules, cream, gel, lotion, foam, pessary, or globules.

9. The method of claim 1, wherein the dose of furazidin is 40 mg.

10. The method of claim 1, wherein the dose of furazidin is 20 mg.

11. The method of claim 1, wherein 10 mg to 300 mg of furazidin is administered daily for ten days.

12. The method of claim 9, wherein 40 mg of furazidin is administered daily for ten days.

13. The method of claim 10, wherein 20 mg of furazidin is administered daily for ten days.

Description

EXAMPLES

1. Analysis of the Antimicrobial Activity

(1) Analysis of the antimicrobial activity of furazidin, nitrofurantoin, nifuratel, clindamycin metronidazole, and nystatin was performed to determine the minimum concentration which inhibits the growth of microorganisms—minimal inhibitory concentration values (MIC) using the method described below. Strains used in the study were Gardnerella vaginalis ATCC 14018, Atopobium vaginae ATCC BAA 55, Candida albicans ATCC 90028, and Candida glabrata ATCC 2001.

(2) A stock solution for all of compounds was 10000 μg/ml, from which 15 serial dilutions were prepared (500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.6 μg/mL, 7.8 μg/mL, and 3.9 μg/mL, 1.95 μg/mL, 0.98 μg/mL, 0.49 μg/mL, 0.24 μg/mL, 0.12 μg/mL, 0.06 μg/mL, and 0.03 μg/mL).

(3) Macro Dilution Method for Bacteria in a Solid Medium

(4) Serial dilution method in a solid medium was carried out to determine the MIC for furazidin, nitrofurantion, nifuratel, clindamycin, metronidazol, and nystatin for the bacteria. To perform the assay, a pure, 48-hour strain reference culture was used, from which a bacterial suspension with a density of 1.0 McFarland in sterile saline was prepared. The suspension was plated onto Schaedler agar medium supplemented with 5% sheep blood (Becton Dickinson) and a test compound at the appropriate concentration. After incubation, the value MIC was read. Colony growth on the plate indicated no activity of the test compound at tested concentration. As the MIC value, the last lowest concentration as taken, at which there was no microbial growth. The incubation conditions used are shown in Table 1.

(5) The synergistic action of the selected compounds was determined using 2-fold lower concentrations of the selected compound than the obtained MIC. Lack of bacterial growth on the plate was interpreted as a synergic action.

(6) TABLE-US-00001 TABLE 1 Incubation conditions for the strains used in the study Incubation Incubation Strain conditions time Gardnerella vaginalis microaerophilic, 72 hours ATCC 14018 37° C. Atopobium vaginae anaerobic, 37° C. 7 days ATCC BAA 55
Macro Dilution Method for Yeast-Like Fungi in a Liquid Medium

(7) Liquid medium RPMI 1640 (Sigma-Aldrich) was used for fungi of the species Candida albicans ATCC 90028, and Candida glabrata ATCC 2001, in which 15 serial dilution were prepared in a range 500 μg/mL-0.03 μg/mL.

(8) From a pure 24-hour culture on Sabouraud solid medium, a fungal suspension having density of 0.5 McFarland in sterile saline. Next, the suspension was diluted, and was introduced into the RPMI 1640 medium yielding final concentrations of fungi 10.sup.5 CFU/mL. Incubation was carried out for 24 hours in aerobic conditions in 37° C. Colony growth on the plate indicated no activity of the test compound at tested concentration. As the MIC value, the last lowest concentration as taken, at which there was no microbial growth

(9) The synergistic action of the selected compounds was determined using 2-fold lower concentrations of the selected compound than the obtained MIC. Lack of fungal growth on the plate was interpreted as a synergic action.

(10) The results are summarized and compared in the following tables.

(11) TABLE-US-00002 TABLE 2 MIC values for the single compounds MIC value [mg/L] Strain Furazidin Nitrofurantion Nifuratel Clindamycin Metronidazol Nystatin Gardnerela vaginalis 0.49 0.98 1.95 1.95 250 500 ATCC 14018 Atopobium vaginae <0.03 1.95 3.9 <0.03 0.98 500 ATCC BAA 55 Candida albicans 500 500 >500 ND 500 1.98 ATCC 90028 Candida glabrata 500 500 500 ND 250 1.98 ATCC 2001 ND—not determined;

