High-efficiency and stable preparation of vaginal lactobacillus

Abstract

The present invention provides a dry preserved lactobacillus preparation suitable for administration to humans as a probiotic or live biotherapeutic treatment, wherein the preparation is stable, has high potency and does not contain excipients of animal origin.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 529,756, filed on July 7, 2017, the entire contents of which are incorporated herein in their entirety for all purposes. Background of the Invention

[0004] All human mucosa are naturally colonized by bacterial microbiota. Recent studies have shown that the microbiota found in the human intestine, oral cavity and vagina closely interact with human cells and tissues to regulate natural biological processes, such as nonspecific host defense. See, for example, Redondo-Lopez et al., (1990) Rev. Infect. Dis. 12: 856-872; Gilbert, JA, et al., Nature 2016 Jul 7, 535 (7610): 94-103; McDermott, AJ et al., Immunology 2014 May, 142 (1): 24-31; Nelson, DB et al., Anaerobe 2016 Dec, 42: 67-73. Typically, healthy vaginal microbiota is dominated by Lactobacillus species, which are Gram-positive rods that play an important role in resisting infection by producing lactic acid and acidifying the vagina or by producing other antimicrobial products such as hydrogen peroxide (H2O2). The most commonly isolated lactobacillus species from the reproductive tracts of healthy women worldwide include Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus 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. Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri are able to produce H2O2, whereas Lactobacillus iners strains generally do not. These species are phylogenetically and functionally distinct from food and / or environmental Lactobacillus species. These facultative anaerobes metabolize glucose to lactic acid, which helps maintain a low vaginal pH (4.0-4.5), which is a major part of the vaginal nonspecific host defense. The 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.

[0005] Strains that produce H2O2 (e.g., Lactobacillus crispatus and Lactobacillus jensenii) are more protective than those that do not (Lactobacillus iners). In fact, it has been shown that women whose vaginal mucosa is colonized with adequate amounts of protective lactobacilli have a 50% lower frequency of gonorrhea, chlamydial infection, trichomoniasis, and bacterial vaginosis. The presence of H2O2-producing lactobacilli in the vagina is associated with a reduced frequency of bacterial vaginosis, symptomatic yeast vaginitis, and sexually transmitted pathogens, including Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. In vitro studies have demonstrated that H2O2-producing lactobacilli have effective bactericidal and virucidal properties against vaginal pathogens, including human immunodeficiency virus (HIV). Therefore, it can be considered that the beneficial lactobacilli associated with the vaginal mucosa provide a protective "biofilm." See, e.g., Falagas et al., (2006) Drugs, 66: 1253-1261.

[0006] Many vaginal and systemic drugs can kill vaginal Lactobacillus, and depletion of the dominant vaginal Lactobacillus species results in a more diverse abnormal microbiota populated by facultative and strict anaerobes, such as Gardnerella vaginalis and Atopobium vaginae, higher vaginal pH and higher levels of proinflammatory cytokines, which may be associated with clinical syndromes such as the development of bacterial vaginosis (BV), the 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. Therefore, treating sexually transmitted diseases with antibiotics may put women at increased risk of recurrent acquisition of the disease. These findings, along with the widespread belief that lactobacilli generally promote vaginal health, have led clinicians to consider recolonizing the vagina with lactobacilli to prevent or treat urogenital tract infections.

[0007] There is considerable interest in developing non-antibiotic ecologically appropriate methods, such as lactobacillus replacement therapy (LRT) to replenish healthy vaginal microbiota and prevent urogenital tract infections. The success of LRT depends in part on the selection of ecologically appropriate lactobacillus strains, cell preservation, recovery of dried lactobacillus preparations after rehydration, and the extent and duration of vaginal colonization. Various methods of 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 more than 100 years in the form of "acidophilus" preparations available in health food stores, and acidophilus milk or yogurt purchased in grocery stores (e.g., these products are generally advertised to contain Lactobacillus acidophilus strains). These products include vaginal tablets, capsules, and vaginal suppositories containing freeze-dried human Lactobacillus acidophilus and various nutritional supplements.

[0008] Since these products cannot colonize the vagina with exogenous lactobacilli, these products are largely ineffective. These failures are usually due to the poor quality of commercially available products, and the lactobacilli species contained in the probiotics are not of vaginal origin and are therefore not suitable for use in the vagina. It has been shown that lactobacilli products sold as food or lactobacilli supplements are often contaminated by other potential pathogens. In addition, lactobacilli obtained from yogurt cannot bind to vaginal epithelial cells. The binding of lactobacilli to epithelial cells is an essential step in establishing colonization of host organisms. In addition, the low percentage of physiologically viable cells reflected by the low recovery in simulated vaginal fluids significantly affects the actual bacterial dose.

[0009] Therefore, there is a need for products for treating vaginal infections that can be manufactured under stringent conditions and use appropriate human strains of Lactobacillus having in vivo microbicidal properties, adherence to vaginal epithelial cells and effective live microbial potency. The present invention addresses these and other needs. SUMMARY OF THE INVENTION

[0011] In one aspect, the invention provides an aqueous bacterial suspension of vaginal lactobacillus species without an excipient of animal origin. The suspension can be produced by a combination of a cell mass of vaginal lactobacillus species and an aqueous storage medium. The aqueous storage medium can contain 5-20% (w / v) trehalose, 2-9% (w / v) xylitol, 0.5-1.5% (w / v) sodium ascorbate, 10-50mM sodium phosphate and optionally 0-5% (w / v) sodium glutamate.

[0012] In another aspect, the present invention provides a method for preserving Lactobacillus species under dry conditions in the absence of excipients of animal origin. The method comprises obtaining a cell concentration of about 10 8 to about 10 10 / ml of an aqueous suspension of vaginal lactobacillus species, centrifuging the solution to form bacterial cell pellets, and resuspending the bacterial cell pellets in an aqueous storage medium. The aqueous storage medium may contain 5-20% (w / v) trehalose, 2-9% (w / v) xylitol, 0.5-1.5% (w / v) sodium ascorbate, 10-50 mM sodium phosphate, and optionally 0-5% (w / v) sodium glutamate. The weight ratio of the wet weight of the cell pellets of the resulting bacterial suspension (i.e., the number of grams of cell pellets after centrifugation and decanting the supernatant) to the storage medium (ml) may be 1:1 to 1:5 of pellets (grams): storage medium (ml).

[0013] Another aspect of the present invention provides a method for treating abnormal vaginal microbiota in women. The method comprises selecting a woman diagnosed with abnormal vaginal microbiota, administering an antibiotic in an amount effective to reduce the level of abnormal vaginal microbiota, and then administering a dry powder derived from an aqueous bacterial suspension of vaginal Lactobacillus species without animal-derived excipients. The aqueous suspension is the result of a combination of cell pellets of vaginal Lactobacillus species with an aqueous preservation medium, which may contain 5-20% (w / v) trehalose, 2-9% (w / v) xylitol, 0.5-1.5% (w / v) sodium ascorbate, 10-50 mM sodium phosphate, and optionally 0-5% (w / v) sodium glutamate.

[0014] In some embodiments, the method of treating abnormal vaginal microbiota in women comprises daily administration of antibiotics for 2 to 7 days, and administration of the dry powder can be started any time between 2 days before stopping administration of the antibiotics and 2 days after administration of the antibiotics. In some cases, the antibiotic can be clindamycin, metronidazole or tinidazole.

[0015] The following embodiments may be combined with any of the above aspects of the invention. For example, in some embodiments, the vaginal Lactobacillus species of the aqueous bacterial suspension can produce greater than 0.5 ppm of hydrogen peroxide under effective culture conditions. In other embodiments, the vaginal Lactobacillus species can be selected from the following species: Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri.

[0016] In some embodiments of the invention, the aqueous storage medium may contain 5-15% (w / v) trehalose, 2-7% (w / v) xylitol, 0.5-1.0% (w / v) sodium ascorbate, 10-30 mM sodium phosphate and optionally 0-5% (w / v) sodium glutamate.

[0017] In another embodiment, the bacterial suspension can be lyophilized to produce a dry powder. The dry powder can have a water activity value of less than 0.220. In some embodiments, the powder can be combined with an inactive excipient at a ratio of 1:1 to 1:10 w / w powder: excipient to adjust the concentration of colony forming units. Alternatively, the dry bacterial powder can be combined with an excipient blend to adjust the potency of the final product. For convenience, the dry powder can be contained in a plastic housing designed for vaginal administration.

[0018] In some embodiments of the invention, the aqueous storage medium of the bacterial suspension does not contain skim milk. In another embodiment, the aqueous storage medium of the bacterial suspension does not contain α-tocopherol. In some embodiments of the invention, the aqueous storage medium of the bacterial suspension does not contain skim milk or α-tocopherol.

