Attorney Ref: # JHU C 18071 PCT VAGINAL PROBIOTIC GEL-FORMING FORMULATIONS, METHODS OF MAKING ANDS USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63/8/568,098, filed March 21, 2024, the contents of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The present invention is generally in the field of probiotic formulations of vaginal microbiota and uses thereof. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants HD103124 and HD108905 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION The introduction of beneficial bacterial communities onto the vaginal surface is effective in biasing the vaginal microbiota toward a predominantly more beneficial population and in treating conditions such as bacterial vaginosis. Some approaches have included transplanting cervicovaginal secretions (“CVS”) from healthy donors (US20160279182). The applicability of this approach would be greatly enhanced by formulations with improved stability, which maintain the viability of the vaginal microbiota, following collection, storage, freezing, lyophilization and/or freeze-drying. It would be beneficial to have vaginal probiotic formulations which are stable following freeze-thawing, and do not need known cryoprotectants such as glucose, glycerol, DMSO, sucrose, dextrose, polyvinylpyrrolidone (PVP), sodium ascorbate, etc., for such stability (U.S. Patent No.9,470,676). It is an object of the present invention to provide vaginal probiotic formulations with improved stability. It is also an object of the present invention to provide methods of treating a subject in need of vaginal probiotics. BRIEF SUMMARY OF THE INVENTION Hypotonic or iso-osmolal vaginal probiotic gel-forming formulations are provided, which include one or more hydrogel forming polymers, a hypotonic carrier and vaginal probiotics. The amount of the hydrogel forming polymer in the formulation is effective to 1 45721826.1
Attorney Ref: # JHU C 18071 PCT improve viability of the at least one vaginal probiotic in the formulation, on thawing after freezing to -80 or -20 oC, when compared to its viability in the absence of the hydrogel forming polymer. Thus, the disclosed formulations are vaginal probiotic formulations with improved stability. Preferably, the hydrogel forming polymer is a poloxamer. The probiotics include bacteria found in healthy vagina (i.e., vaginal microbiota) including, but not limited to Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii. Preferably, at least 50% of the vaginal microbiota is Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, or Lactobacillus jensenii. In some forms, the vaginal probiotics are provided by cervicovaginal secretions (CVS ) obtained from a healthy vagina. The formulations may be in dry (lyophilized) or wet form, having an acidic pH less than 4.0. In some forms, the formulation includes a dissolution agent, for example, one or more acids such as lactic acid or hyaluronic acid, and optionally one or more bioactive agents. In some forms the hypotonic carrier is 1:1 saline:water or water. In some forms, the formulation includes one or more cryoprotectants. Also provided are methods of treating a subject in need thereof. The methods include administering the disclosed formulations to a subject in need thereof. The subject is preferably, human, more preferably, a young adult or an adult human and is not an infant (0- 1yrs old human). In some forms, the subject is an infant, such as a newborn. The subject can be a subject diagnosed with bacterial vaginosis (BV) or a sexually transmitted disease (STD). The hypotonic vaginal probiotic hydrogel forming polymer formulations are administered in a hypotonic solution, with the gel forming polymer at a concentration less than their normal critical gelling concentration. Hypotonically-administered Poloxamer solution below the critical gel concentration is absorbed by the epithelial surface, drawing the Poloxamer into the mucus layers and up against the epithelium where it then becomes concentrated enough to gel. As the Poloxamer is concentrated at the tissue/mucosal interface, it mixes with mucus and gels up against the epithelial surface. The endogenous mucin glycopolymers affect the gelling properties of the hypotonic gelling agents, including the concentration of gelling agent needed to gel and the pore structure of the resulting gel/mucin mixture. The hypotonic gelling vehicles coat the epithelium, including the folds, after vaginal application. In some forms, the vaginal probiotics are administered in combination with one or more bioactive agents. 2 45721826.1
Attorney Ref: # JHU C 18071 PCT BRIEF DESCRIPTION OF THE DRAWINGS FIG.1A shows viscosity as a function of temperature. The viscosity of formulations at (FIG.1B). FIG.1C shows the effect of adding additional excipients on the liquid phase formulation, and at (FIG.1C) 37 oC show the effect of polymer additions on the Pluronic-based hydrogel. Rheological behavior was dominated by the 20% F127 gel properties with minor variations depending on excipient. n=3 for each formulation. One way ANOVA was performed on each data set, *p < 0.05. FIG.2A shows maximum tack force (FIG.2B) adhesion force per unit area for each formulation was measured at 37C. Carbopol 974(P), xanthan gum, and PEG400 significantly increased the maximum tackiness of the 20% F127 gel, and Carbopol 974(P) significantly increased adhesion. n=3 for each formulation. One way ANOVA was performed for each data set, *p < 0.05. FIG.3A shows percent erosion and FIG.3B shows comparative time to full erosion. Only the addition of xanthan gum significantly increases the gel residence time in this constant flow experiment, demonstrating that mechanical integrity is dependent on F127 gel characteristics in most cases. Samples of eluent was taken every 4 minutes and analyzed at 560 nm on a BioTekSynergyMxplate reader. n=3 per formulation, and each data point was run in triplicate and averaged. One way ANOVA was performed on the data set, *p<0.05. FIG.4 shows osmolality of 20% F127 with various excipients. Larger molecular weight excipients increase the osmolality of the solution less than smaller molecule additives, like lactic acid. Each measurement was repeated 3 times. FIG. 5A-5B show that pluronic F127 can solubilize hydrophobic drugs, like estradiol, at clinically relevant concentrations. (FIG.5A) Estracevaginal 0.01% estradiol cream compared to (FIG.5B) 0.01% estradiol fully solubilized in 10% Pluronic F127. FIG.6A-6D show representative images of spots of dye-laden fluid that leaked out after vaginal administration of (FIG.6A) 2% F127, (FIG.6B) 8% F127, (FIG.6C) 10% F127, and (FIG.6D) 18% F127.3/3 mice experienced leakage in both the 2% F127 and 8% F127 groups, while 0/3 mice experienced leakage in both the 10% F127 and 18% F127 groups during 3 minutes of ambulation. The lack of leakage presumably highlights the formation of a hydrogel in vivo. FIG.7. MPT validates in vivo hypotonic gelling concentration of 10% F127 by producing an ensemble averaged mean square displacement (<MSD>) of 200 nm pegylated particles on excised tissue after vaginal administration in various concentrations of F127. It is 3 45721826.1
Attorney Ref: # JHU C 18071 PCT also shown that the addition of excipients, like xanthan gum, do not significantly impact the gelation of the formulation relative to particle mobility as a proxy for drugs. n=5-6 mice per formulation. One way ANOVA was performed on the data set, *p<0.05. FIG.8. Dosing of ProGelprovides effective prevention of PTB in a mouse model. Mifepristone (RU486) was dosed on E15 (black arrow), followed by daily vaginal administration with ProGel (n = 14), Crinone (n = 14), or placebo (n = 9) from E15-E18 (gray arrows). Full- term delivery was considered to occur on E19-20. Statistical significance was determined by log- rank test (Mantel-Cox) with Bonferroni method correction (Bonferri-corrected threshold p=0.017), *p=0.0065. FIG.9. Patients using Crinone and other vaginal creams often complain of clumpy white discharge. After vaginal dosing in mice, clumps could be observed in both lavage fluid and on dissected vaginal tissue as indicated by red circles. In contrast, vaginal dosing with ProGelresulted in more clarity in the lavage fluid similar to mice receiving no gel treatment, and ProGel was not visualizable on excised vaginal tissue. Lavage and dissections were performed 30 minutes after vaginal dosing, n=3 for each group. FIG.10. Exposure to Crinone significantly reduced L. crispatus viability, while ProGel did not impact L. crispatus survival as measured by colony forming units (CFU). Bacteria stock was grown with three biological replicates and plated in triplicate per treatment. One way ANOVA was performed on the data set, *p < 0.05. FIG.11 shows Pluronic F127 (10%) provided cryoprotection for cultured L. crispatus bacteria at both -20 and -80C, with and without the addition of hyaluronic acid (0.5%). FIGs.12A-12C show short-term storage stability of the iso-osmolal formulations. TFV concentration in (FIG.12A) 1% Lactic acid TFV formulation (FIG.12B) No acid TFV formulation and (FIG.12C) 1% Citric acid formulation measured over one week via HPLC in the following storage conditions: 4°C, -20°C, -80°C or flash frozen in liquid nitrogen and then stored at -80°C. n=3 replicates. FIGs.13A-13C show the drug stability under accelerated degradation conditions. Shown are TFV concentration in (FIG.13A) 1% Lactic acid TFV formulation (FIG.13B) No acid TFV formulation and (FIG.13C) 1% Citric acid formulation measured over four weeks via HPLC in the following storage in accelerated degradation or room temperature conditions. n=3 replicates. 4 45721826.1
