Multi-component vaginal ring

Abstract

This disclosure relates to a multi-component intravaginal ring (IVR) that improves the release of non-hormonal substances, which is useful in multi-purpose preventive techniques (MPT) that simultaneously prevent unplanned pregnancies, prevent or treat sexually transmitted infections (STIs), and optimize vaginal health.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority from U.S. Provisional Patent Application No. 63 / 479,848, filed on 13 January 2023, the disclosures of which are incorporated herein by reference.

[0002] This invention was made with government support under grant number P50HD106793, awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] This disclosure relates to a multi-component intravaginal ring (IVR) that improves the release of non-hormonal substances, which is useful as a multi-purpose preventive technology (MPT) product for simultaneously preventing unplanned pregnancies, preventing and / or treating one or more sexually transmitted infections (STIs), and promoting vaginal health. [Background technology]

[0004] Unprotected sex can lead to unplanned pregnancies and STIs (including HIV-1 and HSV-2), which are significant reproductive and sexual health issues for women worldwide.

[0005] While hormonal contraceptives such as oral hormonal contraceptives, hormonal vaginal rings, and hormonal intrauterine devices (IUDs) are very effective in preventing pregnancy, they do not prevent STIs. Furthermore, long-term use of hormonal products may increase the risk of adverse events such as blood clots, and they may be poorly tolerated or contraindicated in some individuals.

[0006] Currently, the only non-hormonal options for preventing pregnancy are copper IUDs and various perinatal devices / products (including male and female condoms, cervical diaphragms / caps, vaginal sponges, vaginal films, and the recently approved acid-buffered Phexxi® gel). However, copper IUDs require insertion by a healthcare provider, and their effectiveness is typically lower with perinatal approaches.

[0007] Women overwhelmingly prefer and are more likely to use MPT products that prevent both unplanned pregnancies and STIs than products that prevent only pregnancy or only STIs. To date, condoms remain the only approved MPT, but they face challenges such as inconsistent use and a typical failure rate of 13%.

[0008] Drug-releasing IVRs are flexible, torus-shaped devices, mostly made from medical-grade thermosetting materials (such as silicone elastomers) or thermoplastic materials (such as ethylene-vinyl acetate copolymers), that provide sustained or controlled release of therapeutic agents into the vagina for clinical benefit. Traditionally, IVRs have provided (i) a convenient formulation option for administering drugs with certain physicochemical properties, (ii) relatively low variability in drug release rates, (iii) minimize systemic absorption of the drug, (iv) avoid first-pass metabolism of the active pharmaceutical ingredient (where drug absorption occurs), and (v) improved user adherence and acceptability / satisfaction compared to other vaginal formulations.

[0009] Currently, controlled release of substances from commercially available IVRs requires that the substance(s) contained within the device's structure first dissolve in the polymer material used, and then diffuse through it. To date, the low solubility and permeability of non-hormonal agents (usually water-soluble or hydrophilic) in the hydrophobic polymers commonly used in IVR manufacturing, and / or the relative lack of efficacy of non-hormonal agents, have limited efforts to advance IVRs toward the release of non-hormonal agents.

[0010] Efforts to overcome these limitations and extend vaginal ring technology to a wider range of active substances include: (i) rings made from biosoluble acacia gum or non-biodegradable hydrogel additives; (ii) rings manufactured using alternative polymers such as hydrophilic / water-swellable polyurethane; (iii) rings containing or made from components having relatively high concentrations of hydrophilic active substances and additives; and (iv) rings containing one or more separate drug-loaded rods or pods that control the release of drug-active substances(s).

[0011] For example, Ovaprene® is a leading non-hormonal IVR currently under development. This device is designed for contraception over several weeks and achieves its contraceptive activity in two ways. The device features a semipermeable polymer mesh barrier that physically blocks sperm from entering the cervix, and also releases ferrous gluconate from the main ring to locally inhibit sperm motility.

[0012] Brij et al. reported the development of an interventional radiology (IVR) consisting of a nanoporous poly(diol citrate) elastomer hydrogel that sustainably releases a mixture of nonhormonal contraceptives and anti-HIV substances, namely ferrous gluconate, L-ascorbic acid, and polyamino-polycarboxylic acid (Ampholines) or poly-L-glutamic acid (PLGA): sodium bicarbonate buffer (Non-Patent Literature 1).

[0013] Inevitably, many of these newer IVR designs require complex, multi-step manufacturing processes that are difficult and costly to scale and produce. High concentrations of active ingredients can induce cytotoxic effects in vaginal epithelial tissue. Including multiple active ingredients in the same compartment within a ring can lead to drug interactions, potentially affecting drug stability and release. Furthermore, combining different drugs within the same IVR presents challenges due to varying solubility and target release rates for each drug.

[0014] With over one million new STI cases every day worldwide, new MPT methods need to cover a wide range of STIs. This is because a narrow scope of STI prevention / treatment can lead to risk compensation behaviors and the defense against or treatment of one pathogen may increase the risk of infection by other pathogens.

[0015] (i) Provide sustained / controlled release of non-hormonal agents, (ii) be practical, (iii) prevent unplanned pregnancies and be highly effective in preventing / treating one or more STIs, and (iv) can be easily manufactured using conventional scalable processes. There is a need to develop a new IVR.

Prior Art Documents

Non-Patent Documents

[0016]

Non-Patent Document 1

Summary of the Invention

[0017] In one aspect, the present disclosure relates to an IVR for intravaginal administration of a therapeutically effective amount of a non-hormonal active agent or a combination thereof to a female subject.

[0018] In one aspect, the present disclosure relates to a method of making an IVR for intravaginal administration of an active ingredient or a combination thereof to a female subject.

[0019] In one embodiment, the disclosure relates to a method for preventing unplanned pregnancies and preventing or treating diseases caused by bacterial and viral infections by applying an interventional radiology (IVR) intravaginally to a female subject for the purpose of administering a pharmaceutically effective amount of an active substance or a combination thereof.

[0020] In one embodiment, the IVR may contain a therapeutically effective amount of a non-hormonal active substance dispersed in an elastomer.

[0021] In one embodiment, the non-hormonal active substance may include at least one selected from copper components, zinc components, or lactide components.

[0022] In one embodiment, the non-hormonal active substance may include a lactide component and at least one selected from a copper component or a zinc component.

[0023] In one embodiment, the non-hormonal active substance may include a lactide component, a copper component, and a zinc component.

[0024] In one embodiment, the copper component may be metallic copper, copper oxide, a copper salt, or a copper ion-ligand complex.

[0025] In one embodiment, the zinc component may be metallic zinc, zinc oxide, or a zinc salt.

[0026] In one embodiment, the lactide component may be D-lactide, L-lactide, DL-lactide, or lactic acid.

[0027] In one embodiment, the elastomer may be selected from silicone, polyethylene vinyl acetate copolymer (EVA), styrene-butadiene-styrene block copolymer, polyphosphazene, poly(isoprene), poly(isobutylene), polybutadiene, polyurethane, nitrile rubber, neoprene rubber, or a combination thereof.

[0028] In one embodiment, the elastomer may be included in an amount of approximately 50% to approximately 99% of the total weight of the IVR.

[0029] In one embodiment, the IVR may be in the form of a matrix, and the elastomer may be silicone.

[0030] In one embodiment, the IVR is in the form of an exposed core, and the elastomer is polyethylene vinyl acetate or polyurethane.

[0031] In one embodiment, the copper salt may be anhydrous copper sulfate or copper sulfate hydrate.

[0032] In one embodiment, the zinc salt may be zinc acetate, zinc formate, zinc lactate, zinc chloride, zinc sulfate, zinc iodide, zinc citrate, or zinc orotate, each in the form of anhydrous or hydrate.

[0033] In one embodiment, the non-hormonal active substance may be present in an amount of about 5% to about 50% by weight relative to the total amount of IVR.

[0034] In one embodiment, the IVR may release copper ions, zinc ions, and lactic acid in a molar ratio of 1:1:1 to 1:6:6 after the first day period.

[0035] In one embodiment, the IVR can release copper ions at a rate of 2 mg / day to 31 mg / day during a 30-day usage period.

[0036] In one embodiment, the IVR can release zinc ions at a rate of 1 mg / day to 17 mg / day during a 30-day usage period.

[0037] In one embodiment, the IVR can release lactate at a rate of 12 mg / day to 105 mg / day during a 30-day period of use. [Brief explanation of the drawing]

[0038] [Figure 1]This figure shows the difference in zinc salts for a 50% decrease in mobility within a 95% confidence interval (CI). [Figure 2] This figure shows the dose-response effect on sperm motility for a single API. [Figure 3] This figure shows the sperm mucus permeability results for a single API and a combination of CS-ZL-LA (CSL). [Figure 4] This figure shows the synergistic effects plotted against the efficacy (shown as the effect rate (Fa)) of combinations of two APIs and combinations of three APIs. [Figure 5] This figure shows the motility inhibition associated with the CS-ZL-LA combination. [Figure 6] This figure shows the percentage of motile sperm at various experimental stages for API (CS-ZL-LA combination) and SVF control. [Figure 7] This figure shows the percentage of sperm that have undergone acrosome exocytosis after incubation for 3 hours under conditions for acquiring fertilization ability. [Figure 8] This figure shows the percentage of dead or damaged organisms after the washing process or incubation for 3 hours under conditions for acquiring fertilization ability, as seen in Sybr14 / propidium iodide staining. [Figure 9] This figure shows the percentage of sperm exhibiting a CTB:488 staining pattern associated with increased membrane fluidity after 3 hours of incubation under conditions of fertilization acquisition (5 mM 2OHCD) and non-fertilization acquisition for untreated control, SVF control, and API (CS-ZL-LA combination). [Figure 10] This figure shows the anti-HIV-1BaL activity of the API alone and in combination. [Figure 11] This figure shows the anti-HIV-1BaL activity of the API in the absence / presence of biological fluids and at concentrations that inhibit sperm motility. [Figure 12] This figure shows the anti-HSV-2 activity of the API alone and in combination. [Figure 13]This figure shows the anti-HSV-2 activity of the API in the absence and presence of biological fluid alone, and at concentrations that inhibit sperm motility. [Figure 14] This figure shows the inhibitory activity of the API against N. gonorrhoeae. [Figure 15] This figure shows the bactericidal activity of its API against C. trachomatis in the presence of biological fluid. [Figure 16] This figure shows the daily and cumulative release of lactide from IVR. [Figure 17] This figure shows the daily and cumulative release of lactate from IVR. [Figure 18] This figure shows the daily and cumulative release of copper ions from IVR. [Figure 19] This figure shows the daily and cumulative release of zinc ions from IVR. [Figure 20] This figure shows the effect of API on pH. [Figure 21] This figure shows the pH of the release medium for the API during a 30-day in vitro release study. [Figure 22] This figure shows the survival rate of cervical exgraves after a single exposure to API. [Figure 23] This figure shows the histological evaluation of cervical exgraves after a single exposure to API. [Figure 24] This figure shows the inflammatory cytokine concentrations after a single exposure of a cervical explant to an API. [Figure 25] This figure shows the survival rate and TEER in VEC-100 tissue after a single exposure to API. [Figure 26] This figure shows the survival rate and TEER in VEC-100 tissue after repeated exposure to API. [Modes for carrying out the invention]

[0039] Before describing in detail at least one embodiment of the present invention, it should be understood that the application of the present invention is not limited to the details shown in the following detailed description or illustrated by the examples. The present invention can be in other embodiments or can be carried out or performed in various ways. Accordingly, it should be understood that numerous modifications can be made to the exemplary embodiments and other configurations can be conceived without departing from the spirit and scope of this disclosure. It should also be understood that the words and terms used herein are for illustrative purposes only and should not be considered as limitations.

[0040] All measurements in this specification are performed at room temperature (25°C ± 5°C) and atmospheric pressure unless otherwise specified. All temperatures used in this specification are in degrees Celsius unless otherwise specified.

[0041] This disclosure may include (open-ended) the components and other components, active substances, or elements described herein, or may consist essentially of them. Unless otherwise noted, the terms “substance,” “component,” “ingredient,” “active substance,” and “active substance” as used herein are interchangeable. As used herein, “comprising” means having one or more other elements not listed, in addition to the listed elements or their equivalents in structure or function. The terms “having” and “including” as used herein should also be interpreted as open-ended unless otherwise indicated by the context. The terms “comprising,” “having,” and “including” encompass the terms “consisting of” and “consisting essentially of.” In this specification, the term "consisting essentially of" means that a composition or method may include additional components and / or steps, provided that the additional components and / or steps do not substantially alter the characteristics of the composition or method described in the claims.

[0042] All ranges enumerated herein include the endpoints and the ranges contained between them. Terms such as “about,” “generally,” and “substantially” are not absolute but should be interpreted as modifying the term or value so as not to correspond literally to the prior art. Such terms are defined in such a way that they are understood by those skilled in the art in the context and terminology they modify. This includes, at a minimum, the degree of expected experimental, technical, and instrumental error with respect to a given technique used to measure the value. Unless otherwise noted, the term “about” as used herein includes all values ​​within a range of ±10% of the specific value shown.

