Live biotherapeutic intravaginal device and methods of use thereof

Abstract

Described herein is an annular intravaginal device including a hydrogel matrix, the hydrogel matrix including a live biotherapeutic agent and a nutrient source for the live biotherapeutic agent, wherein the hydrogel matrix includes a hydrogel-forming polymer crosslinked with a divalent or trivalent cation. Also described is a method of making the annular intravaginal device. The annular intravaginal device is particularly useful in promoting a healthy vaginal microbiome when inserted into the vagina of a woman in need of such treatment as well as to treat infections and disease.

Description

P240285W001107668.332LIVE BIOTHERAPEUTIC INTRAVAGINAL DEVICE AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application 63 / 693,895 filed on September 12, 2024, which is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0001] The present disclosure is related to an intravaginal device comprising a live biotherapeutic agent, and its use to promote a heathy vaginal microbiome as well as to treat infections and diseases.BACKGROUND

[0002] The vaginal microbiome is a complex and dynamic system that serves a unique function of facilitating sexual reproduction while also protecting from harmful pathogens. The vaginal microbiome varies between individuals and can be affected by both internal and external factors. Characterization of the vaginal microbiome is typically dependent on the relative dominance of different microbial species, with Lactobacillus often being the most dominant strain. Lactobacillus uses estrogen-controlled glycogen and other sugars to produce lactic acid, which in turn creates an acidic vaginal environment. In a healthy vaginal microbiome, Lactobacillus helps maintain a vaginal pH of between 3.8 and 4.5, which limits cultivation of foreign pathogens. More specifically, maintenance of an acidic pH produced by this lactic acid content can stimulate im unity from pathogenic bacteria, viruses and fungi by inhibiting their growth. Certain strains of Lactobacillus bacteria are dominant in the vaginal microbiome and have been shown to prevent the propagation of infections, such as bacterial vaginosis (BV) and human papillomavirus (HPV). One of the more common strains is Lactobacillus crispatus, which has been shown to be an effective antimicrobial agent, specifically against urogenital pathogens.

[0003] Current treatments for vaginal infections such as BV and yeast infections include vaginal suppositories and oral treatments. Vaginal suppositories are small, dissolvable capsules that are also inserted in the vagina and typically meant for short term treatment. Users will often need to repeatedly take suppositories to achieve consistent results due to limited dosage and shorter exposure time periods. Live biotherapeutic treatments for these infections are being studied, however those are encapsulated in formulated pills, andP240285W001107668.332 taken orally to allow bacteria to travel through the GI tract, and which are typically seen as less effective than suppositories due to their indirect method of deliver}’ to the vaginal microbiome. No probiotic vaginal suppositories or oral probiotics have FDA approval for treatment of disease at this time. What is needed are alternatives to deliver live biotherapeutics to affect the vaginal microbiome, and evidence of efficacy for treating certain conditions or improving vaginal health.BRIEF SUMMARY

[0004] In an aspect, an annular intravaginal device comprises a hydrogel matrix, the hydrogel matrix comprising a live biotherapeutic agent and a nutrient source for the live biotherapeutic agent, wherein the hydrogel matrix comprises a hydrogel-forming polymer crosslinked with a divalent or trivalent cation.

[0005] In another aspect, a method of making the annular intravaginal device comprises providing a solution comprising the hydrogel-forming polymer, the live biotherapeutic agent, and the nutrient source for the live biotherapeutic agent; adding the divalent or trivalent cation to the solution to provide a crosslinking solution; and casting the crosslinking solution into a mold to provide the annular intravaginal device.

[0006] In another aspect, a method of promoting a healthy vaginal microbiome comprises inserting the annular intravaginal device described above into the vagina of a woman in need of such treatment.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Fig. 1 shows average pH (n=4) of glucose solutions in which samples of ring variations (standard hydrogel, hydrogel with chitosan, hydrogel with polyacrylamide, and corresponding blanks (no probiotics)) were stored for the duration of the testing period. The pH was recorded at weeks 0, 1, 2, 3, and 4 weeks of testing. ANOVA analysis was performed with * indicating a significant difference between groups (p<0.05).

[0008] Fig. 2 shows average peak tensile load (lbs) (n=4) from tensile mechanical testing of standard hydrogel, hydrogel with chitosan, hydrogel with polyacrylamide, and corresponding blank rings (within separate testing groups per time point) at weeks 0, 2, and 4 of ring in vitro testing. Samples were stretched to failure. Sample number n=4 per formula for each time point. ANOVA analysis was performed with * indicating a significant difference between groups (p<0.05).P240285W001107668.332

[0009] Fig. 3 shows mechanical testing peak compressive load (lbs) of standard hydrogel, hydrogel with chitosan, and hydrogel with polyacrylamide at Day 0. Sample number n=4. t-test analysis was performed with * indicating a significant difference between groups (p<0.05).