Attorney Ref: # JHU C 18071 PCT DETAILED DESCRIPTION OF THE INVENTION I. Definitions Bacterial vaginosis (“BV”), as used herein, refers to the overgrowth of one of several non-Lactobacillus types of bacteria normally present in the vagina, upsetting the natural balance of vaginal bacteria. “Cervicovaginal secretions” refers to the mixture of mucus secreted by the cervix, shed epithelial cells, vaginal transudate, and bacteria found in the vagina of a woman. The “critical gel concentration”, or “CGC”, as used herein, refers to the minimum concentration of gel-forming polymer needed for gel formation, e.g. at which a solution-to-gel (sol-gel) transition occurs. The critical gel concentration can be dependent on a number of factors, including the specific polymer composition, molecular weight, temperature, and/or the presence of other polymers or excipients. “Cryoprotectant” is any agent that prevents the formation of ice crystals, which can rupture cell membranes. “Excipient” is used herein to include any other compound that can be contained in or on the microparticle that is not a therapeutically or biologically active compound. As such, an excipient should be pharmaceutically or biologically acceptable or relevant, for example, an excipient should generally be non-toxic to the subject. “Excipient” includes a single such compound and is also intended to include a plurality of compounds. The terms “gel” and “hydrogel”, as used interchangeably herein, refers to a swollen, water-containing network of finely dispersed polymer chains that are water-insoluble, where the polymeric molecules are in the external or dispersion phase and water (or an aqueous solution) forms the internal or dispersed phase. The chains can be chemically crosslinked (chemical gels) or physically crosslinked (physical gels). Chemical gels possess polymer chains that are connected through covalent bonds, whereas physical gels have polymer chains linked by non- covalent bonds or cohesion forces, such as Van der Waals interactions, ionic interaction, hydrogen bonding, or hydrophobic interaction. “Healthy vagina” as used herein refers to vagina having no evidence of sexually transmitted disease or bacterial vaginosis, and pH less than 4.0. “Microbial flora” refers to the microorganisms that normally live in the gastrointestinal tract, skin, nose, etc. In a healthy human, the internal tissues, e.g. blood, brain, muscle, etc., are normally free of microorganisms. However, the surface tissues, i.e., skin and mucous membranes, are constantly in contact with environmental organisms and become readily 5 45721826.1
Attorney Ref: # JHU C 18071 PCT colonized by various microbial species. The mixture of organisms regularly found at any anatomical site is referred to as the normal flora, except by researchers in the field who prefer the term “indigenous microbiota”. Bacteria are the most numerous microbial components of the normal flora. “Microbiota”, a term created by Jeffrey Gordon, refers to the collection of microbial species that form a microbial community. This includes the normal flora and “harmful” ones. The term “osmolarity”, as generally used herein, refers to the total number of dissolved components per liter. Osmolarity is similar to molarity but includes the total number of moles of dissolved species in solution. An osmolarity of 1 Osm/L means there is 1 mole of dissolved components per L of solution. Some solutes, such as ionic solutes that dissociate in solution, will contribute more than 1 mole of dissolved components per mole of solute in the solution. For example, NaCl dissociates into Na+ and Cl− in solution and thus provides 2 moles of dissolved components per 1 mole of dissolved NaCl in solution. Physiological osmolarity is typically in the range of about 280 to about 310 mOsm/L. “Probiotic”, as used herein, utilizes the World Health Organization's 2001 definition of “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host”. Probiotics must be alive when administered, have viability and reproducibility based on in vivo testing, and during use and storage. Sexually transmitted diseases (“STD”) are any of various diseases or infections (such as syphilis, gonorrhea, chlamydia, and genital herpes) that are usually transmitted by direct sexual contact and include some (as hepatitis B and AIDS) that may be contracted by other than sexual means. As used herein, the term “treating” includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition, or disorder being treated. The term “reduce”, “inhibit”, “alleviate” or “decrease” are used relative to a control. One of skill in the art would readily identify the appropriate control to use for each experiment. For example a decreased response in a subject or cell treated with a compound is compared to a response in subject or cell that is not treated with the compound. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, 6 45721826.1
Attorney Ref: # JHU C 18071 PCT immune system health, etc.), the disease or disorder, and the treatment being administered. The effect of the effective amount can be relative to a control. Such controls are known in the art and discussed herein, and can be, for example the condition of the subject prior to or in the absence of administration of the drug, or drug combination, or in the case of drug combinations, the effect of the combination can be compared to the effect of administration of only one of the drugs. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 5%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. II. Hypotonic Vaginal Probiotic Gel-Forming Formulations Hypotonic formulations of hydrogel forming polymers, preferably poloxamers including vaginal probiotics, with enhanced stability, have been developed for delivery to the vagina. The gel-forming formulations contain one or more gel-forming polymers in a hypotonic carrier, and optionally, contain one or more additional agents. In some forms, the formulation includes an additional cryoprotectant. In some forms, the formulation does not include an additional cryoprotectant. A. Vaginal Probiotics The disclosed formulations include populations of vaginally beneficial bacteria (vaginal probiotics), for example, bacteria that is found in a healthy vaginal that is free from sexually transmitted diseases or bacterial vaginosis. In some forms, the vaginal probiotics are provided by vaginal microbiota isolated from a donor, and cultured in vitro. Methods of isolation and in vitro culture of vaginal microbiota are known in the art. DeLong et al. (Front. Cell and Infect. Microbiol., 9(306) (2019)) and O’Hanlon et al., BMC Infect Dis 11, 200 (2011)) disclose how to screen donors to find those with minimal risk of pathogen transmission and “optimal” vaginal microbiota for transplant, including criteria sample collection, and vaginal bacterial culturing. 7 45721826.1
Attorney Ref: # JHU C 18071 PCT In some forms, the subject is not pregnant. In some forms, the donor is a pregnant subject in the third trimester of pregnancy, and the vaginal microbiota can be harvested before or at the time of giving birth. Criteria for screening pregnant donors for subsequent administration of the collected vaginal microbiota to pre-term babies or babies delivered by C-section, are disclosed for example in US Published Application.20160331792. In some forms, the vaginal probiotics are provided by cervicovaginal secretions (“CVS”) from one or more women with vaginal microbiota dominated (>50%) by species typically found in the human vagina, e.g. Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii. The examples demonstrate improved stability of vaginal probiotics formulated as hypotonic gel-forming formulations, characterized by improved the viability on thawing after freezing to -80 or -20 oC. B. HydroGel-Forming Polymers Gel-forming polymers are utilized at a concentration below the normal critical gel concentration of the polymer, e.g. the concentration at which the polymer solution would gel in a test tube when warmed to 37° C. In some forms, the gel-forming polymer is a thermosensitive gel former. Thermosensitive (aka thermoresponsive) hydrogels are solutions that undergo sol-gel transitions when 1) at or above the critical gelling concentration, and 2) at or above the critical gelling temperature. Thermosensitive gelling agents (at or above their critical gel concentration) used for biomedical applications are liquid at room temperature, but form a gel at body temperature. The increase in temperature induces a rearrangement and alignment of the polymer chains, leading to gelation into a 3-dimensional structure. This phenomenon is generally governed by the ratio of hydrophilic to hydrophobic moieties on the polymer chain. A common characteristic is the presence of a hydrophobic methyl, ethyl, or propyl group. Any thermosensitive polymer that fits these criteria can be administered hypotonically below the critical gel concentration to mucosal epithelial and form a uniform gel coating in vivo. In some forms, the he gel forming polymer is a poloxamer. Poloxamers are synthetic triblock copolymers of poly(ethylene oxide)-b- poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO), also commercially known as Pluronics®, Synperonics® or Lutrol®. Poloxamers or Pluronics® are a class of water-soluble nonionic triblock copolymers formed by polar (poly ethylene oxide) and non-polar (poly propylene oxide) blocks, which confer amphiphilic and surface active properties to the polymers. 8 45721826.1