[0043] Unless otherwise noted, as used herein, singular nouns ("a" and "an") that do not specify a quantity are also plural. For example, "a component" may mean not only at least one component, but also multiple components, though not limited to, of different types.

[0044] As used herein, the term "and / or" in a list of two or more items means that any one of the listed properties may be present, or any combination of two or more of the listed properties may be present. For example, if a composition is described as containing active substance A, active substance B, and / or active substance C, the composition may contain A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0045] As used herein, the term “contraceptive” refers to an active substance or combination thereof administered to prevent conception or pregnancy, or to reduce the likelihood thereof.

[0046] As used herein, the term “antibiotic agent” refers to an active substance or combination thereof that can inhibit or eliminate the growth of microorganisms. Antimicrobial agents include, but are not limited to, antibacterial agents, antifungal agents, antiprotozoal agents, and antiviral agents.

[0047] As used herein, the terms “vaginal ring,” “vaginal ring,” “ring,” or “IVR” refer to a toroidal polymer delivery device designed to be inserted into the vagina of a female subject to deliver an active substance to the vagina over an extended period of time (e.g., controlled release).

[0048] As used herein, the terms “matrix ring” or “matrix-type ring” refer to an IVR in which an active substance or combination thereof, along with optionally other additives, is uniformly distributed throughout the elastomer ring. Matrix rings are typically manufactured by injection molding or extrusion of a mixture containing the active substance(s) to result in a uniform distribution of the active substance(s) throughout the elastomer ring. Matrix rings may also be manufactured using advanced 3D printing or additive manufacturing processes. In some embodiments, this design provides exponential or primary release decay, characterized by a rapid initial release of the active substance followed by a slower release rate. More generally, this matrix design provides drug release characterized by a relatively rapid initial burst release followed by a decrease in the drug release rate over time, although other types of kinetics may be observed under certain circumstances.

[0049] As used herein, the terms “reservoir ring,” “exposed core ring,” or “exposed core type ring” refer to an IVR having a reservoir surrounded by a sheath, i.e., an IVR having at least one full-length or partial-length core. The core and sheath may be made of the same material or different materials. For example, the core and sheath may be made of the same elastomer or different elastomers. Various active agents may be incorporated into either the sheath or the exposed core and released from there. In some embodiments, various active agents may be incorporated into the sheath only, the exposed core only, or both and released from there. This design allows for control of the drug release rate compared to matrix rings, often resulting in substantially constant (or zero-order) release of the active agent.

[0050] As used herein, the term “elastomer” refers to a polymer network structure formed when a polymer or a mixture of polymers undergoes crosslinking. Generally, elastomers are formed by chemical crosslinking, and covalent crosslinking ensures that the elastomer returns to its original configuration when stress is removed. However, the term elastomer also applies to certain thermoplastic polymers that exhibit physical crosslinking. Regardless of the type of elastomer, polymers are typically composed of monomer units that are linked together to form a polymer network structure. Monomer units may include carbon, hydrogen, oxygen, silicon, halogens, or combinations thereof. Elastomers are also typically viscoelastic (i.e., both viscous and elastic), have weak intermolecular forces, generally have a low Young's modulus (E), and exhibit high fracture strain compared to other materials.

[0051] As used herein, the terms “prevent,” “prevent,” “treat,” “treat,” or “therapy” mean, for example, intravaginal administration of an active substance or combination thereof via interventional radiology (IVR) to prevent or reduce the likelihood of pregnancy or STI, and / or inhibit or eliminate the growth of microorganisms that increase the risk of STI and other adverse reproductive outcomes.

[0052] As used herein, the terms “active,” “pharmacologically active,” “therapeutically active,” “pharmaceutically active,” or “physiologically active,” used alone or in combination, mean any chemical substance or compound that produces a desired effect (e.g., a systemic effect) suitable for intravaginal administration, for example, via interventional radiology (IVR).

[0053] As used herein, the terms “effective” amount, “pharmacologically effective” amount, “therapeutically effective” amount, “pharmaceutically effective” amount, or “physiologically effective” amount of an active substance used herein mean the amount of a chemical or compound, used alone or in combination with one or more other substances or compounds, that is nontoxic but sufficient to produce the desired therapeutic effect when administered vaginally, for example, through interventional radiology (IVR).

[0054] When used herein, the activity of a “contraceptive,” or its effectiveness in preventing pregnancy, may be determined by the inhibition or reduction of sperm motility by the active substance released from the IVR after it has been administered intravaginally to a female subject.

[0055] As used herein, the terms “synergy,” “synergism,” “synergistic effect,” or “synergistic action” mean the effect of the interaction of the actions of two or more active substances such that the result of the combined action is greater than that expected from a simple additive combination of two or more active substances acting separately.

[0056] As will be understood by those skilled in the art, the effective amount of an active substance, or the percentage or concentration of such an active substance used in an interventional radiology (IVR), can vary depending on a variety of factors, including, for example, the actual contraceptive / antimicrobial activity of each active substance in the IVR against the target of treatment, the synergistic effect of a combination of two or more such active substances, the type of IVR, and the method of delivery of such active substances (e.g., whether the dosage form is intended for sustained release). Such activity can be determined according to conventional methods and, accordingly, the amount of active ingredient formulated.

[0057] As used herein, the terms “pharmaceutically acceptable additive” or “additive” refer to carriers or vehicles, buffers, gel-forming agents or thickeners, suspenders, emollients, humectants, solubilizers, stabilizers, pH adjusters (also called pH modifiers), release enhancers, and preservatives that do not cause significant irritation to living organisms and do not impair the biological activity and properties of the active substance to which they are applied.

[0058] As used herein, the terms “carrier” or “vehicle” refer to carrier materials suitable for intravaginal administration via IVR, and include all such materials known in the art, such as any liquids, gels, solvents, liquid diluents, solubilizers, etc., that are non-toxic and do not cause adverse interactions with other components of the composition. Examples of suitable carriers include water, alcohol, mineral oil, silicone, liquid sugar, wax, petroleum jelly, and various other oils and polymer materials.

[0059] As used herein, the term “release enhancer” refers to a compound that increases the rate of release of the active substance(s) from an IVR. Such compounds include, but are not limited to, polyvinylpyrrolidone (PVP or povidone), modified cellulose ethers (e.g., hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose), microcrystalline cellulose, polyacrylic acid, carbomer, alginic acid, carrageenan, cyclodextrin, dextrin, guar gum, gelatin, xanthan gum, and sugars (e.g., monosaccharides such as glucose, fructose, and galactose, and disaccharides such as lactose, maltose, and fructose).

[0060] As used herein, the terms “release” or “release rate” refer to the amount or concentration of the active ingredient released from an IVR within any specified period. “Sustained release” or “sustained release rate” refers to a release sufficient to provide contraceptive and / or antimicrobial properties over a specific period (e.g., a long period).

[0061] In one embodiment, the disclosure relates to an interventional radiology (IVR) procedure involving the intravaginal administration of a therapeutically effective amount of a non-hormonally active substance or a combination thereof to a female subject.

[0062] Female subjects may be female mammals, including female humans.

[0063] In one embodiment, the IVR may be matrix-type and may contain at least one therapeutically effective amount of a non-hormonal agent dispersed throughout the elastomer.

[0064] In another embodiment, the IVR may be of the exposed core type and may contain at least one therapeutically effective amount of at least one non-hormonal agent dispersed in at least one core elastomer and / or sheath elastomer.

[0065] Examples of elastomers, but not limited to, include silicones (organopolysiloxanes), polyethylene vinyl acetate copolymer (EVA), styrene-butadiene-styrene block copolymer, polyphosphazene, poly(isoprene), poly(isobutylene), polybutadiene, polyurethane, nitrile rubber, neoprene rubber, and mixtures thereof. Silicone elastomers, also known as silicone rubber, are particularly preferred.

[0066] As used herein, the terms “silicone” or “polysiloxane” refer to any of the various compounds that contain alternating silicon and oxygen atoms in either a linear or cyclic arrangement, with one or two organic groups typically attached to each silicon atom. Examples of polysiloxanes include substituted polysiloxanes, diorganopolysiloxanes such as diarylpolysiloxanes, and dialkylpolysiloxanes such as dimethylpolysiloxanes.

[0067] In one embodiment, the elastomer is present at a concentration of about 50% to about 99% based on the total weight of the IVR. In one embodiment, the elastomer is present at a concentration of about 70% to about 99%, preferably about 90% to about 99%, based on the total weight of the IVR. In one embodiment, the elastomer is present at a concentration of about 95%, or about 97%, based on the total weight of the ring.

[0068] In one embodiment, the IVR may be matrix-type and may contain a silicone elastomer (SE) at a concentration of about 50% to about 99%, preferably about 65% to about 90%, more preferably about 70% to about 80%, based on the total weight of the IVR. Final mixing is performed after the non-hormonal active substance is distributed into the SE.

[0069] In one embodiment, the IVR may be of the exposed core type and may contain a thermoplastic substance such as polyethylene vinyl acetate (EVA40) or thermoplastic polyurethane (TPU) containing 40% EVA, at a concentration of about 50% to about 99%, preferably about 55% to about 90%, more preferably about 55% to about 80%, based on the total weight of the IVR, mixed with a non-hormonal substance. The thermoplastic polymer is pulverized using a cryogenic pulverization method to produce a powdered polymer from the raw material granules. The non-hormonal substance powder is weighed in an appropriate ratio with the EVA40 powder or TPU powder and mixed using an appropriate method.

[0070] Examples of non-hormonal substances include, but are not limited to, copper components, zinc components, and lactide components. The copper component may be metallic copper, copper oxide, copper salt, or copper ion-ligand complex. Metallic copper may be in the form of copper microparticles or copper nanoparticles. Examples of copper salts include, but are not limited to, anhydrous copper sulfate and copper sulfate hydrate (CuSO4·5H2O, etc.). The copper component may preferably be anhydrous copper sulfate. The zinc component may be metallic zinc, zinc oxide, or zinc salt. Metallic zinc may be in the form of zinc microparticles or zinc nanoparticles. Examples of zinc salts, though not limited to them, include zinc acetate, zinc acetate hydrate (containing Zn(OAc)2·2H2O), zinc formate, zinc formate hydrate, zinc lactate, zinc lactate hydrate (containing zinc lactate dihydrate), zinc chloride, zinc chloride hydrate, anhydrous zinc sulfate, zinc sulfate hydrate (ZnSO4·H2O, etc.), zinc iodide, zinc iodide hydrate, zinc citrate, zinc citrate hydrate, zinc orotate, and zinc orotate hydrate, in both anhydrous and hydrated forms. The zinc component may preferably be anhydrous zinc lactate, anhydrous zinc acetate, anhydrous zinc sulfate, or zinc sulfate hydrate. The zinc component may most preferably be anhydrous zinc lactate. The lactide component may be lactide, lactic acid, or lactones such as oligomeric or polymeric forms of lactic acid (lactoyl lactic acid or polylactic acid). The lactide component may be in the form of D-lactide ((R,R)-D-lactide or (S,S)-D-lactide, etc.), (R,S)-mesolactide, DL-lactide, or a mixture thereof. The lactide component is preferably DL-lactide.

[0071] In this specification, unless otherwise specifically provided, copper sulfate may also be called copper sulfate (anhydrous), CS, or CSA; zinc acetate may also be called zinc acetate (anhydrous), ZnA, or ZA; zinc lactate may also be called zinc lactate (anhydrous), ZnL, ZL, or ZLA; lactide may also be called DL-lactide, LT, or L; and lactic acid may also be called LA.

[0072] Non-hormonal substances may be milled before being used as materials in IVR manufacturing. In one embodiment, zinc acetate and lactide may require milling before being used as materials for IVR manufacturing. Generally, non-hormonal substances that have not been milled may have a particle size distribution in which 90% of particles are larger than 100 μm and less than 1000 μm, whereas milled non-hormonal substances may have a particle size distribution in which 90% of particles are less than 50 μm, preferably less than 30 μm, more preferably less than 20 μm, for example, 10 μm. In some embodiments, non-hormonal substances that have not been milled may have a particle size distribution in which more than 90% of particles are within the size range of 100 μm to 1000 μm, while milled non-hormonal substances may have a particle size distribution in which more than 90% of particles are less than 100 μm, preferably less than 50 μm, more preferably less than 20 μm, for example, 10 μm. The release rate characteristics of IVRs can be altered by changing the particle size of non-hormonal agents.

[0073] In one embodiment, the IVR may contain at least two non-hormonal agents dispersed throughout the elastomer in therapeutically effective amounts. The non-hormonal agents are selected from copper components, zinc components, or lactide components. In one embodiment, the IVR may contain a lactide component and at least one of the copper component or zinc component dispersed throughout the elastomer in therapeutically effective amounts. In one embodiment, the IVR may contain a copper component, a zinc component, and a lactide component dispersed throughout the elastomer in therapeutically effective amounts.

