Lactobacillus GEL formulation and uses thereof

Abstract

The present disclosure relates to a pharmaceutical composition containing a poloxamer, a stabilization polymer, and Lactobacillus, as well as methods of making and using, and kits containing the composition.

Description

Atty Docket No.053032-548001WO LACTOBACILLUS GEL FORMULATION AND USES THEREOF RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 574,156, filed April 3, 2024, the entire contents of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Given the impact of the maternal microbiome on establishment and development of the infant microbiome, both during and post-delivery, there is potentially a significant benefit in ensuring a healthy microbiome in pregnant and lactating women; one that supports improved health for the mother and helps to pass on those bacteria that are critical to infant health. Recently there has also been compelling evidence that links positive outcomes for reduction in pre-term birth to a healthy vaginal microbiome. Two of the significant barriers to this development are the cost of the potentially complex consortia of bacteria that are required to establish healthy gut / vaginal communities, and the ability to maintain viability of the live microbes during storage, shipment and distribution of the products.

[0003] Storage of many commercially available probiotic foods and supplements is recommended at refrigerated and / or ambient temperatures, and while this is a reasonable approach in countries where ambient temperature is 25°C or in regions with routine access to refrigeration, in many parts of south east Asia and sub-Saharan Africa these conveniences are not as readily available, and consequently distribution under typical conditions to these regions is not practical. Compounding the problem is shipment and distribution, where it is common for materials in transition to experience excursions as high as 50°C-60°C during flight and / or customs layovers. Cryopreservation followed by freeze drying has enabled many products to achieve stability at ambient temperature and above, but these products typically contain only a single or a small number of strains, and therefore the cryopreservation and drying cycles can be tailored to achieve acceptable compatibility. Within larger consortia, cryopreservatives and other excipients as well as drying cycles may have differentiated and variable impact on the individual strains, necessitating a different more holistic approach to stabilization. Encapsulation technologies haveAtty Docket No.053032-548001WO been used to good effect to achieve longer shelf lives, as have novel methods to reduce particle porosity and to drive bacteria into the center of the formed particle where they are less susceptible to exposure to heat, moisture and oxygen. These approaches utilize both traditional freeze drying as well as potentially more cost effective methods such as shelf and spray drying. Opportunities are also present in final product formulation, where optimized preservative matrices, oil based gels and reconstitutable powders are likely to improve stability in hotter / more humid climate conditions and broaden possible distribution and shelf life. SUMMARY

[0004] The instant technology generally relates to a pharmaceutical composition including Lactobacillus and a pharmaceutical gel. In embodiments, the pharmaceutical gel includes a poloxamer and a stabilization polymer. In embodiments, the composition is a gel. In embodiments, the composition is formulated for administration to a vagina of a subject.

[0005] In embodiments, the composition includes about 10 wt% to about 30 wt% Lactobacillus and a pharmaceutical gel comprising: about 10 wt% to about 25 wt% poloxamer; about 1 wt% to about 5 wt% stabilization polymer; and one or more buffers.

[0006] In embodiments, the poloxamer includes poloxamer 407. In embodiments, the poloxamer is present at about 10-25 percent by weight of the composition (wt%).

[0007] In embodiments, the stabilization polymer includes xanthan gum. In embodiments, the stabilization polymer is present at about 1-5 wt%.

[0008] In embodiments, the pharmaceutical gel further includes one or more buffers. In embodiments, the one or more buffers are present at about 0.1% to about 5% w / w. In embodiments, the one or more buffers include citrate and / or a citrate salt. In embodiments, the citrate and / or citrate salt includes one or more of citric acid, citric acid monohydrate, sodium citrate, and sodium citrate dihydrate. In embodiments, the citrate and / or citrate salt is present at about 0.1-3 wt%. In embodiments, the one or more buffers include lactic acid and / or sodium phosphate. In embodiments, the lactic acid is present at about 0.1-3 wt%. In embodiments, the sodium phosphate is present at about 0.1-3 wt%.Atty Docket No.053032-548001WO

[0009] In embodiments, the pharmaceutical gel further includes benzoic acid. In embodiments, the benzoic acid is present at about 0.05-2 wt%. In embodiments, the pharmaceutical gel further includes benzyl alcohol. In embodiments, the benzyl alcohol is present at about 0.5-5 wt%. In embodiments, the pharmaceutical gel does not comprise benzyl alcohol.

[0010] In embodiments, the composition has a viscosity at 37 °C of at least 1.5 million centipoise (cP). In embodiments, the composition has a viscosity at 15°C of at least 50,000 cP. In embodiments, the composition has a viscosity at 15°C of about 50,000 cP to about 5 million cP.

[0011] In embodiments, the Lactobacillus is present at about 1-30 wt%. In embodiments, the Lactobacillus is present at about 2 wt% to about 20 wt%. In embodiments, the Lactobacillus includes one or more Lactobacillus species. In embodiments, the Lactobacillus species comprise one or more of Lactobacillus crispatus. Lactobacillus gasseri, Lactobacillus iners, Lactobacillus vaginalis, and Lactobacillus jensenii.

[0012] In embodiments, the composition includes a prebiotic. In embodiments, the prebiotic includes a sugar. In embodiments, the sugar includes one or more of glucose, dextrose, or maltose. In embodiments, the composition includes glutamine.

[0013] In an aspect is provided methods of using a pharmaceutical composition as described herein. In an aspect is provided a method for providing Lactobacillus to a vagina of a subject, including administering a pharmaceutical composition including a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

[0014] In an aspect is provided a method for treating bacterial vaginosis in a subject, including administering a pharmaceutical composition including a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

[0015] In an aspect is provided a method for preventing recurrence of bacterial vaginosis in a subject, including administering a pharmaceutical composition including a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.Atty Docket No.053032-548001WO

[0016] In an aspect is provided a method for maintaining healthy biota in a vagina of a subject, including administering a pharmaceutical composition including a poloxamer, a stabilization polymer, and Lactobacillus to the vagina subject.

[0017] In embodiments, the composition is administered during or after administration of an antibiotic to the subject.

[0018] In embodiments, the method includes combining the Lactobacillus with the pharmaceutical gel prior to administration, wherein the pharmaceutical gel comprises the poloxamer and the stabilization polymer.

[0019] In an aspect is provided a kit including Lactobacillus and a pharmaceutical gel, wherein the pharmaceutical gel comprises a poloxamer and a stabilization polymer. In embodiments, the pharmaceutical gel is any pharmaceutical gel described herein. In embodiments, the kit includes instructions for mixing the Lactobacillus and the pharmaceutical gel and administration to a vagina of a subject. In embodiments, the kit includes at least one syringe.

[0020] In embodiments, the Lactobacillus is in a first container and the pharmaceutical gel is in a second container. In embodiments, the first container and / or the second container is a syringe.

[0021] In embodiments, the kit includes a connector to connect the first container and the second container. In embodiments, the connector comprises a leur lock connector.

[0022] In an aspect is provided a method of making a Lactobacillus composition, including combining Lactobacillus and a pharmaceutical gel, as described herein. In embodiments, the Lactobacillus and the pharmaceutical gel are combined by: (a) providing the Lactobacillus in a first container; (b) providing the pharmaceutical gel in a second container; (c) connecting the first container and the second container, such that the Lactobacillus is in fluid connection with the pharmaceutical gel; and (d) transferring the Lactobacillus and the pharmaceutical gel between the two containers to make the Lactobacillus composition. In embodiments, the first container is a syringe. In embodiments, the second container is a syringe. In embodiments, step (d) is performed by pushing the Lactobacillus into the second container, or by pushing the pharmaceutical gel intoAtty Docket No.053032-548001WO the first container. In embodiments, the method includes pushing the Lactobacillus and pharmaceutical gel from the first container into the second container, or from the second container to the first container. In embodiments, the Lactobacillus and the pharmaceutical gel are pushed from the first and / or second container into the other container two or more times, for a sufficient number of times to make the Lactobacillus composition. In embodiments, the first container and the second container are connected by a leur lock connector. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG.1 is a photograph showing the appearance of three formulations of base gel (no Lactobacillus or glucose) as described in Example 1. From left to right: Formulations 2303, 2304, and 2305.

[0024] FIG.2 is a graph showing thermal viscosity of Formulations 2303, 2304, and 2305.

[0025] FIG.3 is a graph showing storage modulus and loss modulus (G" and G"(Pa)) at 25 °C of 2303 (Series 1 and Series 4), 2304 (Series 2 and Series 5), and 2305 (Series 3 and Series 6), corresponding to data in Table 4.

[0026] FIG.4 is a graph showing storage modulus and loss modulus (G" and G"(Pa)) at 37 °C of 2303 (Series 1 and Series 4), 2304 (Series 2 and Series 5), and 2305 (Series 3 and Series 6), corresponding to data in Table 5.

[0027] FIG.5 is a photograph showing the appearance of three formulations as described in Example 2. From left to right: 20% w / w Lactobacillus gel, 10% w / w Lactobacillus gel, and placebo.

[0028] FIG. 6 is a graph showing Thermal Viscosity comparison of 20% Lactobacillus gel, 10% Lactobacillus gel, and placebo (Acid Form Gel (Reference)).

[0029] FIG.7 is a graph showing LVER (G’) comparison of 20% Lactobacillus gel, 10% Lactobacillus gel, and placebo.

[0030] FIG.8 is a graph showing LVER (G”) comparison of 20% Lactobacillus gel, 10% Lactobacillus gel, and placebo.Atty Docket No.053032-548001WO

[0031] FIG.9 is a photograph showing the appearance of Lactobacillus gels. From left to right: 2302-01, 2302-02, 2302-03, 2302-04, and 2302-05.

[0032] FIG. 10 is a graph showing an overview of Thermal Viscosity for formulations 2302-01, 2302-02, 2302-03, 2302-04, and 2302-05.

[0033] FIG.11 is a series of graphs showing Main Effects Plot for 2302-01 – 05 Xanthan gum and Poloxamer 407 (%w / w).

[0034] FIG.12 is a photograph showing the appearance of placebo gels. From left to right: 2303, 2304, and 2305.

[0035] FIG.13 is a photograph showing the appearance of Lactobacillus gels. From left to right: 2303, 2304, and 2305.

[0036] FIG. 14 is a graph showing Thermal Viscosity comparison of 178008-2303, 178008-2304, and 178008-2305, without Lactobacillus.

[0037] FIG. 15 is a graph showing Thermal Viscosity comparison of 178008-2303 with Lactobacillus (178008-2303 DP), with Lactobacillus and D-glucose (178008-2303 DP w / D-glu), and placebo.

[0038] FIG. 16 is a graph showing Thermal Viscosity comparison of 178008-2304 with Lactobacillus (178008-2304 DP), with Lactobacillus and D-glucose (178008-2304 DP w / D-glu), and placebo.

[0039] FIG. 17 is a graph showing Thermal Viscosity comparison of 178008-2305 with Lactobacillus (178008-2305 DP), with Lactobacillus and D-glucose (178008-2305 DP w / D-glu), and placebo.

[0040] FIG.18 is a graph showing LVER (25°C) G’ and G’’ comparison for 178008-2303, 178008-2304, and 178008-2305, without Lactobacillus.

[0041] FIG.19 is a graph showing LVER (37°C) G’ and G’’ comparison for 178008-2303, 178008-2304, and 178008-2305, without Lactobacillus.Atty Docket No.053032-548001WO

[0042] FIG.20 is a graph showing Main Effects for Avg. P407 MW vs 16% w / v SPV at 25°C (Left) and 37°C (Right).

[0043] FIG.21 is a graph showing 2320-01 – 2320-05 Placebo Thermal Viscosity Profiles (2X Ramp up).

[0044] FIG. 22 is a graph showing 2320-01 – 2320-05 Drug Product Thermal Viscosity Profiles (2X Ramp up).

[0045] FIG.23 is a graph showing 2320-01 – 2320-05 Drug Product Comparison of Flow Curves.

[0046] FIG.24 is a graph showing 2320-01 – 2320-05 Placebo Gel Comparison of Flow Curves.

[0047] FIG.25 is a graph showing 178008-2306 to 2308 Placebo Gel Overview of Thermal Viscosity.

[0048] FIG.26 is a graph showing 178008-2306 to 2308 Overview of LVER (G’ and G’’).

[0049] FIG.27 is a graph showing the Thermal Viscosity Profile of the placebo gel after 2 weeks, 1 month, and 2 months at 50°C.

[0050] FIG.28 is a graph showing the Thermal Viscosity Profile of the placebo gel after 1 month and 2 months at 40°C and 75% relative humidity (RH).

[0051] FIG.29 is a graph showing the Thermal Viscosity Profile of the placebo gel after 1 month and 2 months at 25°C and 60% relative humidity (RH). DETAILED DESCRIPTION

[0052] After reading this description it will become apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of anAtty Docket No.053032-548001WO example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure as set forth herein.

[0053] Before the present technology is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0054] The detailed description divided into various sections only for the reader’s convenience and disclosure found in any section may be combined with that in another section. Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present disclosure. Definitions

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0056] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0057] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0058] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations whichAtty Docket No.053032-548001WO may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” when used with regard to an amount means that the amount may vary by + / - 10%.

[0059] “Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0060] Terms such as “treat,” “treatment,” “treating,” etc. comprise therapeutic treatment of subjects having already developed a disease, in particular in manifest form. Therapeutic treatment may be symptomatic treatment in order to relieve the signs and / or symptoms of the disease or causal treatment in order to reverse, partially reverse, stop, or slow down the progression of the disease. Thus, the compositions and methods of the present disclosure may be used, for instance, as therapeutic treatment (e.g., for acute or chronic therapy).

[0061] Additionally, terms such as “prevent,” “preventing,” or “prevention” generally refer to the reduction of the occurrence of the disease, and / or a sign and / or symptom thereof, in the treated sample relative to an untreated control sample, or delays the onset of one or more signs and / or symptoms of the disease relative to the untreated control sample, in a statistically significant manner. Preventing the disease, and / or a sign and / or a symptom thereof, includes preventing or delaying the initiation of the disease, sign, and / or symptom. Prevention also includes preventing a recurrence of the disease, sign, and / or symptom.

[0062] In certain aspects, the composition can be applied to a subject, e.g., to the vagina of a subject, and / or to another body cavity, for example, the mouth or the rectum. Any suitable technique may be used to apply the composition to the subject. For instance, the composition may be free or mass flowing, e.g., so that it may be administered through an applicator or other suitable device. Thus, in some embodiments, the composition may be contained within applicator, such as a vaginal applicator or a syringe, which can be applied, e.g., by the subject, or by another person.Atty Docket No.053032-548001WO

[0063] The subject may be, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals (e.g., primates (e.g., monkeys such as cynomolgus monkeys or rhesus monkeys, chimpanzees, etc.); commercially relevant mammals such as cattle, pigs, horses, sheep, rabbits, mice, rats, goats, cats, dogs, etc.) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, turkeys, etc.). In certain embodiments, the subject is a mammal. The subject may be a male or female and at any stage of development. A non-human animal may be a transgenic animal. In embodiments, the subject is a female. In embodiments, the subject is a human. Pharmaceutical Compositions

[0064] In some aspects, provided herein is a pharmaceutical composition comprising Lactobacillus and a pharmaceutical gel comprising a poloxamer and a stabilization polymer. In embodiments, the pharmaceutical gel further comprises one or more buffers. In embodiments, the composition is a gel. In embodiments, the composition is formulated for administration to a vagina of a subject.

[0065] The molecular weight of the poloxamer may be, in one embodiment, from 5 kDa to 25 kDa. In some instances, the molecular weight of the copolymer may be from 9 kDa to 16 kDa. In some cases, the molecular weight of the poloxamer may be at least 1 kDa, at least 2 kDa, at least 3 kDa, at least 4 kDa, at least 5 kDa, at least 7 kDa, at least 9 kDa, at least 10 kDa, at least 15 kDa, at least 16 kDa, at least 20 kDa, at least 25 kDa, at least 50 kDa, etc. In addition, in certain embodiments, the molecular weight of the poloxamer may be no more than 50 kDa, no more than 25 kDa, no more than 20 kDa, no more than 16 kDa, no more than 15 kDa, no more than 10 kDa, no more than 9 kDa, no more than 5 kDa, no more than 4 kDa, no more than 3 kDa, no more than 2 kDa, no more than 1 kDa, etc. Combinations of any of these are also possible. For instance, the poloxamer may have a molecular weight of between 10 kDa and 15 kDa. As other non-limiting examples, the molecular weight may be between 3 kDa and 5 kDa, between 2 kDa and 4 kDa, between 5 kDa and 20 kDa, between 9 kDa and 16 kDa, etc. The molecular weight, in some cases, may be determined as a weight average molecular weight. The molecular weight may be any value or subrange within the recited ranges, including endpoints.Atty Docket No.053032-548001WO

[0066] In embodiments, the poloxamer includes poloxamer 407. The composition may include one or more poloxamers in one set of embodiments. The poloxamer may be used to increase the viscosity of the composition, e.g., as described herein. In some cases, sufficient poloxamer may be present to cause the composition to form a gel, e.g., at room temperature (about 25 ºC) and / or body temperature (about 37 ºC). Furthermore, in some cases, the composition may have a gelling temperature, but the gel temperature may be in a range that is physiologically irrelevant. For instance, the gelling temperature may be above 40 ºC, or below 25 ºC, and thus, the composition does not change phase or gel at normal physiological or body temperatures.

[0067] Poloxamers generally include any of a variety of polyoxyethylene- polyoxypropylene triblock copolymers. In some cases, the poloxamer may be a nonionic block copolymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide). In some embodiments, the poloxamers may be soluble in water and other polar and non-polar solvents.

[0068] Because the lengths of the polymer blocks can be independently customized, many different poloxamers exist that have slightly different properties. For example, the polxoxamer may have a structure:

[0069] HO–[CH2–CH2–O]a–[CH2–CH(CH3)–O]b–[CH2–CH2–O]a–H.

[0070] The structure includes a core of propylene oxide (represented by “b” in the above figure), flanked by ethylene oxide subunits (represented by “a” in the above figure), typically on both sides. The sum of the two a’s may be, for example, from 50 to 500, from 100 to 300, from 150 to 250, or 200. As another example, a may be between 99 and 103, e.g., 101. b may be, for example, from 30 to 100, from 50 to 80, from 60 to 70, or 65. As another example, b may be between 54 and 58, e.g., 56.

[0071] In some embodiments, the ethylene oxide subunits forming the poloxamer may be in molar excess to the propylene oxide subunits. For example, in certain embodiments, the ratio of ethylene oxide subunits to propylene oxide subunits (i.e., a:b) may be, for example, from 3:1 to 5:1, or from 2:1 to 4:1.

[0072] Several suitable poloxamers can be readily obtained commercially, including poloxamer 407, Pluronic®F-127, or the like. The composition may include a single poloxamer,Atty Docket No.053032-548001WO or more than one type of poloxamer. In some cases, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, or at least 90 wt% of the poloxamer within the composition is a single type of poloxamer, for example, poloxamer 407 or Pluronic®F-127.