Attorney Ref: # JHU C 18071 PCT Their aqueous solutions undergo sol-to-gel transition with increasing the temperature above a LCGT (lower critical gelation temperature); moreover, the coexistence of hydrophilic and hydrophobic monomers into block copolymers allows the formation of ordered structures in solution, the most common of these being micelles. The formation of micelles in solution is a reversible and dynamic process useful for encapsulating hydrophobic drugs and delivering them into an aqueous environment (Reviewed in Russo, et al., Pharmaceutics 2019, 11(12), 671). Examples of thermosensitive gel formers that can be used include polyoxyethylene- polyoxypropylene-polyoxyethylene triblock copolymers such as, but not limited to, those designated by the CTFA names Poloxamer 407 (CAS 9003-11-6, molecular weight 9,840- 14,600 g/mol; available from BASF as LUTROL® F127) and Poloxamer 188 (CAS 9003-11-6, molecular weight 7680-9510 g/mol; available from BASF as LUTROL® F68; a copolymer of polyethylene and polypropylene ether glycol); polyoxyethylene-polyoxypropylene- polyoxyethylene triblock polymer with an average molecular weight Mw of 13,000 Da and an average weight percent of polyoxyethylene at 80%; Tetronics tetra-functional block copolymers based on ethylene oxide and propylene oxide available from BASF as Tetronic®; poly(N,N- diethylacrylamide); poly(N,N-dimethylacrylamide); poly(N-vinylcaprolactam); poly(N- alkylacrylamide); poly(N-vinylalkylamide); poly(N-isopropyl acrylamide); polyethylene oxide methacrylate polymers; poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol triblock copolymers (PLGA-PEG-PLGA and PEG-PLGA-PEG); polycaprolactone (PCL)-polyethylene glycol triblock copolymers (PCL-PEG-PCL and PEG-PCL-PEG); chitosan; and combinations thereof. The hydrogels can be formed from individual gel formers or as a combination of gel formers. For example, a poloxamer and another gel former (e.g., a tetronic polymer) may be used in combination to attain the desired characteristics. In addition, various forms of the same gel former (e.g., Poloxamer 188 and Poloxamer 407) can be combined to attain the desired characteristics. The polymer is provided in a concentration less than the concentration that forms a gel in a test tube when heated to 37° C. The concentration must be sufficiently high, but below the critical gel concentration, for the epithelium to absorb enough fluid for the critical gel concentration to be reached in vivo, so gelation can occur on the mucosal epithelial surface. The range of time that it takes for gelation to occur depends on the mucosal surface (the capacity and rate of water absorption), the tonicity of the solution administered (more hypotonic solutions will drive more rapid fluid absorption), and the concentration of polymer administered (if the 9 45721826.1
Attorney Ref: # JHU C 18071 PCT polymer concentration is too low, not enough fluid absorption will occur to concentrate the polymer to its critical gel concentration). However, gelation generally occurs within 1 h in the vagina. C. Hypotonic Carriers Useful hypotonic carriers are biocompatible carriers that preferably cause little to no signs of irritation when administered to human subjects. The carrier can be naturally occurring or non-naturally occurring including both synthetic and semi-synthetic carriers. Preferred carriers are sodium-based. Other solutions, including sugar-based (e.g. glucose, mannitol) solutions and various buffers (phosphate-buffers, tris-buffers, HEPES), may also be used. Hypotonic solution refers to a solution that contains less solute compared to the cytoplasm of the cell. Examples of hypotonic solutions include, but are not limited to, Tris[hydroxylmethyl]-aminomethane hydrochloride (Tris-HCl, 10-100 mM, pH.6-8), (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10-100 mM, pH 6-8) and dilute solutions of PBS, such as a solution containing 0.2 grams KCl, 0.2 grams KH2PO4, 8 grams NaCl, and 2.16 grams Na2HPO4*7H2O in 1000 ml H2O. Hypotonic carriers concentrate the gel-forming polymer at the vaginal epithelial surface, resulting in uniform gel formation on the surface. The hypotonic carrier usually contains water as the major component. The hypotonic carrier can be water, although mixtures of water and a water-miscible organic solvent can also be used. Suitable water-miscible organic solvents include alcohols, such as ethanol, isopropanol; ketones, such as acetone; ethers, such as dioxane and the like; and esters such as ethyl acetate. The hypotonic carrier can be distilled water containing one or more osmolarity modifying excipients. Sodium chloride is the excipient that is most frequently used to adjust osmolarity if a solution is hypotonic. Other excipients used to adjust hypotonic solutions include, mannitol, glycerol, propylene glycol and sodium sulphate glucose. Osmolarity modifying excipients can include pharmaceutically acceptable salts such as sodium chloride, sodium sulfate, or potassium chloride. The hypotonic carrier can have any osmolarity less than the effective isotonic point (the concentration at which fluid is neither absorbed nor secreted by the epithelium) at the vaginal mucosal surface. The isotonic point varies for different mucosal surfaces and different buffers, depending on active ion transport at that epithelial surface; for example, studies have shown that isotonic point in the vagina for sodium-based solutions to be about 300 mOsm/L, but in the colorectum, it is about 450 mOsm/L. In some embodiments the solution has a tonicity from 50 10 45721826.1
Attorney Ref: # JHU C 18071 PCT mOsm/L to 280 mOsm/L, from 100 mOsm/L to 280 mOsm/L, from 150 mOsm/L to 250 mOsm/L, from 200 mOsm/L to 250 mOsm/L, from 220 mOsm/L to 250 mOsm/L, from 220 mOsm/L to 260 mOsm/L, from 220 mOsm/L to 270 mOsm/L, or from 220 mOsm/L to 280 mOsm/L. The io-osmolal formulations have an osmolarity within the physiological range. The hypotonic carrier can include one or more pharmaceutically acidifying agents such acceptable acids, one or more pharmaceutically acceptable bases, or salts thereof. Pharmaceutically acceptable acids include hyaluronic acid, lactic acid, hydrobromic, hydrochloric, and sulphuric acids, and organic acids, such as methanesulphonic acids, tartaric acids, and malic acids. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as pharmaceutically acceptable amines. The hypotonic carrier can include pharmaceutically acceptable buffers such as citrate buffers or phosphate buffers. The hypotonic carrier can include one or more pharmaceutically acidifying agents such acceptable acids, one or more pharmaceutically acceptable bases, or salts thereof. Pharmaceutically acceptable acids include hyaluronic acid, lactic acid, hydrobromic, hydrochloric, and sulphuric acids, and organic acids, such as methanesulphonic acids, tartaric acids, and malic acids. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as pharmaceutically acceptable amines. The hypotonic carrier can include pharmaceutically acceptable buffers such as citrate buffers or phosphate buffers. The preferred concentration range of lactic acid to promote Lactobacillus survival is 1- 1.5% lactic acid. Lactic acid is preferred to other types of food acid such as vinegar, lemon juice, and acetic acid, although these may also be utilized. D. Additives: Dissolution Agents, Cryoprotectants and Diluents As used herein, a “dissolution agent” is an acid, or salt thereof, that is added to the vaginal area, vaginal secretions, cervicovaginal secretions, or formulations containing vaginal probiotics. The formulations may be in dry (lyophilized) or wet form. Diluent is any solution, optionally containing a cryoprotectant and/or a dissolution agent. Diluent may optionally be balanced for a desired osmolarity. Exemplary diluent may be a solution of sodium chloride and lactic acid. Suitable dissolution agents include nitrogen-free organic acid having at least one carboxylic acid group and a total of from 2 to about 20 carbon atoms, a phosphoric acid 11 45721826.1
Attorney Ref: # JHU C 18071 PCT containing compound, a sulfonated polyphosphoric acid compound, a polyphosphonate having three or more phosphonate groups, an enzyme; or salts thereof; or combinations thereof. Examples include lactic acid, citric acid, tartaric acid, gluconic acid, glycolic acid, hydroxysuccinic acid, galactaric acid, hydroxypropionic acid, lactic acid, glyceric acid, hydroxybutyric acid, hydroxyisobutyric acid, hydroxy methylbutyric acid, bis(hydroxymethyl) propionic acid, gibberellic acid, hydroxyoctadecanoic acid, di-tert-butyl hydroxybenzoic acid, benzilic acid, hydroxyl fluorenecarboxylic acid, hydroxydecanoic acid, hydroxynaphthalenecarboxylic acid, hydroxybenzenedicarboxylic acid, hydroxymethylbenzoic acid, hydroxyphenylacetic acid, mandelic acid, hydroxymethoxybenzoic acid, methoxysalicylic acid, hydroxyoctanoic acid, hydroxy cinnamic acid, dihydroxycinnamic acid, dihydroxy- hydrocinnamic acid, hydroxyphenylpropionic acid, dihydroxytartaric acid, hydroxymethoxycinnamic acid, chlorohydroxybenzoic acid, chloromandelic acid, chloro phthalic acid, salicylic acid, chlorosalicylic acid, citrazinic acid, dibromo hydroxybenzoic acid, dichlorohydroxy-benzoic acid, dichlorosalicylic acid, galactouronic acid, glucuronic acid, hydroxypropanedioic acid, hydroxyphenyl propionic acid, lactic acid, methoxysalicylic acid, trihydroxybenzoic acid, or their partial salts and combinations thereof. Another group of organic acids are various hydroxyl free and nitrogen free saturated or unsaturated dicarboxylic acids having from 2 to about 20 carbon atoms and can contain nitrogen atoms. Examples include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, fumaric acid, decanedoic acid, camphoric acid, benzenedicarboxylic acid, phthalic acid, cyclohexanedicarboxylic acid, cyclohexanediacetic acid, octanedioc acid, homophthalic acid, phenylmalonic acid, cyclopentanediacetic acid, nonanedioic acid, benzylmalonic acid, phenylenediacetic acid, phenylsuccinic acid, bromosuccinic acid, carboxyphenacetic acid, cyclobutanedicarboxylic acid, cyclohexanedicarboxylic acid, decanedicarboxylic acid, dibromosuccinic acid, dichlorophthalic acid, diethylmalonic acid, diglycolic acid, dimethylmalonic acid, dimethyl pentanedioic acid, dimethylsuccinic acid, ethylmalonic acid, glutamic acid, hexenedioic acid, imino diacetic acid, methylmalonic acid, methylsuccinic acid, naphthalene dicarboxylic acid, oxalacetic acid, oxopentanedioic acid, undecane dicarboxylic acid, dipicolinic acid, or their partial salts, and combinations thereof. Optionally, the disclosed formulations can include a cryoprotectant. Cryoprotectants include extracelluar cryoprotectants that do not penetrate bacterial cell walls, and intracellular cryoprotectants that penetrate bacterial cell walls. Examples of suitable extracellular cryoprotectants include sucrose, dextrose and polyvinylpyrrolidone (PVP). Examples of suitable 12 45721826.1