[0074] In one embodiment, the IVR contains about 100 mg to about 1600 mg, preferably about 250 mg to about 1600 mg of copper. In one embodiment, the IVR contains about 100 mg to about 1600 mg, preferably about 250 mg to about 1600 mg of zinc. In one embodiment, the IVR contains about 100 mg to about 1600 mg, preferably about 250 mg to about 1600 mg of lactide. In one embodiment, the IVR contains at least 100 mg to about 1600 mg, preferably about 250 mg to about 1600 mg of copper, or 100 mg to about 1600 mg, preferably about 250 mg to about 1600 mg of zinc, in combination with about 100 mg to about 1600 mg, preferably about 250 mg to about 1600 mg of lactide. In one embodiment, the IVR contains approximately 100 mg to approximately 1600 mg, preferably approximately 250 mg to approximately 1600 mg of lactide component, 100 mg to approximately 1600 mg, preferably approximately 260 mg to approximately 1600 mg of copper component, and 100 mg to approximately 1600 mg, preferably approximately 250 mg to approximately 1600 mg of zinc component.

[0075] In one embodiment, the IVR contains copper components in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight. In one embodiment, the IVR contains zinc components in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight. In one embodiment, the IVR contains lactide components in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight. In one embodiment, the IVR contains, in combination with a lactide component in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight, at least 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight, a copper component, or approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight, a zinc component. In one embodiment, the IVR contains a lactide component in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight; a copper component in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight; and a zinc component in amounts of approximately 5% to 50% by weight, approximately 5% to 45% by weight, approximately 5% to 40% by weight, or approximately 5% to 35% by weight, preferably approximately 5% to 30% by weight, or approximately 10% to 20% by weight.

[0076] IVR may contain other pharmaceutically compatible active substances in appropriate amounts. Such active substances include not only pharmacologically active substances but also pharmacologically inactive substances known as pharmacologically acceptable excipients in the art.

[0077] Examples of pharmacologically active substances include, but are not limited to, any suitable antimicrobial agents, including antibacterial, antifungal, antiprotozoan, and antiviral agents known in the art.

[0078] Appropriate antibacterial agents include, but are not limited to, acrosoxacin, amifloxacin, amoxicillin, ampicillin, aspoxicillin, azidocillin, azithromycin, aztreonam, valofloxacin, benzylpenicillin, biapenem, brodimoprim, cefaclor, cefadroxil, cefatolidine, cefcapene, cefdinir, cefetamet, cefmetazole, cefprodil, ceffloxazine, ceftibuten, cefuroxime, cephalexin, cef Aronium, cephaloridine, cephamandol, cefazolin, cefradin, chlorquinaldol, chlortetracycline, cyclacillin, cinoxacin, ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, clofazimine, cloxacillin, danofloxacin, dapsone, demeclocycline, dicloxacillin, difloxacin, doxycycline, enoxacin, enrofloxacin, erythromycin, freloxacin, flomoxef, f Lucloxacillin, Flumequine, Fosfomycin, Isoniazid, Levofloxacin, Mandelic acid, Mesilinum, Metronidazole, Minocycline, Mupirocin, Nadifloxacin, Nalidixic acid, Nifuirtoinol, Nitrofurantoin, Nitroxolin, Norfloxacin, Ofloxacin, Oxytetracycline, Panipenem, Pefloxacin, Phenoxymethylpenicillin, Pipemidic acid, Pyromidic acid, Pivampicillin, Pivmecilina Examples include prulifloxacin, rufloxacin, sparfloxacin, sulbactam, sulfabenzuamide, sulfacithin, sulfametopyrazine, sulfacetamide, sulfadiazine, sulfadimidine, sulfamethizol, sulfamethoxazole, sulfanilamide, sulfisomidine, sulfathiazole, temafloxacin, tetracycline, tetroxoprim, tinidazole, tosufloxacin, trimethoprim, and their salts or esters.

[0079] Appropriate antifungal agents include, but are not limited to, bifonazole, butoconazole, chlordantoin, chlorphenesin, cyclopirox olamine, clotrimazole, everconazole, econazole, fluconazole, flutrimazole, isoconazole, itraconazole, ketoconazole, miconazole, nifuroxime, thioconazole, terconazole, undecenoic acid, and their salts or esters.

[0080] Suitable antiprotozoal agents include, but are not limited to, acetalazole, azanidazole, chloroquine, metronidazole, nifuratel, nimorazole, omidazole, propenidazole, secnidazole, synefungin, tenonitrozol, temidazole, tinidazole, and their salts or esters.

[0081] Appropriate antiviral agents include, but are not limited to, acyclovir, brivudine, cidofovir, curcumin, dapivine, desciclovir, 1-docosanol, edoxudine, Fameyclovir, phiacitabine, ivacitabine, imiquimod, lamivudine, penciclovir, valacyclovir, valganciclovir, and their salts or esters.

[0082] When used, the other active ingredients mentioned above may be present in amounts of about 0.5% to about 40% by weight, preferably about 2.5% to about 15% by weight, of the IVR.

[0083] Examples of pharmaceutically acceptable additives include, but are not limited to, carriers or vehicles, buffers, gel-forming agents or thickeners, suspensions, emollients, humectants, solubilizers, stabilizers, pH adjusters (also called pH modifiers), release enhancers, and preservatives, provided that they do not cause significant irritation to living organisms and do not negate the biological activity and properties of the active substance to which they are applied.

[0084] Suitable release enhancers include, but are not limited to, polyvinylpyrrolidone (PVP or povidone), modified cellulose ethers (e.g., hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose), microcrystalline cellulose, polyacrylic acid, carbomer, alginic acid, carrageenan, cyclodextrin, dextrin, guar gum, gelatin, xanthan gum, and sugars (e.g., monosaccharides such as glucose, fructose, and galactose, and disaccharides such as lactose, maltose, and fructose). When used, the release enhancer may be present in an amount of about 0.5% by weight to about 40% by weight, preferably about 2.5% by weight to about 15% by weight of the IVR.

[0085] The IVR may have a weight of approximately 2g to approximately 15g, preferably approximately 5g to approximately 10g, and more preferably approximately 8g.

[0086] IVR may have any shape and dimensions suitable for intravaginal administration to female subjects. Such a ring can be inserted into the vagina by itself, and within the vagina, the ring is held in place by its shape and inherent elasticity. In one embodiment, the manufactured IVR has an outer diameter of 50 mm to 60 mm. In another embodiment, the IVR has an outer diameter of about 50 mm, about 51 mm, about 52 mm, about 53 mm, about 54 mm, about 55 mm, about 56 mm, about 57 mm, about 58 mm, about 59 mm, or about 60 mm. In another embodiment, the IVR has a cross-sectional diameter of 4.0 mm to 10 mm. In yet another embodiment, the IVR has a cross-sectional diameter of approximately 4.0 mm, 4.5 mm, 5.0 mm, 5.5 mm, 5.6 mm, 6.0 mm, 6.2 mm, 6.5 mm, 7.0 mm, 7.1 mm, 7.2 mm, 7.3 mm, 7.4 mm, 7.5 mm, 7.6 mm, 7.7 mm, 7.8 mm, 7.9 mm, 8.0 mm, 8.5 mm, 9.0 mm, or 9.5 mm.

[0087] IVRs may swell in aqueous media depending on the type and amount of active substance applied.

[0088] IVR can be analyzed for in vitro release by using various release media such as water, buffer solution, or simulated vaginal fluid. For example, the properties of copper and zinc ions released from the IVR can be analyzed by atomic absorption spectroscopy (AAS) or inductively coupled plasma emission spectroscopy (ICP-OES), and the properties of lactide and its hydrolysis product, lactic acid, released from the IVR can be analyzed by high-performance liquid chromatography (HPLC) with spectroscopic detection. In a preferred embodiment, the IVR is placed in deionized water for 30 days (60 rpm, 37°C) to determine copper, zinc, lactic acid, or lactide.

[0089] The IVRs of this disclosure provide long-term controlled release of copper or its ions, zinc or its ions, or lactate. Surprisingly, IVRs containing a combination of at least two components—copper, zinc, or lactide—show increased release of copper or its ions, zinc or its ions, or lactate compared to IVRs containing a single component. In particular, when the IVR contains the non-hormonal agent in salt form, the release amount or rate may be greater compared to when the non-hormonal agent is contained in the form of metal nanoparticles. This is likely due to increased water solubility of the salt.

[0090] The IVR of this disclosure can, upon use, release lactate (or lactide equivalent) into the vaginal cavity at an initial release rate of 1 mg to 30 mg, preferably 1 mg to 25 mg, most preferably 1 mg to 20 mg, as determined in vitro, over the first 24 hours, and subsequently release at a "maintenance" release rate of 0.15 mg to 15 mg, preferably 0.20 mg to 10 mg, most preferably 0.20 mg to 8 mg, on a daily basis over at least the following 28 days. In vitro pH studies have also demonstrated that lactate released from the IVR results in a decrease in pH.

[0091] The IVR of this disclosure can, upon use, release copper or its ions, or zinc or its ions, into the vaginal cavity at an initial release rate of 1 mg to 50 mg, preferably 1 mg to 30 mg, most preferably 1 mg to 25 mg, as determined in vitro, over the first 24 hours; subsequently, on a daily basis for at least the following 30 days, it can release at a "maintenance" release rate of 0.15 mg to 20 mg, preferably 0.20 mg to 15 mg, most preferably 0.20 mg to 10 mg, as determined in vitro; on a daily basis for at least the following 10 days, it can release at a "booster" release rate of 1 mg to 40 mg, preferably 1 mg to 20 mg, most preferably 1 mg to 10 mg, as determined in vitro; and on a daily basis for at least the following 8 days, it can release at another "maintenance" release rate of 0.15 mg to 20 mg, preferably 0.20 mg to 15 mg, most preferably 0.20 mg to 10 mg, as determined in vitro.

[0092] In one embodiment, in combination with any of the features or embodiments described herein, the IVR of the Disclosure can, when used, exhibit a sustained and continuous release of lactate (or lactide equivalent) into the vaginal lumen at a rate of at least 15 mg / day, preferably at least 20 mg / day, at least 25 mg / day, at least 30 mg / day, at least 35 mg / day, or at least 40 mg / day, as determined in vitro, over the first 15 days of a 30-day period. In one embodiment, in combination with any of the features or embodiments described herein, the IVR of the Disclosure can, when used, exhibit a sustained and continuous release of lactate (or lactide equivalent) into the vaginal lumen at a rate of 15 mg / day to 110 mg / day, preferably at least 20 mg / day to 105 mg / day, as determined in vitro, over the first 15 days of a period.

[0093] In one embodiment, the IVR of the Disclosure can, when used, release lactic acid (or lactide equivalent) into the vaginal cavity at a release rate of 5 mg / day to 150 mg / day, preferably 10 mg / day to 110 mg / day, more preferably 12 mg / day to 105 mg / day, or 20 mg / day to 150 mg / day, as determined in vitro, over a period of 30 days. In one embodiment, the IVR of the Disclosure can, when used, release copper ions into the vaginal cavity at a release rate of 1 mg / day to 50 mg / day, preferably 2 mg / day to 35 mg / day, or 5 mg / day to 50 mg / day, as determined in vitro, over a period of 30 days. In one embodiment, the IVR of the Disclosure can, when used, release zinc ions into the vaginal cavity at a release rate of 1 mg / day to 40 mg / day, preferably 1 mg / day to 20 mg / day, or 2 mg / day to 40 mg / day, as determined in vitro, over a period of 30 days.

[0094] In one embodiment, the IVR of this disclosure, comprising 10CSA-10ZLA-10L (a combination of 10% by weight copper sulfate (anhydrous), 10% by weight zinc lactate (anhydrous), and 10% by weight lactide), can, when used, release lactate (or lactide equivalent) into the vaginal cavity at a release rate of 12 mg / day to 60 mg / day, as determined in vitro, release copper ions at a release rate of 2 mg / day to 20 mg / day, and release zinc ions at a release rate of 1 mg / day to 10 mg / day over a period of 30 days. In one embodiment, the IVR of this disclosure, comprising 10CSA-10ZLA-20L (a combination of 10% by weight copper sulfate (anhydrous), 10% by weight zinc lactate (anhydrous), and 20% by weight lactide), can, when used, release lactate (or lactide equivalent) into the vaginal lumen at a release rate of 12 mg / day to 105 mg / day, as determined in vitro, release copper ions at a release rate of 2 mg / day to 35 mg / day, and release zinc ions at a release rate of 1 mg / day to 20 mg / day over a period of 30 days. In one embodiment, the IVR of this disclosure, comprising 5CSA-10ZLA-10L (a combination of 5% by weight copper sulfate (anhydrous), 10% by weight zinc lactate (anhydrous), and 10% by weight lactide), can, when used, release lactate (or lactide equivalent) into the vaginal cavity at a release rate of 5 mg / day to 50 mg / day, as determined in vitro, release copper ions at a release rate of 1 mg / day to 20 mg / day, and release zinc ions at a release rate of 1 mg / day to 8 mg / day over a period of 30 days. In one embodiment, the IVR of this disclosure, comprising 5CSA-20ZLA-20L (a combination of 5% by weight copper sulfate (anhydrous), 20% by weight zinc lactate (anhydrous), and 20% by weight lactide), can, when used, release lactate (or lactide equivalent) into the vaginal cavity at a release rate of 15 mg / day to 100 mg / day, as determined in vitro, release copper ions at a release rate of 2 mg / day to 20 mg / day, and release zinc ions at a release rate of 2 mg / day to 40 mg / day into the vaginal cavity over a period of 30 days.In one embodiment, an IVR of the present disclosure comprising 10CSA-10ZLA-30L (a combination of 10 wt% copper sulfate (anhydrous), 10 wt% zinc lactate (anhydrous), and 30 wt% lactide) can, when used, release lactate (or lactide equivalent) into the vaginal cavity at a release rate of 20 mg / day to 150 mg / day, as determined in vitro, release copper ions at a release rate of 5 mg / day to 50 mg / day, and release zinc ions at a release rate of 2 mg / day to 30 mg / day into the vaginal cavity over a period of 30 days. In this specification, wt% refers to a weight percentage based on the total weight of the IVR.