[0010] Fig. 4 shows results of samples (n=4) of glucose solutions collected by cotton swab, which were then streaked onto Lactobacillus selective agar. Samples were collected on weeks 0, 1, 2, 3 and 4 of incubation with standard hydrogel, hydrogel with chitosan, and hydrogel with polyacrylamide. Colonies were counted after incubating agar plates for 72 hrs. Blank rings had no colony formation (not included). ANOVA analysis was performed with * indicating a significant difference between groups (p<0.05).

[0011] Fig. 5 shows preliminary degradation testing of alginate hydrogel rings (standard hydrogel, hydrogel with chitosan, and hydrogel with polyacrylamide and corresponding blanks) done by taking average (n=4) weight in grams of each variation on weeks 0, 2 and 4 of testing.

[0012] Fig. 6 shows average (n=4) peak tensile strength (lbs) for standard alginate rings with varying Ca2+concentrations. Rings were lyophilized and rehydrated with deionized water before being incubated in a 9% glucose solution. The control group consisted of rings that were rehydrated but not transferred to glucose solution.

[0013] Fig. 7 shows average (n=3) peak tensile strength (lbs) for alginate rings rehydrated with different rehydration solutions. The 75 and 100 mM Ca2+rings were rehydrated in a solution of 50 mM and 25 mM Ca2+, respectively. The control group was rehydrated in deionized water.

[0014] Fig. 8 shows average (n=4) of measured glucose solution pH of positive control and lyophilized rings over 28-day period. A t-test analysis showed no significant difference between average pH values between control and lyophilized samples (p=0.91). pH values were taken from the glucose solution the rings were placed in at time stamps of day 0, 7, 14, and 28.

[0015] The above-described and other features will be appreciated and understood by those skilled in the art from the follow ing detailed description, drawings, and appended claims.DETAILED DESCRIPTION

[0016] Described herein is a multipurpose device incorporating live biotherapeutic agents, e.g., biotherapeutic bacteria, that are locally released within the vaginal microbiomeP240285W001107668.332 to support native Lactobacillus and maintain a healthy acidic vaginal pH (<5). Ring-shaped devices for controlled administration of drug substances such as contraceptive agents into the vagina are known in the art. Several such products are already marketed, such as Estring®, Femring®, and Nuvaring®, each of which provide controlled and sustained release of steroid molecules (substantially water-insoluble drugs) over several days.

[0017] Specifically, described herein is an annular intravaginal device comprising a hydrogel matrix, the matrix comprising a live biotherapeutic agent and a nutrient source for the live biotherapeutic agent. The live biotherapeutic agent and the nutrient source are preferably dispersed throughout the hydrogel matrix. In an aspect, the hydrogel matrix comprises a hydrogel -forming polymer crosslinked with a divalent or trivalent cation.

[0018] As used herein, an "‘intravaginal device” refers to an object having a size, shape and composition for placement in the vaginal and / or urogenital tract of a female, including, e.g., the vagina, cervix, or uterus of a female.

[0019] The device described herein is annular in shape. As used herein, “annular” refers to a shape of, relating to, or forming a ring. Annular shapes include a ring, an oval, an ellipse, a toroid, and the like. In some embodiments, the intravaginal device of the present disclosure is a vaginal ring. The shape of the intravaginal device can be pliable or malleable, and can be altered or deformed temporarily, i.e., can temporarily assume a non-annular shape, e.g., when being inserted into a female. In some embodiments, the device is elastic, i.e., the device spontaneously returns to its original shape after being deformed.

[0020] In some embodiments, the device can be flexible. As used herein, “flexible” refers to the ability of a solid or semi-solid to bend or withstand stress and strain without being damaged or broken. For example, the intravaginal device can be deformed or flexed, such as, for example, using finger pressure (e.g., applying pressure from opposite external sides of the device using the fingers), and upon removal of the pressure, return to its original shape. The flexible properties of the intravaginal device can be useful for enhancing user comfort, and also for ease of administration to the vaginal tract and / or removal of the device from the vaginal tract.

[0021] In an aspect, the device is a monolithic intravaginal device. The term “monolithic intravaginal device” refers to a device that is a matrix device, wherein the matrix device does not comprise a membrane or wall that encloses a reservoir.