Attorney Ref: # JHU C 18071 PCT intracellular cryoprotectants include glycerol (glycerine), at a concentration less than 7%, for example, up to about 1, 2, 3, 4, 5, and 6%. In some forms, the cryoprotectant is a mixture of sodium ascorbate and glucose for example a mixture of about 5% sodium ascorbate mixed 1:1 with glucose. E. Additional Bioactive Agents The hypotonic vaginal probiotic gel-forming formulations can contain one or more agents to be delivered or incorporated into the hydrogel barrier including therapeutic agents, prophylactic agents, diagnostic agents, and/or nutraceuticals. “Bioactive agent” and “active agent” are used interchangeably include without limitation physiologically or pharmacologically active substances that act locally or systemically in the body. A biologically active agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment. Examples can include, but are not limited to, small-molecule drugs, peptides, proteins, antibodies, sugars, polysaccharides, nucleotides, oligonucleotides, aptamers, siRNA, nucleic acids, and combinations thereof. The agents can be a small molecule (e.g., molecular weight less than 2000, 1500, 1000, 750, or 500 atomic mass units (amu)) or a biomolecule, such as peptides, proteins, nucleic acids, polysaccharides, lipids, glycoproteins, lipoproteins, or combinations thereof. The agents can include one or more of those described in Martindale: The Complete Drug Reference, 37th Ed. (Pharmaceutical Press, London, 2011). In some forms, the bioactive agent is a microbicidal drug, i.e., an agent that is destructive to or acting against microbes, such as bacteria, viruses, and other microorganisms. In some forms, the agent is an anti-HIV agent such as tenofovir. The hypotonic gel-forming formulations can contain a therapeutically effective amount of a therapeutic agent to treat, inhibit, or alleviate one or more symptoms of a disease state being treated. The hypotonic gel-forming formulations can contain an effective amount of a prophylactic agent to prevent one or more symptoms of a disease or disorder. Exemplary classes of agents include, but are not limited to, synthetic and natural proteins (including enzymes, peptide-hormones, receptors, growth factors, antibodies, signaling molecules), and synthetic and natural nucleic acids (including RNA, DNA, anti-sense RNA, triplex DNA, inhibitory RNA (RNAi), and oligonucleotides), and biologically active portions thereof. 13 45721826.1
Attorney Ref: # JHU C 18071 PCT Agents may be anti-infective (antibiotics, antivirals, antifungals), anti-inflammatory, for birth control, for treatment of metabolic disorders, for treatment of heartburn or ulcers, for treatment of cardiovascular disorders such as hypertension and atherosclerosis, neuroactive agents, and chemotherapeutics. Examples of useful proteins include hormones such as progesterone, estrogen/estradiol, insulin and growth hormones including somatomedins. Examples of useful drugs include neurotransmitters such as L-DOPA, antihypertensives or saluretics such as Metolazone from Searle Pharmaceuticals, carbonic anhydrase inhibitors such as Acetazolamide from Lederle Pharmaceuticals, insulin like drugs such as glyburide, a blood glucose lowering drug of the sulfonylurea class, synthetic hormones such as Android F from Brown Pharmaceuticals and Testred® (methyltestosterone) from ICN Pharmaceuticals. Representative anti-proliferative (anti-cancer or endometriosis) agents include, but are not limited to, alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites (such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), antimitotics (including taxanes such as paclitaxel and decetaxel and vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, as well as actinomycins such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), topoisomerase inhibitors (including camptothecins such as camptothecin, irinotecan, and topotecan as well as derivatives of epipodophyllotoxins such as amsacrine, etoposide, etoposide phosphate, and teniposide), and combinations thereof. Other suitable anti-cancer agents include angiogenesis inhibitors including antibodies to vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®), other anti-VEGF compounds; thalidomide (THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®); endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib (Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib; transforming growth factor-α or transforming growth factor-β inhibitors, and antibodies to the epidermal growth factor receptor such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®). III. Methods of Making Hypotonic Vaginal Probiotic Gel-Forming Formulations The CVS incorporated into the disclosed formulations can be collected from a donor using standard techniques using commercially available materials such as Instead Soft Cup menstrual fluid device, beaker, syringe, or absorbent matrix. 14 45721826.1
Attorney Ref: # JHU C 18071 PCT The secretions are preferably stored in the refrigerator at 4° C. for up to 1 week, or in certain cases, immediately frozen after collection and stored for up to several months, before being implanted into the recipient. Samples must maintain at least 20% viable bacteria prior to use. The identity and relative abundance of bacteria in the CVS are determined by 16S rRNA pyrosequencing. The sequencing data is then used to identify a community state, and only samples classified within the community states of Lactobacillus that are typically found in the human vagina, including Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii, are considered for transplant, and thus, in some forms, for inclusion into the disclosed formulation. This community state often includes other species of Lactobacillus in smaller fractions. Genetic sequencing techniques and assignment of community states have been defined can be prepared as liquids for administration. Typically these will be prepared as single or multiple dosage units in an appropriate applicator. Powder units may be dual chambered, one containing solvent, with or without excipients to adjust tonicity, and the other containing the hydrogel forming material, typically also including the one or more agents to be administered. Multiple dosage units will typically include a barrel loaded with powder, and a plunger having dosage increments thereon. These will typically be sterilized and packaged in sealed, sterile packaging for storage and distribution. Dosage unit administrators may be designed to fit the anatomic location such as the vagina, to which the vaginal probiotics and optionally one or more active agents is to be delivered. IV. Methods of Using Hypotonic Vaginal Probiotic Gel-Forming Formulations The vaginal probiotic hypotonic gel-forming formulation is applied to the vaginal surface in a subject in need thereof. In one embodiment, the formulations are applied as a liquid to a mucosal coating on an epithelial surface of a subject in need thereof. The gel-forming formulation can be applied in any number of ways known to the skilled artisan as long as the hypotonic solution, or reagents forming the hypotonic solution, contacts the surface. By applying the gel-forming formulations as a hypotonic formulation, water is absorbed into the epithelial tissue. Water absorption provides for concentration of the gel-forming polymer at the surface, resulting in uniform gel formation at the surface. In another embodiment, the gel is applied as it solidifies or in a partially solid form, thereby acting as a barrier, reservoir or depo, or combination thereof. Agents or excipients in the gel-forming formulation can become entrapped in the gel and can be released at or into the surface. 15 45721826.1
Attorney Ref: # JHU C 18071 PCT The polymer-containing formulations are administered at a concentration at or higher than the normal critical gelling concentration of the polymer. A Poloxamer gel administered into the vagina at its critical gel concentration will form a “plug” of gel in the lumen. In contrast, fluid from a hypotonically-administered Poloxamer solution below the critical gel concentration will be absorbed by the epithelial surface, drawing the Poloxamer into the mucus gel and up against the epithelium, thereby enhancing and facilitating delivery of agents to the epithelial cells. As the Poloxamer is concentrated, it mixes with mucus. The endogenous mucin glycopolymers affect the gelling properties of the hypotonic gelling agents, including the concentration of gelling agent needed to gel and the pore structure of the resulting gel/mucin mixture. After vaginal application, the hypotonic gelling vehicles coat the epithelium, including the folds. In some forms, the subject has been diagnosed with bacterial vaginosis (BV), as identified clinically with Amsel's criteria, and confirmed in the laboratory by Nugent scoring (Nugent et al., Journal of Clinical Microbiology, 29(2):297-301 (1991)). In some forms, the subject has recurrent BV. Subjects with recurrent BV (requiring >3 treatment courses in 1 year) can be first be treated with standard antibiotic treatment to reduce the bacterial load in the vagina. Twenty-four hours after the final antibiotic dose, the is then administered an effective amount of the disclosed formulation. The subject may also receive daily vaginal treatment with a food acid such as a lactic acid gel, spray or powder before and/or after transplantation to encourage Lactobacillus growth. Daily treatment may occur for up to 1 week before and/or after transplantation. The preferred concentration range of lactic acid to promote Lactobacillus survival is 1-1.5% lactic acid. Lactic acid is preferred to other types of food acid such as vinegar, lemon juice, and acetic acid, although these may also be utilized. The disclosed formulations can be used to restore normal microbiota in pre-term newborns or newborns delivered by Cesarean section and methods for preventing or ameliorating diseases associated with delivery by Cesarean section or pre-term birth. Maternal vaginal microbes provide the natural seeding to the newborn microbiota (1). Whether vaginal microbes can reach the placenta and the fetus before labor initiates, still unclear (2, 3), but mode of delivery overwhelms any other possible previous signal, and C-section-born babies are microbiologically different from vaginally born infants (1). The maternal vaginal (4) and intestinal (5) microbiota change during the third trimester of pregnancy, but the significance of these changes for the fitness of the baby has not been understood. Early interaction with indigenous microbes is 16 45721826.1