(12) TABLE-US-00003 TABLE 3 Synergy of selected nitrofurans with metronidazole and nystatin against strains of Gardnerella vaginalis, Lactobacillus paracasei, Atopobium vaginae, Candida albicans, Candida glabrata (,,+”synergy, ,,−”no synergy) Test compound Furazidin Nitrofurantoin Nifuratel (concentration) (0.25 mg/L) (0.5 mg/L) (0.98 mg/L) Gardnerella Metronidazole + + − vaginalis (125 mg/L) ATCC 14018 Nystatin + + + (250 mg/L) Test compound Furazidin Nitrofurantoin Nifuratel (concentration) (0.03 mg/L) (0.98 mg/L) (1.95 mg/L) Atopobium Metronidazole + + − vaginae (0.5 mg/L) ATCC BAA 55 Nystatin + + + (250 mg/L) Test compound Furazidin Nitrofurantoin Nifuratel (concentration) (250 mg/L) (250 mg/L) (250 mg/L) Candida Metronidazole + + + albicans (250 mg/L) ATCC 90028 Nystatin − − − (0.98 mg/L) Test compound Furazidin Nitrofurantoin Nifuratel (concentration) (250 mg/L) (250 mg/L) (250 mg/L) Candida Metronidazole + + + glabrata (125 mg/L) ATCC 2001 Nystatin + − − (0.98 mg/L)
The data presented above clearly shows that all of nitrofurans (furazidin, nifuratel, nitrofurantoin), clindamycin and metronidazole exhibit antibacterial activity against Gardnerella vaginalis and Atopobium vaginae, furazidin exhibits the highest antibacterial activity in the group of nitrofuran derivatives against Gardnerella vaginalis and Atopobium vaginae, furazidin exhibits higher antibacterial activity against Gardnerella vaginalis than clindamycin (MIC.sub.furazidin<0.49 mg/L vs. MIC.sub.clindamycin<1.95 mg/L), efficacy of furazidin against Atopobium vaginae is comparable to that for clindamycin (MIC<0.03 mg/L), no activity of nitrofurans were found against yeast-like fungi tested, furazidin and nitrofurantoin, in contrast to nifuratel, show synergistic effects with metronidazole against Gardnerella vaginalis, and Atopobium vaginae, all tested nitrofurans show synergistic effects with nystatin on Gardnerella vaginalis, and Atopobium vaginae, all tested nitrofurans show synergistic effects with metronidazole against Candida albicans, and Candida glabrata, only furazidin shows synergistic effects with nystatin against Candida glabrata.

2. Formulation Examples

(13) a) Vaginal Tablet Formulation

(14) i. Vaginal Tablet with Furazidin

(15) TABLE-US-00004 Component Quantity [mg/tablet] Furazidin 500 Lactose 200 Silicon dioxide 20 Magnesium stearate 2

(16) A mixing vessel is charged with furazidin, lactose, silicon dioxide, and magnesium stearate. After the mixing vessel was closed, the mixture is stirred for 15 minutes. The resulting mixture is compressed into tablets having mass of 722 mg.

(17) ii. Vaginal Tablet with Furazidin and Clotrimazole

(18) TABLE-US-00005 Component Quantity [mg/tablet] Furazidin 250 Clotrimazole 100 Lactose 200 Silicon dioxide 20 Magnesium stearate 2

(19) A mixing vessel is charged with furazidin, clotrimazole, lactose, silicon dioxide, and magnesium stearate. After the mixing vessel was closed, the mixture is stirred for 15 minutes. The resulting mixture is compressed into tablets having mass of 572 mg.

(20) b) Cream Formulation

(21) General preparations for a cream formulation comprising furazidin, optionally with nystatin or metronidazole are as follows. The relevant Phases are defined below.

(22) Phase A and Phase B are heated separately to 60° C. Phase B is added to Phase A, and mixed with a mechanical stirrer. Next, the resulting mixture is homogenized for 1 minute per each 200 g of the mixture at 10000 rpm, followed by addition of Phase C, and the combined phases are mixed with a mechanical stirrer until the mixture reaches room temperature. Yellow cream is obtained.

(23) i. Cream with Furazidin

(24) TABLE-US-00006 Quantity Component [g/100 g of cream] Phase A Cithrol DPHS(PEG-30 2.0 Dipolyhydroxystearate) Arlamol PS15E (PPG-15 Stearyl 6.0 Ether) Cithrol PG32IS (Polyglyceryl - 3 3.0 Diisostearate) Crodamol IPM 8.0 (Isopropyl Myristate) Mineral oil (Paraffinum liquidum) 10.0 Phase B Water To 100.0 MgSO.sub.4•7H.sub.2O 0.7 Phase C Furazidine 20.0
ii. Cream with Furazidin and Nystatin