[0019] In some embodiments of the present invention, the aqueous storage medium may contain 5-20% (w / v) trehalose, 2-9% (w / v) xylitol, 0.5-1.5% (w / v) sodium ascorbate, 10-50mM sodium phosphate and optionally 0-5% (w / v) sodium glutamate, wherein the aqueous storage medium does not contain skim milk. In some embodiments of the present invention, the aqueous storage medium may contain 5-20% (w / v) trehalose, 2-9% (w / v) xylitol, 0.5-1.5% (w / v) sodium ascorbate, 10-50mM sodium phosphate and optionally 0-5% (w / v) sodium glutamate, wherein the aqueous storage medium does not contain α-tocopherol. In some embodiments of the invention, the aqueous storage medium may comprise 5-20% (w / v) trehalose, 2-9% (w / v) xylitol, 0.5-1.5% (w / v) sodium ascorbate, 10-50 mM sodium phosphate, and optionally 0-5% (w / v) sodium glutamate, wherein the aqueous storage medium does not comprise skim milk or α-tocopherol.

[0020] In one aspect, the invention relates to a composition comprising a dry powder derived from an aqueous bacterial suspension of vaginal Lactobacillus species without excipients of animal origin, the composition being used to treat a woman diagnosed with an abnormal vaginal microbiota, wherein the woman has previously been treated with an amount of antibiotics effective to reduce the level of abnormal vaginal microbiota; and wherein the aqueous bacterial suspension is produced by combining a cell mass of vaginal Lactobacillus species with an aqueous preservation medium consisting essentially of: (a) 5-20% w / v trehalose; (b) 2-9% w / v xylitol; (c) 0.5-1.5% w / v sodium ascorbate; and (d) 10-50 mM sodium phosphate. In some embodiments, the aqueous preservation medium optionally comprises 0-5% sodium glutamate.

[0021] Another aspect of the invention relates to a kit for treating a woman diagnosed with an abnormal vaginal microbiota, the kit comprising: a first container comprising one or more antibiotics in an amount effective to reduce the level of abnormal vaginal microbiota; a second container comprising an amount of a composition comprising a dry powder derived from an aqueous bacterial suspension of vaginal Lactobacillus species without animal-derived excipients, wherein the suspension is produced by combining a cell mass of vaginal Lactobacillus species with an aqueous storage medium, the aqueous storage medium consisting essentially of: (a) 5-20% w / v trehalose; (b) 2-9% w / v xylitol; (c) 0.5-1.5% w / v sodium ascorbate; and (d) 10-50 mM sodium phosphate; and optionally, instructions for using the kit to treat a woman diagnosed with an abnormal vaginal microbiota. In some embodiments, the aqueous storage medium optionally comprises 0-5% sodium glutamate.

[0022] In an embodiment, the one or more antibiotics are for administration to the female for, e.g., 2 to 7 days; and wherein the dry powder is for administration any time between two days before completion of antibiotic administration to two days after completion of antibiotic administration.

[0023] Thus, the kit is preferably intended for the sequential administration of i) one or more antibiotics followed by ii) a composition comprising a dry powder derived from an aqueous bacterial suspension of vaginal Lactobacillus species without excipients of animal origin, said composition being used to treat women diagnosed with an abnormal vaginal microbiota.

[0024] In an embodiment, the vaginal Lactobacillus species has the ability to produce greater than 0.5 ppm hydrogen peroxide under effective culture conditions, wherein the Lactobacillus species is, for example, selected from the group consisting of: Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri.

[0025] It should be noted that embodiments and features described in the context of one aspect of the invention may also be applicable to other aspects of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Stability of LACTIN-V formulated with skim milk (lower line, circles) or without skim milk (upper line, inverted triangles). Powder samples were stored at 25°C for one year and activity was determined at different time points by culturing on MRS agar plates and counting colonies.

[0028] Figure 2: Lactobacillus powder formulations with or without monosodium glutamate. The accelerated stability of four LACTIN-V formulations without monosodium glutamate (circle, triangle) or with monosodium glutamate (square, inverted triangle) at 37°C was determined by measuring the viability over time. The powder samples were stored at 37°C for 90 days and the viability was determined at different time points by culturing on MRS agar plates and counting colonies. DETAILED DESCRIPTION OF THE INVENTION

[0030] I. Introduction

[0031] The present invention provides a highly effective and stable dry preserved lactobacillus preparation suitable for administration to humans as a treatment, wherein the preparation has a high colonization efficacy and is free of excipients of animal origin. Specifically, as disclosed herein, the present invention provides methods and compositions for lactobacillus replacement therapy (LRT) to repopulate the vaginal mucosa with protective lactobacillus microflora as a means of correcting dysbiosis and maintaining vaginal health. As described in more detail below, the present invention teaches methods, compositions and reagents for preparing and using instantaneously buffered dry lactobacillus preparations.

[0032] II. Definitions

[0033] As used herein, the terms "vaginal microbiota" or "vaginal microbiome" are used interchangeably and refer to the microorganisms that colonize the vagina, although "microbiota" and "microbiome" are preferred terms. The terms "abnormal vaginal microbiota" or "abnormal vaginal microbiome" or "vaginal dysbiosis" refer to a condition in which the vaginal mucosa lacks protective lactobacilli and is colonized by a large number of different non-lactobacilli. The condition may be symptomatic or asymptomatic.

[0034] As used herein, the terms "aqueous storage medium" or "storage formulation" or "storage medium" are used interchangeably and refer to compositions capable of preserving and maintaining bacterial cell cultures in a metabolically inactive state while minimizing the damaging effects encountered during storage. Typically, when added to a storage medium, frozen, and lyophilized, Lactobacillus strains transition from an actively growing metabolic state to a metabolically inactive state. Therefore, the storage medium can be formulated to obtain optimal cell resilience so that the cells can adhere to the mucosal surface and return to full metabolic activity when rehydrated. The aqueous storage medium used herein is an aqueous solution, which typically contains carbohydrates, polyols (sugar alcohols), antioxidants, buffers, and optional amino acids. The bacterial cell mass is resuspended to about 10 μl using an aqueous storage medium. 8 CFU / mL, where the suspension can be dried, stored at 2-8°C for at least 2 years, and resuspended with less than 15% loss of CFU.

[0035] As used herein, the term "animal-derived excipient" refers to an inert substance derived from an animal that may be included in such a composition containing a substance considered to be an active ingredient. Non-limiting examples include milk, yogurt, butter, chicken fat, lard, gelatin, and animal fat.

[0036] As used herein, the terms "excipient" and "inactive excipient" are used interchangeably and refer to inert substances that are formulated with the active ingredients of a drug, are included for the purpose of long-term stability, provide bulk to a powder formulation (and are therefore often referred to as "bulking agents", "fillers" or "diluents"), or impart therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity or enhancing solubility. Examples of excipients include, but are not limited to, maltodextrin, starch, pregelatinized starch, microcrystalline cellulose, calcium carbonate, dicalcium phosphate, colloidal SiO2, Mannitol, xylitol, trehalose, lactose, sucrose, polyvinylpyrrolidone, crospovidone, glycine, magnesium stearate, sodium stearyl fumarate, cyclodextrin and their derivatives and mixtures.

[0037] As used herein, the term "consisting essentially of" refers to a composition or method that includes the disclosed components or steps, as well as any other components or steps that do not substantially affect the basic and novel properties of the composition or method. A composition consisting essentially of the listed ingredients does not contain additional ingredients that would affect the basic properties of those bacterial compositions. For example, the lactobacillus powder preparations of the present invention may also include pharmaceutically acceptable excipients, such as colorants and / or fillers, and antiviral or antibacterial agents, and / or enzymes, without affecting the viability properties of the dried lactobacillus powder.

[0038] As used herein, the term "Lactobacillus" refers to Gram-positive facultative anaerobic bacteria characterized by the ability to produce lactate (lactic acid) from a carbohydrate source such as glucose. These bacteria may be present in food or may be commensal organisms that colonize the vaginal or gastrointestinal mucosa.

[0039] As used herein, the term "Lactobacillus crispatus" or "Lactobacillus crispatus" refers to a species of the genus Lactobacillus. This species is typically distinguished from other lactobacilli based on the polynucleotide sequence of the ribosomal 16SrRNA gene. "Lactobacillus gasseri" or "Lactobacillus gasseri" and "Lactobacillus jensenii" or "Lactobacillus jensenii" refer to other species of lactobacilli. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii are vaginal species that are capable of producing hydrogen peroxide.

[0040] As used herein, the term "dried composition" refers to a composition from which water has been removed. Air-drying or drying techniques include, for example, heating (e.g., sublimation), applying low pressure or vacuum, lyophilization (i.e., freeze drying), and combinations thereof. The composition is typically dried for ease of storage and transportation.

[0041] As used herein, the term "effective culture conditions" refers to an environment in which lactobacillus cells are placed or exposed to promote the growth of the cells. Thus, the term refers to the culture medium, temperature, atmospheric conditions, substrates, agitation conditions, etc. that can affect cell growth, allowing a proliferation time (doubling rate of cell mass) of about 0.5 to 2.5 hours.

[0042] As used herein, the term "potency" refers to the number of viable microbial cells delivered per drug unit (i.e., drug powder). According to the present invention, the viable cells are capable of growth and reproduction. In order for the lactobacillus dry powder to be effective in vivo, the microbial cells are present in a concentration of at least about 10 8 The potency of CFU / drug unit is necessary for colonization of vaginal epithelial cells and biological effect (e.g., as demonstrated by the absence of an infectious state such as bacterial vaginosis). "High potency" means that each drug unit contains at least 10 8 Vaginal medication containing only viable microbial cells (CFU).