[0073] In embodiments, the poloxamer is present at about 10 to about 25 percent by weight of the composition (wt%). In embodiments, the poloxamer is present at about 15 wt% to about 25 wt%. In embodiments, the poloxamer is present at about 15 wt% to about 20 wt%. In embodiments, the poloxamer is present at about 10 wt%. In embodiments, the poloxamer is present at about 11 wt%. In embodiments, the poloxamer is present at about 12 wt%. In embodiments, the poloxamer is present at about 13 wt%. In embodiments, the poloxamer is present at about 14 wt%. In embodiments, the poloxamer is present at about 15 wt%. In embodiments, the poloxamer is present at about 16 wt%. In embodiments, the poloxamer is present at about 17 wt%. In embodiments, the poloxamer is present at about 18 wt%. In embodiments, the poloxamer is present at about 19 wt%. In embodiments, the poloxamer is present at about 20 wt%. In embodiments, the poloxamer is present at about 21 wt%. In embodiments, the poloxamer is present at about 22 wt%. In embodiments, the poloxamer is present at about 23 wt%. In embodiments, the poloxamer is present at about 24 wt%. In embodiments, the poloxamer is present at about 25 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0074] In embodiments, the stabilization polymer comprises xanthan gum. In embodiments, the stabilization polymer comprises hyaluronic acid, alginic acid, modified celluloses such as hydroxypropyl methylcellulose, or others such as described herein.

[0075] Xanthan gum generally refers to a high molecular weight polysaccharide used as a food additive and rheology modifier, as would be known by those of ordinary skill in the art. In addition, many such xanthan gums are readily available commercially. Xanthan gum may be produced, as a non-limiting example, by a process involving fermentation of glucose or sucrose by the Xanthomonas campestris bacterium. In some embodiments, the backbone of the polysaccharide chain may have two beta-D-glucose units linked through the 1 and 4 positions. The side chains are formed of two mannose and one glucuronic acid, so the chain has repeating modules of five sugar units. The side chain is linked to every other glucose of the backbone at the 3 position. About half of the terminal mannose units have a pyruvic acid group linked as a ketal to its 4 and 6 positions. The other mannose unit has an acetyl group at the 6 positions. Two ofAtty Docket No.053032-548001WO these chains may be aligned to form a double helix, giving a rather rigid rod configuration that accounts for its high efficiency as a viscosifier of water.

[0076] However, it should be understood that not all xanthan gums have precisely the above molecular configuration or properties, and that xanthan gums may vary in molecular composition, e.g., depending on the source of the xanthan gum, especially those arising from different biological sources. In addition, other stabilization polymers instead of (or in addition to) xanthan gum can be used, for example, KELTROL®BT and / or KELTROL®RD, KELZAN®XC, KELZAN®XCD, KELZAN®D, KELZAN®CC, XANTURAL®180, XANTURAL®75, or the like, all of which can be obtained commercially from various suppliers.

[0077] The molecular weight of the xanthan gum or other stabilization polymer can vary. For instance, the xanthan gum or other stabilization polymer may have any suitable molecular weight, for example, at least about 1 million, at least about 2 million, at least about 5 million, at least about 10 million, at least about 25 million, or at least about 50 million. In other embodiments, the molecular weight can vary from about one million to 50 million, e.g., depending upon various factors such as how it is prepared. In some embodiments, the molecular weight can range from approximately 1 million to approximately 25 million, e.g., as measured by a Brookfield Viscometer or other suitable device. In yet other embodiments, the molecular weight may be, for example, 1, 2, 3, 4, or 5 (+ / - 0.5) million, or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19, 20, 21, 22, 23, 24, or 25 (+ / - 2) million. Still other molecular weights are also possible.

[0078] In embodiments, the stabilization polymer is present at about 1 wt% to about 5 wt%. In embodiments, the stabilization polymer is present at about 1 wt% to about 4 wt%. In embodiments, the stabilization polymer is present at about 1 wt% to about 3 wt%. In embodiments, the stabilization polymer is present at about 2 wt% to about 5 wt%. In embodiments, the stabilization polymer is present at about 2 wt% to about 4 wt%. In embodiments, the stabilization polymer is present at about 1 wt%. In embodiments, the stabilization polymer is present at about 1.5 wt%. In embodiments, the stabilization polymer is present at about 2 wt%. In embodiments, the stabilization polymer is present at about 2.1 wt%. In embodiments, the stabilization polymer is present at about 2.25 wt%. In embodiments, the stabilization polymer is present at about 2.5 wt%. In embodiments, the stabilization polymer is present at about 3 wt%. In embodiments, the stabilization polymer is present at about 3.5 wt%. In embodiments, the stabilization polymer isAtty Docket No.053032-548001WO present at about 4 wt%. In embodiments, the stabilization polymer is present at about 4.5 wt%. In embodiments, the stabilization polymer is present at about 5 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0079] In embodiments, the one or more buffers are present at about 0.1% to about 5% w / w (wt%). In embodiments, the one or more buffers are each present at about 0.1% to about 5% w / w. In embodiments, the one or more buffers are present at about 0.1% to about 5% w / w in total. In embodiments, the one or more buffers are present at about 0.1% to about 4% w / w. In embodiments, the one or more buffers are present at about 0.1% to about 3% w / w. In embodiments, the one or more buffers are present at about 0.1% to about 2% w / w. In embodiments, the one or more buffers are present at about 0.1% to about 1% w / w. In embodiments, the one or more buffers are present at about 0.3% to about 5% w / w. In embodiments, the one or more buffers are present at about 0.5% to about 5% w / w. The amount may be any value or subrange within the recited ranges, including endpoints.

[0080] In embodiments, the one or more buffers comprise citrate and / or a citrate salt. In embodiments, the citrate and / or citrate salt comprises one or more of citric acid, citric acid monohydrate, sodium citrate, and sodium citrate dihydrate. In embodiments, the citrate and / or citrate salt comprises citric acid. In embodiments, the citrate and / or citrate salt comprises citric acid monohydrate. In embodiments, the citrate and / or citrate salt comprises sodium citrate. In embodiments, the citrate and / or citrate salt comprises sodium citrate dihydrate. In embodiments, the one or more buffers comprise citric acid or citric acid monohydrate and sodium citrate or sodium citrate dihydrate.

[0081] In embodiments, the citrate and / or citrate salt is present at about 0.1 wt% to about 3 wt%. In embodiments, each citrate and / or citrate salt is present at about 0.1 wt% to about 3 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 3 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 2 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 1.5 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 1 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 0.9 wt%. In citrate and / or citrate salt is present at about 0.1 wt% to about 0.8 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 0.7 wt%. InAtty Docket No.053032-548001WO embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 0.6 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 0.5 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 0.4 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt% to about 0.3 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.1 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.2 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.3 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.4 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.5 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.6 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.7 wt%. In embodiments, a citrate and / or citrate salt is present at about 0.8 wt%. In embodiments, a citrate and / or citrate salt is present at about 1 wt%. In embodiments, a citrate and / or citrate salt is present at about 2 wt%. In embodiments, a citrate and / or citrate salt is present at about 3 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0082] In embodiments, the one or more buffers comprise lactic acid and / or sodium phosphate. In embodiments, the one or more buffers comprise lactic acid. In embodiments, the one or more buffers comprise sodium phosphate. In embodiments, the one or more buffers comprise lactic acid and sodium phosphate.

[0083] In embodiments, the lactic acid is present at about 0.1 wt% to about 3 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 2 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 1.5 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 1 wt%. In embodiments the lactic acid is present at about 0.1 wt% to about 0.9 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 0.8 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 0.7 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 0.6 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 0.5 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 0.4 wt%. In embodiments, the lactic acid is present at about 0.1 wt% to about 0.3 wt%. In embodiments, the lactic acid is present at about 0.1 wt%. In embodiments, the lactic acid is present at about 0.2 wt%. In embodiments, the lactic acid is present at about 0.3 wt%. In embodiments, the lactic acid is present at about 0.4 wt%. In embodiments, the lactic acid is present at about 0.5Atty Docket No.053032-548001WO wt%. In embodiments, the lactic acid is present at about 0.6 wt%. In embodiments, the lactic acid is present at about 0.7 wt%. In embodiments, the lactic acid is present at about 0.8 wt%. In embodiments, the lactic acid is present at about 1 wt%. In embodiments, the lactic acid is present at about 2 wt%. In embodiments, the lactic acid is present at about 3 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0084] In embodiments, the sodium phosphate is present at about 0.1 wt% to about 3 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 2 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 1.5 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 1 wt%. In embodiments the sodium phosphate is present at about 0.1 wt% to about 0.9 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 0.8 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 0.7 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 0.6 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 0.5 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 0.4 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt% to about 0.3 wt%. In embodiments, the sodium phosphate is present at about 0.1 wt%. In embodiments, the sodium phosphate is present at about 0.2 wt%. In embodiments, the sodium phosphate is present at about 0.3 wt%. In embodiments, the sodium phosphate is present at about 0.4 wt%. In embodiments, sodium phosphate is present at about 0.5 wt%. In embodiments, the sodium phosphate is present at about 0.6 wt%. In embodiments, the sodium phosphate is present at about 0.7 wt%. In embodiments, the sodium phosphate is present at about 0.8 wt%. In embodiments, the sodium phosphate is present at about 1 wt%. In embodiments, the sodium phosphate is present at about 2 wt%. In embodiments, the sodium phosphate is present at about 3 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0085] In embodiments, the pharmaceutical gel further comprises benzoic acid. In embodiments, the benzoic acid is present at about 0.05 wt % to about 2 wt%. In embodiments, the benzoic acid is present at about 0.1 wt % to about 2 wt%. In embodiments, the benzoic acid is present at about 0.2 wt % to about 2 wt%. In embodiments, the benzoic acid is present at about 0.05 wt % to about 1 wt%. In embodiments, the benzoic acid is present at about 0.1 wt % to about 1 wt%. In embodiments, the benzoic acid is present at about 0.1 wt%. In embodiments, the benzoic acid isAtty Docket No.053032-548001WO present at about 0.2 wt%. In embodiments, the benzoic acid is present at about 0.3 wt%. In embodiments, the benzoic acid is present at about 0.4 wt%. In embodiments, benzoic acid is present at about 0.5 wt%. In embodiments, the benzoic acid hate is present at about 0.6 wt%. In embodiments, the benzoic acid is present at about 0.7 wt%. In embodiments, the benzoic acid is present at about 0.8 wt%. In embodiments, the benzoic acid is present at about 1 wt%. In embodiments, the benzoic acid is present at about 2 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0086] In embodiments, the pharmaceutical gel further comprises benzyl alcohol. In embodiments, the benzyl alcohol is present at about 0.5 wt% to about 5 wt%. In embodiments, the benzyl alcohol is present at about 1 wt% to about 5 wt%. In embodiments, the benzyl alcohol is present at about 0.5 wt% to about 4 wt%. In embodiments, the benzyl alcohol is present at about 0.5 wt% to about 3 wt%. In embodiments, the benzyl alcohol is present at about 0.5 wt% to about 2 wt%. In embodiments, the benzyl alcohol is present at about 0.5 wt% to about 1 wt%. The amount may be any value or subrange within the recited ranges, including endpoints. In embodiments, benzyl alcohol is expressly excluded.

[0087] In embodiments, the composition has a viscosity at 37 °C of at least 1.5 million centipoise (cP). In embodiments, the composition has a viscosity at 15°C of at least 50,000 cP. In embodiments, the composition has a viscosity at 15°C of about 50,000 cP to about 5 million cP. In some cases, the composition may have a viscosity at room temperature of at least 1 million cP, at least 1.1 million cP, at least 1.2 million cP, at least 1.3 million cP, at least 1.4 million cP, at least 1.5 million, at least 1.6 million cP, at least 1.8 million cP, at least 2 million cP, at least 2.2 million cP, at least 2.4 million cP, at least 2.6 million cP, at least 2.8 million cP, at least 3 million cP, at least 3.5 million cP, at least 4 million cP, etc. In addition, in certain embodiments, the composition may have a viscosity of no more than 4 million cP, no more than 3.5 million cP, no more than 3 million cP, no more than 2.8 million cP, no more than 2.6 million cP, no more than 2.4 million cP, no more than 2.2 million cP, no more than 2.0 million cP, no more than 1.8 million cP, no more than 1.6 million cP, no more than 1.5 million cP, no more than 1.4 million cP, no more than 1.3 million cP, no more than 1.2 million cP, no more than 1.1 million cP, no more than 1.0 million cP, etc. Combinations of any of these are also possible, for example, the composition may exhibit a viscosity of between 1.5 million cP and 2 million cP, between 1.8 million cP and 2.4 million cP,Atty Docket No.053032-548001WO between 1.2 million cP and 3 million cP, etc. The viscosity may be any value or subrange within the recited ranges, including endpoints.

[0088] Such high viscosities can be achieved, in various embodiments, using techniques such as removing air from the composition, which may increase its viscosity and / or cause it to form a gel. Other techniques may also be used, including any of those described herein. For example, in certain embodiments, the composition may be prepared and treated to remove air from the composition, for example, to reduce the composition to 15 vol% air, or less. Techniques for removing air include, but are not limited to, a variety of techniques, such as centrifugation or exposure to relatively high vacuums, e.g., less than 100 mbar.

[0089] In addition, in one set of embodiments, the composition may have a relatively low concentration or amount of air. For instance, in one embodiment, the composition is substantially free of air. In some cases, during manufacture, a large amount of air may be introduced into the composition, e.g., as foam or bubbles, etc. However, this air may be undesirable in accordance with certain embodiments, and accordingly, the composition may be prepared by also including a step of removing air from the composition that has been introduced during manufacture.

[0090] In some embodiments, the composition, after removing at least some of the air may contain no more than 20 vol%, no more than 15 vol%, no more than 12 vol%, no more than 10 vol%, no more than 8 vol%, no more than 6 vol%, no more than 5 vol%, no more than 4 vol%, no more than 3 vol%, no more than 2 vol%, or no more than 1 vol%, etc. of air. In some cases, the air may be removed such that no air bubbles are visually present within the composition.

[0091] Without wishing to be bound by any theory, it is believed that the presence of air may reduce the viscosity of the composition, e.g., making it easier for the composition to flow. Accordingly, in some embodiments, any air that is introduced may be removed, thereby increasing the viscosity of the composition.

[0092] In one embodiment, air may be removed from a composition, e.g., during or after formation, by applying a pressure less than atmospheric or ambient pressure to the composition. For instance, the pressure that is applied may be less than 1 bar, less than 800 mbar, less than 600 mbar, less than 500 mbar, less than 400 mbar, less than 300 mbar, less than 200 mbar, less than 100 mbar, less than 75 mbar, less than 60 mbar, less than 50 mbar, less than 40 mbar, less than 30Atty Docket No.053032-548001WO mbar, less than 20 mbar, less than 10 mbar, less than 5 mbar, less than 3 mbar, less than 2 mbar, less than 1 mbar, etc. It should be noted that 1 atmosphere is approximately 1 bar, and that these pressures are absolute pressures (i.e., a pressure of less than about 1 bar means a pressure lower than atmospheric pressure, i.e., a vacuum pressure). Such pressures may be applied for any suitable length of time, e.g., at least 10 min, at least 20 min, at least 30 min, at least 45 min, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 6 h, at least 24 h, at least 1 day, etc. In addition, in some cases, the pressure may be applied until the composition comprises less than a certain amount of air, e.g., less than 15 vol%, or other percentages such as those described herein. As yet another example, in some cases, a solution may be caused to form a gel by removing a certain amount of air from the solution.

[0093] In certain embodiments, as another example, the air may be removed from the composition using a Versator. In a Versator, a material such as a liquid is spread onto the inside of a rotating Versator disc under vacuum to remove entrapped air, foam, gas, etc. While the liquid travels across the disc, the high vacuum draws off the bubbles, etc., from the liquid. Versators may be obtained from several commercial sources. Accordingly, in some embodiments, a composition such as described herein may be treated using a Versator for any suitable length of time, e.g., at least 10 min, at least 20 min, at least 30 min, at least 45 min, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 6 h, at least 24 h, at least 1 day, etc., and / or until the composition comprises less than a certain amount of air, e.g., less than 15 vol%, or other percentages such as those described herein

[0094] As yet another example, in some cases, the air may be removed from the composition using centrifugation. Without wishing to be bound by any theory, it is believed that by centrifuging the composition, air (being less dense) may be forced out of the composition. Thus, for example, the material may be centrifuged at any suitable speed, e.g., at least at least 500 RPM, at least 1,000 RPM, at least 2,000 RPM, at least 3,000 RPM, at least 5,000, at least 10,000 RPM, or the like, for any suitable length of time, e.g., at least 10 min, at least 20 min, at least 30 min, at least 45 min, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 6 h, at least 24 h, at least 1 day, etc., and / or until the composition comprises less than a certain amount of air, e.g., less than 15 vol%, or other percentages such as those described herein.Atty Docket No.053032-548001WO

[0095] Furthermore, it should be understood that these techniques are non-limiting, and that other methods of removing air, besides centrifuges or Versators, are also possible in still other embodiments.

[0096] In embodiments, the Lactobacillus is present at about 1 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 2 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 3 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 4 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 5 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 6 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 7 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 8 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 9 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 10 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 12 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 15 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 20 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 25 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 1 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 2 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 3 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 4 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 5 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 6 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 7 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 8 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 9 wt% to about 30 wt%. In embodiments, the Lactobacillus is present at about 10 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 12 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 15 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 20 wt% to about 25 wt%. In embodiments, the Lactobacillus is present at about 10 wt% to about 20 wt%. In embodiments, the Lactobacillus is present at about 1 wt%. In embodiments, the Lactobacillus is present at about 2 wt%. In embodiments, the Lactobacillus is present at about 3 wt%. In embodiments, the Lactobacillus is present at about 4 wt%. In embodiments, the Lactobacillus is present at about 5 wt%. In embodiments, the Lactobacillus is present at about 6 wt%. In embodiments, the Lactobacillus is present at about 7Atty Docket No.053032-548001WO wt%. In embodiments, the Lactobacillus is present at about 8 wt%. In embodiments, the Lactobacillus is present at about 9 wt%. In embodiments, the Lactobacillus is present at about 10 wt%. In embodiments, the Lactobacillus is present at about 11 wt%. In embodiments, the Lactobacillus is present at about 12 wt%. In embodiments, the Lactobacillus is present at about 13 wt%. In embodiments, the Lactobacillus is present at about 14 wt%. In embodiments, the Lactobacillus is present at about 15 wt%. In embodiments, the Lactobacillus is present at about 16 wt%. In embodiments, the Lactobacillus is present at about 17 wt%. In embodiments, the Lactobacillus is present at about 18 wt%. In embodiments, the Lactobacillus is present at about 19 wt%. In embodiments, the Lactobacillus is present at about 20 wt%. In embodiments, the Lactobacillus is present at about 21 wt%. In embodiments, the Lactobacillus is present at about 22 wt%. In embodiments, the Lactobacillus is present at about 23 wt%. In embodiments, the Lactobacillus is present at about 24 wt%. In embodiments, the Lactobacillus is present at about 25 wt%. In embodiments, the Lactobacillus is present at about 26 wt%. In embodiments, the Lactobacillus is present at about 27 wt%. In embodiments, the Lactobacillus is present at about 28 wt%. In embodiments, the Lactobacillus is present at about 29 wt%. In embodiments, the Lactobacillus is present at about 30 wt%. The amount may be any value or subrange within the recited ranges, including endpoints.