Attorney Ref: # JHU C 18071 PCT essential for healthy immunological and metabolic programming, and contact with bacterial populations in the vagina during birth marks the beginning of eventual massive bacterial colonization of the newborn's mucosal surfaces. Mucosal immunity is strongly influenced by the microbiota (6), which in the gut mucosa, is subject to continuous surveillance by M cells—from the Peyer's patches of the gut-associated lymphoid tissue (GALT)—for processing by local dendritic cells and subsequently modulate CD4+ to produce Tregs and induce tolerance. In one aspect, the method includes administering to said infant at the time of birth and/or within the first 4 months of life (preferably, within the first 24 hours of life, most preferably within the first hour of life) an effective amount of a vaginal microbiota inoculum, wherein said inoculum is obtained from the subject's mother or from a donor during the third trimester of pregnancy before or at the time of giving birth. The vaginal microbiota formulation can be delivered by a route selected from the group consisting of topical, rectal, mucosal, sublingual, nasal, and via naso/oro-gastric gavage, etc. V. Kits The formulations can be packaged into a dosage unit or applicator for administration to a subject in need thereof, and provided in a kit, with instructions for administration. The disclosed formulations and methods can be further understood through the following numbered paragraphs and examples. 1. A hypotonic or iso-osmolal vaginal probiotic gel-forming formulation comprising: (a) vaginal microbiota, (b) a gel-forming polymer at a concentration at or below above the critical gel concentration of the polymer under isotonic conditions and a temperature between room temperature and body temperature (25 to 37°C), and (c) a hypotonic carrier to form a hypotonic formulation of the polymer, wherein the vaginal microbiota is selected from the group consisting of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, or Lactobacillus jensenii. 2. The formulation of paragraph 1, wherein at least 50% of the vaginal microbiota is one species typically found in the human vagina, selected from the group consisting of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, or Lactobacillus jensenii. 3. The formulation of any one of claim paragraphs 1-2, wherein the gel-forming polymer is a thermo-sensitive gel-forming polymer. 4. The formulation of paragraph 3, wherein the thermosensitive gel-forming 17 45721826.1
Attorney Ref: # JHU C 18071 PCT polymer has a lower critical solution temperature that is below 30°C, preferably below 21 °C. 5. The formulation of any one of paragraphs 1-4, wherein the polymer is a poloxamer, and optionally, wherein the hypotonic carrier is saline:water at a ratio of 1:1. 6. The formulation of any one of paragraphs 1-5, wherein at least 50% of the vaginal microbiota is Lactobacillus crispatus. 7. The formulation of any one of paragraphs 1-6, wherein the gel-forming polymer is between greater than 12 and less than 24% polyoxyethylene-polyoxypropylene- polyoxyethylene triblock polymer with an average molecular weight Mw of 13,000 Da and an average weight percent of polyoxyethylene at 80%.in an aqueous excipient. 8. The formulation of any one of paragraphs 1-7, wherein the gel-forming polymer is between 10 and 18% Poloxamer 407. 9. The formulation of any one of paragraphs 1-8, wherein the vaginal microbiota is provided by an isolated cervicovaginal secretion (CVS) obtained from a healthy vagina. 10. The formulation of paragraph 9, wherein the CVS has been sterile filtered. 11. The formulation of any one of paragraph 1-8, comprising vaginal microbiota isolated and optionally cultured in vitro. 12. The formulation of any of paragraph 1-11 in the form of a dry powder, gel, or liquid. 13. The formulation of any one of paragraph 1-13, wherein the polymer formulation is provided in a single or multiple dosage unit administration. 14. The formulation of paragraph 12, wherein the formulation has been spray dried or lyophilized and formulated for administration to the vagina, optionally in combination with a resuspending or dissolution agent. 15. The formulation of paragraph 12, wherein the formulation has been frozen. 16. The formulation of any one of paragraphs 1-15, further comprising a cryoprotectant selected from the group consisting of glycerol, at a concentration less than 7%, for example, up to about 1, 2, 3, 4, 5, and 6%, or a mixture of about 5% sodium ascorbate mixed 1:1 with glucose. 17. The formulation of any one of paragraphs 1-16, comprising up to 1.5% lactic acid, pH <4.0. 18. The formulation of paragraph 17, wherein the one or more bioactive agents is progesterone, estradiol or tenofovir. 19. The formulation of any one of paragraphs 1-18, packaged into a dosage unit or 18 45721826.1
Attorney Ref: # JHU C 18071 PCT applicator for administration to a subject in need thereof. 20. A method of treating a subject in need thereof, comprising administering the formulation of any one of paragraphs 1-19. 21. The method of paragraph 20, wherein the subject has been diagnosed with bacterial vaginosis or a sexually transmitted disease. 22. The method of paragraph 20 or 21, subsequent daily vaginal administration of one to five mls of a 1-1.5% lactic acid gel (pH < 4.0) for up to one week before and/or after administration of the formulation. 23. The method of any one of paragraphs 20-22, wherein the formulation has been spray dried or lyophilized, the method comprising admixing the formulation with a dissolution agent and adjusting the pH to a pH less than 4.0. 24. The formulation of any one of paragraphs 1-16, further comprising hyaluronic acid and/or one or more bioactive agents. The present invention will be further understood by reference to the following non- limiting examples. EXAMPLES Example 1. Development of a Hypotonic-gel Forming Formulation for Vaginal Drug and Probiotic Delivery Materials and Methods Materials sources Pluronic F127 (P2443, source BCCH3308), DL-Lactic acid (69785), Xanthan gum (G1253, 800-1200 cps 1% solution), Hydroxypropylmethyl cellulose (HPMC) (H9262, 80-120 cP, 2% in H2O (20⁰C), progesterone (P8783), β-estradiol (E8875), Evans Blue (E2129), Rose Bengal (330000) was purchased from Sigma-Aldrich. Normal saline (114-055-101) was purchased from quality biological. Carboxymethylcellulose (CMC, 173 kDa) (CA193), hydroxyethyl cellulose (H1148, 3,400 cps), polyethylene glycol 400 (PEG400) (P0110), was purchased from Spectrum. Hyaluronic acid sodium (HA) (FH145201, MW 1.8-2.5 MDa) was purchased from Biosynth. Polyethylene glycol 6000 (PEG6000) (1546580) and Mifepristone (RU486) (M8046) was purchased from Millipore Sigma. Carbopol974P (CBP1053H, lot 106362, Carbomer Homopolymer Type B USP NF) was gifted from Lubrizol. Crinone (Allergan) and estradiol vaginal cream (USP, 0.01%, Hampton Laine) was purchased from the JHMI clinical pharmacy.200 nm red carboxylate modified Fluospheres (F8786) and AlexaFluor 568 (A20003) was purchased from Invitrogen.35mm glass bottom dishes (P35GC-0-14-C) were 19 45721826.1
Attorney Ref: # JHU C 18071 PCT purchased from MatTek. For gel erosion studies, 30 mL syringes (53548-024) were purchased from VWR, 27Gx1-1/4 PrecisionGlide needles (306136) were purchased from BD, and NE-300 syringe pumps were acquired from New Era Pump Systems, Inc. For bacteria experiments, GasPak jars (260629) and anaerobe satchets (160001) were purchased from BD. MRS agar (110660) and MRS broth (110661) were purchased from Millipore Sigma. For NYCIII media and agar, enzyme grade HEPES (BP310) was purchased from Fisher, Bacto Proteose Peptone No.3 (211693) was purchased from gibco, sodium chloride (J21618.A1), fetal bovine serum (26140079), and glucose (49139) was purchased from Thermo Fisher, Bacto Yeast Extract (212750) and Bacto Agar (0140-01) was purchased from BD. Gardnerella vaginalis (JCP8481B) and Lactobacillus crispatus (EX533959VC06) were obtained from BEI Resources. For bacteria cryopreservation studies, Pluronic F127 (1166, 12500 Da) was purchased from Spectrum Chemicals. Animal welfare statement The experimental protocols involving mice were approved by the Johns Hopkins Animal Care and Use Committee. Unless otherwise specified, CD-1 mice were ordered from Charles River Laboratories at 6-8 weeks and were housed in a reverse light cycle room in cages of 5 if non-pregnant, or individually housed when pregnant. Pregnant animals were ordered timed pregnant to arrive at gestational day E13 and allowed to acclimate in reverse light cycling housing until dosing and experimentation beginning on day E15. Animals were anesthetized with isoflurane prior to euthanasia. Vehicle formulation and rheological characterizations In order to characterize the rheological behavior of F127 gels in vitro, the F127 concentration must be above the critical gel concentration (~15.5-16%). Thus, 20% (w/w) F127, with or without various excipients, was dissolved in water overnight at 4C. Sample osmolality was then measured using an EliTechGroup Vapro Osmometer in triplicate. The linear measurement range of the instrument is 100-1,000 mOsm/kg, so samples were mixed 1:1 with 0.9% normal saline prior to measurement. The formulation osmolality was then calculated from the measured value and the measured osmolality of the normal saline by linear extrapolation. Each measurement was repeated 3 times. To evaluate the effects of excipients on resistance to erosion, an apparatus was constructed to allow for heating the formulations to 37 oC while flowing fluid at a controlled rate tangentially over the surface. Rose Bengal was added at 1 mg/mL for visualization and quantification of gel erosion. Holes were drilled into opposing sides of a 35mm glass bottom 20 45721826.1