[0043] As used herein, the term "lyophilization" refers to the process of freezing a substance and then reducing the concentration of water by sublimation and / or evaporation to a level that does not support biological or chemical reactions.

[0044] As used herein, the term "water activity" and the symbol "a w” means and is defined as being equal to the equilibrium relative humidity (“ERH”) divided by 100. ERH is the equilibrium state where the dry powder neither absorbs moisture from nor releases moisture to the environment. ERH is affected by the composition of all ingredients, particularly those with high water content, which may be present as free water or bound water. The amount of free water may affect the storage stability and purity of the dry powder, which may result in undesired degradation of the activity or growth of contaminating microorganisms during storage.

[0045] As used herein, the term "wet weight" refers to the weight (in grams) of the cell pellet after centrifugation and decanting the supernatant. Typically, after the cell harvesting step, the centrifuge bottle is pre-weighed, the cells are centrifuged, the supernatant is decanted, and the bottle is weighed again. The difference in weight is the wet weight of the pellet.

[0046] III. Compositions and Methods

[0047] Acquiring vaginal bacteria

[0048] The lactobacillus strains that are applicable to medicine of the present invention (that is, medicated powder) can be any lactobacillus strains with identification characteristics as described herein. Suitable screening techniques known in the art can be used to detect and separate lactobacillus strains from natural sources. Specifically, suitable lactobacillus strains that are used for medicine of the present invention can be obtained by publicly available resources such as American Type Culture Collection (ATCC) or biodefense and emerging infection research resource library (BEI, beiresources.org), or are separated from people's healthy vagina. The identification characteristics that are applicable to lactobacillus strains of the present invention and the method for screening these characteristics are discussed in detail below.

[0049] An identifying characteristic of lactobacilli suitable for the present invention is that the lactobacilli strain has a vaginal epithelial cell (VEC) cohesion value percentage of at least about 50%. "VEC cohesion value percentage" is defined as the percentage of VEC to which at least one lactobacilli cell adheres to the total number of VECs in the identified group. According to the present invention, the terms "cohesion" and "adhesion" can be used interchangeably. Adhesion of microbial cells to vaginal epithelial cells is critical for colonization and biological effects. Successful adhesion of lactobacilli cells of the powder to vaginal epithelial cells leads to successful colonization of vaginal epithelial cells. Long-term in vivo colonization is the goal of the products and methods of the present invention, and "VEC cohesion value percentage" is a good prediction of whether a significant number of VECs will accept microbial cells in vitro and in vivo. Methods for determining acceptable VEC cohesion values ​​are well known in the art and can be found in U.S. Patent No. 6,468,526 and U.S. Patent No. 6,093,394. See also Kwok et al., J.Urol.2006,176:2050-2054.

[0050] Another identifying characteristic of Lactobacillus suitable for use in the medicament of the present invention is the ability to produce hydrogen peroxide (H2O2). The H2O2 positive phenotype is also associated with persistent vaginal colonization. See, e.g., Vallor, AC et al., J Infect Dis. 2001 Dec 1; 184 (11): 1431-6. As discussed above, hydrogen peroxide has been shown to be the reason why Lactobacillus kills its microorganisms. Preferably, the Lactobacillus can produce greater than about 0.5 ppm of H2O2 under normal growth conditions. More preferably, under effective growth conditions (defined herein as any culture medium and conditions capable of promoting H2O2 production), the Lactobacillus can produce at least about 10 ppm of H2O2, and even more preferably, the Lactobacillus can produce at least about 20 ppm of H2O2. Effective growth conditions include in vitro growth conditions (e.g., effective culture medium and conditions) and in vivo growth conditions (e.g., successful colonization of the vagina). Vaginal lactobacilli that produce hydrogen peroxide include most strains of Lactobacillus crispatus and Lactobacillus jensenii, and approximately half of the strains of Lactobacillus gasseri, as described in Antonio et al., The Journal of Infectious Diseases 1999, 180: 1950–6.

[0051] The lactobacillus of the present invention produces H2O2 which can be quantified by any method for measuring H2O2 production. Methods for measuring H2O2 production are well known in the art and may include culture methods or direct detection methods. Culture methods may include measuring H2O2 production by quantifying the intensity of the blue pigment formed when the lactobacillus is inoculated on tetramethylbenzidine medium (TMB) and incubated under anaerobic conditions. For example, at 37°C, under anaerobic conditions, the lactobacillus is incubated on a TMB agar plate for about 48 hours. The agar plate is then exposed to ambient air. Exposure to ambient air causes the H2O2 produced by the lactobacillus to react with the horseradish peroxidase in the agar to oxidize the TMB, causing the lactobacillus colonies to turn blue. See, for example, Antonio et al., The Journal of Infectious Diseases 1999; 180: 1950–6. Direct detection methods can be used to measure the amount of H2O2 using commercially available H2O2 detection test strips (e.g., available from EM Sciences or Merck) at a detection scale of 0 to 100 mg / L. See, e.g., Strus, M. et al., The in vitro activity of vaginal Lactobacillus with probiotic properties against Candida. Infect Dis Obstet Gynecol. 2005 Jun; 13(2): 69-75.

[0052] Another identification characteristic of lactobacillus suitable for the medicine of the present invention is the genetic identity and stability of lactobacillus strains in vivo and in vitro over time. According to the present invention, genetic stability refers to that the successive generations of lactobacillus strains can basically maintain the same genetic profile of the parent strain. In other words, the successive generations of genetically stable strains will not obtain significant mutations in their genomic DNA over a period of time. Repetitive sequence polymerase chain reaction (Rep PCR) can be used to confirm the genetic identity and stability of lactobacillus strains over time after in vitro storage or in vivo colonization of vaginal epithelial cells. The Rep PCR method for confirming the genetic identity and stability of lactobacillus strains is well known in the art and can be found in U.S. Patent No. 6,093,394. Also see Antonio & Hillier, J. Clin. Microbiol. 2003, 41: 1881–1887.

[0053] Another identifying characteristic of lactobacilli suitable for use in the medicaments of the present invention is the ability to produce lactic acid. Lactic acid has been shown to inhibit the growth of pathogens in vitro. Preferably, the lactobacilli produce at least about 0.75 mg / 100 mL lactic acid under effective growth conditions, more preferably at least about 4 mg / 100 mL lactic acid, and even more preferably at least about 8.8 mg / 100 mL lactic acid.

[0054] Suitable lactobacillus strains can have relatively large cell sizes. As provided in Bergey's Manual of Determinative Bacteriology, typical lactobacillus has a width of 0.8-1.6 μm and a length of 2.3-11 μm. Preferred lactobacillus strains for the present invention have a cell size of about 1 to about 2 microns in width and about 2 to about 4 microns in length. Without being bound by theory, the inventors of the present application believe that the large size exhibited by the cells of the lactobacillus strains of the present invention can make it better used as a protective agent in biocompetitive exclusion. Biocompetitive exclusion refers to that one or more medicated powder strains of the present invention can competitively inhibit the growth of undesirable bacterial strains. This exclusion is attributed to the fact that beneficial lactobacillus cells (e.g., medicated powder strains) occupy available space on vaginal epithelial cells, thereby preventing the adhesion of pathogenic or undesirable microbial cells.

[0055] In addition to the known species and strains of lactobacillus, newly identified species and strains from nature and mutant strains derived from known or newly identified strains can be used in the medicine of the present invention. Mutants of lactobacillus parent strains with identification characteristics of lactobacillus suitable for medicine of the present invention can be obtained by, for example, the following: subjecting the parent strain to at least one round of chemical and / or radiation mutagenesis to increase the mutagenesis rate, thereby increasing the possibility of obtaining a microorganism with improved desired characteristics. It is obvious to those skilled in the art that the mutant microorganism of the present invention also includes microorganisms that can be obtained by genetically engineering microorganisms to, for example, have an increased VEC cohesion value. As used herein, "mutated microorganisms" are parent microorganisms of such mutations, wherein the nucleotide composition of this microorganism has been modified by mutation, and the mutation is naturally occurring, is the result of exposure to a mutagen, or is the result of genetic engineering.

[0056] Preferred lactobacillus species include Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii, or Lactobacillus species with 95% sequence homology to the 16S rRNA gene sequence of any identified species. Particularly preferred lactobacillus strains are strains with all identification characteristics of Lactobacillus crispatus CTV-05 strains and Lactobacillus crispatus SJ-3C strains. Lactobacillus crispatus CTV-05 is a preferred strain. Methods for distinguishing lactobacillus strains include Rep-PCR (as described in Antonio & Hillier, J. Clin. Microbiol. 2003, 41: 1881–1887), multilocus sequence typing (MLST) (originally developed for identification of pathogenic strains (see, e.g., Maiden, MC et al., 1998, Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA., 95: 3140-2145)) and whole genome sequencing.