[0097] In embodiments, the Lactobacillus comprises one or more Lactobacillus species. In embodiments, the Lactobacillus species comprise one or more of Lactobacillus crispatus. Lactobacillus gasseri, Lactobacillus iners, Lactobacillus vaginalis, and Lactobacillus jensenii. In embodiments, the Lactobacillus species comprises Lactobacillus crispatus. In embodiments, the Lactobacillus species comprises Lactobacillus gasseri. In embodiments, the Lactobacillus species comprises Lactobacillus iners. In embodiments, the Lactobacillus species comprises Lactobacillus vaginalis. In embodiments, the Lactobacillus species comprises Lactobacillus jensenii.

[0098] In embodiments, the composition (or pharmaceutical gel) comprises a prebiotic. In embodiments, the prebiotic comprises a sugar. In embodiments, the sugar comprises one or more of glucose, dextrose, or maltose. In embodiments, the composition (or pharmaceutical gel) comprises glutamine.

[0099] In embodiments, the prebiotic is present at about 0.01 wt% to about 10 wt%. In embodiments, the prebiotic is present at about 0.1 wt% to about 10 wt%. In embodiments, theAtty Docket No.053032-548001WO prebiotic is present at about 1 wt% to about 10 wt%. The amount may be any value or subrange within the recited ranges, including endpoints. Methods of Treatment

[0100] In an aspect is provided a method for providing Lactobacillus to a vagina of a subject, comprising administering a pharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

[0101] In an aspect is provided a method for treating bacterial vaginosis in a subject, comprising administering a pharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

[0102] In an aspect is provided a method for preventing recurrence of bacterial vaginosis in a subject, comprising administering a pharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

[0103] In an aspect is provided a method for maintaining healthy biota in a vagina of a subject, comprising administering a pharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina subject.

[0104] The composition may be any composition as described herein, made as described herein, or made from a kit as described herein.

[0105] In embodiments, the composition is administered during or after administration of an antibiotic to the subject.

[0106] In embodiments, the method includes combining the Lactobacillus with the pharmaceutical gel prior to administration, wherein the pharmaceutical gel comprises the poloxamer and the stabilization polymer.

[0107] In addition, in accordance with certain embodiments, compositions as discussed herein may have relatively high viscosities such as those described herein may be particularly useful to treat subjects having bacterial vaginosis, or other indications. In some embodiments, the bacterial vaginosis may be determined by at least 3 separate diagnoses of bacterial vaginosis within a 365- day period within a subject. Without wishing to be bound by any theory, it is believed that suchAtty Docket No.053032-548001WO compositions may be able to prevent the composition from readily exiting the vagina, which may facilitate treatment of bacteria, e.g., that causes bacterial vaginosis, including recurrent bacterial vaginosis. This may result in improved subject outcomes, and in some cases, over a relatively short period of time (e.g., within 1 or 2 days, or within 3 days, etc.). For instance, such subjects may exhibit reduced vaginal discharge, reduced vaginal odors, increased vaginal pH, lower clue cell counts (which may indicate a smaller bacterial load), lower Nugent scores (which may indicate the degree of bacterial vaginosis), or the like, e.g., as discussed herein.

[0108] For example, in one set of embodiments as discussed herein is generally directed to systems and methods of reducing vaginal discharge. In some cases, subjects having bacterial vaginosis exhibit relatively large amounts of vaginal discharge. While healthy vaginas exhibit some amounts of discharge, those having bacterial vaginosis often exhibit significantly elevated vaginal discharge. In some cases, the discharge may exhibit a change in color, consistency, odor, etc., as compared to normal or healthy vaginal discharge. However, in certain embodiments, a vagina treated as discussed herein may exhibit a significantly less amount of vaginal discharge, e.g., as compared to before treatment. In addition, in some embodiments, there may be a significantly less amount of vaginal discharge that can be determined 1 day after treatment, 2 days after treatment, 3 days after treatment, etc.

[0109] Also, in certain embodiments, systems and methods as described herein may be used to decrease or reduce vaginal odor. Although vaginal discharge has a relatively mild odor, bacterial vaginosis is often associated with a stronger, unpleasant odor, which is sometimes described as a “fishy” smell associated with the discharge. However, in some embodiments, a vagina treated as discussed herein may exhibit a decrease in odor after treatment, e.g., as compared to before treatment. In addition, in some embodiments, there may be a significantly less amount of odor that can be determined 1 day after treatment, 2 days after treatment, 3 days after treatment, etc.

[0110] Without wishing to be bound by any theory, it is believed that such decreases in odor and / or discharge may be caused by action of composition such as discussed herein on bacteria within the vagina, e.g., that cause bacterial vaginosis. In some embodiments, improvements to the vagina may be determinable as improved vaginal function. For example, improved vaginal function may be determinable as an improvement to vaginal pH. Normal pH of the vagina isAtty Docket No.053032-548001WO around 3.8 to 4.5, but in bacterial vaginosis, the pH may be elevated (less acidic), which is more favorable to bacterial infections. Accordingly, in certain embodiments, a vagina treated as discussed herein may exhibit improved functionality, i.e., as evidenced by a more acidic environment and a decrease in pH to less than 4.5. In addition, in some embodiments, such pH changes may be relatively rapid, e.g., determinable 1 day after treatment, 2 days after treatment, 3 days after treatment, etc.

[0111] In another embodiment, improved vaginal function may be determined by determining a decrease in clue cells, which are epithelial cells of the vagina that may exhibit a distinctive stippled appearance when being covered or infected with bacteria. Improved vaginal function and reduced bacterial infection can thus be determined as a decrease in clue cells after treatment, as compared to before treatment. For example, a vagina that is treated as discussed herein may exhibit improved functionality, i.e., as evidenced by a decrease in clue cells. In addition, in some embodiments, such changes in clue cells may be relatively rapid, e.g., determinable 1 day after treatment, 2 days after treatment, 3 days after treatment, etc. Methods of Making

[0112] In an aspect is provided a method of making a Lactobacillus composition, comprising combining Lactobacillus and a pharmaceutical gel, wherein the pharmaceutical gel comprises a poloxamer and a stabilization polymer. The pharmaceutical gel may be any gel as described herein.

[0113] In embodiments, the Lactobacillus and the pharmaceutical gel are combined by: (a) providing the Lactobacillus in a first container; (b) providing the pharmaceutical gel in a second container; (c) connecting the first container and the second container, such that the Lactobacillus is in fluid connection with the pharmaceutical gel; and (d) transferring the Lactobacillus and the pharmaceutical gel between the two containers to make the Lactobacillus composition.

[0114] In embodiments, the first container is a syringe. In embodiments, the second container is a syringe. In embodiments, the first container and the second container are connected by a leur lock connector.

[0115] In embodiments, step (d) is performed by pushing the Lactobacillus into the second container, or by pushing the pharmaceutical gel into the first container. In embodiments, theAtty Docket No.053032-548001WO method includes pushing the Lactobacillus and pharmaceutical gel from the first container into the second container, or from the second container to the first container. In embodiments, the Lactobacillus and the pharmaceutical gel are pushed from the first and / or second container into the other container two or more times, for a sufficient number of times to make the Lactobacillus composition.

[0116] An example method for combining the Lactobacillus and pharmaceutical gel is as follows: • Provide an amount of Lactobacillus in a syringe (e.g., 10 mL syringe). • Syringe tip may be secured with a leur lock or other cap, or with lab tape. Without losing material, a plunger may be used to push the remaining air out of syringe. • Pharmaceutical gel is provided in a second syringe (e.g., 10 mL syringe). • A syringe coupler may be attached to the tips of each syringe. • Syringes are mixed by pushing the contents back and forth between syringes until a uniform gel with no agglomerates is observed. • Optionally, air may be removed after mixing (e.g., by centrifugation, pushing air out of the syringe, etc). Kits

[0117] In an aspect is provided a kit comprising Lactobacillus and a pharmaceutical gel, wherein the pharmaceutical gel comprises a poloxamer and a stabilization polymer. The pharmaceutical gel may be any gel as described herein.

[0118] In embodiments, the kit includes instructions for mixing the Lactobacillus and the pharmaceutical gel and administering the resulting composition to a vagina of a subject.

[0119] In embodiments, the Lactobacillus and pharmaceutical gel are in separate containers. In embodiments, the kit includes one or more applicators. In embodiments, the kit includes one or more vaginal applicators. In embodiments, the Lactobacillus and pharmaceutical gel are formulated to be combined to provide a composition as described herein.Atty Docket No.053032-548001WO

[0120] In embodiments, the Lactobacillus is in a first container and the pharmaceutical gel is in a second container. In embodiments, the first container and / or the second container is a syringe.

[0121] In embodiments, the kit includes at least one syringe. In embodiments, the kit includes two syringes. In embodiments, the kit includes a connector to connect the first container and the second container. In embodiments, the connector comprises a leur lock connector. In embodiments, the one or more vaginal applicators include a syringe.

[0122] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES

[0123] One skilled in the art would understand that descriptions of making and using the compositions described herein is for the sole purpose of illustration, and that the present disclosure is not limited by this illustration. Example 1. Thermal Viscosity and LVER for Gel Base Formulations

[0124] Thermal viscosity and linear viscoelastic region (LVER) of three formulations of base gel were tested, as indicated in Table 1. Results are provided in Tables 2-5 and FIGs.1-4. Table 1. Formulations Formulation 2303 2304 2305 Benzoic Acid (mg / g) 2.5 1.9 1.9 Citric Acid (mg / g) 9.0 9.0 6.0 Sodium Citrate (mg / g) 8.25 8.3 5.5Xanthan gum (mg / g) 20.0 20.0 20.0 Poloxamer 407 (mg / g) 165.0 165.0 165.0 Purified water (mg / g) 745.2 745.2 745.2 5N NaOH / HCl QS to pH 4.5 QS to pH 4.5 QS to pH 4.5Atty Docket No.053032-548001WO Table 2. Thermal Viscosity and LVER Results Formulation Measurement 2303 2304 2305 mPas 20°C 14,815 55,381 247,470 mPas 37°C 639,320 674,260 1,335,700 LVER Limit (Pa) 25°C 7,520 7,680 6,910 LVER Limit (Pa) 37°C 9,340 9,310 9,720 LVER tau (Pa) 25°C 7.79 7.98 35.7 LVER tau (Pa) 37°C 9.58 9.57 0.93 G'G" Crossover (Pa) 25°C 87.0 112.8 249.0 G'G" Crossover (Pa) 37°C 29.8 176.3 284.9 Table 3. Thermal Viscosity: 5-45°C Temp. Viscosity (mPa*s) (°C) 2303 2304 2305 4.91 95 164 95,501 6.58 88 154 92,137 8.36 89 142 88,947 10.15 71 137 86,018 11.94 67 147 83,724 13.72 78 155 84,335 15.5 110 217 93,200Atty Docket No.053032-548001WO 17.27 187 5,657 105,960 19.04 1,840 26,309 158,720 20.79 14,815 55,381 247,470 22.55 30,919 104,640 363,010 24.3 54,553 525,310 632,490 26.05 135,820 609,140 1,383,700 27.8 519,330 661,050 1,564,300 29.54 544,560 678,860 1,567,400 31.29 584,920 686,120 1,551,400 33.03 613,870 685,180 1,500,800 34.77 630,160 679,450 1,411,900 36.52 639,320 674,260 1,335,700 38.26 638,630 669,120 1,263,200 40 631,840 667,440 1,185,600 41.74 619,860 676,090 1,142,900 43.48 601,780 702,050 1,113,400 45.22 579,900 766,860 1,103,200 BA mg / g 2.50 1.90 1.90 CA mg / g 9.00 9.00 6.00 SC mg / g 8.25 8.25 5.50Atty Docket No.053032-548001WO Table 4. LVER at 25°C 2303 2304 2305Point Shear Shear No. Strain Stress Storage Loss Storage Loss Storage Loss Modulus Modulus Modulus Modulus Modulus Modulus [1] [Pa] [Pa] [Pa] [Pa] [Pa] [Pa] [Pa] 1 8.79E-05 0.7578 8490.7 1493.7 7804.6 1172.4 6679.3 1370.3 2 1.55E-04 1.1614 7374.4 1276.1 7712.9 1126.0 6689.2 1262.0 3 2.13E-04 1.6389 7615.7 1167.5 7823.3 1104.3 6800.4 1319.9 4 3.16E-04 2.4273 7594.3 1141.3 7822.3 1110.4 6833.2 1314.4 5 4.59E-04 3.5839 7712.5 1221.4 7878.9 1111.3 6844.3 1292.1 6 6.74E-04 5.2660 7720.3 1181.3 7894.0 1124.8 6923.3 1317.7 7 9.90E-04 7.7136 7699.3 1194.7 7900.4 1126.2 6968.8 1303.6 8 1.46E-03 11.2830 7605.9 1267.6 7870.2 1144.8 7017.4 1310.5 9 2.14E-03 16.2510 7467.2 1369.1 7816.6 1190.1 7047.6 1308.0 10 3.15E-03 23.2890 7251.4 1493.0 7700.6 1262.3 7055.3 1327.0 11 4.63E-03 32.8630 6901.6 1677.6 7486.7 1376.1 7022.4 1356.1 12 6.79E-03 45.0730 6343.5 1939.4 7133.6 1550.0 6916.8 1419.1 13 0.010 58.7420 5454.8 2226.3 6516.1 1805.4 6704.8 1536.7 14 0.015 71.5380 4277.3 2375.2 5202.3 2336.8 6272.7 1734.0 15 0.022 81.2080 3024.2 2254.3 3816.7 2472.5 5544.6 1967.0 16 0.032 87.0050 1955.3 1916.8 2764.4 2224.3 4579.9 2131.9 17 0.047 91.1680 1222.1 1514.3 1927.5 1866.9 3541.3 2142.2 18 0.069 94.7090 757.5 1145.1 1354.4 1525.3 2583.3 1980.2 19 0.101 98.6580 475.8 853.2 930.0 1213.4 1803.9 1704.0 20 0.149 104.8900 308.4 633.7 620.8 943.2 1226.1 1395.5Atty Docket No.053032-548001WO 21 0.218 110.0700 198.6 463.4 409.1 717.2 820.8 1097.8 22 0.320 116.1700 131.2 338.2 258.8 528.1 551.6 844.4 23 0.470 123.5300 85.8 248.6 161.7 380.8 373.0 633.4 24 0.688 132.9300 57.2 184.6 99.4 271.6 254.8 470.3 25 1.010 144.6800 37.0 138.7 63.3 195.3 176.3 346.8 Table 5. LVER at 37°C 2303 2304 2305Point Shear Shear No. Strain Stress Storage Loss Storage Loss Storage Loss Modulus Modulus Modulus Modulus Modulus Modulus [1] [Pa] [Pa] [Pa] [Pa] [Pa] [Pa] [Pa] 1 9.96E-05 0.9095 9012.7 1476.5 9101.0 1345.4 9141.7 939.3 2 1.50E-04 1.3792 9120.9 1346.8 9183.5 1275.1 9510.7 1547.6 3 2.15E-04 2.0038 9234.2 1355.5 9323.5 1397.9 9369.3 1524.3 4 3.09E-04 2.9030 9309.6 1361.5 9320.0 1292.9 9503.6 1534.6 5 4.55E-04 4.3071 9374.8 1336.8 9386.9 1389.4 9678.1 1483.3 6 6.78E-04 6.4430 9413.3 1309.0 9459.2 1329.6 9735.6 1514.4 7 9.96E-04 9.5376 9482.3 1359.7 9473.3 1335.9 9896.5 1498.6 8 1.46E-03 14.0470 9527.6 1369.7 9520.7 1347.4 9971.2 1517.3 9 2.13E-03 20.4810 9502.1 1406.4 9523.7 1369.1 10052.0 1536.0 10 3.14E-03 29.8000 9380.6 1467.9 9449.7 1422.3 10119.0 1542.5 11 0.005 42.8280 9140.7 1556.3 9278.7 1510.5 10097.0 1568.6 12 0.007 60.1980 8653.1 1743.9 8928.2 1696.1 9981.2 1642.5 13 0.010 79.6800 7658.9 2196.5 8202.4 2037.1 9662.0 1797.7 14 0.015 95.9380 5948.1 2718.6 6764.7 2562.9 8974.9 2080.9Atty Docket No.053032-548001WO 15 0.022 110.4300 4290.0 2765.2 5291.6 2710.7 7765.4 2465.7 16 0.032 124.8300 3026.5 2494.2 3871.5 2631.9 6120.7 2740.1 17 0.047 136.8900 2045.4 2110.5 2664.6 2345.0 4432.0 2737.9 18 0.068 146.3800 1330.9 1679.0 1712.4 1910.8 3023.9 2424.7 19 0.101 152.8700 838.7 1257.0 1063.5 1451.8 2045.9 1977.4 20 0.149 156.9100 521.2 917.3 659.2 1067.4 1353.2 1534.8 21 0.218 162.8900 329.6 669.4 407.5 773.1 895.5 1154.7 22 0.320 168.8700 209.1 484.0 250.8 550.1 575.8 834.1 23 0.469 174.2500 130.9 347.7 155.4 389.3 376.5 600.0 24 0.688 181.2400 82.7 250.3 96.5 274.7 239.6 424.9 25 1.010 189.8400 51.7 181.2 60.4 194.1 152.9 300.4

[0125] Appearance for 2303 (2.5 mg / g benzoic acid) indicate that the preservative may not be completely soluble in the gel. The formulation had a hazy appearance with a gel point above room temperature. When sitting undisturbed some partially solubilized material settles towards the bottom giving rise to two distinct layers. This formulation also exhibited an overall low viscosity and weak micro structure at 25 °C indicating the viscous properties were much more prevalent in this batch.

[0126] The two formulations with 1.9 mg / g benzoic acid formed a clear, well-structured gel at room temperature. The lower concentration of benzoic acid in 2304 also appeared to decreased the gel point by approximately 5°C, and increased the viscosity from approximately 14K mPa*s to approximately 55K mPa*s at 20 °C. The LVER for 2303 and 2304 were similar at 25°C and 37°C, but the G’G” cross over shear stress was much higher for 2304.