Attorney Ref: # JHU C 18071 PCT dish to allow for inserting syringes with needles attached, one for fluid entry, and one for fluid exit. Each syringe was connected to a syringe pump set to 100 mL/min flowrate. The glass bottom dish was placed on a 37 oC hot plate, and 50 µL of sample containing Rose Bengal was carefully pipetted in the center. PBS was flowed over the samples, and the eluted fluid was collected every 4 min to measure the amount of Rose Bengal. Samples were read in triplicate on a plate reader at 560 nm absorbance and analyzed against a dye standard curve to calculate the amount of gel eroded.3 technical replicates were run per formulation. Viscosity as a function of temperature and gel tackiness and adhesion force was measured using an Anton Paar cone and plate rheometer (model MCR302) with a PP25 probe and temperature control plates. For viscosity, the loading gap was set to 0.3 mm, and a temperature ramp from 10-40 °C with 40 points (0.25 °C minimum temperature change) were selected with gamma=1 (1/s) (n=3 per sample). For max tack measurements (n=3-4 per sample group), 200 µL of sample was transferred to the sample holder of the rheometer using a 1000 µL Drummond wiretrol plunger pipette. The sample was equilibrated between the probe and sample holder with a 1 mm gap for 2 minutes at 37 °C. The probe was placed in contact with the sample, then moved vertically perpendicular to the sample holder at a controlled rate to measure the normal force. A total of 100 points were measured over 60 seconds with a ramp linear model as the probe was pulled vertically upward at -0.5 mm/s. The peak force on the resulting curve was considered the max tack force. Adhesion was calculated by taking the area under the curve of the tack force. Data was exported from the Anton Parr RheoCompass software and analyzed in Microsoft Excel before being graphed in GraphPad Prism 9. Assessment of formulation leakage As the hypotonic gelling formulations do not form a gel in vitro, the gel-forming potential must be evaluated in vivo. Pluronic F127 solutions at 2%, 8%, 10%, and 18% (w/w) were prepared by mixing overnight at 4C, and 100 μg/mL of Evans blue was added for visualization. To synchronize the estrous cycle stage, naturally cycling mice were staged by visual observation of the external appearance of the introitus and surrounding tissue as previously described [1]. Mice in the estrus stage (n = 3) were dosed vaginally with 20 μL of F127 solution containing Evans blue. The mouse was allowed to rest for 1 minute inverted, then placed in a contained area lined with paper towels. The appearance of blue dye spots on the paper towels was counted as leakage, whereas a lack of dye spots within 3 minutes of ambulation was considered to be an absence of leakage and an indication of gel formation. 21 45721826.1
Attorney Ref: # JHU C 18071 PCT Multiple Particle Tracking (MPT) To quantitatively characterize gel formation, visualization of fluorescent nanoparticles via multiple particle tracking (MPT) was employed. Nanoparticles (200 nm red fluorescent) were chemically modified with 2 kDa polyethylene glycol (PEG) as previously described [2]. Particles were diluted 1:100 (0.02% solids) in 2%, 8%, 10% (with and without 0.125% xantham gum), and 18% (w/w) F127 solutions. Mice (n = 5-6) were staged in the estrus phase as described above [1, 3]. Mice were anesthetized with 3% isoflurane during vaginal dosing and subsequent inversion and sacrifice. Particle-containing solutions (20 μL) were dosed vaginally using a wiretrol. Mice were held inverted for 1 minute and then sacrificed. The vagina was immediately dissected, sliced open vertically, and laid lumen-side-up on a slide with a custom- sized rectangular well cut from multiple layers of electrical tape. The top of the well was sealed with a coverslip and superglue, such that the coverslip contacted the mucosal surface without extensive compression of the tissue. The slide was inverted on a Zeiss Axiovert Observer-D1 and a minimum of 7 videos each at least 30 seconds long were taken per tissue. Videos were then exported and run through a proprietary MATLAB code that tracked the centroids of each particle and produced a mean square displacement (MSD) value for each particle [4]. The ensemble-averaged MSD (〈MSD〉) was calculated as the geometric mean of individual-particle MSDs at a time scale of 1 s. Visualization of in vivo gel distribution F127 was fluorescently labeled by chemical conjugation to AlexaFluor 568 as previously described [5]. The content of fluorescently labeled F127 was kept at 2% (w/w) to ensure that the labeled polymer did not interfere with the gel formation as previously described (ref). For higher concentration F127 solutions, unlabeled polymer was added to reach a total concentration of 10% and 18% (w/w) F127.20 μL of fluorescently labeled polymer solution was administered vaginally to estrus staged mice, and the vagina was dissected out 20 minutes after administration. Tissue was flash frozen in OCT, then sectioned on a Leica Cryostat at 10 µm per section. To prevent smearing of the polymer on the slide, the vaginal tissue sections were not fixed or further stained. Imaging was performed immediately following sectioning on both Texas Red and phase contrast Dark Low (DL) with bright field transmitted light settings on a Zeiss Axiovert. The brightfield and fluorescent images were overlaid using ImageJ. Characterization of cryopreservation properties Lactobacillus crispatus was grown in MRS liquid broth anaerobically at 37 °C. After 3 days, the bacteria were washed with saline and transferred into two sets of cryovials for storage 22 45721826.1
Attorney Ref: # JHU C 18071 PCT at either -20 or -80C. The tubes were centrifuged at 1000g for 5 min at 4°C. The pellet was gently resuspended in 200 µl of either water or a 1:1 mixture of saline diluted with water, which either did or did not contain 10% Pluronic F127 or 10% Pluronic/0.5% HA, and placed in a 37 °C dry bath during the plating procedure. The fresh samples were serially diluted and plated on MRS agar plates. The samples in the cryovials intended for freezing were placed in a CoolCell cell containers and placed in the -20 °C freezer or the -80 °C freezer (cools at a rate of -1 °C/min). The frozen samples were thawed after 2 days of freezer storage by placing in a 37 °C dry bath, and then serially diluted and plated on agar plates. Plates were incubated in anaerobic jars with GasPak EZ anaerobe container system (Becton Dickinson) at 37 °C for 2-3 days before CFUs were counted. Drug loaded formulations To assess the ability of F127 to solubilize hydrophobic drugs, estradiol at various concentrations was added to 20% F127 and mixed at 4 oC overnight. If needed, the solution was vortexed for 1 minute time intervals and/or placed in a sonication bath for 20 minute time intervals and checked regularly. The maximum solubility of estradiol in 10% F127 was 250 µg/mL. A similar process was conducted with progesterone (P4) in 10% F127. The maximum solubility was found to be 120 µg/mL, which was below the clinical equivalent in Crinone of 80 mg/mL. Thus, P4 was wet-milled in 6% F127 at 160 mg/mL for 10 hours, then combined with 14% F127 to yield 10% F127 containing 8% P4 (ProGel). This yielded a P4 nanosuspension instead of solubilized P4, demonstrating that the 10% F127 can be used to deliver nanosuspensions as well as solubilized drug. Characterization of gel discharge Estrus staged 8-week-old CD-1 mice were vaginally dosed with 20 µL of Crinone, ProGel, or saline and allowed to ambulate for 30 minutes. Mice were then either lavaged with 25 uL of sterile saline (n=3 per treatment) and lavage fluid was placed on a slide, or the vagina was dissected, sliced opened, and flattened on a slide (n=3 per treatment). Lavage fluid and tissue were imaged at 10X on a Nikon Eclipse Ni-U light microscope to visualize particulates. Efficacy in RU486 preterm birth model Timed pregnant mice were delivered at E13 and housed in a reverse light cycle room to preserve their native circadian rhythm. Mifepristone (RU486) was dissolved in DMSO at 6.125 µg/100 µL. On E15, each mouse received a subcutaneous dose of 100 µL of RU486 solution. Mice received progesterone treatment received a vaginal dose of either 20 uL of Crinone or 23 45721826.1
Attorney Ref: # JHU C 18071 PCT ProGel via wiretrol [6]. Animals were dosed daily E15-E18 and were monitored for preterm birth on or before E18. There were two technical replicates of this experiment., with half of the animals in each experiment iteration with an additional 3 RU486 subcutaneous control outcomes added in from a separate experiment. H&E and Mucicarmine staining Mice were housed together for 1 week to allow for acclimation. Mice were dosed vaginally once per day for two weeks with 20 µL of either Crinone cream, 10% F127 vehicle, saline, or 10% F127 with 8% progesterone to progesterone dose match Crinone. Animals were housed with others who received the same treatment. One day after the last dose, mice were sacrificed and both vagina and cervix were stored in formalin for 24 hours. Tissues were then sent to the JHMI Reference Histology core where embedding in paraffin wax, sectioning at 6 µm, and both hematoxylin and eosin (H&E) staining and mucicarmine staining was completed. Sections were imaged and analyzed using a Nikon light microscope, and toxicology reports were made in partnership with Hopkins Veterinary Pathology. Pharmacodynamics Two cohorts of 6-8 week old CD-1 mice were used in this study, as hormone fluctuation greatly impacts the vaginal mucosa and drug delivery to it. One cohort of non-pregnant mice were housed together for 1 week to allow for acclimation, and were dosed during estrus as visually determined similar to above. The other cohort consisting of pregnant mice were delivered from Charles River at E13 and were allowed to rest until E15, on which mice were vaginally dosed once with 20 µL of either Crinone cream, 10% F127 with 8% progesterone, 18% F127 with 8% progesterone, 10% F127 with 0.125% xanthan gum and 8% progesterone, or saline. At the given timepoint, mice were sacrificed and tissues were collected and flash frozen. Samples were analyzed by LCMS for progesterone level by the analytical pharmacology core at JHMI. Results were graphed using Prism GraphPad 9. Testing of excipients and gelling vehicle against key vaginal microbiota strains Lab strains of Lactobacillus crispatus and Gardnerella vaginalis were grown in triplicate and sub-cultured during their exponential growth phase. Bacteria in media was mixed 4:1 with drug solutions, including Crinone diluted with saline 1:5, saline, 10% F127, and ProGel. These mixtures were allowed to grow hypoxically with oxygen scavenging satchets for 3 hours, then were diluted and plated. Plates were incubated for 48 hours, then counted. CFUs were counted if they were within the countable range of 35-350 CFUs. CFUs were graphed using Prism Graphpad 9. 24 45721826.1