[0095] In one embodiment, the IVR of the Disclosure may, at the time of use, release lactate (or lactide equivalent) into the vaginal cavity at an initial release rate of 20 mg to 60 mg, preferably 22 mg to 55 mg, most preferably 50 mg to 55 mg, as determined in vitro, over the first week; then, on a daily basis over the following seven days, at a release rate of 30 mg to 105 mg, preferably 50 mg to 90 mg, most preferably 85 mg to 90 mg, as determined in vitro; and on a daily basis over the next seven days, at release rates of 20 mg to 30 mg, preferably 22 mg to 30 mg, most preferably 25 mg to 30 mg, and 12 mg to 25 mg, as determined in vitro.

[0096] In one embodiment, the IVR of the Disclosure may, upon use, release copper ions into the vaginal cavity at an initial release rate of 2 mg to 31 mg, preferably 4 mg to 31 mg, as determined in vitro, over the first week; subsequently, on a daily basis over the following seven days, at a release rate of 2 mg to 20 mg, preferably 15 mg to 20 mg, as determined in vitro; and on a daily basis over the next seven days, at release rates of 2 mg to 10 mg, preferably 4 mg to 10 mg, and 2 mg to 5 mg, as determined in vitro.

[0097] In one embodiment, the IVR of the Disclosure may, upon use, release zinc ions into the vaginal cavity at an initial release rate of 1 mg to 17 mg, preferably 12 mg to 17 mg, as determined in vitro, over the first week; subsequently, on a daily basis over the following 7 days, at a release rate of 5 mg to 9 mg, preferably 7 mg to 9 mg, as determined in vitro; and on a daily basis over the next 7 days, at release rates of 2 mg to 5 mg and 1 mg to 3 mg, as determined in vitro.

[0098] In one embodiment, the rate of copper ion release from a multi-component IVR of the Disclosure (e.g., a ring comprising a copper component and at least one of a zinc component or a lactide component) is increased compared to the rate of copper ion release from an IVR containing a copper component but not a zinc component or a lactide component. For example, in one embodiment, the rate of copper ion release from an IVR of the Disclosure comprising a copper component and either or both a zinc component or a lactide component is increased by at least about 1.5 to about 2 times per day compared to the rate of copper ion release from an IVR containing a copper component but not a zinc component or a lactide component. In another embodiment, the rate of copper ion release from an IVR of the Disclosure comprising a copper component and either a zinc component or a lactide component is increased by at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 times per day compared to the rate of copper ion release from an IVR comprising a copper component but not either a zinc component or a lactide component.

[0099] In one embodiment, the cumulative release rate of copper ions from an IVR of the Disclosure comprising a copper component and either a zinc component or a lactide component increases by at least about 1.5 to about 2 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of copper ions after 7, 14, 21, 30, 60, or 90 days from an IVR comprising a copper component but not containing either a zinc component or a lactide component. In another embodiment, the cumulative release rate of copper ions from an IVR of the Disclosure comprising a copper component and either a zinc component or a lactide component increases by at least about 3 times, 4 times, 5 times, 6 times, or 90 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of copper ions after 7, 14, 21, 30, 60, or 90 days after 3, 14, 21, 30, 60, or 90 days after 3, 14, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 35 times, 40 times, 45 times, or 50 times after 3, 14, 21, 300, 40 times, 45 times, or 50 times after 3, 14, 21, 30, 30, 30, 40 times, 45 times, or 50 times after 3, 14, 21, 30, 30, 30, 40 times, 45 times, or 50 times after 3, 14, 21, 30, 30, In one embodiment, the cumulative release rate of copper ions from an IVR of the Disclosure containing a zinc component or a lactide component increases by approximately 2 to 50 times, approximately 2 to approximately 25 times, approximately 2 to approximately 20 times, approximately 2 to approximately 15 times, approximately 2 to approximately 10 times, approximately 2 to approximately 5 times, approximately 5 to approximately 25 times, approximately 5 to approximately 20 times, approximately 5 to approximately 15 times, approximately 5 to approximately 10 times, approximately 10 to approximately 25 times, or approximately 10 to approximately 20 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of copper ions after 7, 14, 21, 30, 60, or 90 days from an IVR containing a copper component but not a zinc component or a lactide component.

[0100] In one embodiment, the rate of zinc ion release from a multi-component IVR of the Disclosure (e.g., a ring comprising a zinc component and at least one of a copper component or a lactide component) is increased compared to the rate of zinc ion release from an IVR comprising a zinc component but not a copper component or a lactide component. For example, in one embodiment, the rate of zinc ion release from an IVR of the Disclosure comprising a zinc component and either or both a copper component or a lactide component is increased by at least about 1.5 to about 2 times per day compared to the rate of zinc ion release from an IVR comprising a zinc component but not a copper component or a lactide component. In another embodiment, the rate of zinc ion release from an IVR of the Disclosure comprising a zinc component and either a copper component or a lactide component is increased by at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 times per day compared to the rate of zinc ion release from an IVR comprising a zinc component but not a copper component or a lactide component.

[0101] In one embodiment, the cumulative release rate of zinc ions from an IVR of the Disclosure comprising a zinc component and either a copper component or a lactide component increases by at least about 1.5 to about 2 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of zinc ions after 7, 14, 21, 30, 60, or 90 days from an IVR comprising a zinc component but not a copper component or a lactide component. In one embodiment, the cumulative release rate of zinc ions from an IVR of the Disclosure comprising a zinc component and either or both a copper component or a lactide component increases by at least about 10 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of zinc ions after 7, 14, 21, 30, 60, or 90 days from an IVR comprising a zinc component but not a copper component or a lactide component. In another embodiment, the cumulative release rate of copper ions from an IVR of the Disclosure comprising a zinc component and either a copper component or a lactide component increases by at least about 3 times, 4 times, 5 times, 6 times, or 90 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of zinc ions after 7, 14, 21, 30, 60, or 90 days from an IVR comprising a zinc component but not comprising either a copper component or a lactide component. In one embodiment, the cumulative release rate of zinc ions from an IVR of the Disclosure containing a copper component or a lactide component increases by approximately 2 to 50 times, approximately 2 to approximately 25 times, approximately 2 to approximately 20 times, approximately 2 to approximately 15 times, approximately 2 to approximately 10 times, approximately 2 to approximately 5 times, approximately 5 to approximately 25 times, approximately 5 to approximately 20 times, approximately 5 to approximately 15 times, approximately 5 to approximately 10 times, approximately 10 to approximately 25 times, or approximately 10 to approximately 20 times after 7, 14, 21, 30, 60, or 90 days compared to the cumulative release rate of zinc ions after 7, 14, 21, 30, 60, or 90 days from an IVR containing a zinc component but not a copper component or a lactide component.

[0102] In one embodiment, the release rate stabilizes after 3 months of storage. In one embodiment, the release rate stabilizes after 6 months of storage. In one embodiment, the release rate stabilizes after 12 months of storage. In one embodiment, the release rate stabilizes after 24 months of storage. In one embodiment, the release rate stabilizes after 36 months of storage.

[0103] In one embodiment, the present disclosure relates to a method for preparing an interventional radiology (IVR) for intravaginal administration of an active ingredient or a combination thereof to a female subject.

[0104] The matrix-type ring may contain a non-hormonal substance or a combination thereof in the silicone elastomer. Alternatively, the matrix-type ring may contain polyurethane or EVA polymer, or a mixture thereof.

[0105] Silicone matrix rings can be manufactured by mixing a non-hormonal substance, optionally with other active substances and additives, and if applicable, with a suitable crosslinking agent (such as propyl orthosilicate (NPOS)) and catalyst (such as platinum) into various medical-grade silicone materials; injecting the mixture into a properly designed injection mold (such as a Babyplast® injection molding machine fitted with a custom-made ring mold assembly); and optionally applying heat to cure / crosslink the silicone mixture and form an elastomer. More generally, silicone matrix rings can be manufactured by mixing a non-hormonal substance, optionally with other active substances and additives, into a properly formulated silicone elastomer system (which may include a base silicone polymer, a crosslinking agent, a curing catalyst, and other materials suitable for medical applications); and then injecting the mixture into an injection molding machine.

[0106] A thermoplastic matrix ring can be produced by (i) compounding a non-hormonal active substance, optionally together with other active substances and additives, into a thermoplastic polymer (such as ethylene vinyl acetate (EVA) copolymer or polyurethane) using a twin-screw compounding machine; (ii) pelletizing the mixture; and (iii) injection molding the mixture using an injection molding machine fitted with a custom injection mold to form an IVR. In one embodiment, the compounded material can be cooled using air and / or a cooled hole-off belt. The use of a cooling water bath may not be possible due to the water-soluble nature of the non-hormonal active ingredient. Once cooled, the rod output from the extruder can be cut into small pellets for use in downstream injection molding operations. In another embodiment, the thermoplastic matrix ring can be produced by extruding it in a manner similar to the commercial manufacturing method of NuvaRing®, a contraceptive IVR, cutting the extruded material to a predetermined length, and welding the ends to form a ring-shaped device.

[0107] More complex core exposure rings can be manufactured by using different drug loading core segments (either polymer or copper tubes embedded with active substances) and polymer films having holes / windows that partially expose the drug loading core segments.

[0108] In one embodiment, a non-hormonal active substance or a combination thereof can be placed in a core having an empty sheath. In some embodiments, one active substance is placed in the core and the other active substance is placed in the sheath. In one embodiment, a lactide component may be placed in the core, and copper and zinc components may be placed in the sheath. In other embodiments, either a copper component or a zinc component may be placed in the sheath. In other embodiments, two components may be placed in the core and one component may be placed in the sheath.

[0109] A core-exposed ring can be fabricated using a two-step process. In one embodiment, a core ring is first produced in the same manner as the matrix ring described above. This core is then placed in a second mold cavity designed to hold the core in the center of the cavity and to create an opening (window) in the sheath layer during the formation process.

[0110] The curing temperature and curing time vary depending on the specific elastomer(s) used. The curing temperature is preferably below the melting point of the non-hormonal substance used. For example, the curing temperature can vary between room temperature (15°C to 25°C) and about 150°C, preferably within the range of about 60°C to 125°C, and more preferably within the range of about 90°C to 115°C. The curing time can vary from a few seconds to several hours depending on factors such as the elastomer(s) used and the curing temperature.

[0111] The ring can be characterized by microscopic examination, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), atomic absorption spectroscopy (AES), ICP-OES, and HPLC to investigate the properties and behavior of active substances within the instrument, evaluate their release, and assess drug-drug interactions and drug-polymer interactions. Mechanical testing of the IVR can be performed according to established methods, such as using a Shimadzu EZ Test universal tester fitted with a custom-made test jig. For example, various mechanical tests can be optimized to the shape of the IVR and used to mechanically characterize its performance: Shore M hardness, static compression over 28 days, compression from 5 mm to 20 mm, compression over 1000 cycles, and elongation at fracture.

[0112] In one embodiment, the disclosure relates to a method for preventing unplanned pregnancies and preventing and treating diseases caused by bacterial, fungal, and viral infections by applying interventional radiology (IVR) to a female subject, in which a pharmaceutically effective amount of an active substance or a combination thereof is administered intravaginally.

[0113] In one embodiment, the disease may be caused by a sexually transmitted infection.

[0114] IVR is described above. In one embodiment, the IVR of the present disclosure may be useful in inhibiting sperm motility and / or in preventing or blocking infections caused by human immunodeficiency virus (HIV) such as HIV-1, herpes simplex virus (HSV) such as HSV-2, N. gonorea, G. vaginalis (G. vaginalis) (associated with bacterial vaginosis (BV)), Chlamydia, etc.

[0115] In one embodiment, when at least two of copper ions, zinc ions, or lactic acid are released from the IVR used into the vaginal cavity, therapeutic effects including disease prevention and contraceptive effects are achieved.