[0022] In an aspect, the intravaginal device comprises one or more apertures. As used herein, an “aperture” refers to a cavity, hole, depression, indentation, or recess extending from the surface into the device.P240285W001107668.332

[0023] In an aspect, the intravaginal device of the present disclosure is an intravaginal ring, typically comprising an aperture. An exemplary preferred geometry for an intravaginal ring is an overall or outer diameter of from about 45 to about 60 mm, more preferably from about 52 to about 60 mm; an aperture diameter of from about 1 to about 7 mm, more preferably from about 2 to about 6.5 mm, most preferably from about 3 to about 6 mm; a cross-sectional diameter of from about 4 to about 10 mm, more preferably from about 4.5 to about 10 mm, most preferably from about 5.5 to about 9.5 mm; and a core length of from about 2 to about 200 mm.

[0024] The surface area of the device may be more than 800 mm2, more preferably at least 1000 mm2, and will typically be in the order of 1700-2000 mm2, although significantly larger surfaces are possible, provided that the design (physical dimensions) of the vaginal ring is convenient for the subject.

[0025] In some embodiments, the live biotherapeutic agent and / or the nutrient source are homogeneously dispersed in the hydrogel matrix. As used herein, “homogeneous’' refers to a composition, e.g., the hydrogel matrix, that has a substantially uniform distribution of ingredients throughout (i.e. , a homogenous hydrogel matrix invention does not have a composition gradient, is not multi-phasic, or have a multi -laminate structure).

[0026] Exemplary7live biotherapeutic agents comprise Lactobacillus casei, Lactobacillus plantarum, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus casei, Lactobacillus parcasei, Lactobacillus iners, Lactobacillus jessenii, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus hamsteri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bacillus coagulans. Bacillus subtilis, Bifidobacterium lactis, Bacteroides firagilis, and combinations thereof.

[0027] Exemplary nutrient sources for the live biotherapeutic agent include a monosaccharide or a disaccharide, such as glucose, fructose, galactose, sucrose, lactose, maltose, isomaltose, trehalose, lactulose, or a combination thereof. Additional nutrient sources for the live biotherapeutic agent include inulin, maltodextrin, com starch, 0-glucan, apple pectin, and combinations thereof.

[0028] The device described herein includes a hydrogel matrix. Exemplary7hydrogel matrices include hydrogel-forming polymers such as alginate, agarose, poly(ethylene glycol dimethacrylate), polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), gelatin, collagen, agarose, pectin, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide),P240285W001107668.332 poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene). pluronic polyol, polyoxamer, poly(uronic acid), poly(anhydride) and poly(vinylpyrrolidone), a mixture, or a copolymer thereof. In a specific embodiment, the hydrogel matrix comprises alginate.

[0029] The hydrogel matrix is crosslinked with a divalent or trivalent cation, such as Ca2+, Mg2+, Sr2+, Ba2+, Be2+, Al2+, or a combination thereof. In a specific embodiment, the hydrogel matrix is crosslinked with Ca2+.

[0030] In an aspect, the hydrogel matrix further comprises a non-hydrogel-forming polymer. Exemplar}' non-hydrogel-forming polymers include carboxyl polymers, polyacrylamide, polyacrylic acid, polyethylene oxide polymers, acrylates, and combinations thereof.

[0031] In an aspect, the annular intravaginal device may comprise a biopolymer coating. Exemplary biopolymer coatings include starch, a chitosan, a hemicellulose, a lignin, a cellulose, a chitin, a dextran, a fibrin, a cyclodextrin, a protein, a polylactic acid, or a combination thereof.

[0032] In an aspect, the annular intravaginal device further comprises a hormonal or non-hormonal contraceptive, an active agent for hormone replacement therapy, an active agent for treatment of menopausal symptoms, or a combination thereof.

[0033] Exemplary' contraceptives include desogestrel, dienestrol, diethylstilberol, estradiol, estriol, estradiol-3 -acetate, ethinyl estradiol, etonogestrel. gestodene, levonorgestrel, medroxyprogesterone, medroxyprogesterone acetate, mestranol, norethisterone, norgestimate, nonoxynol-9, norethisterone acetate, progesterone, testosterone, testosterone acetate, ST-1435 (a progestin), tibolone, and combinations thereof.

[0034] Exemplary drugs for hormone replacement therapy include dehydroepiandrosterone sulphate, dienestrol, diethylstilberol, estrogens such as estradiol, estriol, estradiol-3-acetate, ethinyl estradiol, gestodene, levonorgestrel, luteinizing hormone releasing hormone, norethisterone, norethisterone acetate, progesterone, ST-1435, testosterone, testosterone acetate, and combinations thereof.