[0127] The buffer concentration in 2305 was decreased to compensate for the increase in ionic species contributed from the benzoic acid. The texture of this formulation was much more cohesive than 2303 or 2304, giving it a “sticky / stringy” texture, whereas the other formulationsAtty Docket No.053032-548001WO were cleaner during handling and transfer. This is demonstrated by the cross over point for 2305 which was more than double the value of 2304.

[0128] Although there was a significant difference in the viscosity at 37°C, the LVER and viscoelastic properties were very similar. It should be noted that the proposed limit for the LVER in 2305 was much lower compared to the other two formulations. This is due to a slight increase in G’ for this formulation. But the actual flow point and cross over were higher for this formulation.

[0129] Conclusion: based on the testing and observations, approximately 2.0 mg / g or less of benzoic acid appears to be soluble in the vehicle, but quantitative analytical analysis would be required to confirm this estimation. It also appears to be possible to adjust the viscosity and texture of the formulation, as well as the gel point, by modifying the ionic balance of the gel. Example 2. Rheological Properties of Gel Formulations with Lactobacillus

[0130] To evaluate the rheological impact of adding Lactobacillus to the gel, 500g of the pharmaceutical gel was prepared as per the composition presented in Table 6. Table 6. Lactobacillus pharmaceutical gel composition Ingredient % w / w mg / g Amount / Batch (g) Poloxamer 407 16.50 165.00 82.50 Xanthan Gum 2.00 20.00 10.00 Citric Acid Monohydrate 0.90 9.00 4.50 Sodium Citrate Dihydrate 0.83 8.25 4.13 Benzyl Alcohol 1.00 10.00 5.00 Purified Water 78.78 787.75 393.88 Total 100.00 1000.00 500.00

[0131] Lactobacillus (20% w / w) Gel sample preparation: Combined 5.0 g ± 0.05 g of Lactobacillus (Lactobacillus crispatus, Creative Enzymes) with 20.0 g of the pharmaceutical gelAtty Docket No.053032-548001WO using a spatula until no agglomerates were observed. About 1.25 g was transferred to a 3 mL syringe and centrifuged to remove air.

[0132] Lactobacillus (10% w / w) Gel sample preparation: Combined 4.0 g ± 0.01 g of Lactobacillus (Lactobacillus crispatus, Creative Enzymes) with 40.0 g of the pharmaceutical gel using a spatula until no agglomerates were observed. About 1.25 g was transferred to a 3 mL syringe and centrifuged to remove air.

[0133] Placebo gel: About 1.25 g of the pharmaceutical gel was transferred to a 3 mL syringe and centrifuged to remove air.

[0134] The results for the analysis of the Lactobacillus batches were compared to the acid- form (placebo) gel as a general reference to any observed changes. Rheology Testing: Thermal Viscosity Testing

[0135] PP 25 Measuring Device Procedure and Parameters: Prior to testing, 1.25g of sample was transferred into a 3 mL syringe and centrifuged for 15 minutes at 6000 RPM to remove any remaining air. Approximately 0.5 g of each sample was transferred to the plate at 25°C. The sample was trimmed and then cooled to 5°C prior to starting the test. Thermal viscosity for the product was performed under the following conditions: ^1 mm gap^ pre-shear of 31 / s for 60 seconds^ 3x temperature ramp 5-45°C, 24 data points, 15 second intervals, 0.22 1 / s shearrate.

[0136] Note: between ramp one and 2 the sample was cooled over the same interval. i.e. 45°C-to-5°C over 6 minutes. This was performed to prevent effects from the polymer thermal memory on the second ramp.

[0137] PP 50 Measuring Device Procedure and Parameters: Prior to testing, the sample was centrifuged in a 50 mL centrifuge tube for 10 minutes at 6000 RPM. Then, 3.0 mL ofAtty Docket No.053032-548001WO sample was transferred from a positive displacement pipette to the sample cup at 25°C. The sample was equilibrated to 5°C prior to starting the test. Thermal viscosity for the product was performed under the following conditions: ^1 mm gap^ pre-shear of 31 / s for 60 seconds^ 3x temperature ramp 5-55°C, 24 data points, 15 second intervals, 31 / s shear rate.

[0138] Note: between ramp one and 2 the sample was cooled over the same interval. i.e. 55°C-to-5°C over 6 minutes. This was performed to prevent effects from the polymer thermal memory on the second ramp. Amplitude Sweep

[0139] Prior to testing, 1.25g of sample was transferred into a 3 mL syringe and centrifuged for 15 minutes at 6000 RPM to remove any remaining air. Then, ~ 0.5 g of sample was transferred to the plate at the target temperature. Two separate tests were performed at two different target temperatures (25°C and 37°C).25 data points, 0.01% - 100% amplitude, 11 / s constant shear rate, 25°C; 25 data points, 0.01% - 100% amplitude, 11 / s constant shear rate, 37°C. Results

[0140] The appearance of the placebo gel is clear, as presented in FIG. 5. After mixing with the Lactobacillus the gel appearance changes to an off-yellow / yellow.

[0141] Addition of Lactobacillus at 10% and 20% had measurable impacts on the rheological properties of the gel. The most significant effects were observed for the thermal viscosity at 37°C and LVER in the 20% w / w sample (Tables 7 and 8 and FIG. 6). Also, for the 20% w / w sample, the gel point was ~8°C lower than the 10% w / w and reference samples. There were effects observed for the 10% w / w sample, but were minimal when compared to the placebo sample.Atty Docket No.053032-548001WO

[0142] Amplitude sweep results of the target formulation with 20% w / w active (Tables 9- 11 and FIGs.7-8), displayed a more “solid like” behavior at low shear rates (higher G’ / G” ratio), but had a less organized microstructure and lower flow point compared to the placebo sample.

[0143] Qualitatively, the stiffness and yield stress of the gel increases with the 20% w / w of Lactobacillus, and the gel takes on a more paste like texture and flow behavior. Table 7: Overview of Rheological results for Lactobacillus and Acid Form (Placebo) Gels Formulation Measurement 20% 10% Acid Form Gel Lactobacillus Lactobacillus (Reference) Viscosity (mPa*s) at 810,570 745,580 445,440 20°C Viscosity (mPa*s) at 595,990 818,510 989,980 37°C Gel Point (°C) 5.0 13.2 13.2 LVER Limit (Pa) 4,430 9,720 9,007 LVER tau (Pa) 4.59 10.1 19.6 G' / G" LVER Ratio 8.8 13.5 6.7 G'G" Crossover (Pa) 151 174 174 Table 8: Thermal Viscosity comparison of Lactobacillus and Placebo Gels Viscosity (mPas) °C 20% w / w Lactobacillus 10% w / w Lactobacillus Reference5.0 18,832 2,875 80,777 13.2 764,430 5,401 85,873 21.1 810,570 745,580 445,440 29.0 716,150 882,190 1,065,300 36.9 595,990 818,510 989,980 44.8 462,040 690,810 1,091,900Atty Docket No.053032-548001WO Table 9: Amplitude Sweep for 20%w / w Lactobacillus Gel Point Shear Shear Storage Loss Loss No. Strain Stress Modulus Modulus Factor Torque [1] [Pa] [Pa] [Pa] [1] [µN·m] 1 0.000100 0.46 4587.90 532.96 0.12 17.02 2 0.000148 0.67 4509.70 517.61 0.12 24.72 3 0.000212 0.98 4577.40 511.34 0.11 35.83 4 0.000316 1.45 4566.80 514.37 0.11 53.24 5 0.000464 2.13 4561.10 513.89 0.11 78.16 6 0.000681 3.13 4563.30 493.91 0.11 114.73 7 0.001000 4.60 4564.70 512.59 0.11 168.77 8 0.001470 6.75 4553.50 524.87 0.12 247.71 9 0.002160 9.86 4531.70 534.48 0.12 361.89 10 0.003180 14.37 4487.30 558.77 0.13 527.32 11 0.004660 20.71 4405.70 600.32 0.14 760.09 12 0.006870 29.62 4260.80 670.27 0.16 1087.10 13 0.010000 41.15 4024.50 771.83 0.19 1510.20 14 0.014700 55.61 3676.10 901.51 0.25 2040.90 15 0.021500 72.70 3213.80 1035.30 0.32 2668.20 16 0.031500 91.41 2672.10 1132.00 0.42 3354.60 17 0.046100 111.00 2110.90 1157.80 0.55 4073.70 18 0.068100 131.60 1584.20 1108.20 0.70 4829.40 19 0.100000 151.34 1144.00 991.79 0.87 5554.20 20 0.147000 170.21 799.16 840.33 1.05 6246.70 21 0.215000 187.92 543.45 681.97 1.26 6896.30 22 0.317000 205.58 365.45 536.84 1.47 7544.50 23 0.465000 223.44 244.10 414.03 1.70 8200.00 24 0.682000 241.79 161.66 315.25 1.95 8873.40 25 1.000000 263.58 107.94 239.89 2.22 9673.00 Table 10: Amplitude Sweep for 10% w / w Lactobacillus Gel Point Shear Shear Storage Loss Loss No. Strain Stress Modulus Modulus Factor Torque [1] [Pa] [Pa] [Pa] [1] [µN·m] 1 0.000097 0.98 10085.00 710.91 0.07 36.07 2 0.000148 1.49 10046.00 676.41 0.07 54.56 3 0.000215 2.16 10036.00 777.29 0.08 79.29Atty Docket No.053032-548001WO 4 0.000317 3.20 10076.00 774.69 0.08 117.46 5 0.000466 4.72 10107.00 792.58 0.08 173.26 6 0.000685 6.95 10114.00 774.95 0.08 254.92 7 0.001000 10.15 10086.00 764.01 0.08 372.38 8 0.001470 14.85 10082.00 773.49 0.08 545.10 9 0.002150 21.72 10046.00 790.34 0.08 796.91 10 0.003170 31.63 9953.20 827.97 0.08 1160.90 11 0.004650 45.65 9774.50 910.77 0.09 1675.40 12 0.006840 64.74 9404.70 1079.20 0.12 2375.90 13 0.010000 88.29 8685.90 1430.20 0.17 3240.20 14 0.014700 113.28 7437.40 2013.30 0.27 4157.30 15 0.021600 134.60 5645.50 2651.10 0.47 4939.80 16 0.031700 153.77 3940.90 2836.50 0.72 5643.30 17 0.046500 173.52 2727.20 2545.10 0.93 6368.10 18 0.068300 191.73 1865.20 2097.50 1.13 7036.40 19 0.100000 206.84 1223.30 1659.30 1.36 7590.70 20 0.147000 219.69 772.41 1274.00 1.65 8062.50 21 0.217000 231.76 475.30 957.32 2.01 8505.50 22 0.319000 242.25 282.22 705.46 2.50 8890.20 23 0.468000 252.06 162.67 512.97 3.15 9250.30 24 0.688000 264.49 93.66 372.99 3.98 9706.60 25 1.010000 277.29 53.21 269.91 5.07 10176.00 Table 11: Amplitude Sweep for Acid Form Gel Point Shear Shear Storage Loss Loss No. Strain Stress Modulus Modulus Factor Torque [1] [Pa] [Pa] [Pa] [1] [µN·m] 1 0.000100 0.90 8974.90 1285.20 0.14 4.16 2 0.000146 1.33 8962.60 1317.70 0.15 6.11 3 0.000214 1.94 8980.20 1374.00 0.15 8.94 4 0.000316 2.86 8970.50 1365.80 0.15 13.19 5 0.000465 4.23 8994.80 1377.80 0.15 19.48 6 0.000679 6.18 9003.40 1377.50 0.15 28.48 7 0.000992 9.03 9002.70 1375.00 0.15 41.61 8 0.001460 13.29 9003.50 1380.10 0.15 61.24 9 0.002150 19.56 9006.70 1387.90 0.15 90.09 10 0.003160 28.76 8999.50 1399.10 0.16 132.46Atty Docket No.053032-548001WO 11 0.004640 42.20 8975.90 1417.30 0.16 194.39 12 0.006820 61.57 8907.40 1466.10 0.17 283.59 13 0.010000 88.85 8715.00 1573.60 0.18 409.25 14 0.014800 125.27 8260.10 1805.80 0.22 577.05 15 0.021700 167.36 7382.80 2193.40 0.30 770.92 16 0.031900 211.15 6089.40 2572.50 0.42 972.60 17 0.046800 251.01 4624.50 2719.50 0.59 1156.20 18 0.068200 280.83 3214.10 2569.30 0.80 1293.60 19 0.100000 311.84 2157.60 2239.40 1.04 1436.40 20 0.147000 338.10 1386.70 1829.80 1.32 1557.40 21 0.216000 368.48 900.39 1448.90 1.61 1697.30 22 0.316000 397.63 580.58 1115.70 1.92 1831.60 23 0.464000 428.22 377.04 842.90 2.24 1972.50 24 0.682000 460.72 248.59 628.64 2.53 2122.20 25 1.000000 491.51 165.54 462.68 2.80 2264.00

[0144] The changes in thermal setting and to some extent the flow point and cross over are likely due to the solid content interfering with the lattice formation. However, the overall rheological profile is maintained (thermal setting, viscosity, and yield stress) and may be adjusted / increased by modifications to the formulation to increase the ratio of poloxamer, xanthangum, or buffer concentration. As such, the next section presents a design of experiment to evaluatethe effects of poloxamer and xanthan gum composition changes on the rheology of the product. Example 3. Formulation Screening

[0145] A small formulation screening was carried out to assess the effect of changes to the %w / w of poloxamer 407 and xanthan gum. The study was intended to determine the feasibility of a formulation containing 20% w / w Lactobacillus with suitable gel and thermal setting properties for retention in the area of application (vaginal canal). For this evaluation, 5 separate 100 g batches were prepared. The %w / w of poloxamer 407 and xanthan gum were varied according to the experimental design (Table 12). The citric acid, sodium citrate, and benzyl alcohol were maintained at constant %w / w; and the water was adjusted based on the poloxamer 407 and xanthan gum composition to maintain the total amount of 100g. Table 12: Overview of Experimental DesignAtty Docket No.053032-548001WO Run ID StdOrder Center Xanthan Poloxamer Pt Blocks Gum %w / w 407 %w / w Water 2302-01 1 1 1 1 16.50 79.78 2302-02 4 1 1 3 19.50 74.78 2302-03 5 0 1 2 18.00 77.28 2302-04 2 1 1 3 16.50 77.78 2302-05 3 1 1 1 19.50 76.78 178008-2301 (Placebo Reference) N / A N / A N / A 2 16.50 78.78 Testing Thermal viscosity: 5-45°C 3x ramp at 0.221 / s. report viscosity over range Amplitude Sweep: 0.1-100% amplitude at 25°C and 11 / s. Report LVER limit, flow point, and cross over

[0146] The formulation compositions of are provided in Tables 13-17. Batch preparation steps, addition of Lactobacillus, and sampling procedures are referenced after the tables. Table 13: 2302-01 Formulation Composition Item Ingred Amount / No. ient % w / w mg / g Batch (g) 1 Poloxamer 407 16.50 165.00 16.50 2 Xanthan Gum 1.00 10.00 1.00 3 Citric Acid Monohydrate 0.90 9.00 0.90 4 Sodium Citrate Dihydrate 0.83 8.25 0.83 5 Benzyl Alcohol 1.00 10.00 1.00 6 Purified Water 79.78 797.75 79.78Atty Docket No.053032-548001WO Total 100.00 1000.00 100.00 Table 14: 2302-02 Formulation Composition Item I Amount / No. ngredient % w / w mg / g Batch (g) 1 Poloxamer 407 19.50 195.00 19.50 2 Xanthan Gum 3.00 30.00 3.00 3 Citric Acid Monohydrate 0.90 9.00 0.90 4 Sodium Citrate Dihydrate 0.83 8.25 0.83 5 Benzyl Alcohol 1.00 10.00 1.00 6 Purified Water 74.78 747.75 74.78 Total 100.00 1000.00 100.00 Table 15: 2302-03 Formulation Composition Item Ingr Amount / No. edient % w / w mg / g Batch (g) 1 Poloxamer 407 18.00 180.00 18.00 2 Xanthan Gum 2.00 20.00 2.00 3 Citric Acid Monohydrate 0.90 9.00 0.90 4 Sodium Citrate Dihydrate 0.83 8.25 0.83 5 Benzyl Alcohol 1.00 10.00 1.00 6 Purified Water 77.28 772.75 77.28 Total 100.00 1000.00 100.00 Table 16: 2302-04 Formulation CompositionAtty Docket No.053032-548001WO Item o. Ingr Amount / N edient % w / w mg / g Batch (g) 1 Poloxamer 407 16.50 165.00 16.50 2 Xanthan Gum 3.00 30.00 3.00 3 Citric Acid Monohydrate 0.90 9.00 0.90 4 Sodium Citrate Dihydrate 0.83 8.25 0.83 5 Benzyl Alcohol 1.00 10.00 1.00 6 Purified Water 77.78 777.75 77.78 Total 100.00 1000.00 100.00 Table 17: 2302-05 Formulation Composition Item Ingr Amount / No. edient % w / w mg / g Batch (g) 1 Poloxamer 407 19.50 195.00 19.50 2 Xanthan Gum 1.00 10.00 1.00 3 Citric Acid Monohydrate 0.90 9.00 0.90 4 Sodium Citrate Dihydrate 0.83 8.25 0.83 5 Benzyl Alcohol 1.00 10.00 1.00 6 Purified Water 76.78 767.75 76.78 Total 100.00 1000.00 100.00

[0147] Batches were prepared as follows: 500 mL jacketed vessel was set up, filled with coolant, and equilibrated to 2-5°C. Water was added to the vessel and equilibrated to 5-10°C with slow mixing. Citric acid and sodium citrate were added and the solution mixed between 120-150 RPM until completely dissolved. Benzyl alcohol was added and the solution mixed between 120- 150 RPM until completely dissolved. Poloxamer 407 was added to the solution and mixed slowlyAtty Docket No.053032-548001WO between 50-80 RPM until completely dissolved (~4-6 hours). Xanthan gum was added to the solution and mixed slowly between 50-80 RPM until completely dissolved (~4-6 hours). For placebo gel, approximately 1.25g was transferred to a 3 mL syringe and centrifuged for 30 minutes to remove air.

[0148] For Lactobacillus (20% w / w) Gel sample preparation, 5.0 g ± 0.05 g of Lactobacillus was added to 20.0 g of the placebo gel using a spatula and mixed (~1-2 minutes) until no particulates were observed. Approximately 1.25g Lactobacillus gel was transferred to a 3 mL syringe and centrifuged for 30 minutes to remove air. Rheology Testing

[0149] Rheology testing was performed as described in Example 2. Thermal rheology testing was performed with 3x temperature ramp 5-45°C, 6 points, 1-minute intervals, 0.221 / s shear rate.