(25) TABLE-US-00007 Quantity Component [g/100 g of cream] Phase A Cithrol DPHS(PEG-30 2.0 Dipolyhydroxystearate) Arlamol PS15E (PPG-15 6.0 Stearyl Ether) Cithrol PG32IS 3.0 (Polyglyceryl - 3 Diisostearate) Crodamol IPM 8.0 (Isopropyl Myristate) Mineral oil (Paraffinum liquidum) 10.0 Phase B Water To 100.0 MgSO.sub.4•7H.sub.2O 0.7 Phase C Furazidin 20.0 Nystatin 1.28 g (6 312 000 j.m./g) (8000000 j.m.)
iii. Cream with Furazidin and Metronidazole

(26) TABLE-US-00008 Quantity Component [g/100 g of cream] Phase A Cithrol DPHS(PEG-30 2.0 Dipolyhydroxystearate) Arlamol PS15E (PPG-15 Stearyl 6.0 Ether) Cithrol PG32IS (Polyglyceryl - 3 3.0 Diisostearate) Crodamol IPM 8.0 (Isopropyl Myristate) Mineral oil (Paraffinum liquidum) 10.0 Phase B Water To 100.0 MgSO.sub.4•7H.sub.2O 0.7 Phase C Furazidin 20.0 Metronidazole 1.0
c) Vaginal Globules

(27) General preparation for vaginal globules comprising furazidin, optionally with nystatin. The relevant Phases are defined below.

(28) Phase A is melted, and Phase B is added. After homogenization, globules having 3 grams are formed.

(29) i. Globules with Furazidin

(30) TABLE-US-00009 Globule 1 Globule 2 Quantity Quantity Component [mg/globule] Component [mg/globule] Phase A Phase A Cocoa butter To 3000.0 Cocoa butter To 1000.0 Phase B Phase B Furazidin 300.0 Furazidin 40.0
ii. Globules with Furazidin and Nystatin

(31) TABLE-US-00010 Globule 3 Globule 4 Quantity Quantity Component [mg/globule] Component [mg/globule] Phase A Phase A Cocoa butter To 3000.0 Cocoa butter To 1000.0 Phase B Phase B Furazidin 300.0 Furazidin 40.0 Nystatin  38.4 Nystatin 15.8 (6 312 000 (8 000 000 (6 312 000 (100 000 j.m./g) j.m.) j.m./g) j.m.)

3. Observation of In Vivo Efficacy

(32) Further, in vivo efficacy of furazidin in treatment of bacterial vaginal infections was studied.

(33) A group of 29 women with symptoms of bacterial vaginal infection (abnormal vaginal discharge of characteristic fish-like smell, presence of clue cells, and in several cases itching and/or pain during urination) was screened using PCR techniques for the presence of Gardnerella vaginalis, and/or Atopobium vaginae as predominant causatives of bacterial vaginal infections. The selection was conducted as described in Kusters J. G. et al., Eur J Clin Microbiol Infect Dis. 2015; 34(9): 1779-1785. In 26 out of 29 women presence of Gardnerella vaginalis or Atopobium vaginae or both causatives was conformed. This selected group of 26 women was further screened as to the presence of accompanying Candida albicans infection. The selection was performed using method described for example in Trama J. P. et al., Infect Dis Obstet Gynecol., June 2005; 13(2): 63-67 and additionally confirmed by commercial kit Candida albicans TaqMan PCR Kit (form Norgenbiotek).

(34) The group was divided into 6 subgroups as follows:

(35) TABLE-US-00011 No. of Group Causative patients 1 GV 3 2 AV 4 3 GV + AV 11 4 GV + CA 1 5 AV + CA 2 6 GV + AV + CA 5 GV: Gardnerella vaginalis AV: Atopobium vaginae CA: Candida albicans

(36) Five patients in Group 3, one patient in Group 4 and two patients in Group 6 reported previous treatment of bacterial vaginal infections with metronidazole and/or clindamycin.

(37) Patients in Group 1 to Group 3 received intravaginally Globule 2. Patients in Group 4 to 6 received intravaginally Globule 4 (with nystatin), all Groups following the dosing regimen: one globule daily for 10 days. The globules were deposited high in the vagina.

(38) In the Group 6, 3 out of 15 patients was receiving Globule 2 for the first 3 days, but due to lack of improvement, further treatment with Globule 2 was discontinued and Globule 4 was instead administered for further 7 days. Change of this regimen didn't cause change in the amount of administered furazidin comparing to Globule 2 but additionally nystatin was provided to control Candida growth.

(39) After 10 days of treatment, all patients showed no symptoms of bacterial vaginal infection such as abnormal vaginal discharge of characteristic fish-like smell, and presence of clue cells. None of the patients reported itching or pain, including pain during urination. Those patients were deemed to be cured and the patients reported improvement of general quality of life. PCR analysis for Gardnerella vaginalis, and/or Atopobium vaginae showed lack of this bacteria in samples obtained from patients two days after the described treatment had been completed.