[0035] Exemplary drugs for treatment of menopausal symptoms include estrogen, estradiol, progesterone, testosterone, fezolinetant (Veozah®), paroxetine (Brisdelle®), venlafaxine, paroxetine, escitalpopram, fluoxetine, gabapentin, clonidine, and combinations thereof.

[0036] In an aspect, the annular intravaginal device is stable, for example, the annular intravaginal device maintains a pH of less than 4 after 4 weeks in a 9% w / v glucose solution at human body temperature.P240285W001107668.332

[0037] In an aspect, the annular intravaginal device is freeze-dried.

[0038] In an aspect, a method of making the annular intravaginal device described herein comprises providing a solution comprising the hydrogel-forming polymer, the live biotherapeutic agent, and the nutrient source for the live biotherapeutic agent; adding the divalent or trivalent cation to the solution to provide a crosslinking solution; and casting the crosslinking solution into a mold to provide the annular intravaginal device.

[0039] The method optionally further comprises coating the device with a biopolymer coating. Coating can include spraying the biopolymer, or dipping the device into a solution comprising the biopolymer.

[0040] Also included herein are methods of promoting a healthy vaginal microbiome comprising inserting the annular intravaginal device described herein into the vagina of a woman in need of such treatment.

[0041] In an aspect, the woman in need of such treatment has bacterial vaginosis. Bacterial vaginosis (BV) is the most common cause of vaginitis symptoms among women of childbearing age. Previously called non-specific vaginitis or Gardnerella-associated vaginitis, BV is associated with sexual activity and can be sexually transmitted. In bacterial vaginosis the normal Lactobacillus-dominated vaginal microflora is replaced by high concentrations of mixed anaerobic and facultative flora. Typically, this includes Peptostreptococcus sp., Gardnerella vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum. BV is a common vaginal infection and has been associated with an increased risk of preterm labor and delivery, premature rupture of membranes, chorioamnionitis, and pelvic inflammatory disease.

[0042] In another aspect, the woman in need of such treatment has a sexually transmitted disease such as human papilloma virus (HPV) infection, human immunodeficiency virus (HIV) infection, chlamydia, genital herpes, gonorrhea, pubic lice, syphilis, trichomoniasis, Mycoplasma genitalium infection, Ureaplasma infection, cytomegalovirus infection, molluscum contagiosum infection, or zika virus infection.

[0043] C urrent standards in the treatment of sexually transmitted diseases have focused on oral and intravenous administration of antibiotics and antiviral agents. The device described herein can promote a healthy vaginal microbiome and prevent diseases such as BV and STDs.

[0044] In an aspect, the woman is in need of treatment for a vaginal yeast infection. Vaginal yeast infection or valvovaginal candidiasis is a common cause of vaginal irritation. Several factors are associated with increased symptomatic yeast infection in women,P240285W001107668.332 including pregnancy, uncontrolled diabetes mellitus, and use of oral contraceptives or antibiotics. Other factors that may increase the incidence of yeast infection include using douches, perfumed feminine hygiene sprays, and topical antimicrobial agents, and wearing tight, poorly ventilated clothing and underwear. The device described herein may be particularly well-suited to treat and / or prevent vaginal yeast infections.

[0045] In an aspect, the woman in need of promoting a healthy vaginal microbiome may be a pregnant woman at risk of preterm labor. Preterm birth is a leading cause of neonatal morbidity and mortality. Women with an imbalance in their vaginal microbiome can have an increased risk of preterm labor.

[0046] In an aspect, the woman in need of promoting a healthy vaginal microbiome is suffering from symptoms of menopause such as vaginal dryness, itching, vaginal epithelium atrophy, and combinations thereof.

[0047] The invention is further illustrated by the following non-limiting examples.ExamplesMethods

[0048] Live biotherapeutic preparation: Live biotherapeutic bacteria w ere cultured and centrifuged to form pellets. Live biotherapeutics were grow n by adding an AZO® Lactobacillus live biotherapeutic blend to sterilized Lactobacillus specific MRS broth. The live biotherapeutic mixture was then gently shaken and the container placed into an orbital incubator set to 37°C and 75 RPM. Bacteria were allowed to grow for >24 hours. Live biotherapeutic pellets w ere formed by centrifuging a sample of MRS-live biotherapeutic solution at 4000 rpm for 20 minutes at 4°C. After centrifugation, MRS supernatant was aspirated. Centrifuged pellets were then gently washed with 0.9% w / v saline solution. After washing, the remaining saline solution was aspirated, and pellets resuspended with 1 mL of the same saline wash solution. The entire volume of resuspended pellets w as added to precrosslinked alginate solutions and mixed thoroughly. Excess or unused pellets were stored at 4°C up to a week.