[0150] Visually, incorporation of the Lactobacillus was fairly rapid. The gel appeared uniform with no observable large clumps or dry powder after mixing with a spatula. The appearance of the gels (FIG.9) was very similar in color. However, there were some qualitative differences in the texture for each sample. Batch 2302-01 did not form a stiff gel prior to adding the Lactobacillus; this sample was closer to a viscous fluid. The remaining samples did form typical gels, but samples 2302-03 and 2302-04 were notably more viscous at room temperature. Additionally, batch 2302-04 presented as a much more cohesive material compared to the other samples. Batch 2302-02 formed the most elegant gel but also appeared to have a fairly low yield point and spread more like a fluid when small amount of shear was applied. The results are presented in comparison to the pilot prototype (178008-2301) of the 20% Lactobacillus gel which was used for the initial evaluation. The target for the study was to assess the degree of improvement to the thermal viscosity and rheological properties which could be achieved based on minor adjustments to the formulation.

[0151] Thermal Viscosity Results Comparison of the thermal viscosity indicated that most of the gels demonstrated significant thermal setting between 5 - ~20°C; and there was a sharp decline above ~30°C. This was most likely due to rapid dehydration of the polypropylene oxideAtty Docket No.053032-548001WO and decreased solubility disrupting the pseudo micelle lattice. This is evidenced by the most significant drops being observed in samples 2302-02 and -05 which contained the highest amounts of poloxamer. Compared to the reference sample, batch 2302-04 demonstrated the greatest improvement in the thermal viscosity, and the smallest drop off after 30 °C (Table 18 and FIG. 10). Table 18: Summary of Thermal Viscosity for 2302-01 - 05 Temp Batch / Viscosity (mPas) 178008-2301 (°C) 2302-01 2302-02 2302-03 2302-04 2302-05 20% Ref. 4.9 14,322 43,770 26,740 11,246 29,498 31,419 13.2 12,571 624,980 703,470 875,580 511,890 740,370 21.0 21,711 505,860 768,940 919,840 551,530 788,460 28.9 94,655 375,380 693,420 953,500 460,030 680,340 36.9 50,933 288,870 612,100 839,250 348,710 564,790 44.7 17,694 216,030 472,550 717,340 214,340 444,290 Note: The thermal viscosity for 178008-230120% Lactobacillus is referenced in the table above for comparison purposes.

[0152] Amplitude Sweep Results Review of the G’G” and LVER data (Table 19) mirrored some of the qualitative observations from appearance and texture. The amplitude sweep profiles of G’ and G’’ for 2302-01 – 05 are compared to the reference batch 178008-2301 (20% Lactobacillus) in Figures 11-15. Sample 2302-01 exhibited a low G’G” ratio with a poorly defined yield point, and a relatively low transition between the viscoelastic solid and viscous fluid state. Batch 2302-02 had the highest LVER but a sharp transition to the viscous fluid state with a low yield stress (tau), which indicated a relatively weak microstructure. Samples 2302-03 and 04 present the highest yield stress with a relatively similar limit to the LVER compared to the reference. Table 19: Summary of LVER and Flow Point for 2302-01 – 05Atty Docket No.053032-548001WO LVER Flow Batch Limit Point G’ FP tau FP % G’:G” (Pa) (Pa) (Pa) gamma Ratio 2302-01 1050 190 58.75 21.89 4.4 2302-02 5690 1524 120 5.56 8.9 2302-03 3700 485 163 23.78 6.8 2302-04 4200 573 183 22.53 7.0 2302-05 3710 799 68 6.03 7.9 178008-230120% Reference 4430 876.9 165.3 13.33 8.8

[0153] Due to this being a screening study, in which no replication occurred, an evaluation of significance for the effects was not possible. Review of the main effects (FIG.11) suggested a non-linear correlation between the input factors and viscosity or flow point shear stress. Generally, 2% w / w xanthan gum and 18% w / w poloxamer 407 produced the highest values for viscosity at 37°C and flow point. However, the LVER and G’G” ratio improved linearly in relation to both xanthan gum and poloxamer %w / w.

[0154] Based on this screening, sample 2302-04 demonstrated sufficient thermal setting (~800k mPas) and viscoelastic properties (gamma 22.5%) to support retention of the gel in the vaginal canal following administration. The formulation texture may be improved by optimizing the ratio of poloxamer and xanthan gum. Additionally, from the main effects a formulation composed of 18.0-18.5% poloxamer and 2.5-3.0% xanthan gum may yield further improvement to the rheological properties. Alternatively, using a mixture of poloxamer 407 or 188 may increase thermosetting behavior. Example 4. Formulation Screening with Benzoic Acid

[0155] It was determined that benzyl alcohol may cause adsorption to the applicator plastic of applicator barrels. Due to the potential applicator barrel incorporation during the end-of- production filling process, benzyl alcohol would not be suitable for stability studies.

[0156] The previous formulation was modified for the incorporation of benzoic acid (2.5 and 1.9 mg / g) as a substitute for benzyl alcohol. For this evaluation, 3 separate 500 g batches of the placebo gel were prepared with the base formulation compositions of 16.5% w / w poloxamerAtty Docket No.053032-548001WO and 2.0% w / w xanthan gum for direct comparison to the previous formulation to assess the impact of the change. Additionally, the formulation procedure was modified for monitoring / adjusting the pH of benzoic acid as it slowly dissolved in a 2-8°C environment.

[0157] Batch 178008-2303 was prepared with 2.5 mg / g of benzoic acid and 1.73% w / w total buffer concentration. Batches 178008-2304 and -2305 were prepared with 1.9 mg / g of benzoic acid and with different total buffer concentrations (1.73% and 1.15% w / w). The third formulation was prepared to assess impact on the total buffer concentration on the rheology These placebo batches were tested for thermal viscosity and amplitude sweep.

[0158] To improve the mixing process of Lactobacillus crispatus, a syringe coupler was incorporated. The syringe coupler allows two leur-lok tip syringes to be connected such that a powder and a semi-solid material can be mixed efficiently. Using the syringe coupler, two separate drug products were prepared: 2% w / w Lactobacillus and 2% w / w Lactobacillus and 2% w / w D- Glucose. After mixing the drug product with and without d-glucose, samples were tested for thermal viscosity and compared with the placebo gel.

[0159] The formulation composition of batches 178008-2303 – 2305 are provided in Tables 20-22. Batch preparation steps, mixing Lactobacillus with and without d-glucose, and sampling procedures are described after the tables. Table 20: 178008-2303 Formulation Composition Item Ingredient % w Amount / No. / w mg / g Batch (g) 1Lactobacillus crispatus 0.00 0.00 0.002 Poloxamer 407 16.50 165.00 82.50 3 Xanthan Gum 2.00 20.00 10.00 4 Citric Acid Monohydrate 0.90 9.00 4.50 5 Sodium Citrate Dihydrate 0.83 8.25 4.13 6 Benzoic Acid 0.25 2.5 1.25 7 5N NaOH / HCl QS to pH 4.5 QS to pH 4.5 QS to pH 4.5 8 Purified Water 74.52 745.2 372.6 9 Purified Water QS 5.00 50.00 25.00 Total 100.00 1000.00 500.00Atty Docket No.053032-548001WO Table 21: 178008-2304 Formulation Composition Item Ingredi Amount / No. ent % w / w mg / g Batch (g) 1Lactobacillus crispatus 0.00 0.00 0.002 Poloxamer 407 16.50 165.00 82.50 3 Xanthan Gum 2.00 20.00 10.00 4 Citric Acid Monohydrate 0.90 9.00 4.50 5 Sodium Citrate Dihydrate 0.83 8.30 4.13 6 Benzoic Acid 0.19 1.90 0.95 7 5N NaOH / HCl QS to pH 4.5 QS to pH 4.5 QS to pH 4.5 8 Purified Water 74.52 745.2 372.6 9 Purified Water QS 5.06 50.6 25.3 Total 100.00 1000.00 500.00 Table 22: 178008-2305 Formulation Composition Item o. Ingredie Amount / N nt % w / w mg / g Batch (g) 1Lactobacillus crispatus 0.00 0.00 0.002 Poloxamer 407 16.50 165.00 82.50 3 Xanthan Gum 2.00 20.00 10.00 4 Citric Acid Monohydrate 0.60 6.00 3.00 5 Sodium Citrate Dihydrate 0.55 5.50 2.75 6 Benzoic Acid 0.19 1.90 0.95 7 5N NaOH / HCl QS to pH 4.5 QS to pH 4.5 QS to pH 4.5 8 Purified Water 74.52 745.2 372.6 9 Purified Water QS 5.64 56.4 28.2 Total 100.00 1000.00 500.00

[0160] To prepare the batches, a jacketed vessel was set up and equilibrated to 2-8°C. Purified water was added and citric acid added with mixing (130-150 RPM) until dissolved. Sodium citrate was added to the solution and mixed until completely dissolved. Benzoic acid was added to the solution and pH monitored; pH was adjusted with 5N sodium hydroxide to maintain a pH of 4.5. The solution was mixed until fully dissolved. Mixing speed was reduced and poloxamer 407 added, mixing until fully dissolved. Xanthan gum was added to the solution and mixed until completely dissolved. pH was measured and adjusted with 5N sodium hydroxide to pH 4.5. Added purified water as needed to bring to 500 g total per batch (“QS”) and mixed for 15Atty Docket No.053032-548001WO minutes. Transferred approximately 20 g of sample to a 50 mL centrifuge tube and centrifuged for 30 minutes at 6000 RPM to remove air.

[0161] For placebo gel, approximately 1.25g of centrifuged gel was transferred to a 3 mL syringe and centrifuged for 15 minutes to remove air. For Lactobacillus (2% w / w) gel formulation, 100 mg Lactobacillus was mixed with approximately 4.9 g of centrifuged gel composition until a uniform yellow / off-yellow gel with no agglomerates was observed. Approximately 1.25 g of the Lactobacillus (2% w / w) gel formulation was transferred to a 3 mL syringe and centrifuged for 30 minutes at 6000 RPM to remove air. For Lactobacillus (2% w / w) and D-Glucose (2% w / w) sample preparation, 100 mg of Lactobacillus and 100 mg of d-glucose were mixed with approximately 4.9 g of centrifuged gel composition until a uniform yellow / off-yellow gel with no agglomerates was observed. Approximately 1.25 g of the Lactobacillus (2% w / w) and D-Glucose (2% w / w) gel formulation was transferred to a 3 mL syringe and centrifuged for 30 minutes at 6000 RPM to remove air.

[0162] To mix Lactobacillus into the gel, the following procedure was used: Lactobacillus (2% w / w) sample preparation:^ Weighed 100 mg of Lactobacillus and transferred into a 10 mL syringe.^ Secured syringe tip with lab tape. Without losing material, used a plunger and pushed theremaining air out of syringe. Note: For batch 178008-2303, the placebo gel formulation exhibited two distinct layers when sitting undisturbed at room temperature. The formulation was mixed with a spatula for 2 minutes prior to transferring to a centrifuge tube. After centrifuging for 10 minutes at 6000 RPM, the placebo gel was a clear, viscous gel.^ Transferred ~ 5g of centrifuged placebo gel into a 5 mL syringe.^ From the 5 mL syringe, transferred 4.9 g of centrifuged placebo gel into a tared 10 mLsyringe.^ Set plunger to ~ 5-6 mL without losing material.^ Attached syringe coupler to the tips of each 10 mL syringe.^ Began mixing syringes until a uniform yellow / off-yellow gel with no agglomerates wasobserved.^ Transferred ~ 1.25g to a 3 mL syringe and centrifuged for 30 minutes at 6000 RPM toremove air. Note: Prior to centrifugation of the sample, the gel gradually changed to a liquid after ~ 1-2 minutes at room temperature.Atty Docket No.053032-548001WO Lactobacillus (2% w / w) and D-Glucose (2%w / w) sample preparation:^ Weighed 100 mg of Lactobacillus and 100 mg of d-glucose and transferred into the same 10mL luer-lok tip syringe.^ Secured syringe tip with lab tape. Pushed remaining air out of syringe with plunger withoutlosing material.^ Note: For batch 178008-2303, the formulation was mixed with a spatula for 2 minutes priorto transferring to a centrifuge tube. After centrifuging for 10 minutes at 6000 RPM, the placebo gel was a clear, viscous gel.^ Transferred ~ 5g of centrifuged placebo gel into a 5 mL syringe.^ From the 5 mL syringe, transferred 4.9 g of centrifuged placebo gel into a tared 10 mLsyringe.^ Set plunger to ~ 5-6 mL without losing material.^ Attached syringe coupler to the tips of each 10 mL syringe.^ Began mixing syringes until a uniform yellow / off-yellow gel with no agglomerates wasobserved.^ Transferred ~ 1.25g to a 3 mL syringe and centrifuged for 30 minutes at 6000 RPM toremove air. Note: Prior to centrifugation of the sample, the gel gradually changed to a liquid after ~ 1-2 minutes at room temperature. Rheology Testing

[0163] Rheology testing was performed as described in Example 2.

[0164] When sitting undisturbed at room temperature, the appearance for batch 178008- 2303 (2.5 mg / g benzoic acid) was visually observed to contain some partially solubilized material settled towards the bottom and yielding two distinct layers. Based on the visual observations, it appeared that at least a portion of the benzoic acid was not completely dissolved. After mixing with a spatula for 2 minutes and centrifuging the sample, the gel was well-structured and clear (FIG.12). Batches 2304 and 2305 (1.9 mg / g benzoic acid) formed a clear and well-structured gel at room temperature. After centrifuging the placebo gel and mixing 178008-2303 with the Lactobacillus, the drug product would begin to liquify at room temperature (FIG. 13). The drug product for batches 2304 and 2305 did not exhibit this behavior and was a well-structured yellow / off-yellow gel at room temperature.

[0165] Thermal Viscosity: The gel point in batch 178008-2303 exhibited an increase of ~5°C in the gel point and a decrease in the viscosity from 55K mPa*s to 14K mPa*s at 20°C whenAtty Docket No.053032-548001WO compared to batch 178008-2304 (Table 23).The thermal viscosity for placebo batch 178008-2305 (FIG. 14) was significantly higher in viscosity and the gel point was at least 2°C lower when compared to the other batches. This formulation had a decreased buffer concentration to compensate for the increase in ionic species contributed from the benzoic acid. For reference, Table 24 provides an overview of viscosity for 2303-2305 placebo gels. Table 23: 178008-2303 – 2305 Overview of Thermal Viscosity and Amplitude Sweep Measurement Formulation 178008-2303 178008-2304 178008-2305 mPas 20°C 14,815 55,381 247,470mPas 37°C 639,320 674,260 1,335,700Table 24: 178008-2303 – 2305 (Placebo) Thermal Viscosity (5-45°C) Temp. (°C) Viscosity (mPa*s) 2303 2304 2305 4.91 95 164 95,5016.58 88 154 92,1378.36 89 142 88,94710.15 71 137 86,01811.94 67 147 83,72413.72 78 155 84,33515.50 110 217 93,20017.27 187 5,657 105,96019.04 1,840 26,309 158,72020.79 14,815 55,381 247,47022.55 30,919 104,640 363,01024.30 54,553 525,310 632,49026.05 135,820 609,140 1,383,70027.80 519,330 661,050 1,564,30029.54 544,560 678,860 1,567,40031.29 584,920 686,120 1,551,40033.03 613,870 685,180 1,500,80034.77 630,160 679,450 1,411,90036.52 639,320 674,260 1,335,70038.26 638,630 669,120 1,263,20040.00 631,840 667,440 1,185,60041.74 619,860 676,090 1,142,90043.48 601,780 702,050 1,113,40045.22 579,900 766,860 1,103,200Benzoic Acid mg / g 2.50 1.90 1.90Citric Acid mg / g 9.00 9.00 6.00Atty Docket No.053032-548001WO Sodium Citrate mg / g 8.25 8.25 5.50

[0166] For the drug product with d-glucose, an increase in viscosity was observed when compared to the drug product after ~18°C in batch 2304 and overall, in batches 2303 and 2305 (Table 25 and FIGs.15-17). Additionally, significantly higher viscosity was observed overall in batch 2305 when compared to batches 2303 and 2304 with and without addition of d-glucose. Table 25: 178008-2303 – 2305 Thermal Viscosity Comparison of Placebo and DP 2303 2304 2305 mPa*s mPa*s mPa*s Temp (°C) DP DP DP with D- Placeb with D- Placeb with D- DP DP DP Placebo Glucos o Glucos o Glucos e e e 15 163 875 110 6,913 2,312 217 28,596 36,167 93,200 21 10,322 41,079 14,815 93,556 107,060 55,381 92,154 207,300 247,470 541,26 648,59 801,23 37 608,790 639,320 682,530 674,260 864,330 1,335,700 0 0 0

[0167] Amplitude Sweep: The placebo batches were tested for LVER limit, flow point, and cross-over point (Table 26) to evaluate their viscoelastic properties. Batch 2303 exhibited an overall low viscosity and weak microstructure at 25°C (FIG.18), indicating the viscous properties were much more prevalent in this batch. The LVER for 2303 and 2304 were similar at 25°C and 37°C, but the G’G’’ cross over shear stress was much higher for batch 2304. Table 26: 1780082303 - 2305 Overview of LVER (25°C and 37°C) Formulation Measurement 178008-2303 178008-2304 178008-2305LVER Limit (Pa) 25°C 7,520 7,680 6,910 LVER Limit (Pa) 37°C 9,340 9,310 9,720 LVER tau (Pa) 25°C 7.79 7.98 35.7Atty Docket No.053032-548001WO LVER tau (Pa) 37°C 9.58 9.57 0.93 G'G" Crossover (Pa) 25°C 87.0 112.8 249.0 G'G" Crossover (Pa) 37°C 29.8 176.3 284.9

[0168] The texture of batch 2305 was much more cohesive compared to batches 2303 and 2304 giving it a “sticky / stringy” texture. Whereas the other formulations were cleaner during handling and transfer. This is demonstrated by the crossover point for batch 2305, which was more than twice the value of batch 2304. Although there was a significant difference in the viscosity at 37°C, the LVER and viscoelastic properties (FIG.19) were very similar. It should be noted that the proposed limit for the LVER in batch 2305 was much lower compared to the other two formulations. This is due to a slight increase in G’ for this formulation. However, the flow point and cross over were higher for this formulation.

[0169] Based on the testing and observations ~2.0 mg / g or less of benzoic acid appears to be soluble in the vehicle, but quantitative analytical analysis would be required to confirm this estimation. It also appears to be possible to adjust the viscosity and texture of the formulation, as well as the gel point, by modifying the ionic balance of the gel. This was evidenced by the increase in viscosity which was inversely correlated to the buffer concentration.