Attorney Ref: # JHU C 18071 PCT Statistical Analysis One-way analysis of variance (ANOVA) was used for comparing two groups in Graphpad Prism 9, and was followed by Tukey’s multiple comparison test when comparing three or more groups in Graphpad Prism 9. Sample sizes were similar in all experiments using ANOVA/Tukey. The statistically significant threshold for these comparisons was p<0.05. Log- rank test (Mantel-Cox) was used for comparing survival statistics during the preterm birth study in Graphpad Prism 9 testing the null hypothesis that all samples come from populations with the same survival and differences are due to chance. The statistically significant threshold for these comparisons was adjusted using the Bonferroni method (Bonferroni-corrected threshold p = 0.017). Results Hypotonic gel-forming formulations are compatible with excipients for vaginal application Pluronic solutions formulated below the critical gel concentration do not form a gel in vitro, so for in vitro characterization of rheological properties, concentrations about the critical gel concentrationmust be used. Thus, 20% (w/w) Pluronic F127 solutions were formulated with and without “generally regarded as safe” (GRAS) excipients used in gel and cream products to assess compatibility. Studies then assessed how the addition of excipients affected the rheological properties at various temperatures. As shown in Figure 1A, all compositions containing 20% F127 transitioned from a liquid to a gel in the range of 17-22 oC. The effect of the polymer additives was more evident when evaluating the viscosity in the liquid phase, as shown at 17 oC in Figure 1B. For example, the addition of 0.5% HA and 0.5% Carbopol 974P increased the viscosity of the 20% F127, whereas 0.5% hydroxyethylcellulose (HEC) did not significantly increase the viscosity at 17 oC (Figure 1B). However, at 37 oC, the F127 gel largely dominated the rheological properties with a few exceptions with changes in viscosity. For example, the addition of 0.25% xantham gum (XG) and 0.5% Carbopol 974P increased the gel viscosity compared to 20% F127 alone (Figure 1C). However, it was evident that none of the excipients added interfered with the formation of a gel when the temperature was appropriately increased. Further, only the XG and Carbopol 974P had significant effects on either max tack force (Figure 2A) and adhesion force (Figure 2B). Studies then characterized how the rheological properties of the Pluronic-based gels would influence the in vitro erosion time (Figure 3A). The studies showed that 0.25% xanthan gum significantly extended the control formulation’s erosion time (Figure 3B). Overall, the F127 25 45721826.1
Attorney Ref: # JHU C 18071 PCT gel properties dominate the erosion characteristics, with only xanthan gum significantly increasing the resistance to erosion and altering the gel’s mechanical integrity over time. As the osmolarity is an important feature of a hypotonic formulation, the effect of various excipients on the osmolarity were measured. The addition of other polymers, including hyaluronic acid (HA), carboxymethylcellulose (CMC), and xantham gum, did not have a significant impact on the osmolality (Figure 4). However, the addition of small molecules like lactic acid and citric acid had a larger effect on osmolality (Figure 4). An important aspect to consider for vaginal dosage forms is the discharge that may occur, particularly with daily use. It has been noted that some vaginal products, such as Estrace®, can cause a clumpy, white discharge that is unpleasant and potentially concerning for patients [7, 8]. As shown in Figure 5, Estrace vaginal estradiol cream is a thick, opaque white material. However, the same concentration of estradiol (0.01%) can be fully solubilized by 10- 20% Pluronic F127 (Figure 5). Hypotonic formulation below the critical gel concentration improves retention and uniformity of distribution As the gelation phenomena of formulations below the critical gel concentration cannot be assessed in vitro, in vivo characterizations was performed to identify Pluronic F127 compositions that would form a gel when dosed hypotonically. First, a qualitative experiment was performed to visualize leakage, which would not occur if there was a liquid to gel transition after vaginal dosing. Using dye-loaded solutions, leakage was consistently observed after dosing 2% and 8% (w/w) F127, while no leakage was observed after dosing 10% and 18% F127 (Figure 6). This indicated that while the 10% F127 formulation was below the critical gel concentration, dosing in a hypotonic solution led to gelation similar to 18% F127 dosed above the critical gel concentration. To quantitatively characterize the gelation phenomenon, F127 solutions containing polyethylene glycol (PEG) coated nanoparticles were vaginally dosed. As previously described, nanoparticles become physically entrapped within the gel, which is reflected in low mobility observed via multiple particle tracking (MPT) [5, 9]. Thus, when excising the vaginal tissue and directly observing the nanoparticles on the vaginal tissue surface, high particle mobility would be indicative of a liquid environment, whereas trapped nanoparticles would reflect the presence of a gel. The studies showed that while the nanoparticles dosed vaginally in the 2% and 8% F127 showed high mean squared displacement (MSD) indicative of thermal motion in a liquid, nanoparticles dosed vaginally in the 10% (with and without XG) F127 were immobilized similar 26 45721826.1
Attorney Ref: # JHU C 18071 PCT to dosing in the 18% F127 (Figure 7). This indicates that despite being below the critical gel concentration, the hypotonic 10% F127 formulations formed a gel in the vagina in vivo. Subsequent studies to assess the vaginal distribution of the hypotonic 10% F127 gel compared to the standard 18% F127 formulated above the critical gel concentration. Fluorescently-labelled F127 was utilized to visualize the gel inside the vaginal lumen and the uniformity of contact with the highly folded vaginal epithelium. As shown in Figure 8A and B, the 10% F127 formed a distinct uniform coating of the vaginal epithelium, whereas the 18% F127 was excluded from vaginal folds (denoted by arrows in Figures 8Dand E). Similarly, the 10% F127 appeared to accumulate on the tissue surface due to the hypotonically-driven absorption and concentration of the polymer (Figures 8A, B, C), whereas the 18% F127 immediately formed a gel in the lumen (denoted by asterisks in Figures 8D, E, F). The hypotonic gel-forming formulation does not negatively impact Lactobacillus crispatus survival The vaginal microbiome is crucial for overall female reproductive health, and disruption and dysbiosis in the vaginal microbiota is associated with an increased risk for preterm birth, pelvic inflammatory disease, etc. [10, 11]. Thus, it is important that vaginal products do not negatively affect the endogenous Lactobacillus bacteria associated with reproductive tract health. L. crispatus is often found as the dominant bacteria in a healthy bacterial community, so the impact of the disclosed formulations on L. crispatus survival in vitro was assessed. Exposure to Crinone resulted in a 100-fold decrease in the number of colony forming units (CFUs), whereas ProGel had no effect on L. crispatus survival (Figure 12). The hypotonic gel-forming formulation demonstrates Lactobacillus crispatus cryopreservation F127 did not have a negative effect on L. crispatus, so subsequent experiments explored its compatibility as a probiotic delivery system. Bacterial survival is of paramount importance during storage, so the potential for cryopreservation was assessed. As shown in Figure 13A, freezing L. crispatus in 10% F127 improved the viability on thawing after freezing to -80 or - 20C. Further, 10% F127 could be combined with 0.5% HA, and either 1:1 saline:water or water could be used as a hypotonic medium for cryopreservation (Figure 13B). HA is a hygroscopic agent, thus adding it to the F127 results in a potential moisturizing benefit along with the delivery benefit of the F127. A hypotonic gel-forming formulation containing progesterone shows equivalent efficacy in preventing preterm birth with enhanced product profile 27 45721826.1