[0049] Alginate ring preparation: A 5% w / v sodium alginate solution was created with sterilized DI water. Dissolution of sodium alginate was promoted through use of a hot plate set to approximately 100°C and magnetic stir bar set at 750 rpm. Once the alginate mixture homogenized, CaCOs, powder was added to achieve the desired concentration. The hot plate was set to approximately 40° C, and 2% w / v of maltose and dosage of live biotherapeutic suspension were added to the beaker and mixed thoroughly. After theP240285W001107668.332 pre / probiotics homogenized, crosslinking was initiated with a 0.5 molar ratio of glucono- delta-lactone (GDL) to CaCO? (250 mM GDL) to maintain a neutral pH. After mixing, the solution was immediately poured into a ring casting mold which was 3D printed with polylactic acid (PLA). The casting trays were then covered with parafilm and placed into a 4°C refrigerator overnight to complete crosslinking. Rings were stored in the refrigerator until used for testing. Four rings were selected for pH and bacteria diffusion / viability testing. Eight rings were fabricated for mechanical testing (four for Day 0 and four for Day 14), with the four rings used in pH / bacteria testing used for final mechanical testing on Day 28.

[0050] Alginate-polyacrylamide ring preparation: Fabrication began using the prior protocol for creating a standard 5% w / v sodium alginate solution until the step prior to GDL addition with 16.8% w / v of acrylamide (AAM) added. After mixing, N,N’- methylenebisacrylamide (MBAA) was added at 0.0006 times weight of acrylamide (w / AAM), 125 mM of CaCOs, 2% w / v maltose, and the suspension of live biotherapeutics. A separate solution of 0.006 w / AAM of ammonium persulfate (APS), 0.055 w / AAM of tetramethyl-ethylenediamine (TEMED), and 250 mM GDL created for crosslinking. A small volume (~ 0.5 mL) of crosslinking solution was added into the casting mold, followed by addition of alginate-acrylamide solution to fill mold, with manual mixing to initiate crosslinking. Casting molds were again covered in parafilm and refrigerated at 4°C overnight to gel until testing. Four rings were selected for pH and bacteria diffusion / viability testing. Eight rings were fabricated for mechanical testing (four for Day 0 and four for Day 14), with the four rings used in pH / viability testing used for final mechanical testing on Day 28.

[0051] Alginate-chitosan coating ring preparation: A standard hydrogel protocol was used to create 5% w / v alginate hydrogel rings crosslinked with 125 mM Ca2+. After completing crosslinking overnight at 4°C, rings were allowed to return to room temperature and submerged in a 1% w / v chitosan hydrochloride (CH-CI) solution with gentle stirring for 1 hour in an orbital incubator (set at 37°C, 75 rpm). After removal, the rings were blotted to remove excess coating solution and refrigerated again at 4°C until used in testing. Four rings were selected for pH and bacteria diffusion / viability testing. Eight rings were fabricated for mechanical testing (four for Day 0 and four for Day 14), with the four rings used in pH / viability testing used for final mechanical testing on Day 28.

[0052] Glucose solution preparation: To keep rings hydrated and provide a nutrient source for bacteria, the rings were fully submerged in 9% w / v glucose solution. The solution had a slightly neutral pH (~6), allowing for better analysis of effect of biotherapeutics duringP240285W001107668.332 the testing period. Prior research has shown increased survival rates for Lactobacillus bacteria in acidic environments in the presence of metabolizable sugars, such as glucose.

[0053] Ring incubation: After both the rings and glucose solution were fabricated, samples were placed into labeled 100 mm x 10 mm Petri dishes. Glucose solution was added to each container to fully submerge the rings (approximately 10 mL). Each Petri dish was covered with parafilm to create an anaerobic environment and protect the ring from possible contamination. Rings of the same formula were kept together in labeled bioassay trays, with sterile DI water added for humidity. Trays were then placed in a VWR® 3500 incubated orbital shaker set at 37°C and 75 rpm for the duration of testing to match human physiological conditions.

[0054] pH testing: The pH of glucose solution for each sample was measured at weeks 0, 1, 2, 3, and 4 using a Fisherbrand™ Accumet™ AB 150 calibrated pH meter. To prevent cross-contamination, the probe was sterilized with 70% EtOH between measurements. "Blank" rings containing no live biotherapeutics of each variation were also tested as a negative control.

