Compositions for treating candidiasis infections

Abstract

The present invention relates to a composition comprising as antifungal active agent at least 2.Math.10.sup.10 CFU of lactobacilli and a sulfur-containing compound for use as a first-line treatment for candidiasis and for recurrent candidiasis.

Claims

1. A method for first-line treatment of candidiasis in a patient in need thereof, the method comprising vaginally administering to said patient a pharmaceutical composition comprising: i) as therapeutic active agent at least 5×10.sup.10 CFU of Lactobacillus rhamnosus 35 (Lcr35); and ii) at least 60 mg of thiosulfate, wherein the pharmaceutical composition is used as a substitute for a chemical antifungal.

2. The method of claim 1, wherein candidiasis is recurrent candidiasis.

3. The method of claim 1, wherein pathogen is selected from the group consisting of: Candida albicans, Candida glabrata and Candida tropicalis.

4. The method of claim 1, wherein the pharmaceutical composition comprises from 60 mg to 300 mg of thiosulfate.

5. The method of claim 1, wherein the pharmaceutical composition comprises from 60 to 120 mg of thiosulfate.

6. The method of claim 1, wherein the pharmaceutical composition comprises from 60 to 80 mg of thiosulfate.

7. The method of claim 1, wherein the pharmaceutical composition comprises at least 1×10.sup.11 CFU of Lactobacillus rhamnosus 35 (Lcr35).

8. The method of claim 1, wherein the pharmaceutical composition comprises from 1×10.sup.11 to 1×10.sup.12 CFU of Lactobacillus rhamnosus 35 (Lcr35).

9. The method of claim 1, wherein the candidiasis is vulvovaginal candidiasis.

10. The method of claim 1, wherein the pharmaceutical composition is formulated to be administered in one dose, once per day.

11. The method of claim 1, wherein the pharmaceutical composition is in the form of an immediate-release tablet or capsule.

12. The method of claim 1, wherein the pharmaceutical composition is in the form of a multilayer tablet comprising: at least one immediate-release layer comprising, in relation to the total weight of all the immediate-release layers, at least 5×10.sup.10 CFU of Lactobacillus rhamnosus 35 (Lcr35) and 60 to 300 mg of thiosulfate; and at least one sustained-release layer, comprising, in relation to the total weight of all the sustained-release layers, from 1×10.sup.7 to 1×10.sup.10 CFU of Lactobacillus rhamnosus 35 (Lcr35).

13. The method of claim 12, wherein in the multilayer tablet the sustained-release layer comprises from 10 to 40% by weight of hydroxypropylmethylcellulose (HPMC) in relation to the total weight of said layer.

14. A method for first-line treatment of vulvovaginal candidiasis and its recurrence in a patient in need thereof comprising vaginally administering a pharmaceutical composition comprising: i) as therapeutic active agent at least 5×10.sup.10 CFU of Lactobacillus rhamnosus 35 (Lcr35); and ii) at least 60 mg of thiosulfate, optionally repeating the administration of the same pharmaceutical composition 2, 3, 4 or 5 days after the first dose is taken and administering a composition comprising Lactobacillus rhamnosus Lcr35 at a concentration of 10.sup.7 to 10.sup.10 CFU/g and thiosulfate to promote the prevention of recurrence of vulvovaginal candidiasis, wherein the pharmaceutical composition is used as a substitute for a chemical antifungal.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Impact of a standard concentration (WO2014/009349 versus the invention)

(2) Loss of viability of C. albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(3) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.9 CFU+30 mg of thiosulfate as described in WO 2014/009349;

(4) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+60 mg of thiosulfate;

(5) FIG. 2. Effect of the combination of L. rhamnosus Lactobacillus Rhamnosus LCR35® at 10.sup.11 CFU+thiosulfate at various concentrations

(6) Loss of viability of C. albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(7) Control C. albicans (30 mg of thiosulfate);

(8) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+0 mg of thiosulfate;

(9) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+30 mg of thiosulfate;

(10) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+60 mg of thiosulfate;

(11) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+150 mg of thiosulfate;

(12) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+225 mg of thiosulfate;

(13) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.11 CFU+300 mg of thiosulfate

(14) FIG. 3. Effect of the combination of Lactobacillus Rhamnosus LCR35® at various concentrations+60 mg of thiosulfate

(15) Loss of viability of C. albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(16) Control C. albicans (60 mg of thiosulfate);

(17) Lactobacillus Rhamnosus LCR35® at a concentration of 2.5.Math.10.sup.19 CFU+60 mg of thiosulfate

(18) Lactobacillus Rhamnosus LCR35® at a concentration of 5.Math.10.sup.10 CFU+60 mg of thiosulfate;

(19) Lactobacillus Rhamnosus LCR35® at a concentration of 7.5.Math.10.sup.10 CFU+60 mg of thiosulfate;

(20) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate;

(21) FIG. 4. Effect of the combination of Lactobacillus spp. at 10.sup.11 CFU+60 mg of thiosulfate on Candida albicans.