[0057] Culturing vaginal bacteria

[0058] Lactobacillus strains useful for the present invention can be grown in liquid or solid culture media (e.g., agar). Bacterial culture media for growing Lactobacillus strains useful for the present invention are known, commercially available (e.g., from BDDifco), and include, for example, de Man, Rogosa and Sharpe (MRS) culture media and Rogosa culture media. Lactobacillus is preferably cultured anaerobically or microaerophilically, and the temperature of the culture media can be any temperature suitable for the growth of Lactobacillus. Lactobacillus strains of the present invention can be cultured under anaerobic conditions and are typically grown at about 37°C. Effective culture conditions for vaginal Lactobacillus strains useful for the present invention are well known in the art. Specific culture conditions, culture media, and methods for culturing Lactobacillus strains, particularly Lactobacillus crispatus and Lactobacillus gasseri, can be found in, for example, U.S. Patent No. 8,329,447, U.S. Patent No. 6,093,394, and Davis, C. Enumeration of probiotic strains: Review of culture-dependent and alternative techniques to quantify viable bacteria. J Microbiol Methods. 2014; 103: 9-17.

[0059] The medium is inoculated with an actively growing culture of a Lactobacillus strain in an amount sufficient to produce a suitable cell density (or potency) for transfer to a storage medium after a reasonable growth period. A non-limiting example of a reasonable growth period for Lactobacillus as used herein is a generation time of 1-2.5 hours. The cells are grown to a range of about 10 8 CFU / mL to about 10 10 The preferred cell density of CFU / mL. Cell density is determined using a culture-based method in which serial dilutions of lactobacillus cultures are plated onto MRS agar plates and incubated anaerobically at 37°C for 48 hours. The colonies on the plates are counted and the number of CFU (colony forming units) in the sample is calculated as CFU / mL or CFU / gram. The method for determining CFU is described in detail below.

[0060] In case the cells grow to a preferred cell density, any suitable method can be used to remove cells from the substratum to gather in the crops the bacterial cells. Non-limiting exemplary methods of gathering in the crops cultured cells include filtration, centrifugation and precipitation. In some instances, the cells gathered in the crops cultured may include hollow fiber filtration and washing via diafiltration. The method for the lactobacillus cells gathered in the crops cultured is well known in the art, and is described in detail in the embodiments. After separating cells from the substratum and / or washing the biomass, centrifugal cells are to form cell masses, to prepare to be suspended in the preservation medium.

[0061] Preparation of aqueous storage medium

[0062] The bacterial cell mass formed by the above method is resuspended in a suitable aqueous storage medium, wherein the weight ratio of the wet weight of the cell mass (grams) to the storage medium (mL) can be 1:1 to 1:8 of cell mass (grams): storage medium (mL). In some embodiments, the bacterial cell mass is resuspended in a suitable aqueous storage medium, wherein the weight ratio of the wet weight of the cell pellet (grams) to the storage medium (mL) can be 1:1 to 1:7 of cell pellet (grams): storage medium (mL), or 1:1 to 1:6, or 1:1 to 1:5, or 1:1 to 1:4, or 1:1 to 1:3, or 1:1 to 1:2, or 1:2 to 1:6, or 1:3 to 1:5 of cell pellet (grams): storage medium (mL). In some embodiments, the bacterial cell mass is resuspended in a suitable aqueous storage medium, wherein the weight ratio of the wet weight of the cell mass (grams) to the storage medium (mL) can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8 of cell mass (grams): storage medium (mL). In some embodiments, the bacterial cell mass is resuspended in a suitable aqueous storage medium, wherein the weight ratio of the wet weight of the cell mass (grams) to the storage medium (mL) can be 1:1 to 1:5 of cell pellet (grams): storage medium (mL). In some embodiments, the bacterial cell mass is resuspended in a suitable aqueous storage medium, wherein the weight ratio of the wet weight of the cell mass (grams) to the storage medium (mL) can be 1:1 to 1:3 of cell pellet (grams): storage medium (mL).

[0063] Aqueous storage medium includes ingredients that minimize the damaging effects encountered during storage. Storage medium of the present invention includes carbohydrates, polyols, antioxidants, buffers and optional amino acids. The carbohydrates used in the storage medium function as freeze-drying protectants to protect and stabilize cells during storage and thereafter during freeze drying. Non-limiting exemplary carbohydrates suitable for the present invention include trehalose, dextrose, lactose, maltose, sucrose and / or any other disaccharides or polysaccharides. In some embodiments, the storage medium includes about 0.5% to about 30% by weight of carbohydrates / volume (w / v) of the storage medium, or about 1% to about 25%, or about 5% to about 20%, or about 10% to about 15% by weight of carbohydrates / volume of the storage medium. In some embodiments, the storage medium includes about 0.5% by weight of carbohydrates / volume (w / v) of the storage medium, or about 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25% or 30% by weight of carbohydrates / volume of the storage medium. In some embodiments, the storage medium comprises about 5% to about 20% trehalose by weight / volume (w / v) of the storage medium. In some other embodiments of the invention, the storage medium comprises about 5% to about 15% trehalose by weight / volume of the storage medium.

[0064] The polyol (that is, polyol) in the preservation medium is a lyoprotectant, which helps to protect cells from the stress of dehydration during freeze drying. Non-limiting exemplary polyols suitable for the present invention include xylitol, ribitol, glycerol, galactitol, inositol, mannitol, maltitol, isomalt, lactitol, erythritol, sorbitol and / or arabitol. In some embodiments, the preservation medium comprises the preservation medium of the polyol / volume (w / v) of about 0.1% to about 12 % by weight, or about 1 % by weight to about 10 % by weight, or about 2 % by weight to about 9 % by weight, or about 3 % by weight to about 7 % by weight of the polyol / volume. In some embodiments, the storage medium comprises about 0.1% by weight of polyol / volume (w / v) of the storage medium, or about 0.5%, 1%, 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12% by weight of polyol / volume of the storage medium. In some embodiments, the storage medium comprises about 2% to about 9% by weight of xylitol / volume of the storage medium. In some other embodiments of the present invention, the storage medium comprises about 2% to about 7% by weight of xylitol / volume of the storage medium.

[0065] The antioxidant in the preservation medium delays the oxidative damage to the microbial cells during preservation and storage. Non-limiting exemplary antioxidants suitable for the present invention include sodium ascorbate, ascorbic acid, ascorbyl palmitate, propyl gallate and vitamin E (alpha-tocopherol). In some embodiments, the preservation medium comprises an antioxidant / volume (w / v) of about 0.1% by weight to about 5% by weight, or an antioxidant / volume (w / v) of about 0.5% by weight to about 3.0% by weight, or an antioxidant / volume of about 1.0% by weight to about 2.0% by weight. In some embodiments, the preservation medium comprises an antioxidant / volume (w / v) of about 0.1% by weight, or an antioxidant / volume (w / v) of about 0.3%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% by weight or 5.0% by weight. In some embodiments, the storage medium comprises about 0.5 wt % to about 1.5 wt % sodium ascorbate per volume of storage medium. In some other embodiments of the invention, the storage medium comprises about 0.5 wt % to about 1.5 wt % sodium ascorbate per volume of storage medium.

[0066] Buffers suitable for storage media enhance the stability and recovery of bacterial cells. Buffers suitable for storage media are physiological agents that do not produce any toxic effects on bacteria, vaginal epithelial cells or female patients using pharmaceutical compositions. Non-limiting exemplary buffers suitable for the present invention include sodium phosphate, disodium phosphate, potassium phosphate, sodium bicarbonate, histidine, arginine and sodium citrate. In some embodiments, the pKa of the buffer can be from about 4.3 to about 8.0, or from about 4.6 to about 7.7, or from about 5.0 to about 7.3, or from about 5.4 to about 7.0, or from about 6.0 to about 6.7. In some other embodiments, the storage medium comprises a buffer solution with a pKa of at least 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or more. In some embodiments, the storage medium comprises a buffer solution with a pKa in the physiological range. In other embodiments, the storage medium comprises a buffer solution having a pKa of about 6.7 to about 7.8.

[0067] In other embodiments, the storage medium comprises a buffer of about 5mM to about 70mM, or about 10mM to about 65mM, or about 15mM to about 60mM, or about 20mM to about 55mM, or about 25mM to about 50mM, or about 30mM to about 45mM, or about 35mM to about 40mM. In some embodiments, the storage medium comprises about 5mM buffer, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70mM. In some embodiments, the storage medium comprises about 10mM to about 50mM sodium phosphate. In some other embodiments of the present invention, the storage medium comprises about 10mM to about 30mM sodium phosphate.