[0055] Bacterial viability and diffusion testing: The glucose solution for samples at Day 0, 3, 7, 14, 21, and 28 was swabbed with a sterile cotton swab and streaked onto Lactobacillus selective agar. Agar plates were incubated for 72 hours anaerobically in an orbital shaker (37°C, 75 rpm) before the number of colonies formed on each plate was counted. A solution of “blank7’ rings with no live biotherapeutics also tested as a control.

[0056] Mechanical testing: Hydrogel rings were loaded onto an MTS Insight™ machine for tensile strength testing to simulate removal of the ring from the vagina, with rings stretched at a rate of 0.1 mm / sec until failure. Rings also underwent compressive strength testing, to both 50% and 90% of initial diameter, to simulate insertion of the ring into the vagina (which typically involves compressing and twisting the ring). A 50 N load cell was used and customized grips 3D printed out of PLA for loading the rings.

[0057] Degradation testing: Hydrogel rings had an initial weight taken on Day 0 for rings utilized in tensile testing on Day 14 and Day 28 to give an average weight. The weight was measured again when the rings were removed from solution and gently blotted dry prior to loading the rings for mechanical testing.Example 1 : pH testing results

[0058] The average pH from samples (n=4) of each ring formula (including ‘blank’ rings with no live biotherapeutics) collected at weeks 0, 1 , 2, 3, and 4 plotted over time isP240285W001107668.332 shown in Fig. 1. ANOVA analysis showed significant differences between groups by week 1. The results indicated the standard ring formula maintained the most consistent acidic pH over the duration of testing.Example 2: Mechanical testing results

[0059] The average ultimate tensile load (lbs) from samples (n=4) of each ring formulation were collected at specified time points (week 0, 2 and 4) as shown in Fig. 2. Compressive strength testing was performed on a separate sample of rings (n=4) fabricated on Day 0 for 50% and 90% compression as shown in Fig. 3. During compression testing, all rings returned to their original shape after compression, with no signs of permanent deformation. Analysis using standard ANOVA showed blank formulations had no significant difference in tensile load between their respective formulations that included bacteria. Standard and chitosan rings show ed significant differences from the polyacrylamide rings. For compressive testing, chitosan showed a significantly higher compressive strength than both the standard and polyacrylamide formulations as seen in Fig. 3.Example 3: Bacterial viability’ and diffusion results

[0060] After sw abbing glucose solutions on Days 0, 7, 14, 21, and 28 then streaking onto Lactobacillus selective agar plates, the number of colonies for each formulation were counted after incubating the agar plates for 72 hours and plotted over time as shown in Fig. 4. For quality assurance of agar, a stock glucose solution was used as a negative control and a resuspend sample of a bacterial pellet w as used as a positive control. As expected, stock glucose showed no signs of colony formation and the live biotherapeutic solution yielded a high colony count (data not shown). 'Blank' rings with no live biotherapeutics were also used as additional negative control and showed no colony formation on agar. By Day 7, it was found that standard formula rings had the highest number of colonies, w ith a significantly lower number of colonies in other variations.Example 4: Degradation testing

[0061] Average weight from samples (n=4) of each ring formulation collected on Days 0, 14, and 28 plotted over time is shown in Fig. 5. No significant change in ring weight was observed, but some batch-to-batch variation in weight was observed.P240285W001107668.332Discussion of Examples 1-4

[0062] Preliminary testing of the rings demonstrated that rings fabricated with higher concentrations of CaCOs or coated with chitosan had greater maximum tensile strengths than lower CaCOg concentration rings or those without a chitosan coating. The results indicated the chosen standard formula performed similarly to chitosan-coated rings in tensile testing, with chitosan-coated rings having significantly greater compressive strength compared to the other two formulations. However, tensile strength is only one measure of mechanical properties, and achieving tensile strength in a hydrogel that is similar to that of a plastic based contraceptive rings is unlikely due to chemical differences. Because all ring formulas were able to return to shape following compression , ther eis promise for the ability to insert rings without them breaking apart.