[0116] In one embodiment, a therapeutic effect is achieved when copper ions and zinc ions are released from the IVR used into the vaginal cavity in a molar ratio of 5:1 to 1:5, preferably 1:1 to 1:3, more preferably 1:2, for 24 days after the first day of use, as determined in vitro. In one embodiment, a therapeutic effect is achieved when copper ions and lactate are released from the IVR used into the vaginal cavity in a molar ratio of 5:1 to 1:5, preferably 1:1 to 1:3, more preferably 1:2, for 24 days after the first day of use, as determined in vitro. In one embodiment, a therapeutic effect is achieved when zinc ions and lactate are released from the IVR used into the vaginal cavity in a molar ratio of 5:1 to 1:5, preferably 2:1 to 1:2, more preferably 1:1, for 24 days after the first day of use, as determined in vitro. In one embodiment, a therapeutic effect is achieved when copper ions, zinc ions, and lactate from the IVR used are released into the vaginal fluid daily for 24 days after the first day of use in a molar ratio of 1:0.5:2 to 1:4:8, preferably 1:0.8:3, 1:1:2, 1:1:3, 1:1.25:5, 1:1.5:5, 1:3:7, more preferably 1:1.5:5.25, as determined in vitro.

[0117] In one embodiment, preferably when the IVR includes a CSA-ZLA-L combination, in combination with any of the features or embodiments described herein, a therapeutic effect is achieved when copper ions and zinc ions are released from the IVR used into the vaginal lumen in a molar ratio of 1:1 to 1:6, preferably 1:3 to 1:6, more preferably 1:3.5 to 1:5.5, daily for 24 days after the first day of use, as determined in vitro. In one embodiment, preferably when the IVR includes a CSA-ZLA-L combination, in combination with any of the features or embodiments described herein, a therapeutic effect is achieved when copper ions and lactate are released from the IVR used into the vaginal lumen in a molar ratio of 1:1 to 1:6, preferably 1:3 to 1:6, more preferably 1:1.66 to 1:5.25, daily for 24 days after the first day of use, as determined in vitro. In one embodiment, preferably when the IVR includes a CSA-ZLA-L combination, in combination with any of the features or embodiments described herein, a therapeutic effect is achieved when zinc ions and lactate are released from the IVR used into the vaginal lumen in a molar ratio of 3:1 to 1:1, preferably 2.5:1 to 1.1:1, more preferably 2.2:1 to 1.05:1, for 24 days after the first day of use, as determined in vitro. In one embodiment, a therapeutic effect is achieved when copper ions, zinc ions, and lactate are released from the IVR used in a molar ratio of 1:1:1 to 1:6:6, preferably 1:3:3 to 1:6:6, more preferably 1:3.5:1.66 to 1:5.5:5.25, and even more preferably 1:5.5:5.25, daily over 24 days following the initial day of use, as determined in vitro, into the vaginal fluid.

[0118] In one embodiment, preferably when the IVR includes the CSA-ZLA-L combination in combination with any of the features or embodiments described herein, the IVR contains copper ions and zinc ions in a molar ratio of 1:1 to 1:6, preferably 1:3 to 1:6, and more preferably 1:3.5 to 1:5. In one embodiment, preferably when the IVR includes the CSA-ZLA-L combination in combination with any of the features or embodiments described herein, the IVR contains copper ions and lactic acid in a molar ratio of 1:1 to 1:6, preferably 1:3 to 1:6, and more preferably 1:1.66 to 1:5.25. In one embodiment, preferably when the IVR includes the CSA-ZLA-L combination, in combination with any of the features or embodiments described herein, the IVR contains zinc ions and lactic acid in a molar ratio of 3:1 to 1:1, preferably 2.5:1 to 1.1:1, more preferably 2.2:1 to 1.05:1 (in vaginal fluid daily for 24 days after the initial day of use). In one embodiment, preferably when the IVR includes the CSA-ZLA-L combination, in combination with any of the features or embodiments described herein, the IVR contains copper ions, zinc ions, and lactic acid in a molar ratio of 1:1:1 to 1:6:6, preferably 1:3:3 to 1:6:6, more preferably 1:3.5:1.66 to 1:5.5:5.25, and even more preferably 1:5.5:5.25.

[0119] In one embodiment, the IVR of this disclosure may be useful for maintaining vaginal health.

[0120] The IVR of this disclosure may be administered intravaginally to a female subject about one week to six weeks before sexual intercourse. In one embodiment, the IVR may be administered intravaginally to a female subject about one day to fifteen days before sexual intercourse. [Examples]

[0121] How to fabricate an interventional radiology (IVR) device. The IVRs of this disclosure can be manufactured by any method known to those skilled in the art, but are preferably manufactured using injection molding or extrusion techniques, more preferably by reaction injection molding of silicone elastomers. The term "injection molding" refers to a manufacturing process of producing components / devices from either thermoplastic or thermosetting materials using appropriately designed injection molds. Examples of thermoplastic materials include polyethylene, polyethylene vinyl acetate (PEVA), and certain polyurethanes, while examples of thermosetting materials include silicone rubber / elastomers. Matrix-type silicone elastomer rings containing an active substance can be manufactured by (i) mixing the active substance with one or more components of a silicone system (e.g., a base, crosslinking agent, catalyst, additive, dispersant, etc.), (ii) injecting the mixture into an appropriately designed injection mold, and (iii) optionally applying heat to cure / crosslink the silicone mixture to form an elastomer.

[0122] These methods generally involve dispersing an active substance and an elastomer, such as a polysiloxane, in a suitable solvent or dispersant, such as a silicone liquid, and curing the ring using a platinum catalyst, such as a platinumsiloxane complex, thereby producing a platinum-catalyzed ring. The IVRs of this disclosure can be produced using any well-known elastomer, such as the aforementioned polysiloxanes. In one embodiment, the elastomer used in the methods of this disclosure, such as a polysiloxane, is an addition-cured dimethylsiloxane silicone elastomer comprising a mixture of hydrogenated-vinyl-terminated polydimethylsiloxane molecules. In another embodiment, the elastomer used in the methods of this disclosure, such as a polysiloxane, is a condensation-cured silicone elastomer comprising hydroxy-terminated dimethylpolysiloxane molecules and a tetraalkoxysilane crosslinking agent. In yet another embodiment, the elastomer used in the methods of this disclosure, such as a polydimethylsiloxane, is MED-8470. In certain embodiments, the method further includes the use of a crosslinking agent, such as a hydrogenated polydimethylsiloxane or dimethylmethylhydrogenpolysiloxane crosslinking agent. In another embodiment, the silicone elastomer may contain organic groups other than methyl groups, such as fluorine or phenyl.

[0123] In one embodiment, the method further includes catalyzing a ring in a ring mold. The mold is then opened, followed by the removal and trimming of the IVR. The ring mold is preferably coated with, for example, Teflon® or an electroplated metallized coating. The ring mold may be made of hardened carbon steel, stainless steel, aluminum, or any other material deemed appropriate. It is understood that the dimensions and design of the mold give the physical shape of the IVR, for example, a partial or complete ring, or any other desired shape. Preferably, the device has a partially or complete toroidal shape, more preferably a partially or complete torus shape, or a substantially cylindrical shape. A toroid means a ring-like body produced by rotating any closed loop (including ellipses, circles, or any irregular curves) around a fixed line outside the loop. The toroidal shape may be a complete toroid or a partial toroid. A torus means a ring-like body produced by rotating a circle around a fixed line outside the circle. The torus shape may be a complete ring-like shape or a partial ring-like shape. The geometric characteristics of the mold and IVR can be modified depending on the application.

[0124] Alternatively, IVR devices or their components may be manufactured by extrusion processes well known to those skilled in the art, such as co-extrusion or blend extrusion (see, for example, U.S. Patent No. 5,059,363, the entire disclosure of which is incorporated herein by reference). In a preferred embodiment, IVR devices can be manufactured from a matrix-type LSR4350 silicone elastomer produced at 115°C for 3 minutes, as used in the examples described later herein.

[0125] In vitro study of sperm motility reduction with CS-ZA-LA Active pharmaceutical ingredients (APIs): CS-CuSO4, ZA-Zn(OAC)2, LA-Lactic Acid Sperm motility assay Fresh human sperm samples were obtained on the day of the experiment. Stock solutions of each API in 100 mM saline were prepared in advance. Immediately before the experiment, solutions of each API concentration to be tested were prepared by dilution with saline. These included solutions containing multiple APIs added together.

[0126] The effects of each API on sperm motility and viability were determined using human donors, by adding 200 μL of API diluted in sterile saline to 50 μL of fresh semen. For motility assessment, after incubation of the incubation mixture for 5 minutes, two andrology-trained researchers counted 100 sperm under a microscope and classified them as either non-motile or motile. In the case of motile sperm, they were further classified according to the nature of their motility, from those that did not move forward and barely twitched to those with forward motility. To account for natural variations in sperm characteristics, each experiment was repeated at least three times on different days using different donors.

[0127] Data Analysis The percentage of motility for each experimental condition was calculated by normalizing the percentage of motile sperm in the experimental group relative to the percentage of motile sperm in the saline control group, and setting the average percentage of motile sperm from the control experiment to 100%. For example, the percentage of motility at concentration c is: motility c % = motile sperm c Number of motile sperm 生理食塩水 The number This is the result.

[0128] After obtaining percentage data on motility, synergistic effects were measured using CompuSyn software. (See Chou et al., "CompuSyn for Drug Combinations: PC Software and User's Guide: For the Quantification of Synergistic and Antagonistic Effects in Drug Combinations, and IC") 50 Value, ED 50 Value, and LD 50Computer program for value determination (CompuSyn for drug combinations: PC software and user's guide: a computer program for quantitation of synergism and antagonism in drug combinations, and the determination of IC 50 , ED 50 and LD 50 values)」(incorporated herein by reference in its entirety) was referred to. The motility data for the individual API concentrations and all binary and ternary API solutions were input into the program and analyzed according to the Chou-Talalay principle of half-maximal effects. Table 1 shows the calculated EC values.

[0129]

Table 1

[0130] Table 1 shows that among the APIs, CS is the most effective, followed by ZA and then LA. However, LA has the smallest % difference between its EC 50 concentration and EC 90 concentration, indicating that its motility inhibitory efficacy rapidly begins to increase beyond a certain threshold.

[0131] Furthermore, it was shown that all the APIs act quickly on sperm immobilization and their activities are recognized within 30 seconds.

[0132] Next, the synergism between each API was tested to determine whether any synergistic effect between the compounds could be seen to enable a reduction in dosage. Three possible combinations of two compounds were each tested, followed by testing of all combinations of the three APIs. The combinations of two compounds were tested at their EC 50The tests were conducted using serially diluted APIs in fixed ratios corresponding to the approximate ratios of the values ​​(2:1 for ZA:CS and LA:CS, and 1:1 for ZA:LA). Synergistic effects were calculated using Compusyn with the Chau-Talaley half-effects approach and expressed as a Combination Index (CI) value. A CI value < 1 indicates synergy, while a CI value > 1 indicates antagonism. A smaller CI value for a combination indicates a greater degree of synergy. Table 2 shows the EC values ​​for combinations of two compounds, along with their corresponding CI values ​​at their concentrations. It is important to note that the EC concentration refers to the sum of the concentrations of the two components (e.g., the EC of 13.97 mM ZA:LA in a 1:1 ratio). 50 (This refers to 6.985 mM LA + 6.985 mM Zn). Table 2 shows the EC and CI values ​​calculated for each combination of the two APIs.

[0133] [Table 2]

[0134] Table 2 shows that all three combinations exhibited significant synergy, resulting in a decrease in the concentration of each individual API required to reach the same effect level. LA is also considered the most important API for enhancing synergy, yielding significantly lower CI values ​​in two of its combinations compared to the ZA:CS combination. Table 2 also shows that the CI value for the LA combination increases with increasing API concentration and effect level.

[0135] After the combination of the two APIs was completed, the synergistic effect among all three was tested using a 3x3x3 checkerboard style assay, testing all 27 permutations of the three selected concentrations for each of the three APIs. Based on the results of the previous assay of the two compounds and a practical examination of the release rate of the API rings, it was decided to use a higher lactate concentration than CS and ZA, expecting to maximize the effect of lactate on the synergistic effect and reduce the required release levels of CS and ZA. In the case of CS and ZA, the three selected concentrations were the EC of each API. 50 These corresponded to 1 / 4, 1 / 3, and 1 / 2 of the value. In the case of LA, these proportions were doubled, and EC 50 The concentrations were set to 1 / 2, 2 / 3, and 1 times the original value. The concentrations actually used are listed in Table 3.

[0136] [Table 3]

[0137] From the obtained data, the estimated effect of each individual API was isolated by averaging the percentage decrease in motility and the CI value for each API concentration tested. This 3×3×3 checkerboard assay tested each single concentration against nine permutations of the other two APIs. That is, each cell in Table 4 represents the average of nine combinations containing the specified API concentration.