[0068] In some embodiments, the storage medium may optionally include amino acids, which contribute to improving the stability of lactobacillus cells at elevated temperatures without significantly affecting cryopreservation during freeze drying. In some embodiments, the optional amino acid may be a salt form of a suitable amino acid. Non-limiting exemplary amino acids and / or their salts suitable for the present invention include sodium glutamate, glutamine, glycine, arginine, histidine and lysine. In some embodiments, the storage medium optionally includes an amino acid / volume (w / v) of about 0% to about 5% by weight, or an amino acid / volume (w / v) of about 0.5% to about 3.0%, or an amino acid / volume (w / v) of about 1.0% to about 2.0% by weight. In some embodiments, the storage medium optionally includes an amino acid / volume (w / v) of about 0.1% by weight, or an amino acid / volume (w / v) of about 0.3%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or 5.0% by weight of an amino acid / volume (w / v) of a storage medium. In some embodiments, the amino acid optionally included in the storage medium is the amino acid salt sodium glutamate, preferably monosodium glutamate. In some embodiments, the storage medium optionally includes about 0% to about 5% by weight of sodium glutamate / volume of the storage medium. In some other embodiments of the present invention, the storage medium optionally includes about 0% to about 5% by weight of monosodium glutamate / volume of the storage medium. In some embodiments, the storage medium optionally includes about 1% to about 4% by weight of sodium glutamate / volume of the storage medium. In some other embodiments of the present invention, the storage medium optionally includes about 1% to about 4% by weight of monosodium glutamate / volume of the storage medium.

[0069] The storage medium of the present invention comprises carbohydrates at about 5% to 20% by weight / volume of the storage medium, polyols at about 2% to 9% by weight / volume of the storage medium, antioxidants at about 0.5% to 1.5% by weight / volume of the storage medium, and 10mM-50mM of buffer. In other embodiments, the storage medium suitable for the present invention may comprise carbohydrates at about 5% to 15% by weight / volume of the storage medium, polyols at about 2% to 7% by weight / volume of the storage medium, antioxidants at about 0.5% to 1.0% by weight / volume of the storage medium, and buffers at about 10mM to 30mM.

[0070] In some embodiments, the storage medium of the present invention comprises carbohydrates at about 5% to 20% by weight / volume of the storage medium, polyols at about 2% to 9% by weight / volume of the storage medium, antioxidants at about 0.5% to 1.5% by weight / volume of the storage medium, 10 mM to 50 mM of buffer, and optionally amino acids at about 0% to 5% by weight / volume of the storage medium. In other embodiments, the storage medium suitable for the present invention may comprise carbohydrates at about 5% to 15% by weight / volume of the storage medium, polyols at about 2% to 7% by weight / volume of the storage medium, antioxidants at about 0.5% to 1.0% by weight / volume of the storage medium, 10 mM to 30 mM of buffer, and optionally amino acids at 0% to 5% by weight / volume of the storage medium.

[0071] An example of a particularly useful preservation medium of the present invention comprises trehalose as a carbohydrate, about 5% to 20% by weight / volume of the preservation medium; xylitol as a polyol, about 2% to 9% by weight / volume of the preservation medium; sodium ascorbate as an antioxidant, about 0.5% to 1.5% by weight / volume of the preservation medium; and sodium phosphate as a buffer, 10 mM to 50 mM. In some embodiments, a particularly useful preservation medium of the present invention comprises trehalose as a carbohydrate, about 5% to 20% by weight / volume of the preservation medium; xylitol as a polyol, about 2% to 9% by weight / volume of the preservation medium; sodium ascorbate as an antioxidant, about 0.5% to 1.5% by weight / volume of the preservation medium; sodium phosphate as a buffer, 10 mM to 50 mM and optionally, sodium glutamate as an amino acid, about 0% to 5% by weight / volume of the preservation medium. In other embodiments, the storage medium suitable for the present invention comprises about 5% to 15% trehalose by weight / volume of the storage medium, about 2% to 7% xylitol by weight / volume of the storage medium, about 0.5% to 1.0% sodium ascorbate and 10mM to 30mM sodium phosphate by weight / volume of the storage medium. In some other embodiments, the storage medium suitable for the present invention comprises about 5% to 15% trehalose by weight / volume of the storage medium, about 2% to 7% xylitol by weight / volume of the storage medium, about 0.5% to 1.0% sodium ascorbate by weight / volume of the storage medium, 10mM to 30mM sodium phosphate and optionally sodium glutamate, about 0% to 5% by weight / volume of the storage medium. Representative storage medium compositions (which are by no means intended to be limiting) are included in Table 1 below.

[0072] Table 1. Exemplary preservation medium compositions and ingredient ratios.

[0073]

[0074] *The amount of sodium phosphate is measured in mM

[0075] Before adding the above-mentioned harvested lactobacillus cells to the culture medium, the cells can be washed in saline buffer. After the harvested lactobacillus cells are introduced into the storage medium described herein, the resulting mixture is referred to as a cell storage medium slurry. In some embodiments, the cell storage medium slurry may have 10 8 CFU / mL to 10 11 CFU / mL activity. More preferably, the cell storage medium slurry may have at least about 10 10 Activity in CFU / mL It is to be understood that variations of the basic culturing, harvesting, and suspending steps disclosed herein will be appreciated by one of ordinary skill in the art, and thus, the present invention incorporates such variations.

[0076] Dry cell storage medium slurry

[0077] The cell preservation medium slurry can be dried using any suitable drying method known in the art to produce the resulting bulk drug powder. Typically, the effect of drying is to place the bacteria in a dormant state to protect the bacteria from environmental factors that negatively affect bacterial viability. The standard method for inducing dormancy is by removing water. Typically, enough water is removed so that normal cellular processes (e.g., enzyme activity) are stopped or at least significantly reduced.

[0078] Any of a variety of methods known in the art for drying bacterial preparations can be used to dry the cell preservation medium slurry to increase its stability for long-term storage. Drying methods and protective agents are disclosed in the review of Morgan et al. (2006) J.Microbiol.Meth.66:183-193. Suitable drying methods include air drying, vacuum drying, oven drying, spray drying, flash drying, fluidized bed drying, controlled atmosphere drying, and freeze drying (i.e., freeze drying). In some embodiments, a desiccant is used to assist the drying process, and / or to prevent moisture from being reabsorbed into the dried preparation. In some embodiments, a freeze dryer (i.e., Virtis, SP Scientific) is used for drying. Detailed freeze drying methods are known to those skilled in the art and are disclosed in U.S. Patents Nos. 6,093,394; 8,329,447; and 8,642,029. The efficacy of the resulting dried preparation, referred to as raw material powder, was tested using the following method. The efficacy of the dried raw material drug powder can be 10 9 CFU / g to 10 12 CFU / g. More preferably, the raw material powder may have at least about 10 10 Activity of CFU / g.

[0079] Measuring residual water

[0080] Any suitable method known in the art can be used to test the presence of residual water in the dried preparation. In some cases, the residual water in the dried preparation can be measured by gravimetric analysis, as described in U.S. Patent No. 8,329,447 and No. 8,642,029. Alternatively, an instrument such as IR-120 moisture analyzer (Denver Instruments, Denver, Colo.) for measuring water content in the powder can be used to monitor the moisture content of the preparation in the drying process. It is also possible to measure residual moisture by performing well-known coulometric titration or volumetric titration techniques, such as Karl Fischer titration.

[0081] The water content in the lactobacillus powder can also be measured as free water or water activity (a) using a water activity meter, such as the AquaLab CX-2 Model series (Decagon Instruments, Pullman, Wash.) or the Rotronic Model series (Rotronic Instrument Corp., Huntington, NY). w The water activity meter (AquaLab CX-2, Decagon Instruments) uses the chilled mirror dew point technique to measure the product’s a w When a sample is placed in the Aqualab, the stainless steel mirror inside the chamber is repeatedly cooled and heated while dew forms and is driven off. The instrument's fan circulates air in the sensing chamber, speeding up the equilibration process. Each time dew forms on the mirror, the Aqualab measures the temperature and calculates the sample's a w , saving these values ​​to compare with previous values. w When the values ​​differ by less than 0.001, the measurement process is completed.

[0082] Water level or water activity (a w ) determines the overall stability of the resulting dry raw material lactobacillus drug powder. A person skilled in the art will understand that the water activity of a drug, such as a of the drug powder of the present invention, w By maintaining a low water activity in the drug product, degradation of the active drug ingredient (i.e., the lactobacillus drug powder) can be avoided. In addition, drug products with low water activity, such as the lactobacillus drug powder of the present invention, are less prone to crystallization, agglomeration, and caking, which lead to degradation and ineffectiveness of the drug. These are time-dependent reactions, the rates of which are affected by water activity. wDetails on the effects on product formulation can be found in the USP Methods <1112> Microbiological attributes of non-sterile pharmaceutical products - Application of water activity determination.

[0083] In some embodiments, the dry raw Lactobacillus drug substance may have a measured a of about 0.001 to about 0.220, or about 0.005 to about 0.200, or about 0.010 to about 0.150, or about 0.025 to about 0.100, or about 0.050 to about 0.075. w In other embodiments, the dried raw Lactobacillus drug substance may have a measured a of about 0.001, 0.003, 0.005, 0.007, 0.010, 0.030, 0.050, 0.070, 0.100, 0.150, 0.170, 0.200, 0.220. w In certain embodiments, the dry raw Lactobacillus drug substance may have a measured a of less than 0.220. w .

[0084] Measuring performance

[0085] Use any suitable method known in the art to test the effectiveness of lactobacillus preparations of the present invention (wet and / or dry) at different times throughout the preparation process. This method for measuring the effectiveness of lactobacillus preparations includes but is not limited to the method based on cultivation. The light scattering method for measuring the cell density of lactobacillus is used to monitor the fermentation process and relates to the optical density of the bacterial sample at 600nm.

