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EP-3648778-B1 - HIGH POTENCY STABLE FORMULATIONS OF VAGINAL LACTOBACILLUS

EP3648778B1EP 3648778 B1EP3648778 B1EP 3648778B1EP-3648778-B1

Inventors

  • PARKS, THOMAS P.
  • MARCOBAL, Angela

Dates

Publication Date
20260513
Application Date
20180705

Claims (13)

  1. An aqueous bacterial suspension of vaginal Lactobacillus species, having no animal-derived excipients, where the suspension results from a combination of a cell pellet of vaginal Lactobacillus species with an aqueous preservation medium consisting essentially of: (i) trehalose at between 5-20%, w/v; (ii) xylitol at between 2-9%, w/v; (iii) sodium ascorbate 0.5-1.5%, w/v; and (iv) sodium phosphate at between 10-50 mM.
  2. The bacterial suspension of claim 1, wherein: (a) the aqueous preservation medium optionally comprises sodium glutamate at between 0-5%; (b) the vaginal Lactobacillus species has the ability to produce greater than 0.5 ppm of hydrogen peroxide under effective culture conditions; and/or (c) the vaginal Lactobacillus species is selected from the species consisting of Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri.
  3. The bacterial suspension of claim 1 or 2, wherein the aqueous preservation medium consists essentially of: (i) trehalose at between 5-15%, w/v; (ii) xylitol at between 2-7%, w/v; (iii) sodium ascorbate 0.5-1.0%, w/v; and (iv) sodium phosphate at between 10-30 mM.
  4. A dry powder obtained by lyophilizing the bacterial suspension of any one of claims 1 to 3.
  5. The dry powder of claim 4, wherein the powder: (a) has a water activity value of less than 0.220; and/or (b) is combined with an inactive excipient at a ratio of powder: excipient of between 1:1 and 1:10 w/w, preferably between 1:1 and 1:5 w/w.
  6. The dry powder of claim 5, wherein the excipient is maltodextrin.
  7. The dry powder of any one of claims 4 to 6, wherein the powder is contained within a plastic housing designed for vaginal administration.
  8. A method of preserving Lactobacillus spp. under dry conditions without animal-derived excipients, the method comprising: (i) obtaining an aqueous suspension of vaginal Lactobacillus species having a cell concentration between 10 8 to 10 13 per ml; (ii) centrifuging the solution to form a bacterial cell pellet; and (iii) resuspending the bacterial cell pellet in an aqueous preservation medium consisting essentially of: (a) trehalose at between 5-20%, w/v; (b) xylitol at between 2-9%, w/v; (c) sodium ascorbate 0.5-1.5%, w/v; and (d) sodium phosphate at between 10-50 mM; where the weight ratio of cell pellet wet weight (grams) to preservation media (mL) is between 1:1 and 1:5 grams of cell pellet to milliliter of preservation media to yield a bacterial suspension.
  9. The method of claim 8, wherein the aqueous preservation medium consists essentially of: (a) trehalose at between 5-15%, w/v; (b) xylitol at between 2-7%, w/v; (c) sodium ascorbate 0.5-1.0%, w/v; and (d) sodium phosphate at between 10-30 mM.
  10. The dry powder of any one of claims 4 to 7 for use in a method of treating abnormal vaginal microbiota in a woman, said method comprising the steps of: (i) selecting a woman having a diagnosis of abnormal vaginal microbiota; (ii) administering an antibiotic in an amount effective to reduce the level of abnormal vaginal microbiota; (iii) following step ii, administering to the woman the dry powder.
  11. The dry powder for use of claim 10, wherein step ii includes daily administration of antibiotic for between 2 and 7 days and wherein step iii begins at any time between two days before the completion of antibiotic administration and two days after the administration of antibiotic in step ii ends.
  12. The dry powder for use of claim 10 or 11, wherein: (a) the vaginal Lactobacillus species has the ability to produce greater than 0.5 ppm of hydrogen peroxide under effective culture conditions, (b) the vaginal Lactobacillus species is selected from the species consisting of Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri; (c) the antibiotic is clindamycin, metronidazole, or tinidazole; and/or (d) the dry powder is combined with an inactive excipient at a ratio of powder: excipient of between 1:1 and 1:10 w/w, preferably between 1:1 and 1:5 w/w.
  13. The dry powder for use of claim 12, wherein the excipient is maltodextrin.