[0063] Another design aspect was bacterial viability’ and diffusion to ensure changes in pH can be contributed to Lactobacillus. The hydrogel rings appear to have bursts of bacterial diffusion out of the rings into the surrounding solution, with the highest colonyformation observed on Day 7 and Day 14. A notable decrease in the number of colonies from samples taken at Day 21 is believed to indicate the bacteria may potentially not remain viable once diffused out of the ring, however, further testing and sequencing of the bacterial samples collected would need to be done to confirm. pH measurements of the solution have consistently been used as secondary data to indicate bacteria's ability to produce an environmental pH below 5. The results indicated the standard ring formula achieved a pH below 5 by Day 7 and maintained this pH level for the entire testing period. In comparison, both the chitosan and polyacry lamide rings had significantly more alkaline pH levels. Without being held to theory, chitosan has been shown to be a more alkaline-like chemical and typically does not have a high molecular weight, which may cause it to dissolve into the solution faster and increase environmental pH. It is also possible that the coating and the secondary mesh network of polyacrylamide limited nutrient diffusion to bacteria (along w ith diffusion of bacteria out of the ring), causing bacteria to die and release acidic by-products. When assessing the overall potential effect of bacteria diffusion on pH, the standard formula had a relatively high number of colonies corresponding with an average lower pH level.Example 5: Lyophilization and long-term storage

[0064] Lyophilization (or freeze-drying) has been shown to be an effective means of preserving certain strains of anaerobic bacteria. Lyophilization is a low temperature dehydration process that freezes the material while lowering the atmospheric pressure, thusP240285W001107668.332 removing ice by sublimation. Freeze-dried rings can then be shipped to distributors under normal shipping conditions. Prior to use, the hydrogel must be rehydrated to allow flexibility and revive bacteria from cryopreservation. Rehydration can be done using a standard neutral buffer solution. Once received, distributors may then potentially rehydrate rings prior to giving them to users. Alternatively, packaging can be designed that allows users to rehydrate the rings using a provided buffer solution at-home. The packaging must also maintain sterility for users pnor to insertion. For packaging, there are various methods used to safely store and transport medical devices and solutions, including medical blister packaging and dual pliable pouches.

[0065] To test the feasibility of lyophilization, after rings were fabricated, individual rings were placed into labeled 100 mm x 25 mm Petri dishes and frozen at -20°C for 24 hours. Once frozen, the petri dishes containing the rings were placed into a lyophilizer without the petri dish lid for another 24 hours. Freeze-drying was conducted at a condenser temperature of-80°C at 80 mTorr for 6 hours after which petri dishes were flame sealed under vacuum and stored at 4°C until further testing.

[0066] To improve the potential of rehydration, variations in the concentration of crosslinker were utilized with four replicates of 75, 100, and 125 mM Ca2+. Rings were freeze-dried and rehydrated for 24 hours in deionized water at room temperature to serve as a control group. Another set of rings was rehydrated for 24 hours and transferred to a glucose solution and incubated in a heated orbital shaker for a week. Both room temperature and incubated rings were then tested for tensile strength on the MTS machine.

[0067] Additionally, tensile strength testing was conducted on rings rehydrated in vary ing solutions. Four samples prepared with the two lower concentrations of Ca2+(75 and 100 mM), were rehydrated for 24 hours in either deionized water (as control group) or a solution of calcium carbonate to increase the concentration of the rings post-freeze drying. The 75 mM rings were rehydrated in a 50 mM calcium carbonate solution, and the 100 mM rings were rehydrated in a 25 mM calcium carbonate solution to ideally equalize concentration at 125 mM.

[0068] The average peak tensile load (lbs) from samples (n=4) of each ring formulation collected at specified time points of incubation (Day 1 and 7) is shown in Fig. 6. The average peak tensile load (lbs) from sample (n=4) rings rehydrated in respective Ca2+solution compared to rehydration in water shown in Fig. 7.

[0069] In order to determine the effect of lyophilization on the viability of bacteria embedded within the hydrogel, four ring samples were prepared using lyophilization andP240285W001107668.332 subsequent rehydration. These rings were then submerged and tested using pH analysis as an indicator of bacterial viability as shown in Fig. 8. Both freeze-dried and positive control rings that were not lyophilized were submerged in glucose solution and incubated over a 28- day period. pH values were taken directly from the glucose solution at time stamps of days 0, 7, 14, and 28. These values were averaged and compared at the discrete time stamps on the plot shown in Fig. 7. The difference in pH between the control and freeze-dried rings was found to be statistically insignificant, with a p-value of 0.91 calculated from a two-tailed t- test.

[0070] Preliminary testing of bacteria viability using CFU calculation involved using sterilized inoculating loops to spread samples of the glucose solution, storing 125 mM rings onto Lactobacillus selective agar plates on Days 0, 1, 2, and 3. Agar plates were incubated for 24 hours. No bacterial growth was observed throughout the duration of testing. Positive control group of non-freeze dried 125 mM rings also showed no bacterial growth. ‘Blank’ rings with no live biotherapeutics were also used as a negative control and showed no colony formation on agar.