[0138] [Table 4]

[0139] Table 4 shows that, of the three APIs, CS is considered to be the most effective and synergistic at relatively low concentrations, showing a stable increase in effect and CI over a very narrow concentration range. ZA shows a similar, though less pronounced, ability to reduce sperm motility, but shows almost no dose-response effect on CI values. LA shows similar effects to CS and ZA at doses 1 and 2, but at the highest concentration, a significant decrease in motility and especially a large leap in CI is observed (its EC 50 (It was 20.54 mM). Overall, this data shows that CS and ZA are their EC 50 While LA is significantly more effective at inducing synergistic effects and reducing sperm motility at lower percentage values, it requires higher relative concentrations to achieve the same effect. However, at these high lactate concentrations, LA becomes remarkably potent in enhancing the synergistic effects between all three APIs.

[0140] We selected combinations of three APIs that resulted in a motility reduction of over 90%, and used them in conjunction with our previous insights into their synergistic properties to determine the final concentration combinations and proceed. Table 5 shows all of these combinations.

[0141] [Table 5]

[0142] Based on the data, the target intravaginal concentrations of 4 mM CS, 6 mM ZA, and 21 mM LA were determined to be the ideal mixture to be aimed for in further ring formulations. 4 mM CS exhibits a significant ability to inhibit sperm motility, which is due to its original EC 50It was observed at less than half the concentration, significantly reducing its potential toxicity and lowering the required ring release rate. Since ZA proved to be the least important API in terms of reduced motility and induction of synergies, a low level of 6 mM ZA was chosen to minimize the ring release challenges while still expecting enough ZA to synergistically interact with other APIs, thus keeping their concentrations to a minimum. Ultimately, LA was selected at a relatively high level of 21 mM because it showed a significant increase in efficacy and synergies at high concentrations, exhibited the lowest toxic dose, and was found to have a much higher ability to be released rapidly from the ring in preliminary studies.

[0143] Various zinc salt studies Several zinc salts were tested for sperm motility reduction using the sperm motility assay described above, and their activity is shown in Figure 1. Zinc acetate produced an unpleasant odor upon release from the ring, and zinc formate was found to have solubility issues. Zinc lactate (ZL) was selected based on its overall profile regarding sperm motility reduction, odor, solubility, and pH.

[0144] In vitro study of sperm motility reduction and sperm mucus permeability with CS-ZL-LA API: CS-CuSO4, ZL-Zinc Lactate, LA-Lactate The effects of each API on sperm motility and viability were determined according to the method described above. Figure 2 shows the results of sperm motility tests for each API (CS, ZL, and LA), all of which inhibit sperm motility at mM concentrations. Table 6 shows the calculated EC values ​​for each API (CS, ZL, and LA).

[0145] [Table 6]

[0146] Dose-response curves and EC of a single API 50After determining the values, the synergistic effects of combinations of two and three APIs were investigated using a checkerboard assay that tested all permutations of 3 to 4 concentrations of each API. The synergistic effects were calculated using the Chow-Talaley half-effect principle and Compusyn software, which quantifies the synergistic effect as a combination index (CI) for each combination. CI < 1 indicates synergy, CI > 1 indicates antagonism, and CI = 1 indicates a pure additive effect.

[0147] The sperm-cervical mucus penetration test (SMPT) experiment (Ivic et al., "Critical evaluation of methylcellulose as an alternative medium in sperm migration tests," Human Reproduction, Vol. 17, No. 1, January 2002, pp. 143-149) was performed by placing semen + API solution into capillaries filled with 1% methylcellulose (methylcellulose is an effective substitute currently used in diagnostic laboratories) and measuring sperm migration after 30 minutes. An SMPT test is used with fresh human ejaculate (semen) treated with the test API or vehicle only (control) to determine how well treated sperm migrate through cervical mucus or several substitute media (such as methylcellulose, which does not change in concentration and characteristics from cervical mucus from different women or different cycle times, thus enabling a high degree of precision, accuracy, and reproducibility) filled with glass capillaries, compared to untreated sperm.

[0148] Figure 3 shows the SMPT results for a single API and a combination of CS-ZL-LA. Calculated data for decreased sperm motility and inhibition of sperm mucus permeability are shown in Table 7. Sperm permeability through cervical mucus (methylcellulose) could be inhibited at lower API concentrations compared to those required to inhibit sperm motility.

[0149] [Table 7]

[0150] All tested CS / ZL / LA combinations showed some degree of synergistic effect on sperm motility reduction and inhibition of sperm mucus permeability. See Figure 4, which plots the synergistic effect against efficacy (shown as effect rate (Fa)) (where Cu refers to CS and Zn refers to ZL), Figure 5, which shows the inhibition of motility for the CS-ZL-LA combination (where each bar represents the CS-ZL-LA combination, filled bars indicate forward motility, and hashed bars indicate no forward motility based on the average of four experimental replicates), and Table 8, which shows the calculated reduction in motility and CI values ​​(where CI < 1 indicates synergistic effect, and a smaller CI indicates a greater magnitude of synergy).

[0151] [Table 8]

[0152] As can be seen in Figure 4, CS+LA and ZL+LA showed the highest levels of synergistic effect, with an average CI of less than 0.2. CS+LA and ZL+LA also showed almost identical CI / Fa curves, suggesting similar mechanisms of action. CS+ZL showed little synergistic effect, especially at low concentrations. CS+ZL+LA showed a significant synergistic effect at high Fa levels, but at lower concentrations, it showed a slightly lower synergistic effect than CS+LA or ZL+LA.

[0153] As can be seen from Table 8, a significant correlation was observed between increased LA dose and increased synergistic effect (p<0.0001), whereas no such correlation was observed with ZL and CS. With the CS-ZL-LA combination, the API concentration required for 90% motility inhibition was reduced to 1 / 5 of that required for CS and ZL. The ability to inhibit forward motility started at even lower concentrations for individual APIs and combinations. Figure 3 and Table 7 show that the SMPT results showed a similar reduction in the required API concentration for the CS-ZL-LA combination, and suggested that contraceptive efficacy may be sufficient even at concentrations lower than those required to inhibit motility.

[0154] Research on sperm fertilization ability acquisition regarding CS-ZL-LA API: CS-CuSO4, ZL-Zinc Lactate, LA-Lactate Acquisition of fertilization is the process by which sperm mature within the female reproductive tract and is crucial for acrosome exocytosis and fertilization. Therefore, acquisition of fertilization is an attractive candidate for contraceptive development.

[0155] Sperm processing and API treatment Fresh semen samples were obtained by masturbation from healthy men aged 21–40 who gave their consent. The samples were liquefied at 37°C for at least 30 minutes. Next, 200 μL of semen was combined with either 50 μL of API (CS-ZL-LA combination: 3.2 mM CS, 9.6 mM ZL, 16.8 mM LA) or 50 μL of artificial vaginal fluid (SVF) (vehicle control) (Owen, "A Vaginal Fluid Simulant," Contraception 1999, 59, 91-95, doi:10.1016 / S0010-7824(99)00010-4) and mixed by gently triturating with a wide-mouth pipette. An untreated negative control group was also maintained. The samples were incubated at 37°C for 5 minutes.

[0156] To remove seminal plasma and API, sperm were first centrifuged at 300 g for 10 minutes through enhancement S-Plus cell isolation medium (Vitrolife), and the supernatant was removed to leave a loose sperm pellet. The sperm pellet was resuspended by gently triturating in the remaining 100 μL of medium, then transferred to a new tube and resuspended in 4 mL of modified human tubal fluid (mHTF, Fujifilm Corporation), and centrifuged at 600 g for 10 minutes. The resulting pellet was then resuspended in 150 μL of mHTF, and sperm concentration was calculated using a hemocytometer. Depending on the experiment, sperm were subsequently incubated for 3 hours at a concentration of 10 million / mL under either fertilization-acquiring conditions (5 mM 2-hydroxypropyl-β-cyclodextrin, 2-OHCD) or non-fertilization-acquiring conditions (no cyclodextrin). These conditions for acquiring fertilization ability have been shown to be effective in human sperm (Cardona, "Ganglioside G in Human Sperm") M1 Localization patterns of the Ganglioside GM1 in Human Sperm are an indicator of male fertility and independent of traditional semen measures (Mol Reprod Dev 2017, 84, 423-435, doi:10.1002 / mrd.22803).

[0157] Assessment of motor skills Sperm motility was evaluated in untreated ejaculate after incubation for 5 minutes with each treatment (API, SVF, and untreated control), followed by washing, and then incubation for 3 hours under conditions for fertilization ability acquisition. To assess motility, 15 μL of semen was examined at 40x magnification on a slide warmed to 37°C. The number of forward-motile sperm was visually evaluated and recorded.

[0158] Sperm membrane fluidity evaluation Changes in membrane fluidity were evaluated using a modified version of the Cap-Score test (Androvia Lifesciences, Mountainside, New Jersey). To test male fertility, the Cap-Score test involves incubating sperm with 2-OHCD, a mediator of sterol outflow and a stimulant for acquiring fertilization capacity, followed by membrane ganglioside G M1 By quantifying sperm with different localization patterns, the percentage of sperm responding to these conditions was established (Moody et al., "Validation of a Laboratory-Developed Test of Human Sperm Capacitation," Mol Reprod Dev 2017, 84, 408-422, doi:10.1002 / mrd.22801). This test used cholera toxin B (CTB) conditions and proprietary fixed conditions, G M1 This induced a rearrangement of these patterns. In the more rigid membranes that have not undergone sterol outflow, CTB:488 is G M1 It binds to the posterior acrosome cell membrane and lower equatorial segment of the sperm head, and G M1 This causes rearrangement. In more fluid membranes that signal a response to 2-OHCD via sterol efflux, CTB:488 is G on the acrosome. M1 Labeling the posterior acrosome cell membrane or subequatorial region of G M1 It does not cause a rearrangement of G M1 These cells may remain across the entire cell membrane on the acrosome, or they may even be concentrated at the tip of the acrosome.

[0159] After incubation for 3 hours under either a fertilization-acquiring or non-fertilization-acquiring condition, sperm were fixed overnight in mHTF at room temperature. Immediately before imaging, 1 μL of CTB:488 (Invitrogen) was added to the sperm. Then, 5 μL of sperm stained with CTB:488 was placed on a slide and imaged using a Nikon Eclipse TE2000-U fluorescence microscope. For each experimental treatment, the number of sperm showing a CTB:488 staining pattern consistent with a fluid or rigid membrane was counted. Then, the percentage of sperm with a CTB:488 staining pattern that indicates high membrane fluidity was calculated.

[0160] Coomassi staining of acrosome exocytosis Coomassi staining is a commonly used method for evaluating the acrosome status of fixed sperm. After incubation under viability acquisition conditions for 3 hours, sperm were fixed in 2% paraformaldehyde for 10 minutes. The sperm were then centrifuged at 500 g for 10 minutes. The supernatant was removed, and the sperm were resuspended in 500 μL of 100 mM ammonium acetate (pH 9.0) and centrifuged again at 500 g for 10 minutes. The supernatant was removed, and the sperm pellet was further resuspended in 500 μL of 100 mM ammonium acetate (pH 9.0). To place the sperm on a slide, a circular region outline was drawn on a clean glass slide using a PAP pen. 40 μL of semen was added to the enclosed region and air-dried on a slide warmer at 37°C. To stain the sperm, 150 μL of Coomassi working solution (0.22% Coomassi R-250, 50% methanol, 10% acetic acid) was added to the dried sperm for 6 minutes. Excess dye was removed, and the slides were washed by immersion in ddH2O. The washed slides were air-dried on a slide warmer. Immediately before scoring, one drop of aqueous mounting medium and a coverslip were placed on the slide. The number of sperm with intact or reacted acrosomes was counted at 40x magnification using a Nikon Eclipse E200 optical microscope.

[0161] Live / Dead Staining Sperm viability was evaluated using Sybr-14 / propidium iodide staining (LIVE / DEAD Sperm Viability kit, Invitrogen). In this assay, SYBR-14 labeled live sperm with green fluorescence, while propidium iodide labeled sperm with damaged membranes with red fluorescence. First, the SYBR-14 stock was diluted 50-fold in mHTF. Then, 1.6 μL of diluted SYBR-14 was added to the sperm and incubated at 37°C for 10 minutes. Subsequently, the sperm were treated with 1.6 μL of propidium iodide and incubated for another 10 minutes at 37°C. The sperm were then immobilized by plating onto a glass-bottomed dish (Mattek) coated with poly-D-lysine. Images were taken using an Olympus IX3-FP confocal microscope equipped with a heating stage. The number of sperm exhibiting either a staining pattern for living sperm or a staining pattern for dead / damaged sperm was evaluated in ImageJ, and the percentage of sperm exhibiting a staining pattern for dead / damaged sperm was calculated for each group.