(22) Loss of viability of C. albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(23) Control C. albicans (60 mg of thiosulfate);

(24) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate;

(25) L. casei at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate;

(26) L. crispatus at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate

(27) FIG. 5. Effect of the combination of Lactobacillus Rhamnosus LCR35® at 10.sup.11 CFU+60 mg of thiosulfate on Candida spp.

(28) 5A: Loss of viability of clinical Candida albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(29) Control clinical C. albicans (60 mg of thiosulfate);

(30) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate

(31) 5B: Loss of viability of clinical Candida glabrata (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(32) Control clinical C. glabrata (60 mg of thiosulfate);

(33) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate

(34) 5C: Loss of viability of Aspergillus fumigatus (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(35) Control A. fumigatus (60 mg of thiosulfate);

(36) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate

(37) 5D: Loss of viability of Saccharomyces cerevisiae (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(38) Control S. cerevisiae (60 mg of thiosulfate);

(39) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate

(40) FIG. 6. Effect of the combination of Lactobacillus Rhamnosus LCR35® at 10.sup.11 CFU+sulfur-containing molecules on Candida albicans.

(41) Loss of viability of C. albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours)

(42) 6A: Effect of the combination of Lactobacillus Rhamnosus LCR35® at 10.sup.11 CFU+60 mg of thiosulfate

(43) Control C. albicans (60 mg of thiosulfate);

(44) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of thiosulfate

(45) 6B: Effect of the combination of Lactobacillus Rhamnosus LCR35® at 10.sup.11 CFU+60 mg of cysteine

(46) Control C. albicans (60 mg of cysteine);

(47) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+60 mg of cysteine

(48) 6C: Effect of the combination of Lactobacillus Rhamnosus LCR35® at 10.sup.11 CFU+150 mg of glutathione

(49) Control C. albicans (60 mg of glutathione);

(50) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+150 mg of glutathione

(51) FIG. 7. Activity of the invention in tablet form

(52) Loss of viability of C. albicans (log.sub.10 (CFU/mL)) as a function of co-culture time (hours) for a tablet form

(53) Control C. albicans (60 mg of thiosulfate);

(54) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+220 mg of thiosulfate (invention in lyophilized powder form);

(55) Lactobacillus Rhamnosus LCR35® at a concentration of 1.Math.10.sup.11 CFU+180 mg of thiosulfate (invention in tablet form);

(56) Lactobacillus Rhamnosus LCR35® at a concentration of 10.sup.9 CFU+140 mg of thiosulfate as described in WO 2014/009349, formulated in tablet form.

EXAMPLES

(57) Effect of the combination of Lactobacillus Rhamnosus LCR35®+ thiosulfate on pathogens of various Candida species

(58) Materials and Method:

(59) After pre-culture in Sabouraud broth, the pathogen is placed with a count of about 1.Math.10.sup.8 CFU/mL in 30 mL of SVF (medium for simulating the vaginal environment pH 4.2) and put in direct contact with the lactobacilli in lyophilized form following a step of fermentation and of lyophilization+sulfur-containing molecule (thiosulfate or cysteine) (without pre-culture so as to approach the conditions of use in vivo). The pathogen is counted on agar at T.sub.0, T.sub.12 h, T.sub.16 h, T.sub.20 h, T.sub.24 h and, in addition, at T.sub.36 h for FIGS. 1, 3 and 7. The results are expressed as the logarithmic decrease between the initial count (To) and the sampling points. Antifungal activity is determined to be specific as of a decrease of 3 log. A pathogen viability control is prepared in parallel under the same conditions without the presence of compounds of the invention.

(60) The expression “Formulation A of the invention WO2014/009349” refers to a formulation of the Lactobacillus Rhamnosus LCR35® strain as prepared by the process described in Example 4 of the application WO2014/009349, i.e., in the presence of 113 g/L of sodium thiosulfate during lyophilization. In the following examples, Formulation A comprises 1.Math.10.sup.9 CFU of Lactobacillus Rhamnosus LCR35® (versus 1.Math.10.sup.8 CFU for the examples of WO2014/009349).

(61) Four concentrations of lactobacilli were tested: 2.5.Math.10.sup.19 CFU, 5.Math.10.sup.10 CFU, 7.5.Math.10.sup.19 CFU and >1.Math.10.sup.11 CFU, or per mL: 8.Math.10.sup.8 CFU/mL, 1.6.Math.10.sup.9 CFU/mL, 2.5.Math.10.sup.9 CFU/mL and 3.3.Math.10.sup.9 CFU/mL, as well as the concentration found in Formulation A of the invention WO2014/009349 of 10.sup.9 CFU or 3.3.Math.10.sup.7 CFU/mL.