[0086] The preferred method for measuring the efficacy of lactobacillus preparations is a culture-based method involving serial dilutions. A sample of the lactobacillus preparation to be tested is obtained and serially diluted. Serial dilutions of small aliquots (i.e., 100 μL) are spread onto MRS agar plates. The samples are incubated anaerobically at 37°C for 48 hours. After an appropriate period of time, the plates are illuminated by placing the culture dishes in transmitted light. Commercially available bacterial counting software is used to count individual colonies manually or with a camera and computer, and the number of CFU in the sample is calculated as CFU / ml or CFU / gram. More details related to culture-based methods are disclosed in Brugger, SD et al., Automated Counting of Bacterial Colony Forming Units on AgarPlates.PLOS ONE2012; 7(3): e33695.

[0087] Purity and properties

[0088] In addition to measuring potency and water activity, the bulk drug powder produced by the above drying process can be tested for purity and identity. Methods well known in the art, such as the U.S. Pharmacopeia method <61> Microbial Enumeration Tests and USP Methods <62> The purity of the raw drug powder is determined as described in the test for specific microorganisms. Genetic identification of Lactobacillus species in raw powder and final drug product is performed by isolating genomic DNA using a commercially available kit (e.g., Power Soil DNA Isolation Kit, Mo Bio), amplifying the 16S rRNA gene by PCR using specific primers, sequencing the gene using a commercial DNA sequencing service (MCLAB), and comparing the sequence to a reference standard. The characteristics of the Lactobacillus strains in the raw drug powder are determined using methods well known in the art, such as repetitive sequence polymerase chain reaction (Rep PCR) and as described in U.S. Patents Nos. 6,093,3941, 8,329,447, and 8,642,029.

[0089] Diluting bacterial powder with inactive excipients

[0090] In order to comply with the potency and dosage guidelines agreed upon and developed by the U.S. Food and Drug Administration (FDA), the activity of the raw Lactobacillus drug powder is diluted with a pharmaceutically acceptable excipient. Any suitable inactive pharmaceutically acceptable excipient (i.e., diluent) known in the art can be used to dilute the potency of the Lactobacillus drug powder. In some embodiments, the diluent can be maltodextrin, pregelatinized starch, lactose, Mannitol, xylitol, microcrystalline cellulose, sugar or their combination. In other embodiments, inactive filler can be used in combination with another diluent. In other embodiments, maltodextrin or pregelatinized starch can be used to dilute raw lactobacillus drug powder. In some embodiments, maltodextrin is used to dilute raw lactobacillus drug powder.

[0091] In some embodiments, raw material lactobacillus drug powder is diluted 3 times to 10 times with inactive excipient.In other embodiments, raw material lactobacillus drug powder and inactive excipient can be 1:1 to 1:12w / w combination with the ratio of powder and inactive excipient.In some embodiments, raw material lactobacillus drug powder can be with inactive excipient with the ratio of powder and inactive excipient of 1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9,1:10,1:11 or 1:12w / w and inactive excipient.In specific embodiments, raw material lactobacillus drug powder and inactive excipient can be 1:1 to 1:10w / w combination with the ratio of powder and inactive excipient.In specific embodiments, raw material lactobacillus drug powder and inactive excipient can be 1:1 to 1:5w / w combination with the ratio of powder and inactive excipient. In some embodiments, the raw Lactobacillus drug powder may be combined with inactive excipients at a ratio of 1:1 to 1:3 w / w of powder to inactive excipients.

[0092] In a specific embodiment, the raw lactobacillus drug powder can be combined with maltodextrin at a ratio of 1:1 to 1:10 w / w of powder to maltodextrin. In a specific embodiment, the raw lactobacillus drug powder can be combined with maltodextrin at a ratio of 1:1 to 1:5 w / w of powder to maltodextrin. In some embodiments, the raw lactobacillus drug powder can be combined with maltodextrin at a ratio of 1:1 to 1:3 w / w of powder to maltodextrin. The potency of the diluted dry raw lactobacillus drug powder, referred to as the drug product, can be 10 8 CFU / g to 10 11 CFU / g, or 10 8 CFU / g to 10 10 CFU / g. More preferred drug products may have greater than 10 9 Activity of CFU / g.

[0093] Drug product can be with the dosage package of about 100mg to 600mg.In some embodiments, drug product dosage can be with the dosage package of about 100mg or about 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg or 600mg.In other embodiments, drug product can be with the dosage package of about 150mg to about 450mg or about 150mg to about 400mg or about 150mg to about 350mg.In some embodiments, drug product can be with the dosage package of about 150mg to 250mg.In specific embodiments, drug product can be with the dosage package of about 200mg.

[0094] The drug product can be placed in a medical powder applicator (referred to as the final drug product) and packaged to prevent moisture and oxygen during transportation and storage. The packaging can include any suitable material for such protection, such as Mylar or metal film bags. In some embodiments, the final drug product is sealed into individual packages, such as for individual doses.

[0095] Use of dried Lactobacillus final drug product

[0096] As described herein, the final drug product (i.e., dry powder) of any suitable lactobacillus species and strain can be used to prevent and / or treat vaginal infection (i.e., abnormal vaginal microbiome). This vaginal infection includes but is not limited to bacterial vaginosis, yeast vaginitis, gonorrhea, chlamydia, trichomoniasis, human immunodeficiency virus infection, herpes simplex virus type 2 (HSV-2), urinary tract infection and pelvic inflammatory disease. In some embodiments, the final drug product can be used to prevent and / or treat bacterial vaginosis, yeast vaginitis or urinary tract infection. In a specific embodiment, the dry powder can be used to prevent and / or treat bacterial vaginosis (BV).

[0097] Abnormal vaginal microbiota can be detected and diagnosed using any suitable means known in the art. Vaginal infection can be symptomatic or asymptomatic. Symptoms generally include an abnormal odor and / or discharge, and discomfort of itching and / or pain. Depending on the vaginal infection, a woman may be able to detect it without medical advice or diagnostic equipment or kits. For example, several inexpensive over-the-counter kits are available for detecting yeast vaginitis (e.g., Vagisil TM ).

[0098] In some cases, the practitioner will detect and diagnose vaginal infection. Clinical criteria require the presence of at least three symptoms, including those mentioned above, vaginal fluid pH>4.5 and microscopic examination of clue cells (e.g., vaginal epithelial cells covered with adherent coccobacilli). For example, bacterial vaginosis can be detected by, for example, Amsel clinical criteria or Gram-stained vaginal smears (Nugent scoring system). Gram-stained vaginal smears are used to measure the relative concentration of lactobacilli (Gram-positive bacteria), Gram-negative and Gram-variable bacilli and cocci (i.e., Gardnerella vaginalis, Prevotella, Porphyromonas and Peptostreptococci) and the unique curved Gram-negative bacilli of BV (i.e., Mobiluncus). Detection and diagnostic methods for various vaginal infections are well known in the art and are described in U.S. Patent No. 8,329,447. See also https: / / www.cdc.gov / std / tg2015 / bv.htm.

[0099] The dry powder of the present invention can be applied alone or in combination (for example, at the same time, before and / or after) with any other therapy for preventing and / or treating vaginal infection. The application of any other therapy for preventing and / or treating vaginal infection can be applied in an amount that effectively reduces the level of abnormal vaginal microbiome. Other therapies for preventing and / or treating vaginal infection can include antibiotics or antiviral agents well known in the art. In some embodiments, other therapies for preventing and / or treating vaginal infection can be antibiotics. Suitable antibiotics for preventing and / or treating abnormal vaginal microbiome are well known in the art. Such antibiotics include but are not limited to clindamycin, metronidazole and tinidazole. The antibiotic used in combination with the dry powder of the present invention can be any suitable form for application. For example, antibiotics can be delivered locally (as a gel or cream), or delivered with oral or vaginal tablets, capsules or suppositories. In a specific embodiment, antibiotics are applied with a local gel.

[0100] Antibiotic treatment can be administered 1-2 times per day. In specific embodiments, antibiotic treatment can be administered once per day.

[0101] In some embodiments of the present invention, antibiotics can be used for 2 to 7 days. In other embodiments, antibiotics can be used for 2, 3, 4, 5, 6 and 7 days. In some other embodiments, antibiotics can be used for 2 to 7 days, or 3 to 6 days, or 4 to 5 days, or 4 to 7 days. In specific embodiments, antibiotics can be used for 2 to 7 days. In another embodiment, antibiotics can be used for 7 days. In another embodiment, antibiotics can be used for 5 days.

[0102] In some embodiments, the application of dry powder is during the last few days of the antibiotic application scheme (i.e., 2 to 4 days before the antibiotic application scheme is completed). In other embodiments, the dry powder is applied after the antibiotic application scheme is completed. The dry powder can be applied once or twice a day after the antibiotic application scheme is completed. In some embodiments, the dry powder can be applied twice a day after the antibiotic application scheme is completed. In a specific embodiment, the dry powder can be applied once a day after the antibiotic application scheme is completed.