[0162] To understand how motility changed throughout the experiment, the percentage of sperm exhibiting forward motility was evaluated at various stages: in raw ejaculate, after incubation for 5 minutes with API (CS-ZL-LA combination: 3.2 mM CS, 9.6 mM ZL, 16.8 mM LA in SVF) or SVF, after washing, and after incubation for 3 hours under viability conditions. Figure 6 shows the percentage of motile sperm at various experimental stages for the four experiments. API-treated sperm showed varying degrees of motility recovery after washing. However, even after initial motility recovery, API-treated sperm showed almost complete cessation of forward motility after incubation for 3 hours under viability conditions. This decrease in motility after viability was a result of exposure to API, as a similar dramatic decrease in motility was not observed in the SVF vehicle control. As shown in Figure 6, API-treated sperm showed almost complete loss of motility after incubation for 3 hours under viability conditions. These data revealed that even short-term exposure to APIs can have long-term effects on sperm motility.

[0163] Figure 7 shows that short-term incubation with API (CS-ZL-LA combination: 3.2 mM CS, 9.6 mM ZL, and 16.8 mM LA in SVF) resulted in increased spontaneous acrosome exocytosis after 3 hours of incubation under fertilization-acquiring conditions, compared to SVF vehicle controls and untreated controls.

[0164] Sperm viability was examined using SYBR-14 / propidium iodide staining after the washing process and after incubation for 3 hours under viability conditions. Figure 8 shows that API-treated sperm showed decreased viability after incubation for 3 hours under viability conditions compared to the SVF vehicle control and the untreated control. No effect of API treatment on sperm viability was observed after washing. However, after incubation for 3 hours under viability conditions, an increase in the percentage of dead / damaged sperm was observed in the API-treated group compared to the untreated control and the vehicle (SVF) control. This indicates that short-term exposure to API has a long-term effect on sperm viability.

[0165] Cholera toxin B (CTB) is a membrane ganglioside G2 of up to 5 molecules. M1 It can bind to G. In sperm with a more rigid cell membrane because it has not undergone sterol efflux, CTB is G M1 By binding to the posterior acrosome cell membrane and the subequatorial region, G M1 This triggers rearrangement. In sperm that have acquired fertilizing ability after sterol outflow, the CTB is located on the G on the acrosome. M1 Label G M1 It does not cause redistribution. After incubation with API for 5 minutes and then incubation for 3 hours under fertility acquisition conditions, a very high percentage of sperm showed a CTB:488 staining pattern associated with increased membrane fluidity. These percentages far exceeded the published percentage (mean = 35.3 ± 7.7%) for a group of 76 men recently proven to be fertile. In fact, this mean percentage was higher than any percentage recorded in over 7,000 patient samples in Androvia (Travis, private communication). The vehicle (SVF) control group and the untreated control group showed percentages consistent with those expected from a population of fertile men. These results also demonstrate that short-term exposure to API has long-term effects on sperm physiology beyond motility.

[0166] This increase in membrane fluidity was neither a result of 2-OHCD-mediated sterol efflux nor a result of physiologically relevant acquisition of fertilization ability. This is because a very high percentage of API-treated sperm incubated under non-fertilization conditions also showed high membrane fluidity. Therefore, API-treated sperm did not physiologically acquire fertilization ability (they did not undergo acrosome exocytosis or sterol efflux strictly required for fertilization), but instead showed increased membrane fluidity as a result of pathological processes induced by API. Figure 9 shows that the increase in membrane fluidity was unrelated to 2-OHCD-induced sterol efflux. The pathological increase in membrane fluidity is expected to lead to an increase in spontaneous acrosome exocytosis, which is also thought to contribute to decreased motility at the population level. Coomassi staining revealed an increase in the percentage of exocytotic sperm in our API-treated group. As evidenced by the changes in membrane fluidity, and considering that these sperm were not exposed to any exocytosis-related triggers, this increase in exocytosis reflected a non-physiological process.

[0167] This disclosure reveals a novel mechanism of action for CS-ZL-LA IVR. Short-term exposure to the CS-ZL-LA-IVR API (3.2 mM CS, 9.6 mM ZL, and 16.8 mM LA) demonstrated numerous long-term effects on sperm fertilization capacity, including decreased motility, reduced viability, increased membrane fluidity, and increased spontaneous acrosome exocytosis. These effects are likely to be a result of dysregulation of membrane fluidity, leading to increased acrosome exocytosis and cell death.

[0168] Research on antiviral / antibacterial activity Experiments analyzing API interactions in anti-infection assays were conducted using fixed API ratios.

[0169] In vitro anti-HIV activity assay API: CS-CuSO4, ZA-zinc acetate, LA-DL-lactic acid, either alone or in combination (where CZL refers to CS-ZA-LA) The MAGI assay was performed to detect anti-HIV-1 BaL Activity was tested. Each API (CS, ZA, and DL-LA(LA)) at various concentrations, either alone or in combination, was prepared in physiological saline (0.9% NaCl) and tested for HIV-1 activity for 30 minutes at 37°C, 5% CO2, and 98% humidity. BaL Incubated together with HIV-1 in physiological saline. BaL A viral control containing API-HIV-1 was incubated under the same conditions. After incubation, API-HIV-1 BaL The mixture was diluted 10-fold and added to TZMbl cells. After incubation for 72 hours, the cells were washed, stained with X-gal, and fixed. The virus-only control well contained approximately 200 infected cells. Infected cells were counted using a CTL instrument. The API was potent anti-HIV-1 at concentrations from submM to low mM. BaL The compounds showed activity, with CSL(CS-ZA-LA) being the most potent (Figure 10, Table 9).

[0170] [Table 9]

[0171] HIV-1 BaL The active substance was incubated with ±2% VF and 20% SF. The mixture was then serially diluted and applied to TZM-bl cells, and viral titers were determined based on FFU. The graph shows the results of three experiments (mean ± SEM) (Figure 11).

[0172] The three active substances (individually and in combination) significantly inhibited infection in the absence of body fluids. The activity of the individual active substances decreased in the presence of body fluids. However, the combinations retained their activity, and the ternary combination showed the most potent activity.

[0173] in vitro anti-HSV activity Assay of the virucidal activity of APIs against HSV-2 API: CS-CuSO4, ZA-zinc acetate, LA-lactic acid, either individually or in combination (where CZL refers to CS-ZA-LA) An HSV-2 plaque reduction assay was performed to test the anti-HSV-2 activity of ZA, CS, and LA. Each API, either alone or in combination, was prepared in physiological saline (0.9% NaCl) at various concentrations and incubated at 37°C, 5% CO2, and 98% humidity for 10 minutes. 3 HSV-2 G infected particles were pre-incubated for 30 minutes. Virus controls containing HSV-2 were incubated in physiological saline under the same conditions. After incubation, all dilutions and controls were diluted 1 / 10 in DMEM and titrated using a plaque assay. The results are shown in Figure 12 and Table 10. Figure 12 shows the percentage of plaque-forming units (PFU) ± SE relative to the virus control at each API concentration. As shown in Figure 12, ZA, CS, and LA, individually or in combination, showed dose-dependent HSV-2 inhibition in the plaque reduction assay. Data represent the mean of two independent experiments. In Table 10, the molar ratio of CS:LA:ZA in ZS-CA-LA is 1:20:100. All APIs, individually and in combination, exhibited potent viricidal activity against HSV-2, with CS-ZA-LA being the most potent API.

[0174] [Table 10]

[0175] HSV-2 G was incubated with 1.25 mM CS, 12.5 mM ZA, 16.6 mM LA ± 2% VF, and 20% SF at 37°C, 5% CO2, and 98% humidity for 30 minutes. The mixture was then serially diluted by 1 / 10 and applied to Vero cells to determine the viral titer (PFU / mL). The control condition included Vero cells exposed to HSV-2 G in the absence of the active substance. Figure 13 shows the results of three experiments (log, mean ± SEM, plaque-forming units per milliliter (PFU / mL)).

[0176] In vitro assays of anti-vaginalis activity and anti-gonorea activity API: CS-CuSO4, ZA-zinc acetate, ZL-zinc lactate, LA-lactic acid, either individually or in combination (CZL refers to CS-ZL-LA). The minimum cidal concentrations (MCCs) for G. viginalis and N. gonorea were determined. The bacteria were resuspended, and approximately 3 × 10⁻⁶ bacteria were used. 8 The concentration was adjusted to colony-forming units (CFU) / ml (1 MacFarland). Serial dilutions of API (2x concentration) were prepared in broth. Approximately 5 × 10⁻⁶ 5 CFU / ml samples were exposed to API for 30 minutes at 37°C in 5% CO2 (in a tightly sealed tube), and then plated undiluted in double or triple layers onto agar plates. The plates were cultured aerobically (N. gonorea) or anaerobically (G. vicinalis). Diluted, untreated bacteria were plated to confirm the start of inoculation. API concentrations in the sample that resulted in a decrease of more than 3 log (99.9%) of colony-forming units (CFU) were considered bactericidal (less than 500 CFU / ml, fewer than 12 colonies on the plate).

[0177] In the case of N. gonorea, the MCC was 10 mM in the CS, 100 mM in the ZA, and 111 mM in the LA. In the case of G. vaginalis, the MCC was 5 mM in the CS, over 100 mM in the ZA, and 111 mM in the LA. No changes in MCC were observed after testing in the presence of human AB serum.

[0178] To determine API interactions, a 3D checkerboard assay was employed using microliquid dilution. A 4-fold concentrated stock of the API in broth was prepared, and 10 5It was added to a 96-well plate containing CFU / well. N. gonorrhoeae was aerobically cultured for 24 hours, and G. vaginalis was anaerobically cultured for 48 hours in an anaerobic jar. At the end of the incubation, the bacteria were gently mixed and fixed with PFA. The optical density (OD) at 650 nm was obtained. The OD from the control wells containing broth only and broth containing API was subtracted from the OD in the wells containing bacteria and API. The fractional inhibitory combination index (FICI) was calculated. FICI = (MIC when drugs are combined / MIC of drug A alone) + (MIC when drugs are combined / MIC of drug B alone) + (MIC when drugs are combined / MIC of drug C alone). FICI ≤ 0.5 - synergism, 0.5 < FICI < 4 - no interaction, ≥ 4 - antagonism. For both pathogens, no interaction / weak synergism was observed.

[0179] 5×10 5 5×10 CFU / mL of N. gonorrhoeae was incubated for 30 minutes at 37 °C, 5% CO2, and 98% humidity with these active substances of 2.5 mM CS, 25 mM ZA, 50 mM ZL, and 27.75 mM LA in single combinations, binary combinations, and ternary combinations ± 30% AB serum. The selected concentrations were one-third to one-fourth of the MCC determined for each single API. Untreated bacteria and bacteria exposed to polymyxin B (64 μg / mL) were the positive control and negative control, respectively. After incubation, the bacteria were plated in duplicate on GC agar plates supplemented with Isovitalex (25 μL / plate). The untreated control was plated after dilution to 1:100. After incubation at 37 °C with 5% CO2 for 24 hours, the colonies were counted using Image J software. Dilutions that resulted in a decrease of more than 3 logs in bacterial viability (99.9%) were considered bactericidal. These figures represented four experiments (Log of CFU count, mean ± SD).

[0180] Figure 14 and Table 11 show the results for N. gonorea. As shown in Figure 14, bactericidal effects were induced by CS / ZA combinations, CS / LA combinations, and CS-ZA-LA combinations below bactericidal concentrations. 10 mM CS and 111 mM DL-LA were MCCs against N. gonorea ± human serum, but ZL did not show inhibitory activity. Bactericidal activity was induced by binary and ternary active substance combinations below MCC. Table 11 shows the MCCs of single active substance ± 10% AB serum and 30% AB serum.

[0181] [Table 11]

[0182] Assay of in vitro bactericidal activity against Chlamydial trachomatis (C. trachomatis) API: CS-CuSO4, ZA-zinc acetate, ZL-zinc lactate, LA-DL-lactic acid, either individually or in combination (where CZL refers to CS-ZL-LA). C. trachomatis was incubated with ±2% VF and 20% SF of the active substance at room temperature for 2 hours. The mixture was serially diluted to 1 / 10 in SPG solution and applied in triplicate to HeLa cells. Images were acquired using a Cytation-5 instrument, and the number of inclusions was analyzed using Gen5 software in Cytation-5. The control condition included HeLa cells exposed to C. trachomatis in the absence of the active substance (Figure 15).

[0183] A ternary combination of selected concentrations of CS (approximately 1 mM to 3 mM), zinc salt (approximately 3 mM to 13 mM), and DL-LA (approximately 16 mM to 80 mM) yielded the most potent antiviral and anti-C. trachomatis activity in the presence of vaginal fluid (VF) and semen (SF).

[0184] LA concentrations were higher than in other models, and no activity was observed at lower concentrations. The tested concentration (80 mM) was within the physiological range and the target release range. CS and LA significantly inhibited infection, with the exception of ZL, regardless of the presence of body fluids. Body fluids did not have a significant effect on activity.

[0185] Research on ring formulations API: CSA-CuSO4 (anhydrous), ZLA-zinc lactate (anhydrous), L-DL-lactide alone or in combination (where CZL refers to CSA-ZLA-L) This disclosure provides the formulation development and testing of a novel non-hormonal multipurpose interventional radiology (IVR) technology (also known as the "CZL" ring) that sustains the release of three active substances: anhydrous copper sulfate (CSA), anhydrous zinc lactate (ZLA), and DL-lactide (L; which hydrolyzes to lactic acid (LA)).