(62) Various concentrations of sodium thiosulfate in the test culture medium were tested: 0, 1, 2.5, 7.5 and 10 g/L.

(63) Several pathogens derived from clinical samples were tested: Candida albicans, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, as well as a reference yeast, Saccharomyces cerevisiae.

(64) Several lactobacilli species were tested: L. crispatus, L. casei.

(65) The results are presented in FIGS. 1 to 6.

(66) Preparation of Tablets

(67) The tablet of the present invention is a single-layer tablet formulated to obtain a controlled release of the Lactobacillus Rhamnosus LCR35® strain at a concentration greater than or equal to 1.Math.10.sup.11 CFU/g and a quantity of thiosulfate of 180 mg. The weight of the tablet is about 950 mg.

(68) The tablets are prepared using an industrial-size tablet press with a compression force on the order of 20 kN.

Conclusions

(69) FIG. 1: The invention makes it possible to have an inhibition higher than 3 log as of 20 hours of co-culture whereas the product from the prior invention (Formulation A described in WO 2014/009349) induces no antifungal effect on the pathogen Candida albicans ATCC10231.

(70) FIG. 2. As of 1 g/L of thiosulfate (range tested from 1 to 10 g/L), the invention makes it possible to have an inhibition higher than 3 log as of 20 hours of co-culture. At the optimal concentration of 2 g/L, this level of antifungal effect is reached as of 16 hours of co-culture between the invention and the pathogen Candida albicans ATCC10231.

(71) FIG. 3: An antifungal activity of 3 log is obtained in 24 hours starting with an Lactobacillus Rhamnosus LCR35® concentration of 5.Math.10.sup.10 CFU (or 1.6.Math.10.sup.9 CFU/mL). For a concentration of 2.5.Math.10.sup.10 CFU (or 8.Math.10.sup.8 CFU/mL), this antifungal activity is obtained after 36 hours of co-culture between the invention and the pathogen Candida albicans ATCC10231.

(72) FIG. 4: The antifungal activity of the invention is demonstrated as of 20 hours for various lactobacilli species for the pathogen Candida albicans ATCC10231 in co-culture.

(73) FIG. 5: The antifungal activity of the invention is demonstrated as of 16 hours for various species: two pathogenic clinical strains: Candida albicans and Candida glabrata; one pathogenic strain: Aspergillus fumigatus; and one yeast strain: Saccharomyces cerevisiae.

(74) FIG. 6: The antifungal activity of the invention is demonstrated as of 16 hours for various sources of sulfur-containing molecules (thiosulfate, cysteine, glutathione) compared with the pathogen Candida albicans ATCC10231 in co-culture.

(75) FIG. 7: The antifungal activity is retained after formulation as controlled-release tablets.

BIBLIOGRAPHICAL REFERENCES

Publications

(76) 1. Sobel J D. Recurrent vulvovaginal candidiasis. A prospective study of the efficacy of maintenance ketoconazole therapy. N Engl J Med 1986; 315: 1455-58. 2. Fischer G. Chronic vulvovaginal candidiasis: what we know and what we have yet to learn. Australas J Dermatol, 2012 November; 53(4):247-54. doi: 10.1111/j.1440-0960.2011.00860.x. Epub 2012 Sep. 24 3. Petrova et al. Lactobacillus species as biomarkers and agents that can promote various aspects of vaginal health. Frontiers in Physiology. 2015. 4. Kern et al. Preventive treatment of vulvovaginal candidosis with vaginal probiotic (Gynophilus®-Lcr Regenerans®) results of the observational study candiflore. La letter du Gynecologue no. 370—March 2012 5. Yue et al. The dynamic changes of vaginal microecosystem in patients with recurrent vulvovaginal candidiasis; a retrospective study of 800 patients. Arch Gynecol Obstet DOI 10.1007/s00404-015-3774-2 6. Murina et al. Can Lactobacillus fermentum LF10 and Lactobacillus acidophilus LA02 in a slow-release vaginal product be useful for prevention of recurrent vulvovaginal candidiasis? A clinical study. J Clin Gastroenterol. Volume 48, sup. 1; November/December 2014 7. Sophie Coudeyras et al. Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group, Appl. Environ. Microbiol. doi:10.1128/AEM.02286-07, 2008.

PATENTS AND PATENT APPLICATIONS

(77) U.S. Pat. No. 6,093,394 U.S. Pat. No. 6,468,526 U.S. Pat. No. 7,807,440 US 2010/0151026 WO 84/04675 WO 2000/035465 US 2002/0044926 WO 2006/045475 WO2014/009349 WO 2014/009330