[0103] In some embodiments of the present invention, the dry powder of 1 to 14 days can be used after the antibiotic application scheme is completed. In other embodiments, the dry powder of 1, 2, 3, 4, 5, 6, 7, 10, 12 or 14 days can be used after the antibiotic application scheme is completed. In some other embodiments, the dry powder of 2 to 12 days, or 3 to 10 days, or 4 to 7 days, or 5 to 6 days can be used after the antibiotic application scheme is completed. In a specific embodiment, the dry powder of 5 to 7 days can be used after the antibiotic application scheme is completed. In another embodiment, the dry powder of 5 days can be used after the antibiotic application scheme is completed.

[0104] After the antibiotic application scheme and the initial dry powder application scheme are completed, the dry powder of the present invention can be applied for a period of time. For example, after the initial treatment (that is, 1 dose per day for 7 days) is completed using the dry powder, the dry powder can be applied 1 to 5 times per week. In some embodiments, the dry powder can be applied 1 time, 2 times, 3 times, 4 times or 5 times per week after the initial dry powder treatment scheme is completed. In other embodiments, the dry powder can be applied 1 to 4 times per week, or 2 to 5 times per week, or 1 to 3 times per week, or 1 to 2 times per week after the initial dry powder treatment scheme is completed. In a specific embodiment, the dry powder can be applied 1 time per week after completing the initial dry powder treatment scheme.

[0105] In some embodiments of the present invention, 1 to 26 weeks of dry powder can be used after completing the initial dry powder treatment regimen. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 weeks of dry powder can be used after completing the initial dry powder treatment regimen. In some other embodiments, 2 weeks to 26 weeks, or 3 weeks to 24 weeks, or 4 weeks to 22 weeks, or 5 weeks to 20 weeks, or 6 weeks to 18 weeks, or 7 weeks to 16 weeks, or 8 weeks to 14 weeks, or 10 weeks to 12 weeks of dry powder can be used after completing the initial dry powder treatment regimen. In a specific embodiment, 5 weeks to 10 weeks of dry powder can be used after completing the initial dry powder treatment regimen. In another embodiment, 10 weeks of dry powder can be used after completing the initial dry powder treatment regimen.

[0106] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0107] Although the above invention has been described in detail by way of illustration and example for the purpose of clear understanding, it will be apparent to one of ordinary skill in the art based on the teachings of the present invention that certain changes and modifications may be made to the present invention without departing from the spirit or scope of the appended claims.

[0108] IV. Examples

[0109] The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that a variety of noncritical parameters can be changed or modified to produce essentially similar results.

[0110] Example 1 Preparation of dry composition of Lactobacillus

[0111] This example details the general strategy for preparing a dry composition of lactobacillus in powder form as a medical product, including bacterial culture, suspension in a preservation medium, drying, dilution and packaging. The procedures described herein for culturing and processing Lactobacillus crispatus SJ-3C can be applied to any microorganism suitable for the present invention.

[0112] Original Lactobacillus crispatus SJ-3C (SJ-3C) cells are available from the American Type Culture Collection (ATCC) under ATCC No. PTA- 10138. Master and working cell banks of these cells are prepared, which can then be used to prepare dried lactobacillus compositions.

[0113] SJ-3C cells were first plated onto modified de Man, Rogosa and Sharpe (MRS) agar plates and grown under anaerobic conditions at 37°C for 72 hours. Cells from the plates were inoculated into 10 mL of modified MRS and incubated anaerobically at 37°C for 24 hours. The culture was then transferred to 490 mL of growth medium and incubated at 37°C for 24 hours before being transferred to 4.5 L of medium in a 5L Bellco bioreactor. The 5 liters of culture were incubated anaerobically at 37°C for another 24 hours to serve as fermentor inoculum.

[0114] Fermentation was carried out in a fermenter (100 L fermenter) at pH 6.0 in the presence of a modified MRS medium bubbling with nitrogen. Fermentation was started by adding an inoculum and completed after about 15 hours when the cells reached an early stationary phase and stopped growing. At this point, glucose was exhausted, lactic acid production stopped, and the optical density (OD600) of the culture at 600 nm remained constant and cells were harvested.

[0115] The cells were harvested, concentrated, and washed by exchanging the buffer into phosphate buffered saline (diafiltration) using a tangential flow membrane in a sterile closed hollow fiber system. When the residual lactate concentration reached 10% of the starting value at harvest and the pH of the permeate remained constant, the cells were aseptically removed from the harvest system and collected by centrifugation at 1500 x g for 20 minutes (2-8° C.).

[0116] The cell pellets were resuspended in a preservation medium solution, using 2.5 mL of preservation solution per gram of cell paste. The preservation medium solution contained 15% trehalose, 6% xylitol, and 1% sodium ascorbate in 10 mM sodium phosphate buffer (pH 7.4), which was used to prepare multiple batches of harvested SJ-3C slurries. The resulting batches of preservation medium cell slurries had a 1×10 10 CFU / mL to 5×10 10 Calculate activity in CFU / mL. Transfer the slurry to a sterile Lyoguard TM The cell paste was plated and lyophilized in a VirtisGenesis lyophilizer. The viability of the cell slurry was determined by plate count prior to lyophilization. TM The disks were placed in heat-seal bags with desiccant, purged with nitrogen, and kept at 2-8°C until ground.

[0117] The SJ-3C bulk drug substance was produced by grinding the lyophilized cell cake with 0.5% colloidal silicon dioxide as an anti-caking agent using a cone mill. The bulk powder was purged with nitrogen (N2) and stored in heat-sealed bags at 2-8°C with a desiccant until used to manufacture the drug product. The purity, potency (CFU), identity, and residual moisture of the SJ-3C bulk drug substance were tested using methods as previously described and those known to those skilled in the art. The ideal activity of the resulting batch of dried SJ-3C bulk drug substance should be 5×10 10 CFU / g to 1.0×10 11 CFU / g. The ideal water activity of the dried SJ-3C bulk drug substance should be <0.220. When tested for purity, the resulting SJ-3C bulk drug substance will contain a total aerobic count of <200 CFU / g, total yeast and mold of <20 CFU / g, and no objectionable organisms. The identity of the resulting SJ-3C bulk drug substance was confirmed by 16S rRNA gene sequence.

[0118] The raw drug substance was diluted 3 to 10 times with maltodextrin to obtain 2 × 10 9 CFU / dose up to 5×10 9 The final dose of CFU / dose is 200 mg. A dose of the diluted drug substance is placed in a medical powder applicator and packaged as the final drug product.

[0119] Example 2 Formulation studies of storage media with and without excipients of animal origin

[0120] The following example shows the development and characterization of a skim milk-free storage medium formulation that exhibits good room temperature stability for dry powdered Lactobacillus crispatus. This example illustrates that the stability of the dried Lactobacillus powder is increased when animal-derived excipients are removed from the storage medium. The following procedure describing the preparation and development of a storage medium culture using Lactobacillus crispatus CTV-05 can be applied to any microorganism suitable for use in the present invention.

[0121] For development studies of storage formulations, Lactobacillus crispatus CTV-05 was grown in modified MRS medium at pH 6.0 on a 1-liter scale in a stirred bioreactor BioFlo 110 fermentor and bioreactor (New Brunswick Scientific, Edison, NJ). A batch fermentation method was used, and cells were harvested in the early stationary phase when glucose consumption and lactic acid production were complete. The fermentation process typically produced 1.0×10 9 -1.5×10 9CFU / mL, cell viability>90%. Cells were recovered by centrifugation, washed with phosphate buffered saline, and mixed with one or more storage matrices. The mixture was then freeze-dried in a Virtis Advantage freeze dryer.

[0122] Initially, the cryopreservation and accelerated stability of CTV-05 were evaluated in 16 storage matrices containing different concentrations of excipients for preserving lactobacilli, including skim milk, trehalose, xylitol, ascorbic acid, α-tocopherol, and phosphate buffer (Table 2).

[0123] Table 2 Composition of the storage medium containing skim milk

[0124]

[0125] *The amount of sodium phosphate is measured in mM

[0126] The samples were placed in serum vials and frozen at -40°C for 5 hours, followed by primary drying at -40°C for 30 hours and secondary drying at 25°C for 20 hours in a vacuum. The samples were packaged in foil bags with desiccant and stored at 37°C. Powder samples were taken out regularly and viability (i.e., activity) was measured by plating on MRS agar and performing colony counts at 0, 10, and 30 days. As shown in Table 3, formulations #6 and #16 showed the best storage stability at 37°C (70%-75% retention of initial vigor).

[0127] Table 3 Stability of CTV-05 in a storage matrix containing skim milk at 37°C

[0128]

[0129] Similar experiments were performed using the same 16 formulations as above, with the skim milk component removed (Table 4).

[0130] Table 4 Composition of the storage medium without skim milk

[0131]

[0132]

[0133] *The amount of sodium phosphate is measured in mM

[0134] As shown in Table 5 below, Formulations #6 and #16 without skim milk exhibited similar initial potency and the highest storage stability at 37°C (approximately 25% of initial viability).