[0170] The drug product with d-glucose exhibited an increase in the thermal viscosity profile when compared to the drug product without d-glucose. D-glucose was added to the formulation to support / act as a probiotic for the Lactobacillus. Example 5. Poloxamer 407 and Xanthan Gum Material Evaluation

[0171] To investigate the impact of the raw material attributes, and provide some formulation design space concerning these attributes, an evaluation for 5 different lots of poloxamer 407 and xanthan gum was performed. Each lot was compared for correlations between the differences in the raw material physicochemical properties (provided by the manufacturer’s certificate of analysis) and the measured rheological properties which might impact the formulation efficacy.Atty Docket No.053032-548001WO Poloxamer 407 Evaluation

[0172] For the poloxamer 407 evaluation, each lot was prepared as a 14%, 16%, and 20% w / v solution in water. Rheological testing was then performed on these solutions and compared to observe any significant correlations between the lots and the effect of concentration. MiniTab statistical software was used to assess correlations between the properties listed on the manufacturer’s CoA and the rheological results using linear and non-linear regression models, as well as main effect analysis. An ancillary analysis of poloxamer 407 was also performed with 2 separate HPLC methods: size exclusion chromatography (SEC), and reverse-phase chromatography / CCAD. Each method was used to assess differences in the molecular weight distribution from lot-to-lot.

[0173] Poloxamer 407 samples were tested for single point viscosity and thermal rheology. The thermal profiles for each concentration of poloxamer were used to determine the critical micelle temperature (CMT). The CMT is the temperature at which the poloxamer 407 becomes more structured and forms a gel. From a visual standpoint, the temperature at which the shear stress (Pa) of the solution begins to increase significantly is the CMT. For each concentration of poloxamer 407, the CMT was determined and plotted versus the corresponding %w / v. The resulting graph provided a linear correlation for which the slope is representative of the CMT coefficient for that lot of poloxamer.

[0174] Poloxamer 407 was added to purified water and mixed (in Camframo BDC overhead mixer with the Rushton mixer blade) at 200 RPM until completely dissolved. Product was centrifuged at 6000 RPM for 10 minutes to remove air. If visible air pockets remained, repeated this step until all visible air has been removed from the product. Rheology testing was performed as described in Example 2.

[0175] A review / comparison of the CoAs (Table 27) evidenced only some small differences between most ot the physicochemical properties; the most significant difference noted was the average molecular weight. An overview of the rheological properties is provided in Table 28. The properties for the 16% w / v are referenced here to have a similar comparison to the base Lactobacillus formulation composition (16.5% w / w).Atty Docket No.053032-548001WO Table 27: Poloxamer 407 Certificate of Analysis Physicochemical Properties un % pH Un Congea R satura Name Supplier Lot# M (2.5% ling ID W Oxyethy tion lene soluti temp on) (mEQ / g) (°C) 2313- Kolliphor Sigma- BCCH14 119 01 P407 Aldrich 73 36 72.5 6.7 0.0430 54.0 2313- Kolliphor 02 P407 BASF GNG065 110 21B 00 72.5 6.9 0.0380 52.0 2313- Kolliphor 03 P407 BASF GNF096 125 21B 81 72.5 6.5 0.0430 54.0 2313- Kolliphor 04 P407 BASF GND113 139 21B 36 72.3 6.8 0.0390 54.0 2313- Poloxamer 05 407 BASF GNF222 119 21B 36 72.7 6.7 0.0410 51.0 Table 28: Results for Tested Poloxamer 407 Rheological Properties CMT 16 Ma Max 16%S 16%S Coef Tau Run f % x PV PV N Tem ID ame Supplier Lot# . GP Tau (mPa* (mPa* (Slop (°C 16 p s) 31 / s s) 31 / s e) ) % 16 % 25°C 37°C 2313- Kolliphor Sigma- BCCH14 01 P407 Aldrich 73 -2.50 37. 121. 8 2 44.3 67.0 38,346 2313- Kolliphor GNG0652 42. 115. 02 P407 BASF -2.89 5 48.7 41.2 9,946 Geismar 1B 1 2313- Kolliphor 124. 03 P407 BASF GNF0962 1 -2.19 37. 1 46.5 69.6 39,571 Geismar B 8 2313- Kolliphor BASF GND1132 - 31. 156. 04 P407 1B 1.41 2 6 53.1 138.8 50,967Atty Docket No.053032-548001WO 2313- Poloxamer GNF22 05 407 BASF 22 1B -2.11 35. 139. 6 0 44.3 87.0 44,672 CMT Coeff.: Critical Micelle Temperature Coefficient GP: Gelling Point Tau: Shear Stress (Pa) SPV: Single Point Viscosity

[0176] Prior to analysis of the data a review of the probability distribution plots indicated a normal distribution of data for the gel point of the 16% solution, the CMT coefficient, max shear stress (tau), max shear stress temperature; because of the normal distribution of the data, analysis of variance and linear regression modeling can be used to predict outcomes based on the model.

[0177] An exponential increase in the single point viscosity was observed in correlation with an increase in the %w / v of P407 in solution (Table 29). These data suggest that small changes in the concentration of the polymer may have an exaggerated impact on viscosity. Additionally, an increase in temperature (25°C to 37°C) correlated to an increase in viscosity which follows the expected thermal properties of the material. The SPV at 25°C and 37°C was determined to have a semi-linear correlation with the average molecular weight, as observed from the main effects plots (FIG.20). Table 29: Single Point Viscosity and Single Point Shear Stress (25 and 37°C) SPV (mPa Single Point Single Point Batch %w / v *s) Sh SPV (mPa*s) 25°C ear Stress Shear Stress (Pa) 25°C 37°C (Pa) 37°C 14% 35.0 0.1 172.4 0.5 2313-01 16% 67.0 0.2 38,346.0 115.0 20% 46,393.0 139.2 96,929.0 290.8 14% 20.3 0.1 43.7 0.1 2313-02 16% 41.2 0.1 9,945.9 29.8 20% 285.0 0.9 100,220.0 300.7 14% 31.7 0.1 106.8 0.3 2313-03 16% 69.6 0.2 39,571.0 118.7 20% 53,819.0 161.5 103,030.0 309.1 14% 45.2 0.1 20,496.0 61.5 2313-04 16% 138.8 0.4 50,967.0 152.9 20% 85,042.0 255.1 110,320.0 330.9 2313-05 14% 34.5 0.1 219.5 0.7Atty Docket No.053032-548001WO 16% 87.0 0.3 44,672.0 134.0 20% 58,035.0 174.1 105,490.0 316.4

[0178] Thermal Rheology and CMT: The thermal profiles illustrate the change in viscosity relative to temperature, and at lower concentration a marked impact based on the average molecular weight. As previously described, these data were used to determine the CMT for each sample, (Table 30), and subsequently, the CMT coefficient for each lot of material. Table 30: CMT for P407 Solutions Run ID CMT (14%) CMT (16%) CMT (20%) 2313-01 44.3 37.8 29.02313-02 48.7 42.1 31.22313-03 42.1 37.8 29.02313-04 35.6 31.2 26.82313-05 42.1 35.6 29.0

[0179] The R2 value for the CMT coefficient was greater than 0.96 for all the lots included in this study which demonstrates a good fit for the data. It is important to note that the difference for the observed CMT and maximum shear stress decreased significantly between the low (14% w / v) and high (20% w / v) concentration. For example, at 14% w / v run 2313-02 (11,000 da) had a CMT of ~49°C and a maximum shear stress of 0.2 Pa. Run 2313-04 (13,936 Da) resulted in 36°C and 39.9 Pa respectively at 14% w / v. However, the results for these samples at 20% w / v were: 2313-02 [31.2°C, 298.8 Pa] and 2313-04 [26.8°C, 297.8 Pa]. The observed difference for the CMT and maximum shear stress decreased significantly at the higher concentrations. This trend was consistent for all the lots included in this study and suggests that increasing the amount of poloxamer in the drug product formulation may reduce the variance derived from lot-to-lot differences in the average molecular weight.

[0180] A regression model for the CMT coefficient vs the average MW of P407 evidenced a strong correlation between the material attribute and the corresponding rheology. The p-value for the model was 0.012 and the R2value is 90.99%, which indicates the model may be considered predictive. Both the regression model and the main effects plot illustrated a linear correlation suchAtty Docket No.053032-548001WO that the critical micelle temperature decreases in relation to both the concentration of poloxamer in solution, and the average molecular weight. These data do not directly correlate to the drug product / gel formulation, but it can be inferred that the gel point and maximum viscosity would be significantly impacted by both the concentration and molecular weight of the excipient.

[0181] A separate regression model evaluated the gelling point at 16% w / v vs the average molecular weight of poloxamer. This was evaluated because it was the closest data point to the drug product formulation (16.5% w / w). These data demonstrated an inverse correlation for the CMT; and an increase in the maximum observed shear stress with the molecular weight of the polymer. From this it can be expected that the gel point of the drug product may decrease up to 10°C, and the viscosity may increase 10-50% as the molecular weight of the excipient increases.

[0182] No other trends / correlations were found between the material attributes listed on the certificates on analysis and the measured rheological properties.

[0183] The results of this study evidenced that the molecular weight of poloxamer 407 had a significant effect on the rheological properties of formulations prepared from this material. Strong correlations were observed between the average molecular weight and the single point viscosity, CMT, and thermal rheology. It was also noted that the impact on the thermal rheology was reduced as the concentration of poloxamer in the solution increased.

[0184] For the SEC / HPLC analysis no correlations were found between the peak %area of the peaks and any of the physical properties or rheological results. For the CAD / HPLC analysis a loose correlation was observed between the peaks at retention times ~8.9 and ~12.9 and the average molecular weight provided on the certificate of analysis. However, there is no direct effect on the rheological properties based on the percent area for either of these peaks. Xanthan Gum Evaluation

[0185] The purpose of this study was to conduct a material analysis of five different lots of xanthan gum from 2 different manufacturers (CP Kelco for 3 lots and Vanderbilt for 2 lots). Each of these lots were assessed for differences in their physicochemical properties that might impact the formulation. Xanthan gum solutions were prepared at concentrations of 1%, 2%, andAtty Docket No.053032-548001WO 3% w / w in purified water. Rheological testing was then performed on these solutions. Moisture content was also tested in parallel with the rheology to determine if there were any significant differences from the CoA.

[0186] Xanthan gum was added to purified water and mixed (in Camframo BDC overhead mixer with the Rushton mixer blade) at 200 RPM until completely dissolved. Product was centrifuged at 6000 RPM for 10 minutes to remove air. If visible air pockets remained, repeated this step until all visible air has been removed from the product. Rheology testing was performed as described in Example 2. Moisture content testing and loss on dry testing were also performed.

[0187] For this analysis, a flow curve was generated from each sample, plotting shear rate against viscosity. From these data, shear-thinning coefficients and eta-0 values (theoretical viscosity at 0 shear) were derived. These values were then compared among the lots and against the test criteria specified in the certificates of analysis (CoAs) to determine if there were differences that could potentially affect the base viscosity or rheology of the raw material. For reference, Table 31 provides the certificate of analysis values for the physicochemical properties of all the lots tested.

[0188] A comparison of the rheological data (as presented in Table 32) revealed that all lots examined in this study exhibited consistent results for shear-thinning coefficients, viscosity, and eta-0. The data presented reasonable levels of variation. Furthermore, when the relationship with solution concentration (1-3% w / w) was examined, very similar properties were observed.

[0189] Additionally, moisture content was assessed to confirm and evaluate any variations. Each lot of xanthan gum exhibited LOD values (Table 32) similar to those in the CoA and consistent with the USP monograph. Table 31: Reported Certificate of Analysis Physicochemical Properties Particl Assay Particl Assay e Size e Size IP Viscosit Run Manf. (% Pyruvic (80 A LO pH (1% Lot# Xantha (%CO Acid % mesh ( y Viscosit ID 200 pp D (mPa*s y Rat Solutio um) 2) 180µm mes io n G h m % n) 75 ) ) µm)Atty Docket No.053032-548001WO 2313 -06 7B5794K 102.8 4.8 4.5 100.0 99.0 60.0 6.7 1445.0 1.2 7.2 2313 -07 0E0782K 95.0 4.4 4.3 100.0 NA 56.0 8.9 1544.0 1.2 6.7 2313 -08 2524439 Conform s NA 6.1 99.0 NA 89.0 10.3 1380.0 1.0 7.4 2313 -09 2553930 Conform s 4.8 Conform s 99.0 NA 34.0 8.2 1714.0 N / A 6.9 2313 -10 22-001655 99.4 4.6 4.2 100.0 NA 38.0 10.2 1419.0 1.1 6.9 Table 32: 2313-06 to 2313-10 Rheological Properties and Physical Testing 2% w / w Avg. Shear Eta-0 Log xanthan Moisture Run ID Lot# Thinning. Slope gum Eta LOD (%) Content (%) Coeff. (mPas) 0.221 / s (mPas) 2313-06 7B5794K -0.883 8206 47347 9.2 11.4 2313-07 0E0782K -0.858 10509 48854 8.0 10.7 2313-08 2524439 -0.893 7633.5 51895 11.4 26.8 2313-09 2553930 -0.876 8637.5 44196 7.5 9.3 2313-10 22-001655 -0.846 10521 44678 9.0 11.6 Average -0.871 9101.4 47394 9.0 14.0 StDev 0.019 1338.68 3162.15 1.5 7.2 Note: The viscosity (mPa*s) for the Eta 0.221 / s of 2%w / w xanthan gum was calculated by the viscosity equation (y=k*xb)

[0190] Screening for the main effects indicated no significant correlation between the differences observed in the CoA values and the rheological properties of the xanthan gum lots. Based on the results of this study, there were no apparent differences among the lots of xanthan gum that were likely to have an impact on the drug product formulation. Example 6. Formulation Selection Ranging

[0191] This example was performed to assess the effect of changes to the %w / w of poloxamer 407, total buffer (citric acid monohydrate and sodium citrate dihydrate), and xanthan gum. In addition, high-shear mixing was incorporated in the placebo production process. ThisAtty Docket No.053032-548001WO change does not impact the rheology of the drug product, but increases the rate of excipient dissolution. The purpose of the study was to determine a suitable formulation to support filling into a vaginal applicator and retain suitable viscosity and thermal setting properties for retention in the physiological space.

[0192] For this evaluation, 5 separate 300 g batches of the gel were prepared. Lactobacillus and D-glucose were then mixed with the gel using a leur-lok syringe. The % w / w of poloxamer407, total buffer, and xanthan gum were varied according to the experimental design (Table 33).A lot of poloxamer with an average molecular weight of 11,936 g / mol was used for the study because the MW was most representative of the typical raw material as obtained from BASF. Table 33: Experimental Design for ST-2023-20 StdOrder RunID CenterPt BlocksPoloxamer% %w / w Total %w / w w / w Buffer Xanthan Gum 12320-01 1 1 17.50 0.60 2.254 2320-02 1 1 20.00 1.73 2.255 2320-03 0 1 18.75 1.17 2.132 2320-04 1 1 20.00 0.60 2.003 2320-05 1 1 17.50 1.73 2.00

[0193] The formulation composition of batches 2320-01 to 2320-05 are provided in Table 34. Batch preparation steps, mixing Lactobacillus with d-glucose, and sampling procedures are described after the tables. Table 34: Formulation Compositions % w / w Item No. Ingredient 2320-01 2320-02 2320-03 2320-04 2320-051 Poloxamer 407 17.50 20.00 18.75 20.00 17.50 2 Xanthan Gum 2.25 2.25 2.13 2.00 2.00 3 Citric Acid Monohydrate 0.31 0.90 0.61 0.31 0.90 4 Sodium Citrate Dihydrate 0.29 0.83 0.56 0.29 0.83 5 Benzoic Acid 0.19 0.19 0.19 0.19 0.19 6 5N HCl QS to pH QS to pH QS to pH QS to pH QS to pH 4.5 4.5 4.5 4.5 4.5Atty Docket No.053032-548001WO 7 Purified Water 73.52 73.52 73.52 73.52 73.52 8 Purified Water QS 5.94 2.32 4.24 3.69 5.07 Total 100.00 100.00 100.00 100.00 100.00

[0194] Batches were prepared in a 1 L jacketed vessel with recirculating water bath and Caframo BDC overhead mixer with a 1.0” pitch blade turbine impeller. Purified water was added to the vessel and mixing begun at 650 RPM, 2-8°C. Citric acid and sodium citrate were added to the water and mixed until completely dissolved. Benzoic acid was added to the solution and pH adjusted with 5N sodium hydroxide to maintain a pH of 4.5; mixed until completely dissolved. Changed to ross mixer with the 2” Cowles blade, mixing at 650 RPM, 2-8°C. Poloxamer 407 was added to the solution and mixed until completely dissolved. Xanthan gum was added to the solution and mixed for ~ 5 minutes (increased viscosity). Mixing speed was increased to 2500 RPM and mixed for 15 minutes. pH was adjusted to 4.5 with 5N HCl while mixing at 2500 RPM. Purified water was added QS and mixed at 2500 RPM for 15 minutes. Gel was passed through the ointment mill 3 times at 45 RPM with gap settings (front / back): pass 1 (2mm / 7mm), pass 2 (1mm / 4mm) and pass 3 (0mm / 2mm). ~ 20 g of sample was transferred to a 50 mL centrifuge tube and centrifuged for 60 minutes at 6000 RPM to remove air. If air pockets remain, centrifuged for an additional 60 minutes at 6000 RPM to remove air.

[0195] For placebo gel, approximately 1.25g of centrifuged gel was transferred to a 3 mL syringe and centrifuged for 15 minutes to remove air. For Lactobacillus (2% w / w) and D-Glucose (2% w / w) sample preparation, 100 mg of Lactobacillus and 50 mg of d-glucose were mixed with approximately 4.85 g of centrifuged gel composition until a uniform yellow / off-yellow gel with no agglomerates was observed. Approximately 1.25 g of the Lactobacillus (2% w / w) and D- Glucose (2% w / w) gel formulation was transferred to a 3 mL syringe and centrifuged for 15 minutes to remove air.

[0196] Rheology testing was performed as described in Example 2. Rheology was tested for the placebo and drug product. Single point viscosity, thermal rheology, amplitude sweep, and flow curve were evaluated for any significant differences between 2320-01 to -05. Minitab was utilized to provide statistical analysis for the results by main effect plots and interaction plots of multiple components. An overview of the results is provided in Table 35.Atty Docket No.053032-548001WO Table 35: 2320-01 – 2320-05 Overview of Rheological TestingAtty Docket No.053032-548001WO

[0197] Results for batches 2320-01, -03, and -05 indicated slight decreases in viscosities at 0.221 / s from the placebo to the drug product, and 2320-04 a significant decrease (Table 35). However, 2320-04 exhibited the highest viscosity, and the poloxamer composition was at the highest limit with the lowest buffer concentration. Batch 2320-05 exhibited the lowest viscosity, and the poloxamer composition was at the lowest limit with the highest buffer concentration. Overall, the viscosities at 25°C were improved when compared to previous formulations.

[0198] A main effects plot illustrated that a non-linear correlation existed between %w / w of poloxamer 407 and the single point viscosity (mPa*s) at 25°C and both shear rates for the placebo and drug product. An inverse non-linear correlation was also observed for the buffer concentration and the single point viscosities at 0.221 / s and 31 / s. At 0.221 / s, a significant impact on viscosity was observed from the interaction plot of the %w / w poloxamer and %w / w of xanthan gum for the placebo and drug product. A significant interaction was also observed between total buffer and %w / w of xanthan gum for the placebo and drug product. These findings were the same for the 31 / s shear rate. Although, at 31 / s poloxamer had a slightly increased interaction with the total buffer for the drug product when compared to the drug product at 0.221 / s.