Attorney Ref: # JHU C 18071 PCT Progesterone was formulated into a nanosuspension for dosing in the hypotonic gel- forming formulation. Progesterone at 8% was nanomilled and suspended in 10% Pluronic F127 (ProGel) to dose match to Crinone 8% vaginal cream. In a mouse model of system progesterone withdrawal using a mifepristone (RU486) injection, 65% preterm birth was observed in the control animals injected with RU486 (Figure 9). When animals were dosed vaginally with either Crinone or ProGel daily starting on E15, only 15% of animals delivered preterm (Figure 9, *p < 0.05). However, when assessing the local toxicity, the data showed that the Crinone gel had a negative effect on cervical mucus cells, leading to cervical mucus layer depletion (Figure 10A). In contrast, the ProGel (Figure 10B) had no observable effect distinguishable from treatment with the F127 vehicle (Figure 10C) or saline (Figure 10D). Further, Crinone is a thick, opaque white cream that is known to cause unpleasant discharge in patients. Similarly, thick chunks of cream were observed in lavage fluid and on the vaginal tissue surface after dosing in mice, whereas the ProGel did not have any apparent residue or visually observable material in the mouse vagina (Figure 11), indicating it would not have the same issues with discharge. Discussion Many existing vaginal products are gels or creams that can be messy, difficult to administer, and lead to unpleasant discharge. Further, highly viscous products have limited distribution throughout the collapsed, folded vaginal tissue surface. These products often contain high amounts of excipients that render them hypertonic to the vaginal epithelium, which causes epithelial toxicity, drives fluid excretion and product leakage, and may be harmful to the endogenous microbiota. A liquid-to-gel vaginal drug delivery approach has been developed to transport both hydrophilic and hydrophobic drugs through cervicovaginal mucus to the underlying vaginal epithelium for uptake and distribution throughout the female reproductive tract. The formulation is developed as a hypotonic solution below the critical gel concentration of the polymer, allowing it to remain in liquid form until contact with the cervicovaginal epithelia. Upon contact, water absorption by the epithelia facilitates uniform spreading and concentration of the polymer solution, which gels upon surpassing the critical gel concentration, facilitating sustained drug absorption. The formation of a hydrogel reduces leakage, while the hypotonic gel-forming formulation provides uniform epithelial coverage. The solubilization properties of the gel- forming polymer help maintain drug dosage while minimizing the appearance of clumpy or colored discharge. As a proof of principle, progesterone delivered in the gel-forming vehicle 28 45721826.1
Attorney Ref: # JHU C 18071 PCT (ProGel) demonstrated therapeutic prevention of preterm birth (PTB) without epithelial toxicity in a mouse model and did not induce the in vitro Lactobacillus depletion observed with the commercial cream (Crinone® 8%). Additionally, estradiol was fully solubilized at 0.01%, matching the dosage of the commercially available estradiol cream (Estrace®). References 1. Byers, et al., PLoS One, 2012.7(4): p. e35538. 2. Mert, et al., J Control Release, 2012.157(3): p.455-60. 3. Ensign, et al., Mol Pharm, 2013.10(6): p.2176-82. 4. Lai, et al., Proc Natl Acad Sci U S A, 2010.107(2): p.598-603. 5. Kim, et al., Nat Biomed Eng, 2020.4(11): p.1053-1062. 6. Hoang, et al., J Control Release, 2019.295: p.74-86. 7. Crinone® 4% and Crinone® 8%, (progesterone gel) PHYSICIAN INFORMATION. 2013: p. https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/020701s026lbl.pdf. 8. Package leaflet: Information for the user. Crinone 8% w/w Vaginal Gel, progesterone. 2020: p. https://www.medicines.org.uk/emc/files/pil.1283.pdf. 9. Yu, et al., Adv Healthc Mater, 2016.5(21): p.2745-2750. 10. Chen, et al., Front Cell Infect Microbiol, 2021.11: p.631972. 11. Hoang, et al., PLoS Pathog, 2020.16(1): p. e1008236. Publications cited herein and the material for which they are cited are specifically incorporated by reference. Example 2: Compositional Analysis of Probiotic and Tenofovir-Containing Formulations for Multipurpose Vaginal Administration: Impact on Drug Stability, Lactobacillus Viability, and Infection Prevention Additional compositional data are provided for formulations for vaginal administration. These multipurpose formulations incorporate probiotics with a microbicidal (anti-HIV) drug to provide therapeutic treatment for dysbiosis-related conditions, such as bacterial vaginosis (BV), while also offering protection against sexual transmission of HIV infection. Exemplary formulations contain lactic acid to support Lactobacillus survival and maintain compatibility with the vaginal environment. Tenofovir (TFV), a microbicidal drug, has been developed and evaluated for HIV prevention across various settings and dosing approaches, including vaginal administration. Research suggests that bacteria associated with BV can degrade TFV, potentially reducing its efficacy in vaginal dosing for HIV prevention. This exemplary multipurpose approach can improve the efficacy of infection prevention. 29 45721826.1
Attorney Ref: # JHU C 18071 PCT The formulations described in this example were evaluated to determine whether acidic pH and lactic acid, intended to support Lactobacillus, affect drug concentration during storage. The hypotonic formulations achieved osmolalities within a range suitable for maintaining high Lactobacillus viability. Materials and Methods Materials Tenofovir (147127-20-6, lot no. B19LN1009) was purchased from BOC Sciences. Poloxamer 407 (9003-11-6), Lactic Acid (50-21-5), Sodium DL- Lactic Solution (S0179), and Citric Acid (C1296) was purchased from Spectrum Chemical. USP Water (9190-1) was purchased from RICCA Chemical Company. HPLC grade acetonitrile (A998–4) and HPLC grade water (W5–4) were purchased from Fisher Chemical. Dulbecco's Phosphate Buffered Saline (DPBS) (21–031-CV) was purchased from Corning Cellgro.5M NaOH (SX0607L-6) was purchased from Millipore Corporation. Characterization Osmolarity was measured on a Vapro osmometer and pH was measured on a Fisher Scientific accumet XL200 pH meter. Measurements were performed in triplicate and averaged. Iso-osmolal Tenofovir Formulations To make 50 mL of the 1% lactic acid TFV formulation, 264mg of TFV, 25.66g of 20% w/w Pluronic F127 in water 8.629g of 2% lactic acid, and 16.36g of 2% sodium lactic acid was added to a 50ml conical tube and placed on a tube rotator at 4°C overnight. To make 50 mL of the no acid TFV formulation, 264 mg of TFV, 25.66g of 20% w/w Pluronic F127 in water, 25g of DPBS, and 200ul of 5M NaOH was added to a 50ml conical tube and placed on a tube rotator at 4°C overnight. To make 50 mL of the 1% Citric acid TFV formulation (pH control) formulation, 264 mg of TFV, 25.66g of 20% w/w Pluronic F127 in water, 1g of citric acid, and 980ul of 5M NaOH was added to a 50ml conical tube and placed on a tube rotator at 4°C overnight. Hypotonic Tenofovir Formulations To make 50 mL of the 1% lactic acid theoretical pH 4.13 formulation, 264mg of Tenofovir and 5 g of Pluronic F127 were mixed with 8.63g of 2% lactic acid, 16.36g of 2% sodium lactic acid and 20g of USP grade water in a 50ml conical tube and placed on a tube rotator at 4°C overnight. To make 50 mL of the 1% lactic acid theoretical pH 4.0 formulation, 264mg of Tenofovir and 5 g of Pluronic F127 were mixed with 11.6g of 2% lactic acid, 13.4g of 2% 30 45721826.1
Attorney Ref: # JHU C 18071 PCT sodium lactic acid and 20g of USP grade water in a 50ml conical tube and placed on a tube rotator at 4°C overnight. To make 50 mL of the 1% lactic acid theoretical pH 3.85 formulation, 264mg of Tenofovir and 5 g of Pluronic F127 were mixed with 12.56g of 2% lactic acid, 12.43g of 2% sodium lactic acid and 20g of USP grade water in a 50ml conical tube and placed on a tube rotator at 4°C overnight. To make 50 mL of the 0.5% lactic acid theoretical pH 3.85 formulation, 264mg of Tenofovir and 5 g of Pluronic F127 were mixed with 6.28g of 2% lactic acid, 6.21g of 2% sodium lactic acid and 32.5g of USP grade water in a 50ml conical tube and placed on a tube rotator at 4°C overnight. Storage stability The TFV concentration in the formulations was measured over 1 week in various storage conditions. The formulations were prepared and aliquoted into HPLC vials and stored at one of the following temperatures: 4°C, -20°C, -80°C or flash frozen in liquid nitrogen and then stored at -80°C. The TFV in the sample was measured via HPLC upon formulation and then after 1 week. Acute accelerated stability The TFV concentration in the formulations was measured over 4 weeks in accelerated degradation or room temperature conditions. The formulations were prepared and aliquoted into HPLC vials and stored in either an environmental chamber maintained at accelerated degradation conditions (40 °C with 25% relative humidity) or maintained at room temperature conditions (25 °C with 75% relative humidity). The TFV in the sample was measured via HPLC upon formulation and then after 1 week, 2 weeks and 4 weeks. HPLC quantification The concentration of tenofovir was quantified via HPLC. The HPLC mobile phase included 30:70 (v/v) acetonitrile:water containing 0.1% phosphoric acid with a flow rate of 1 mL/min with a run time of 8 min. Detection was performed at a λmax = 260 nm. Chromatographic separation was achieved using a Luna reversed phase C18 column (150 × 4.6 mm, 5 μm particle size, OOF-4252-EO). A standard curve was created and used to calculate the concentration of TFV in the samples. Results The data demonstrates that TFV remains stable in the iso-osmolal formulations tested. FIGs.12A-12C show short-term storage stability. TFV concentration in (FIG.12A) 1% Lactic 31 45721826.1
Attorney Ref: # JHU C 18071 PCT acid TFV formulation, (FIG.12B) No acid TFV formulation and (FIG.12C) 1% Citric acid formulation, measured over one week via HPLC in the following storage conditions: 4°C, -20°C, -80°C or flash frozen in liquid nitrogen and then stored at -80°C. n=3 replicates. FIGs.13A-13C show the drug stability under accelerated degradation conditions. The data shows TFV concentration in (FIG.13A) 1% Lactic acid TFV formulation, (FIG.13B) No acid TFV formulation, and (FIG.13C) 1% Citric acid formulation, measured over four weeks via HPLC in the following storage in accelerated degradation or room temperature conditions. n=3 replicates. Table 1: Characterization of Hypotonic Formulations Formulation 1% Lactic acid 1% Lactic acid 1% Lactic acid pH 0.5% Lactic acid pH pH 4.13 pH 4.0 3.85 3.85 for
ma n an ng g aco ac us ce v a y. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 32 45721826.1