[0071] The use of the terms “a” and '‘an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values 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. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0072] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may beP240285W001107668.332 made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

P240285W001107668.332Claims1. An annular intravaginal device, comprising a hydrogel matrix comprising a live biotherapeutic agent and a nutrient source for the live biotherapeutic agent, wherein the hydrogel matrix comprises a hydrogel-forming polymer crosslinked with a divalent or trivalent cation.

2. The annular intravaginal device of claim 1, wherein the device comprises an aperture.

3. The annular intravaginal device of claim 1, wherein the hydrogel-forming polymer comprises alginate, agarose, poly(ethylene glycol dimethacrylate), polylactic acid, poly glycolic acid, poly(lactic-co-glycolic acid), gelatin, collagen, agarose, pectin, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, a polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), polyethylene oxide), poly(allylamine), poly(acrylate), poly(4- aminomethylstyrene), a pluronic polyol, a poly oxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), a mixture, or a copolymer thereof.

4. The annular intravaginal device of claim 1, wherein the divalent or trivalent cation comprises Ca2+, Mg2+, Sr2+, Ba2+, Be2+, Al2+, or a combination thereof.

5. The annular intravaginal device of claim 1 , wherein the nutrient source comprises a monosaccharide or a disaccharide.

6. The annular intravaginal device of claim 5, wherein the monosaccharide or disaccharide comprises glucose, fructose, galactose, sucrose, lactose, maltose, isomaltose, trehalose, lactulose, or a combination thereof.

7. The annular intravaginal device of claim 1, wherein the nutrient source comprises inulin, maltodextrin, com starch, P-glucan, apple pectin, or a combination thereof.

8. The annular intravaginal device of claim 1, wherein the hydrogel matrix comprises a non-hydrogel -forming polymer selected from carboxyl polymers,P240285W001107668.332 polyacrylamide, poly acrylic acid, polyethylene oxide polymers, acrylates, and combinations thereof.

9. The annular intravaginal device of claim 1, further comprising a biopolymer coating.

10. The annular intravaginal device of claim 8, wherein the biopolymer coating comprises a starch, a chitosan, a hemicellulose, a lignin, a cellulose, a chitin, a dextran, a fibrin, a cyclodextrin, a protein, a polylactic acid, or a combination thereof.

11. The annular intravaginal device of claim 1 , wherein the live biotherapeutic agent comprises Lactobacillus casei, Lactobacillus plantarum, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus casei, Lactobacillus parcasei. Lactobacillus iners, Lactobacillus jessenii, Lactobacillus rhamnosus. Lactobacillus reuteri. Lactobacillus helveticus, Lactobacillus hamsteri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bacillus coagulans, Bacillus subtilis, Bifidobacterium lactis, Bacteroides fragilis, or a combination thereof.

12. The annular intravaginal device of claim 1, further comprising a hormonal or non-hormonal contraceptive, an active agent for hormone replacement therapy, an active agent for treatment of menopausal symptoms, or a combination thereof.

13. The annular intravaginal device of claim 1, wherein the annular intravaginal device maintains a pH of less than 4 after 4 weeks in a 9% w / v glucose solution at human body temperature.

14. The annular intravaginal device of claim 1, wherein the device is freeze-dried.

15. A method of making the annular intravaginal device of claim 1, comprising providing a solution comprising the hydrogel-forming polymer, the live biotherapeutic agent, and the nutrient source for the live biotherapeutic agent; adding the divalent or trivalent cation to the solution to provide a crosslinking solution; and casting the crosslinking solution into a mold to provide the annular intravaginal device.P240285W001107668.33216. The method of claim 15, further comprising coating the annular intravaginal device with a biopolymer coating.

17. A method of promoting a healthy vaginal microbiome, comprising inserting the annular intravaginal device of claim 1 into the vagina of a woman in need of such treatment.

18. The method of claim 17, wherein the woman in need of such treatment has bacterial vaginosis.

19. The method of claim 17, wherein the woman in need of such treatment has a sexually transmitted disease such as human papilloma virus (HPV) infection, human immunodeficiency virus (HIV) infection, chlamydia, genital herpes, gonorrhea, pubic lice, syphilis, trichomoniasis, Mycoplasma genitalium infection, Ureaplasma infection, cytomegalovirus infection, molluscum contagiosum infection, or zika virus infection.

20. The method of claim 17, wherein the woman in need of such treatment has a vaginal yeast infection.

21. The method of claim 17, wherein the woman in need of such treatment is a pregnant woman at risk of preterm labor.

22. The method of claim 17, wherein the woman is in need of such treatment to decrease symptoms of menopause including vaginal dryness, itching, vaginal epithelium atrophy, and combinations thereof.

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