Description

BACKGROUND The mucosal membranes of all humans are naturally colonized by bacterial microbiota. Recent studies have indicated that the microbiota found in the human gut, mouth and vagina, interact closely with cells and tissues of the body to regulate natural biological processes such as non-specific host defense. See, e.g., Redondo-Lopez, et al. (1990) Rev. Infect. Dis. 12:856-872; Gilbert, J.A., et al. Nature 2016 Jul 7, 535(7610):94-103; McDermott, A. J., et al. Immunology 2014 May, 142(1):24-31; Nelson, D. B., et al. Anaerobe 2016 Dec, 42:67-73. Generally, healthy vaginal microbiota is dominated by Lactobacillus species, which are gram positive rods that play an important role in resisting infection via production of lactic acid and acidification of the vagina, or by production of other antimicrobial products, such as hydrogen peroxide (H2O2). The species of Lactobacillus most commonly isolated from the reproductive tracts of healthy women worldwide include L. crispatus, L. jensenii, L. gasseri, and L. iners. See, e.g., Antonio et al., (1999) J. Infect. Dis. 180:1950-1956; Vasquez et al., (2002) J. Clin. Microbiol. 40:2746-2749; Vallor, A. C., et al. J Infect Dis. 2001 Dec 1, 184(11):1431-6; Ravel, J., et al. Proc Natl Acad Sci, USA. 2011 Mar 15, 108 Suppl 1:4680-7. L. crispatus, L. jensenii, and L. gasseri are capable of producing H2O2, whereas L. iners strains generally do not produce H2O2. These species are phylogenetically and functionally different from food and/or environmental Lactobacillus species. These facultative anaerobes metabolize glucose to lactic acid, contributing to the maintenance of a low vaginal pH (4.0-4.5) that accounts for a major part of the non-specific host defense of the vagina. An acidic pH has a significant antagonistic effect on the growth of opportunistic commensal and pathogenic organisms, and lactic acid has antiviral activity against HIV and HSV-2. The H2O2-producing strains (e.g. L. crispatus and L. jensenii) are more protective than those that do not produce H2O2 (L. iners). Indeed, it has been demonstrated that women with vaginal mucosa colonized with sufficient amounts of protective Lactobacillus spp. have a 50% lower frequency of gonorrhea, chlamydial infections, trichomoniasis and bacterial vaginosis. The presence of H2O2-producing lactobacilli in the vagina have been linked to a decreased frequency of bacterial vaginosis, symptomatic yeast vaginitis and sexually transmitted pathogens including Neisseria gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis. In vitro studies have demonstrated that H2O2-producing lactobacilli have potent bactericidal and viricidal properties against vaginal pathogens, including human immunodeficiency virus (HIV). Therefore, beneficial lactobacilli associated with the vaginal mucosa can be considered to provide a protective "biofilm". See e.g., Falagas et al., (2006) Drugs, 66:1253-1261. Many vaginal and systemic medications may kill vaginal Lactobacillus, and the depletion of the dominant vaginal Lactobacillus species leads to a more diverse abnormal microbiota populated with facultative and strict anaerobes, such as Gardnerella vaginalis and Atopobium vaginae, higher vaginal pH, and higher levels of proinflammatory cytokines, which can be associated with the development of clinical syndromes, such as bacterial vaginosis (BV), establishment of opportunistic infections, and an increased risk of acquiring HIV-1 and Herpes simplex virus type 2 (HSV-2) in women. See, e.g., Sha et al. (2005) J. Infect. Dis. 191:25-32; Taha et al. (1998) AIDS 12:1699-1706; Bolton, M., et al. Sex Trans Dis 2008 Mar 35(3):214-215 Hence, treatment of sexually transmitted diseases with antibiotics may place women at increased risk for repeated acquisition of the diseases. These findings, along with the widespread belief that lactobacilli generally promote vaginal health, have suggested to clinicians that women should recolonize the vagina with Lactobacillus to prevent or treat urogenital tract infections. There has been considerable interest in the development of non-antibiotic, ecologically appropriate approaches, such as Lactobacillus Replacement Therapy (LRT) to replenish the healthy vaginal microbiota and to prevent urogenital infections. The success of LRT depends in part on selection of an ecologically appropriate Lactobacillus strain, cell preservation, recovery of the dry Lactobacillus formulation following rehydration, as well as the extent and duration of vaginal colonization. Various methods for administering beneficial bacteria and other substances to the vaginal mucosa are known. In fact, Lactobacillus products for intravaginal or oral use have been available for over 100 years in the form of "acidophilus" preparations available in health food stores, and acidophilus milk or yogurt bought in grocery stores (e.g., these products typically advertise the inclusion of a strain of Lactobacillus acidophilus). These products have included vagina