[0135] Table 5 Stability of CTV-05 in a storage matrix without skim milk at 37°C

[0136]

[0137] While skim milk in formulations #6 and #16 appeared to improve storage stability, higher concentrations of sodium ascorbate and xylitol also appeared to improve storage stability. However, higher concentrations of α-tocopherol did not appear to improve the storage stability of CTV-05, and subsequent experiments showed that removal of α-tocopherol (and its vehicle, Tween 20) resulted in improved stability of CTV-05.

[0138] After removing α-tocopherol (and Tween 20) from the storage matrix of Formulation 6 in Table 2 above, it was determined that skim milk could also be removed without adversely affecting cryoprotection during freeze drying or long-term storage stability at 25°C ( Figure 1 Therefore, a storage solution containing 15% trehalose, 6% xylitol, 1% sodium ascorbate and 10 mM sodium phosphate (pH 7.4) was used ( Figure 1 , upper line) achieves the best combination of low temperature protection and room temperature stability. This formulation provides better stability ( Figure 1 , the line below).

[0139] Example 3 Use of Monosodium Glutamate to Improve Stability

[0140] According to the procedure of Example 2, cultured Lactobacillus crispatus CTV-05 (LACTIN-V) cells were formulated in four different storage media: 1) 15% trehalose, 6% xylitol, 1% sodium ascorbate and 10 mM sodium phosphate, pH 7.4 (triangle); 2) the same storage medium as 1), and additionally 5% monosodium glutamate (inverted triangle); 3) 12% trehalose, 8% xylitol, 1% sodium ascorbate and 10 mM sodium phosphate, pH 7.4 (circle); and 4) the same storage medium as 3), and additionally 5% monosodium glutamate (square). Figure 2 As shown in , monosodium glutamate improved the stability of two powder formulations at elevated temperature (37°C) while having no effect on initial cryopreservation.

[0141] Example 4 Use of the dry composition to treat abnormal vaginal microflora

[0142] This example details in vivo studies showing that the lactobacilli of the invention can be used in vivo, delivered to patients in powder form, to recolonize the vagina of women with recurrent bacterial vaginosis.

[0143] A 25-year-old female patient with recurrent bacterial vaginosis (BV) had symptoms of BV infection, such as abnormal vaginal odor and discharge, and discomfort with itching and pain. BV was detected in the female patient by Amsel clinical criteria (fulfilling ≥3 criteria) and confirmed microbiologically by the Nugent scoring system (Nugent score of 7-10).

[0144] Patients received topical metronidazole 0.75% Initial standardized antibiotic therapy was performed once a day for 5 days. After the completion of metronidazole treatment, the female patient began treatment with the Lactobacillus SJ-3C drug product of the present invention. A 200 mg dose of 2×10 9 CFU / dose of active SJ-3C drug product. Patients received one dose of SJ-3C drug product once daily before sleep for 5 consecutive days, followed by twice weekly treatment for 10 weeks.

Claims

1. An aqueous bacterial suspension of vaginal Lactobacillus species without excipients of animal origin, wherein the vaginal Lactobacillus species is Lactobacillus crispatus, and wherein the suspension is produced by the combination of a cell mass of the vaginal Lactobacillus species with an aqueous preservation medium consisting of: (i) 15% w / v trehalose; (ii) 6% w / v xylitol; (iii) 1% w / v sodium ascorbate; and (iv) 10 mM sodium phosphate.

2. An aqueous bacterial suspension of vaginal Lactobacillus species without excipients of animal origin, wherein the vaginal Lactobacillus species is Lactobacillus crispatus, and wherein the suspension is produced by the combination of a cell mass of the vaginal Lactobacillus species with an aqueous preservation medium consisting of: (i) 12-15% w / v trehalose; (ii) 6-8% w / v xylitol; (iii) 1% w / v sodium ascorbate; (iv) 10 mM sodium phosphate; and (v) 5% w / v sodium glutamate.

3. The bacterial suspension of claim 1 or 2, wherein the vaginal Lactobacillus species has the ability to produce greater than 0.5 ppm hydrogen peroxide under effective culture conditions.

4. A dry powder produced by freeze-drying the bacterial suspension according to claim 1 or 2.

5. The dry powder of claim 4, wherein the powder has a water activity value of less than 0.

220.

6. The dry powder of claim 5, wherein the powder is combined with an inactive excipient in a ratio of 1:1 to 1:10 w / w.

7. The dry powder of claim 6, wherein the inactive excipient is maltodextrin.

8. The dry powder of claim 6, wherein the ratio of powder: inactive excipient is 1:1 to 1:5 w / w.

9. The dry powder of claim 4, wherein the powder is contained within a plastic housing designed for vaginal administration.

10. A method for preserving lactobacilli under dry conditions and in the absence of excipients of animal origin, the method comprising: (i) Obtain a cell concentration of 10 8 / ml to 10 13 / ml of an aqueous suspension of a vaginal Lactobacillus species, wherein the vaginal Lactobacillus species is Lactobacillus crispatus; (ii) centrifuging the aqueous suspension to form a bacterial cell pellet; and (iii) resuspending the bacterial cell mass in an aqueous storage medium to produce a bacterial suspension, wherein the aqueous storage medium consists of: (a) 15% w / v trehalose; (b) 6% w / v xylitol; (c) 1% w / v sodium ascorbate; and (d) 10 mM sodium phosphate; The weight-to-volume ratio of the wet weight of the cell mass to the aqueous storage medium is 1:1 g / mL to 1:5 g / mL.

11. A method for preserving lactobacilli under dry conditions and in the absence of excipients of animal origin, the method comprising: (i) Obtain a cell concentration of 10 8 / ml to 10 13 / ml of an aqueous suspension of a vaginal Lactobacillus species, wherein the vaginal Lactobacillus species is Lactobacillus crispatus; (ii) centrifuging the aqueous suspension to form a bacterial cell pellet; and (iii) resuspending the bacterial cell mass in an aqueous storage medium to produce a bacterial suspension, wherein the aqueous storage medium consists of: (a) 12-15% w / v trehalose; (b) 6-8% w / v xylitol; (c) 1% w / v sodium ascorbate; (d) 10 mM sodium phosphate; and (e) 5% w / v sodium glutamate; The weight-to-volume ratio of the wet weight of the cell mass to the aqueous storage medium is 1:1 g / mL to 1:5 g / mL.

12. The method of claim 10 or 11, wherein the vaginal Lactobacillus species has the ability to produce greater than 0.5 ppm hydrogen peroxide under effective culture conditions.

13. The method of claim 10 or 11, further comprising freeze-drying the bacterial suspension to produce a dry powder.

14. The method of claim 13, wherein the powder has a water activity value of less than 0.

220.

15. The method of claim 14, wherein the dry powder is combined with an inactive excipient in a ratio of 1:1 to 1:10 w / w.

16. The method of claim 15, wherein the inactive excipient is maltodextrin.

17. The method of claim 15, wherein the ratio of powder: inactive excipient is 1:1 to 1:5 w / w.

18. Use of a dry powder derived from an aqueous bacterial suspension of vaginal Lactobacillus species without excipients of animal origin for the preparation of a medicament for treating abnormal vaginal microbiota in a female, wherein the female has been diagnosed as having abnormal vaginal microbiota; and the female has previously been administered an antibiotic in an amount effective to reduce the level of abnormal vaginal microbiota; and the suspension is produced by combining a cell mass of a vaginal Lactobacillus species with an aqueous preservation medium consisting of: (a) 15% w / v trehalose; (b) 6% w / v xylitol; (c) 1% w / v sodium ascorbate; and (d) 10 mM sodium phosphate; wherein the vaginal Lactobacillus species is Lactobacillus crispatus.

19. Use of a dry powder derived from an aqueous bacterial suspension of vaginal Lactobacillus species without excipients of animal origin in the preparation of a medicament for treating abnormal vaginal microbiota in a female, wherein the female has been diagnosed as having abnormal vaginal microbiota; the female has previously been administered an antibiotic in an amount effective to reduce the level of abnormal vaginal microbiota; and the suspension is produced by combining a cell mass of vaginal Lactobacillus species with an aqueous preservation medium consisting of: (a) 12-15% w / v trehalose; (b) 6-8% w / v xylitol; (c) 1% w / v sodium ascorbate; (d) 10 mM sodium phosphate; and (e) 5% w / v sodium glutamate; wherein the vaginal Lactobacillus species is Lactobacillus crispatus.

20. The use of claim 18 or 19, wherein the vaginal Lactobacillus species has the ability to produce greater than 0.5 ppm hydrogen peroxide under effective culture conditions.

21. The use according to claim 18 or 19, wherein the dry powder has a water activity value of less than 0.

220.

22. The use of claim 21, wherein the dry powder is combined with an inactive excipient in a ratio of 1:1 to 1:10 w / w.

23. The use according to claim 22, wherein the inactive excipient is maltodextrin.

24. The use of claim 22, wherein the ratio of powder: inactive excipient is 1:1 to 1:5 w / w.

25. The use according to claim 18 or 19, wherein the antibiotic is clindamycin, metronidazole or tinidazole.

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