[0199] Overall, the thermal rheology profiles for batches 2320-02, -03, and -05 were relatively similar for the placebo and drug product when compared to -01 and -04 (FIG. 21). Interactions between the %w / w of poloxamer and xanthan gum and %w / w total buffer and xanthan gum impact the viscosity and the thermal profile. Batches -01 and -04 have 0.60% w / w total buffer which exhibited an overall significant viscosity profile increase for the placebo and drug product (FIG.22) when compared to the 1.17% and 1.73% w / w batches. However, when comparing the placebo, batches -02, -03, and -05 exhibited a slight decrease in thermal viscosity profiles for the drug product when compared to the significant decrease for -01 and -04. These significant decreases from the placebo to the drug product may be attributed from the overall composition of %w / w poloxamer, total buffer, and xanthan gum.

[0200] For the placebo and drug product at 10°C, the most significant impact on the viscosity was the buffer concentration between 0.60 and 1.17 %w / w. The interactions between the 17.5% poloxamer, 0.60% total buffer, and 2.25% w / w xanthan gum at 10°C had the mostAtty Docket No.053032-548001WO significant impacts on increased viscosity for the placebo and drug product. For the placebo and drug product at 37°C, the most significant impact on the viscosity was the buffer concentration between 0.60 and 1.17 %w / w. Placebo interactions with %w / w xanthan gum and the total buffer had slight impacts at 37°C, whereas %w / w xanthan gum and poloxamer had major impacts on the viscosity. For the drug product interactions, major impacts occurred with the poloxamer and total buffer, and poloxamer with xanthan gum.

[0201] CMT was determined visually from the thermal profiles for the placebo and drug products. Overall, the CMT values ranged from 17.3°C to 20.8°C for the placebos and 15.5°C to 19.1°C for the drug products. Overall, the most significant interaction on the CMT for the placebo was between the total buffer and xanthan gum. Additionally, the interaction appeared to be non- linear as the center point for the total buffer / xanthan gum correlated with the high point for the buffer. These findings indicated a more stable thermal setting for the interactions with the total buffer / xanthan gum at 1.17% / 2.00-2.13% w / w. For the drug product, the most significant interaction on the CMT was between the poloxamer and xanthan gum. Another interaction was non-linear and between the total buffer / xanthan gum, with the center point for the total buffer / xanthan gum correlating with the low point for the buffer. This finding represents the CMT would exhibit a more stable setting for the interactions with the total buffer / xanthan gum at 0.60%- 1.17% / 2.13% w / w.

[0202] The LVER limit (Pa) and the flow point at 25°C and 37°C (G’G’’) for the placebo of batches 2320-01 and -02 were the lowest and highest, respectively, when compared to the other batches. Since this relates to the structural rigidity of the product, the shear stress (log Pa) that must be applied to the -02 product to induce flow would be significantly higher than the -01 product. This quality may be due to the compositions of poloxamer and total buffer, as -02 product has the highest %w / w of each component and -01 product has the lowest %w / w.

[0203] When compared to the other batches at 25°C, 2320-01 exhibited a significantly weaker microstructure with a slow transition into a viscoelastic fluid. The G’ / G’’ ratio of -01 and -02 are the lowest and highest, respectively, at 25°C and 37°C (Table 35), indicating a stiffer and less rigid structure for -01 and a softer, more rigid structure with a clean break for -02. On the otherAtty Docket No.053032-548001WO hand, the LVER limit, flow points, and microstructures at 25°C and 37°C for 2320-03 to -05 were similar.

[0204] The purpose of this viscosity curve is to measure how the gel behaves when subjected to increasing shear rates. As such, results demonstrated a significant decrease in viscosity as the shear rate approaches 21 / s (FIGs.23 and 24). All flow curves exhibited an R2 value of at least 0.975 which demonstrates a good fit for the data. For both the placebo and drug products, batch 2320-04 had the highest flow curve constant, followed by 2320-01. At any shear rate, -04 will exhibit a higher viscosity when compared to the remaining batches. In addition, the flow curves of all placebo batches, except for -05, exhibited higher flow curve constants than the drug products.

[0205] Based on these results, a more viscous product can result from a combination of 20%w / w poloxamer, 0.60 % w / w to 1.17 %w / w total buffer, and 2.25% w / w xanthan gum at temperatures of 25°C and above. At 10-15°C, batches 2320-02, 03, and 05 exhibited a viscosity of 50-100K mPa*s, which is an ideal viscosity for the purpose of filling into an applicator. All CMT values were between 15.5 – 20.8°C and is an ideal range for future formulations. As such, the following recommendations for future formulation prototypes are provided below. The formulation selection was based on several key targets for the drug product: • A viscosity of <50-100K mPa*s below 15°C to assist with air removal and filling operation during the manufacturing. • A CMT between 18-25°C to reduce the viscosity during the mixing and provide sufficient temperature range to meet the previous target. • A viscosity of NLT 1.5 million mPa*s at 37°C to ensure vaginal retention after application. • Minimize changes in viscosity after mixing with the active ingredient. • Stablize the viscosity of the drug product over time. • Provide sufficient preservtive for microbiological stability in the drug product, without negatively affecting the activity of Lactobacillus after addition-prior to use. • Reduce the impact on viscosity / CMT from lot-lot-variations in the molecular weight of poloxamer 407.Atty Docket No.053032-548001WO Table 36: Recommended Formulations Formulation (% w / w) Ingredient 123Poloxamer 407 19.00 19.00 19.00Xanthan Gum 2.10 2.00 2.00Citric Acid Monohydrate 0.50 0.42 0Lactic Acid 0 0 0.26Sodium Citrate Dihydrate 0.45 0.38 0Sodium Phosphate Monobasic 0 0 0.23Benzoic Acid 0.19 0.19 0.195N HCl / NaOH QS to pH 4.5 QS to pH 4.5 QS to pH 4.5Purified Water 73.52 73.52 73.52Purified Water QS 4.24 4.49 4.80Total 100.00 100.00 100.00

[0206] With consideration of these criteria the selected formulations all target 19.0% w / w for poloxamer 407. This is because higher concentrations, above 17-18%, have demonstrated reduced impact for both viscosity changes after mixing with the active component, and changes on the molecular weight of the excipient. The formulation also reduced the concentration of the buffer from the previous level(s) to help improve the overall viscosity. The alternative formulation (3) incorporates an additional change to the buffer components to evaluate the impact of lactic acid on the long term rheological stability. Example 7. Evaluation of Alternative Formulations

[0207] The formulations in Table 36 were made as described above and tested for rheological properties. Placebo and Lactobacillus (2% w / w) and D-Glucose (1% w / w) samples were prepared as described above and air removed.

[0208] The appearances of 2306 – 2308 were similar to previous formulations. Placebo gels were clear with no particulate matter and the drug product gels were white to off-white / opaque with no visible agglomerates. Rheology was tested for the placebo and drug product. Single point viscosity, thermal rheology, and amplitude sweep were evaluated.Atty Docket No.053032-548001WO

[0209] Placebo gels for all batches (2306-2308) were above 2 million mPa*s, and 2308 exhibited an increase in viscosity of ~200K more than the others (Table 37). However, at a 0.22 1 / s shear rate, the viscosity has more variability when compared to 1-31 / s, as the high point on the exponential graph is exceptionally larger. This was determined from the flow curves in the previous study. The drug product gel for 2308 resulted in a slight decrease in viscosity when compared to the significant decrease for -06 and -07. Table 37: 2306-2308 Overview of Single Point Viscosity Sample Viscosity (mPa*s) 178008-2306 178008-2307 178008-2308 Placebo 2,011,000 2,075,000 2,277,000 Drug Product 1,521,000 1,745,000 2,197,600

[0210] The thermal viscosity profiles of batches 178008-2306 to 2308 exhibited an increase which correlated to the buffer concentration. (FIG. 25), as 2308 exhibited the highest profile. At 15 °C and below, the viscosities for -06 and -07 (~130,000 – 186,000 mPa*s) were slightly higher than the target of 50,000-100,000 mPa*s. Additionally, batch -08 had a significantly higher viscosity (~230,000 – 250,000 mPa*s) than the target. However, these data met the predictive target of no less than 1.5 million mPa*s at 37°C.

[0211] The CMT of the products were 19.1°C-20.8°C and met the target range of 18 -25°C (Table 38). Table 38: CMT (°C) Batch 178008-2306 -2308 Batch CMT (°C) 178008-2306 20.8178008-2307 19.1178008-2308 19.1

[0212] A sharp transition to the crossover indicated a soft but stiff material with a clean break for 2306 (Table 39 and FIG.26). Batches 2307 and 2308 exhibited slightly longer transitions to the crossover compared to -06, which indicated that both are slightly softer and less rigid yetAtty Docket No.053032-548001WO stiff with a clean break. A pronounced LVER and high G’:G” ratio in the LVER suggested distinct solid phase behavior at low shear for all batches. Table 39: 178008-2306 to 2308 Overview of LVER and Crossover Formulation Measurement 178008-2306178008-2307 178008-2308 LVER Limit (Pa) 25°C 7,910 5,820 7,620 LVER tau (Pa) 25°C 40.6 30.1 39.4 Cross Over (Pa) 25°C 1,860 1,516 1,970 G’G’’ Ratio 5.6 5.2 5.8

[0213] The products meet the CMT and 37°C thermal viscosity requirements from the predictive range. Viscosities from the placebo to the drug product were significantly decreased for 178008-2306, while 2307 and 2308 exhibited the least decrease.

[0214] Below 15°C, the thermal viscosity resulted in a higher viscosity than the predictive range of 50K-100K mPa*s. For the filling process, batch 178008-2306 may be slightly viscous to fill into an applicator tube at 5°C-10°C but should be manageable. Batches 178008-2307 and 2308 may be too viscous for the filling process and some filling evaluations at to assess this further are recommended.

[0215] There were several significant findings based on the studies conducted to evaluate the formulation components in their overall impact on the rheology of the acid form and drug products. 1. The average molecular weight of poloxamer 407 significantly impacted the single point viscosity, thermal rheology, and CMT of solutions. Concentration (%w / w) of P407 also reduced the impact on thermal rheology. 2: The formulation composition of the total buffer and poloxamer 407 significantly impacted the viscosity, thermal rheology, and CMT of the overall product. Adjusting the total buffer to 0.8 – 0.95% w / w and 19.0% w / w poloxamer 407 resulted in an increased viscosity compared to the earlier formulations.Atty Docket No.053032-548001WO 3. Interactions with xanthan gum and the citric acid / sodium citrate buffer were significantly impacting the CMT of the placebo gel while slightly impacting the drug product. 4. An additional formulation was prepared with a buffer of lactic acid and sodium phosphate that substituted for the citric acid and sodium citrate buffer. The overall viscosities of the acid form and drug product was higher than the formulations with the original buffer, and the drug product was closer in viscosity to the acid form. Example 8. Short Term Stability

[0216] Two formulations (Formulations 2 and 3 from Table 36, referred to in this Example as 178008-2307 and 178008-2308, respectively) of Placebo gel (without Lactobacillus) were prepared and evaluated over 2 months at 50°C, 40°C / 75% RH, and 25°C / 60% RH. Included in this study was a comparison of 2 different buffer systems which were assessed for any impact on the rheological stability.

[0217] The schedule and testing is outlined in Table 40. Table 40: Stability Schedule and Testing Time PointsTargetTest Method Description Criteria T=0 2 wks 1M 2M Report Results: Single Point A at 2 minutes Rotational Rhe ,B, A,B, Viscosity ometery X A C C and 31 / s at 25°C LVER and Flow A, Report Results: Oscillating RheB, A,B, Pointometery X AC C at 0.01%-100% and 51 / s Report Results: Average of two Thermal Viscosity thermal ramps and Gel Point (°C) Rotational Rheometery X A A,B, A,B, C C (5°C-45°C) at 1 minute intervals and 0.221 / s pH USP <791> X A A,B, A,B, 4.4-4.6 C CAtty Docket No.053032-548001WO Clear Gel with no indication of phase Appearance Visual X A A,B, A,B, C C separation, agglomerates, or particulate matter Assay of Sodium HPLC A,B, A,B, 90.0%-110.0% Benzoate (RD-0314) X A C C T=0 X 50°C A 40°C / 75%RH B 25°C / 60%RH C

[0218] The samples were stored at the upright position under the following conditions for 2 months: 25°C / 60%RH: 8 applicators total (for each placebo formulation) ^4 applicators at T=1 and 2 months40°C / 75%RH: 8 applicators total (for each placebo formulation) ^4 applicators at T=1 and 2 months50°C: 12 applicators total (for each placebo formulation) ^2 applicators at T=0.5, 1, and 2 months

[0219] Samples (5.0g) were packaged in vaginal applicators and overwrapped in foil with the plunger. Packaging Information: Vaginal Applicator Barrel: Manufacturer: HTI Plastics Part Number: 109777 PC00282, REC2301109 Vaginal Applicator Plunger:Atty Docket No.053032-548001WO Manufacturer: HTI Plastics Part Number: 109776 P00280, REC230110 Cap, Pink: Manufacturer: HTI Plastics Part Number: 104698 PC00281, REC2301108 Foil Sachet: 4” x 7” O.D. Mylar Foil Pouch, bottom tear notch Manufacturer: PAKVF4 PC00283, REC2301147

[0220] These data (Table 41) suggested that for all storage conditions pH had no significant change over 2 months of stability under all storage conditions. The visual appearance for the gels remained clear and colorless without any phase separation, and no agglomerates or particulate matter was observed. Table 41: pH and Appearance Results Appearance Storage Time p Pha Agglomerates Batch se Condition Point H Color Separatio / n Particulate Matter Ambient T=04.4 Clear / Colorless No Absent 2 Weeks 4.4 Clear / Colorless No Absent 50°C 4. Clear / 1M 4 Colorless No Absent 178008- 2307 2M 4.3 Clear / Colorless No Absent 40°C / 75%R 4. Clear / 1M 4 Colorless No Absent H 2M 4.4 Clear / Colorless No Absent 25°C / 60%R C H1M4.4 lear / Colorless No AbsentAtty Docket No.053032-548001WO 2M 4.4 Clear / Colorless No Absent 4.4 Clear / Ambient T=0Colorless No Absent 2 Weeks 4.3 Clear / Colorless No Absent 50°C Clear / 1M 4.3 Colorless No Absent 178008- M 4 Clear / 2 .2 Colorless No Absent 2308 40°C / 75%R 1M 4.3 Clear / Colorless No Absent H Clear / 2M 4.3 Colorless No Absent 25°C / 60%R 1M 4.3 Clear / Colorless No Absent H Clear 2M 4.4 / Colorless No Absent

[0221] Both prototype formulations were evaluated for assay of benzoic acid. For this analysis, a target criterion of 90.0%-110.0% and the absolute percent difference from time zero were used to evaluate the stability of benzoic acid in the placebo gels. A percentage difference of ≤ 5.0% was considered acceptable. The % assay for all time points / batches was between 95.0- 105.0%, and the absolute percent difference for benzoic acid was NMT ± 5.0% from time zero. These results demonstrated acceptable stability for benzoic acid in both prototype formulations at all storage conditions. There were no trend associated with the storage temperature, or the batch formulation. Benzoic acid appears to be stability in the proposed formulations. Single Point Viscosity:

[0222] These data (Table 42) suggested a significant decrease in viscosity at 50˚C from the initial time point to 2 months for both formulations. However, both formulations exhibited better stability at 40°C / 75% RH and CRT conditions. There was one out of trend (high) result was reported for the 2 months for batch 2308. But, because of the limited number of data points, no trend analysis could be executed. Table 42: Single Point Viscosity Batch Storage Condition Time Point Viscosity (mPa*s)Atty Docket No.053032-548001WO Ambient T=0 145,9002 Weeks 131,60050°C1M 119,840178008-23072M 93,91040°C / 75%RH1M 131,3802M 135,39025°C / 60%RH1M 144,9402M 144,860Ambient T=0 171,7302 Weeks 154,38050°C1M 135,000178008-23082M 107,14040°C / 75%RH1M 147,6502M 175,62025°C / 60%RH1M 151,6602M 141,390Amplitude Sweep:

[0223] These data evidenced a significant change at 50˚C for both batches (Tables 43 and 44). For 178008-2307, the linear viscoelastic region (LVER) and flow point increased significantly at 2 weeks and 2 months, and 178008-2308 decreased significantly after 2 weeks. Conversely, there were no significant changes suggested in these data for the flow point and LVER at the 40˚C / 75%RH and 25˚C / 60%RH stability conditions at 2 months. Table 43: Amplitude Sweep for 178008-2307 LVER Limit Flow Point Storage Condition Time Point Tau (Pa) Storage Modulus Storage Modulus G' (Pa) Tau (Pa) G' (Pa) RT T=0 53 5,050 337 1,2482 Weeks 35 6,740 358 1,56950°C1M 27 5,130 312 1,2322M 32 6,090 212 1,91540°C / 75%RH1M 51 4,930 331 1,1202M 25 4,680 313 1,20225°C / 60%RH 1M 53 5,110 347 1,197Atty Docket No.053032-548001WO 2M 29 5,480 345 1,328Table 44: Amplitude Sweep for 178008-2308 LVER Limit Flow Point Storage Time Point Storage Storage Condition Tau (Pa) Modulus G' Tau (Pa) Modulus G' (Pa) (Pa) RT T=0 58 5,570 360 1,3232 Weeks 62 5,960 363 1,43950°C1M 43 4,170 295 9132M 11 2,100 192 45440°C / 75%RH1M 55 5,240 353 1,1922M 51 4,900 341 1,19225°C / 60%RH1M 57 5,510 363 1,2622M 56 5,390 368 1,292Thermal Viscosity:

[0224] At the 50°C storage temperature the thermal profiles (Tables 45-46, FIG. 27) indicated a significant decrease in the viscosity for both formulations at measuring temperatures >21°C. These changes were reduced at the 40°C / 75%RH storage temperature (Tables 45-46, FIG. 28) to a minimal difference (~300K-400K) at measuring temperatures >21 °C. But when compared to the initial time point, although continues to exhibit greater than 1.5 million mPa*s. No significant changes in viscosity were observed from at measuring temperatures < 21˚C. These data suggested 25˚C / 60%RH for both batches were similar with no significant changes between the initial time point and 2 months (FIG. 29). However, 178008-2308 exhibited a higher thermal viscosity profile from the initial time point until 2 months when compared to 178008-2307. Table 45: Thermal Viscosity for 178008-2307 Viscosity (mPa*s at 0.221 / s) °C 50˚C 40°C / 75%RH 25°C / 60%RH Initial 2 Weeks 1M 2M 1M 2M 1M 2M 5.0 172,340 113,000 91,635 3,209 161,995 136,185 172,070 170,070 13.2 185,590 136,000 115,255 3,858 176,685 152,285 184,310 180,470Atty Docket No.053032-548001WO 21.0 331,820 316,000 291,640 104,901 336,060 305,085 321,260 324,17528.9 2,118,150 1,910,000 1,715,750 1,056,200 1,964,350 1,773,500 2,081,050 2,064,90036.8 2,353,300 2,070,000 1,914,100 1,110,800 2,200,600 1,956,550 2,320,350 2,306,55044.7 1,868,300 1,550,000 1,467,250 1,074,400 1,749,000 1,518,250 1,805,200 1,797,750Table 46: Thermal Viscosity for 178008-2308 Viscosity (mPa*s at 0.221 / s) °C 50˚C 40°C / 75%RH 25°C / 60%RH Initial 2 Weeks 1M 2M 1M 2M 1M 2M 5.0 223,925 247,000 245,520 176,515 235,700 235,115 232,230 234,615 13.2 235,765 261,000 256,860 201,720 251,455 248,750 245,080 247,56021.0 391,755 443,000 413,470 283,260 396,840 378,180 422,230 384,03028.9 2,468,050 2,360,000 2,231,850 896,635 2,332,250 2,148,600 2,450,500 2,446,30036.8 2,956,800 2,730,000 2,653,950 1,207,200 2,763,850 2,576,400 2,940,500 2,920,50044.7 2,646,550 2,440,000 2,308,550 976,930 2,465,850 2,261,200 2,578,000 2,647,500

[0225] The gelling point of 21˚C was exhibited for all conditions for both batches. AET:

[0226] AET (Table 47) demonstrated passing results for both formulations at T=14 and 28 days. These results meet the USP acceptance criteria for antimicrobial effectiveness. Table 47: AET Results Organism Initial 14-Day 28-Day 178008-2307 178008-2308 178008-2307 178008-2308 P. aeruginosa 4.4 x 10^5 CFU / g >4.0 >4.0 NI NIS. aureus 2.2 x 10^5 CFU / g >4.0 >4.0 NI NIE. coli 1.4 x 10^5 CFU / g >4.0 >4.0 NI NIC. albicans 4.1 x 10^5 CFU / g NI NI NI NIA. brasiliensis 1.5 x 10^5 CFU / g NI NI NI NINI: No increase

[0227] These data suggested that the Lactobacillus placebo gels (178008-2307 and 178008-2308) at 40˚C / 75%RH and 25˚C / 60%RH demonstrated acceptable stability for 2 monthsAtty Docket No.053032-548001WO and did not exhibit significant changes. The results of this study indicate that either formulation has a high potential for long term rheological, preservative, and antimicrobial stability. Example 9. Mixing of Lactobacillus Gel Formulations

[0228] Lyophilized Lactobacillus crispatus (powder) and placebo gel were provided in separate __ mL syringes. Two syringe configurations were tested with the key parameter being the smallest diameter hole between syringes: -Config 1 (Samples 4, 5, and 6): Off-the-shelf standard luer lock syringes docked with afemale-to-female connector. This makes the smallest diameter about 1.75 mm in the male cone on each syringe. -Config 2 (Samples 1, 2, and 3): Modified luer lock syringes to an “open bore”configuration with a hole of about 4 mm diameter docked with a female-to-female connector. This makes the smallest diameter about 3.8 mm in the center of the connector.

[0229] The plunger of the gel syringe was compressed until all the gel transferred into the coupled lactobacillus powder packed tube. The tube set was then flipped 180° and recompressed. Crosshead speed: 100 mm / min (~4 in / min); Laboratory conditions: +21.4°C and 55.5% relative humidity (RH). Shimadzu Universal Test Machine (AG-IS) and Shimadzu SLBL-100N Load Cell (339-83459-06) were used.

[0230] Compression values of the samples ranged from 9.2 N to 23.0 N. Table 48: Compression Test Data Tube Set Number Compression Tube Set Compression (Open Bore) Peak Force Number Peak Force (N) (N) (Standard Leur Lock) 1 A 9.2 4 A 22.1 1 B 11.4 4 B 15.5 2 A 11.5 5 A 18.6 2 B 10.1 5 B 12.4 3 A 17.4 6 A 23.0 3 B 11.7 6 B 10.8 Mean 11.8 17.1 Standard Deviation 2.9 5.0 Maximum 17.4 23.0 Minimum 9.2 10.8Atty Docket No.053032-548001WO

[0231] All forces were within acceptable ranges. Example 10. Viability of Lactobacillus in Gel Formulations

[0232] One gram (1g) of lyophilized Lactobacillus crispatus will be mixed manually using a spatula for a minimum of 60 sec with the gel. If the mixtures do not appear uniform after 60 sec, longer times will be investigated.

[0233] The gel / Lactobacillus mixtures will be held at room temperature with sampling times of 1 min, 15 min, 30 min, 60 min, and 120 min post mixing. The mixtures will be incubated at 37°C, stationary as spread into empty Petri dishes, in the presence of 5% CO2 to reflect the vaginal environment. The CO2incubator will be maintained in high humidity to prevent undue evaporation from the Petri dishes. At the designated times, samples will be removed after 30 sec of mixing (with use of sterile plastic spreaders at 50 revolutions per minute for 30 sec to remove bacteria from Petri dish bottoms). At the pre-specified times above, 1 ml of the gel / Lactobacillus mixtures will be serially diluted (in 10-fold increments) and placed on chocolate agar plates to determine the number of colony-forming units (CFUs).

[0234] The goal will be to attain >80% of the number of CFUs initially mixed into the gel remaining viable at least over the first time-points evaluated. The goal for later iterations will be to maintain >90% viability for a minimum of 15 min post-mixing.

[0235] All gels will also be tested by mixing and storing at temperatures of 30°C and 37°C and held for up to 15 min post mixing.

Claims

Atty Docket No.053032-548001WO WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising Lactobacillus and a pharmaceutical gelcomprising a poloxamer and a stabilization polymer.

2. The composition of claim 1, wherein the poloxamer comprises poloxamer 407.

3. The composition of claim 1 or 2, wherein the poloxamer is present at about 10-25 percentby weight of the composition (wt%).

4. The composition of any one of claims 1-3, wherein the stabilization polymer comprisesxanthan gum.

5. The composition of any one of claims 1-4, wherein the stabilization polymer is present atabout 1-5 wt%.

6. The composition of any one of claims 1-5, the pharmaceutical gel further comprising oneor more buffers.

7. The composition of claim 6, wherein the one or more buffers are present at about 0.1% toabout 5% w / w.

8. The composition of claim 7, wherein the one or more buffers comprise citrate and / or acitrate salt.

9. The composition of claim 7 or 8, wherein the citrate and / or citrate salt comprises one ormore of citric acid, citric acid monohydrate, sodium citrate, and sodium citrate dihydrate.

10. The composition of claim 8 or 9, wherein the citrate and / or citrate salt is present at about0.1-3 wt%.Atty Docket No.053032-548001WO11. The composition of any one of claims 6-10, wherein the one or more buffers compriselactic acid and / or sodium phosphate.

12. The composition of claim 11, wherein the lactic acid is present at about 0.1-3 wt%.

13. The composition of claim 11 or 12, wherein the sodium phosphate is present at about 0.1-3 wt%.

14. The composition of any one of claims 1-13, the pharmaceutical gel further comprisingbenzoic acid.

15. The composition of claim 14, wherein the benzoic acid is present at about 0.05-2 wt%.

16. The composition of any one of claims 1-15, the pharmaceutical gel further comprisingbenzyl alcohol.

17. The composition of claim 16, wherein the benzyl alcohol is present at about 0.5-5 wt%.

18. The composition of any one of claims 1-15 which does not comprise benzyl alcohol.

19. The composition of any one of claims 1-18, wherein the composition is a gel.

20. The composition of any one of claims 1-19, wherein the composition has a viscosity at 37°C of at least 1.5 million centipoise (cP).

21. The composition of any one of claims 1-20, wherein the composition has a viscosity at15°C of at least 50,000 cP.

22. The composition of claim 21, wherein the composition has a viscosity at 15°C of about50,000 cP to about 5 million cP.Atty Docket No.053032-548001WO23. The composition of any one of claims 1-22, wherein the Lactobacillus is present at about1-30 wt%.

24. The composition of claim 23, wherein the Lactobacillus is present at about 2 wt% to about20 wt%.

25. A composition comprising about 10 wt% to about 30 wt% Lactobacillus and apharmaceutical gel comprising: (a) about 10 wt% to about 25 wt% poloxamer;(b) about 1 wt% to about 5 wt% stabilization polymer; and(c) one or more buffers.

26. The composition of claim 25, wherein the one or more buffers comprise citrate and / or acitrate salt.

27. The composition of claim 26, wherein the citrate and / or citrate salt comprises one or moreof citric acid, citric acid monohydrate, sodium citrate, and sodium citrate dihydrate.

28. The composition of claim 26 or 27, wherein the citrate and / or citrate salt is present at about0.5-3 wt%.

29. The composition of any one of claims 25-28, the pharmaceutical gel further comprisingbenzoic acid.

30. The composition of claim 29, wherein the benzoic acid is present at about 0.05-2 wt%.

31. The composition of any one of claims 25-30, wherein the composition is a gel.

32. The composition of any one of claims 25-31, wherein the composition has a viscosity at37 °C of at least 1.5 million centipoise (cP).Atty Docket No.053032-548001WO33. The composition of any one of claims 25-32, wherein the composition has a viscosity at15°C of at least 50,000 cP.

34. The composition of claim 33, wherein the composition has a viscosity at 15°C of about50,000 cP to about 5 million cP.

35. The composition of any one of claims 1-34, wherein the Lactobacillus comprises one ormore Lactobacillus species.

36. The composition of claim 35, wherein the Lactobacillus species comprise one or more ofLactobacillus crispatus. Lactobacillus gasseri, Lactobacillus iners, Lactobacillus vaginalis, and Lactobacillus jensenii.

37. The composition of any one of claims 1 to 36, further comprising a prebiotic.

38. The composition of claim 37, wherein the prebiotic comprises a sugar.

39. The composition of claim 38, wherein the sugar comprises one or more of glucose,dextrose, or maltose.

40. The composition of any one of claims 1 to 39, further comprising glutamine.

41. The composition of any one of claims 1 to 40 which is formulated for administration to avagina of a subject.

42. A method for providing Lactobacillus to a vagina of a subject, comprising administering apharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.Atty Docket No.053032-548001WO43. A method for treating bacterial vaginosis in a subject, comprising administering apharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

44. A method for preventing recurrence of bacterial vaginosis in a subject, comprisingadministering a pharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina.

45. A method for maintaining healthy biota in a vagina of a subject, comprising administeringa pharmaceutical composition comprising a poloxamer, a stabilization polymer, and Lactobacillus to the vagina subject.

46. The method of any one of claims 42-45, wherein the composition is administered duringor after administration of an antibiotic to the subject.

47. The method of any one of claims 42-46, wherein the composition is a composition of anyone of claims 1-41.

48. The method of any one of claims 42-47, comprising combining the Lactobacillus with thepharmaceutical gel prior to administration, wherein the pharmaceutical gel comprises the poloxamer and the stabilization polymer.

49. A kit comprising Lactobacillus and a pharmaceutical gel, wherein the pharmaceutical gelcomprises a poloxamer and a stabilization polymer.

50. The kit of claim 49, wherein the poloxamer comprises poloxamer 407.

51. The kit of claim 49 or 50, wherein the poloxamer is present at about 10-25 percent byweight of the pharmaceutical gel (wt%).Atty Docket No.053032-548001WO52. The kit of any one of claims 49-51, wherein the stabilization polymer comprises xanthangum.

53. The kit of any one of claims 49-52, wherein the stabilization polymer is present at about 1-5 wt%.

54. The kit of any one of claims 49-53, the pharmaceutical gel further comprising one or morebuffers.

55. The kit of claim 54, wherein the one or more buffers are present at about 0.1% to about 5%w / w.

56. The kit of claim 54 or 55, wherein the one or more buffers comprise citrate and / or a citratesalt.

57. The kit of claim 56, wherein the citrate and / or citrate salt comprises one or more of citricacid, citric acid monohydrate, sodium citrate, and sodium citrate dihydrate.

58. The kit of claim 56 or 57, wherein the citrate and / or citrate salt is present at about 0.1-3wt%.

59. The kit of any one of claims 49-58, wherein the one or more buffers comprise lactic acidand / or sodium phosphate.

60. The kit of claim 59, wherein the lactic acid is present at about 0.1-3 wt%.

61. The kit of claim 59 or 60, wherein the sodium phosphate is present at about 0.1-3 wt%.

62. The kit of any one of claims 49-61, the pharmaceutical gel further comprising benzoic acid.

63. The kit of claim 62, wherein the benzoic acid is present at about 0.05-2 wt%.Atty Docket No.053032-548001WO64. The kit of any one of claims 49-63, the pharmaceutical gel further comprising benzylalcohol.

65. The kit of claim 64, wherein the benzyl alcohol is present at about 0.5-5 wt%.

66. The kit of any one of claims 49-63, wherein the pharmaceutical gel does not comprisebenzyl alcohol.

67. The kit of any one of claims 49-66, wherein the Lactobacillus comprises one or moreLactobacillus species.

68. The kit of claim 67, wherein the Lactobacillus species comprise one or more ofLactobacillus crispatus. Lactobacillus gasseri, Lactobacillus iners, Lactobacillus vaginalis, and Lactobacillus jensenii.

69. The kit of any one of claims 49-68, wherein the pharmaceutical gel further comprises aprebiotic.

70. The kit of claim 69, wherein the prebiotic comprises a sugar.

71. The kit of claim 70, wherein the sugar comprises one or more of glucose, dextrose, ormaltose.

72. The kit of any one of claims 49-71, wherein the pharmaceutical gel further comprisesglutamine.

73. The kit of any one of claims 49-72, wherein the pharmaceutical gel is formulated foradministration to a vagina of a subject.Atty Docket No.053032-548001WO74. The kit of one of claims 49-73, further comprising instructions for mixing the Lactobacillusand the pharmaceutical gel and administration to a vagina of a subject.

75. The kit of any one of claims 49-74, wherein the Lactobacillus is in a first container and thepharmaceutical gel is in a second container.

76. The kit of claim 75, wherein the first container and / or the second container is a syringe.

77. The kit of any one of claims 49-76, further comprising at least one syringe.

78. The kit of any one of claims 75-77, further comprising a connector to connect the firstcontainer and the second container.

79. The kit of claim 78, wherein the connector comprises a leur lock connector.

80. A method of making a Lactobacillus composition, comprising combining Lactobacillusand a pharmaceutical gel, wherein the pharmaceutical gel comprises a poloxamer and a stabilization polymer.

81. The method of claim 80, wherein the poloxamer comprises poloxamer 407.

82. The method of claim 80 or 81, wherein the poloxamer is present at about 10-25 percent byweight of the pharmaceutical gel (wt%).

83. The method of any one of claims 80-82, wherein the stabilization polymer comprisesxanthan gum.

84. The method of any one of claims 80-83, wherein the stabilization polymer is present atabout 1-5 wt%.Atty Docket No.053032-548001WO85. The method of any one of claims 80-84, the pharmaceutical gel further comprising one ormore buffers.

86. The method of claim 85, wherein the one or more buffers are present at about 0.1% to about5% w / w.

87. The method of claim 85 or 86, wherein the one or more buffers comprise citrate and / or acitrate salt.

88. The method of claim 87, wherein the citrate and / or citrate salt comprises one or more ofcitric acid, citric acid monohydrate, sodium citrate, and sodium citrate dihydrate.

89. The method of claim 86 or 87, wherein the citrate and / or citrate salt is present at about 0.1-3 wt%.

90. The method of any one of claims 80-89, wherein the one or more buffers comprise lacticacid and / or sodium phosphate.

91. The method of claim 90, wherein the lactic acid is present at about 0.1-3 wt%.

92. The method of claim 90 or 91, wherein the sodium phosphate is present at about 0.1-3 wt%.

93. The method of any one of claims 80-92, the pharmaceutical gel further comprising benzoicacid.

94. The method of claim 93, wherein the benzoic acid is present at about 0.05-2 wt%.

95. The method of any one of claims 80-94, wherein the pharmaceutical gel does not comprisebenzyl alcohol.

96. The method of any one of claims 80-95, wherein the Lactobacillus comprises one or moreLactobacillus species.Atty Docket No.053032-548001WO97. The method of claim 96, wherein the Lactobacillus species comprise one or more ofLactobacillus crispatus. Lactobacillus gasseri, Lactobacillus iners, Lactobacillus vaginalis, and Lactobacillus jensenii.

98. The method of any one of claims 90-97, wherein the pharmaceutical gel further comprisesa prebiotic.

99. The method of claim 98, wherein the prebiotic comprises a sugar.

100. The method of claim 99, wherein the sugar comprises one or more of glucose, dextrose, ormaltose.

101. The method of any one of claims 80-100, wherein the pharmaceutical gel further comprisesglutamine.

102. The method of any one of claims 80-101, wherein the pharmaceutical gel is formulated foradministration to a vagina of a subject.

103. The method of any one of claims 80-102, wherein the Lactobacillus and the pharmaceuticalgel are combined by: (a) providing the Lactobacillus in a first container;(b) providing the pharmaceutical gel in a second container;(c) connecting the first container and the second container, such that the Lactobacillusis in fluid connection with the pharmaceutical gel; and (d) transferring the Lactobacillus and the pharmaceutical gel between the twocontainers to make the Lactobacillus composition.

104. The method of claim 103, wherein the first container is a syringe.

105. The method of claim 102 or 103, wherein the second container is a syringe.Atty Docket No.053032-548001WO106. The method of any one of claims 103-105, wherein step (d) is performed by pushing theLactobacillus into the second container, or by pushing the pharmaceutical gel into the first container.

107. The method of claim 106, further comprising pushing the Lactobacillus and pharmaceuticalgel from the first container into the second container, or from the second container to the first container.

108. The method of claim 107, wherein the Lactobacillus and the pharmaceutical gel are pushedfrom the first and / or second container into the other container two or more times, for a sufficient number of times to make the Lactobacillus composition.

109. The method of any one of claims 103-108, wherein the first container and the secondcontainer are connected by a leur lock connector.

110. The method of any one of claims 103-109, wherein a force applied to transfer theLactobacillus and the pharmaceutical gel between the two containers is between about 9.2 N to about 23.0 N.

111. A method of treating a subject with a Lactobacillus composition, comprising administeringthe Lactobacillus composition made by the method of any one of claims 80-110 or from a kit of any one of claims 49-79 to a subject.

112. The method of claim 111, wherein the Lactobacillus composition is administered to thevagina of the subject.

113. A Lactobacillus composition made by the method of any one of claims 80-110.

114. The method of any one of claims 42-46, wherein the composition was made by the methodof any one of claims 80-110 or from a kit of any one of claims 49-79.

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