[0186] Method for fabricating a matrix-type LSR-4350 IVR A matrix-type silicone (Silbione® LSR-4350) vaginal ring (Table 12) was manufactured using a custom-made ring mold (outer diameter 57.6 mm, cross-sectional diameter 7.9 mm) attached to an electrically heated laboratory-scale injection molding machine.

[0187] [Table 12]

[0188] All ring formulations were cured at 115°C for 3 minutes.

[0189] Each ring was packaged in a foil pouch and sealed within 2 minutes of demolding.

[0190] In vitro release test (IVRT) In vitro release tests were conducted over 30 days using three rings per formulation. Each ring was placed in a 100 mL plastic container, each individually labeled. 20 mL of Milli-Q ultrapure water was added to each container. The containers were capped and placed in a SciQuip Incu-Shake FL16-2 orbital shaking incubator (37°C, 60 rpm, 25 mm orbital throw). IVR samples were analyzed for lactide and lactic acid using high-performance liquid chromatography (HPLC), and for copper and zinc ions using inductively coupled plasma atomic emission spectrometry (ICP-OES). After 24 hours ± 15 minutes, the containers were removed from the incubator, shaken for approximately 10 seconds, and then 1 mL to 2 mL of release medium was collected for HPLC analysis, and approximately 10 mL was collected for ICP-OES analysis (or as needed). Next, the release medium was completely replaced with 20 mL of fresh MilliQ water (except for 60 mL added over the weekend to maintain the release rate), and the sealed container was returned to the incubator.

[0191] HPLC analysis IVR samples were analyzed for DL-lactide and lactic acid using a Waters HPLC system (Waters Limited, Ireland). In short, 50 μL of each in vitro release sample was injected into a Thermo Scientific BDS Hypersil® C18 column (150 mm × 4.46 mm, 3 μm particle size) maintained at 35°C and fitted with a guard column. Isocratic elution was performed at a rate of 1.3 mL / min for 5 minutes using a mobile phase of 4% (volt / volt) acetonitrile and 96% (volt / volt) potassium phosphate buffer (7.7 mM, pH 3.0). Lactic acid and DL-lactide were detected at a wavelength of 210 nm at 1.8 minutes and 3.7 minutes, respectively.

[0192] ICP-OES analysis In vitro released samples were analyzed for copper and zinc ions using an ICP-OES (PerkinElmer Avio® 220 Max ICP-OES, UK) equipped with a glass MEINHARD® K1 nebulizer and a baffled cyclonic spray chamber (standard).

[0193] result Ring characteristics Each IVR measured approximately 57.4 mm x 7.3 mm and weighed between 7.8 g and 9.3 g. Its mechanical properties were similar to those of commercially available VR products (compressive stress over 20 mm ranging from 0.8 N to 4.3 N, 41 to 67, and 248% to 564% Shore M hardness values, as well as percentage elongation at fracture).

[0194] In vitro release of lactide (LT) and lactic acid (LA) Figure 16 shows the daily and cumulative release of lactide (LT), and Figure 17 shows the daily and cumulative release of lactic acid (LA) from IVR to deionized water over 30 days.

[0195] All IVRs loaded with lactide in deionized water released lactide in the form of LT and LA. Since lactide is readily hydrolyzed to lactic acid (both in vitro and in vivo), it is more beneficial to evaluate and measure the release of lactic acid.

[0196] A burst release of lactide was observed over the first five days, after which little to no release occurred. The average burst release of lactide on day 1 in 20 mL of deionized water at 37°C exceeded 122 mg. The 10CSA-10ZLA-20L ring released 248.5 mg of lactide and 48.4 mg of lactide on day 1. This is almost twice as high as the total lactide and lactide released from the 10CSA-10ZLA-10L ring on the same day.

[0197] Towards the end of the second week, the daily LA release from the 10CSA-10ZLA-20L IVR (86.4 mg / day) was almost twice as high as the LA released from the 10CSA-10ZLA-10L ring (48.9 mg / day), compared to 27.7 mg / day for the 30L ring and 34.3 mg / day for the 10CSA-20L ring.

[0198] In week 4, the average daily release of LA from the 30L ring and the 10CSA-20L ring ranged from 22.5 mg / day to 23.5 mg / day, while the daily release of LA from the 10CSA-10ZLA-10L ring and the 10CSA-10ZLA-20L ring decreased to the range of 11.0 mg / day. This indicates that, over the first two weeks of the 30-day study, the ring formulations loaded with these three active substances released LT and LA at relatively higher rates than the other formulations.

[0199] Regarding the ternary active substance IVR (10CSA-10ZLA-20L), a sustained and continuous release of lactic acid (12 mg / day to 105 mg / day) was observed over a 30-day period.

[0200] [Table 13]

[0201] Over a 30-day study, the total cumulative LT release percentage (nearly 100% L released) from ring formulations loaded with three active substances (10% (weight / weight) or 20% (weight / weight) regardless of LT loading) was higher than that of the 10CSA-20LT ring (62.9%) and even higher than that of the 30L ring (44.7%).

[0202] In vitro release of zinc and copper ions Figure 19 shows the daily and cumulative release of zinc ions from IVR to deionized water over a 30-day period.

[0203] Regarding zinc ion release, no characteristic osmotically controlled burst release on day 1 was observed in the 10CSA-10ZLA-10L (3.7 mg) and 10CSA-10ZLA-20L (12.5 mg) formulations. The 10CSA-10ZLA-20L formulation (ring loaded with 40% (wt / wt) active substance) showed a three-fold higher day 1 zinc ion release than the 10CSA-10ZLA-10L (ring loaded with 30% (wt / wt) active substance), which was enhanced by an additional 10% (wt / wt) DL-lactide in the ring. The average week 1 (days 2-4) zinc ion release (16.6 mg) of the 10CSA-10ZLA-20L formulation was higher than that of the 10CSA-10ZLA-10L formulation (2.3 mg). The subsequent daily release of zinc ions from both the 10CSA-10ZLA-20L and 10CSA-10ZLA-10L formulations decreased over time after the first week. The significant difference in the cumulative release of zinc ions from the two formulations was mainly due to the release in the first week. In comparison, the 10CSA-10ZLA-20L formulation showed the highest release rate in the first week, while the 10CSA-10ZLA-10L formulation showed the highest release rate in the second week. A delay effect was observed in the release of the 10CSA-10ZLA-10L formulation in the first week.

[0204] Figure 18 shows the daily and cumulative release of copper ions from IVR to deionized water over a 30-day period.

[0205] Regarding copper ion release, the 10CSA-10ZLA-10L and 10CSA-10ZLA-20L formulations showed similar daily and cumulative release profiles to those of zinc ions. As shown in Table 15, generally, daily copper ion release was always higher than zinc ion release for each formulation. 56% zinc ions and 78% copper ions were released from the 10CSA-10ZLA-10L ring, and 78% zinc ions and 99% copper ions were released from the 10CSA-10ZLA-20L ring. Theoretically, the same drug load of CSA and ZLA should show the same uniform distribution within the silicone elastomer ring. Since the reported water solubility of CSA and ZLA is 201 g / L and 48.2 g / L (20°C), respectively, the higher copper ion release is likely due to a faster dissolution rate into the release medium (deionized water).

[0206] Both the 30CSA and 10CSA-20L formulations are rings loaded with 30% (weight / weight) of the active substance. Both showed burst releases of 14.7 mg and 8.5 mg of copper ions on day 1, respectively, during week 1. The copper ion release rate of 30CSA decreased in week 2 and then increased in weeks 3 and 4, while 10CSA-20L showed a relatively consistent copper ion release rate in week 2 and then increased in weeks 3 and 4. The highest release rate was observed in week 4 for both formulations. Regarding the order of the highest release rates over 30 days, the delay time for 30CSA and 10CSA-20L was longer than that of 10CSA-10ZLA-10L, followed by the 10CSA-10ZLA-20L ring. Compared to the 10CSA-10ZLA-10L ring, the 30CSA ring and the 10CSA-20L ring released only 10% and 30% copper ions, respectively.

[0207] For the ternary active substance IVR (10CSA-10ZLA-20L), sustained and continuous release of copper ions and zinc ions (2 mg / day to 31 mg / day and 1 mg / day to 17 mg / day, respectively) was observed over a 30-day period.

[0208] [Table 14]

[0209] In vitro release pH profile Since low pH in vivo is expected to significantly contribute to sperm immobilization, we investigated the effect of APIs on the pH of the medium.

[0210] Figure 20 shows the pH of 1 mg / mL solutions of various salts containing anhydrous copper sulfate, zinc sulfate monohydrate, zinc lactate dihydrate, and anhydrous zinc acetate.

[0211] Figure 21 shows the pH of the release media during a 30-day in vitro release study for five IVRs (30CSA, 10CSA-10ZLA-10L, 10CSA-10ZLA-20L, 30L, and 10CSA-20L). The pH of all release media for 10CSA-10ZLA-10L, 10CSA-10ZLA-20L, 30L, and 10CSA-20L was maintained below 4.6 over the 30-day period. The decrease in pH for these rings is thought to be consistent with the release of lactide, which is rapidly hydrolyzed to form lactic acid. Copper and zinc salts further adjust the pH of the media according to the well-established Lewis acid properties of these metal ions and the acid-base behavior of their counterions.

[0212] Over a 30-day release in deionized water, the pH range for lactide-loaded IVRs was between 2.1 and 3.3, which is consistent with lactide hydrolysis to lactic acid in the presence of water, although the average pH value for 30 CSA IVRs was 4.5 ± 0.1 (Table 15). The pH of a healthy vagina was found to be between 3.8 and 4.5. The pH of deionized water should be between 5.5 and 6.0.

[0213] [Table 15]

[0214] Toxicity assay API: Copper sulfate (CS), DL-lactic acid (LA), zinc acetate (ZA), or zinc lactate (ZnLA or ZL), or combination thereof. Single exposure to an API Cervical explants were exposed to APIs [copper sulfate (CS), DL-lactate (LA), zinc acetate (ZA), or zinc lactate (ZnLA or ZL), or a combination thereof] for 24 hours (hr) under control (saline ± 5% VF) and gyno-oil conditions. Tissue viability (%) compared to the saline control is shown (mean ± SEM) in the absence of 5% VF (n=3 to 11 experiments) or in its presence (n=3 experiments). The p-value represents the comparison between the indicated treatment and saline. * <0.05, *** <0.0005). Dunn's multiple comparison test along with the Kruskal-Wallis test (Figure 22). Table 16 shows the amount of API used in a single treatment.

[0215] [Table 16]

[0216] In Figure 23, cervical explants were incubated for 24 hours with selected concentrations of API in or without 5% VF. The explants were fixed, paraffin-embedded, sectioned, and stained with H&E.

[0217] Inflammatory cytokine concentrations after single exposure of cervical explants to API Cervical explants were exposed to API for 24 hours in or without 5% VF. The supernatant was collected and analyzed for pro-inflammatory cytokines. Mean ± SEM values ​​for IL-1α and IL-1β (n=3-6 experiments) are shown (Figure 24).

[0218] Survival rate and TEER in VEC-100 tissue after single exposure to API The VEC-100 tissue was incubated with the API for 24 hours. Tissue viability was analyzed by MTT assay and LDH assay, and TEER measurements were recorded. Untreated conditions, Gynol conditions, and Triton conditions were included as controls. Data from four individual experiments are shown (Figure 25).

[0219] Table 17 shows an overview of the findings for the indicated concentrations of API after single exposure.

[0220]

Table 17

[0221] Exposure to CS (up to 10 mM) alone, zinc salts (up to 100 mM) alone, and LA (up to 222 mM) alone did not induce toxicity. After 24-hour exposure to a single active substance, no histopathological changes, decrease in tissue viability (MTT, LDH), decrease in TEER, or increase in cytokine concentration were observed. A ternary active substance combination of 10 mM CS, 25 mM ZA, and 222 mM LA in saline caused epithelial damage in cervical vaginal explants. However, no damage was detected in the presence of 5% VF.

[0222] Multiple exposures to API The VEC-100 tissue was incubated daily for 1 hour over 5 days with the API (alone and in combination) at selected concentrations (showing synergistic action in the anti-sperm assay: 4 mM CS, 21 mM ZL, 22 mM LA), rinsed in PBS, and incubated for an additional 23 hours. A single experiment is shown. As shown in Figure 26, tissue viability and TEER were analyzed on the indicated days (mean ± SEM of replicates). Table 18 shows that the indicated concentrations are safe and have anti-sperm activity.

[0223]

Table 18

[0224] Table 19 shows an overview of the findings for the indicated concentrations of API after multiple exposures.

[0225] [Table 19]

[0226] Tissue viability (MTT, LDH) and TEER did not decrease with individual active substances and their combinations (4 mM CS, 21 mM ZL, 22 mM LA).

Claims

1. A vaginal ring containing a therapeutically effective amount of a non-hormonal active substance dispersed in an elastomer.

2. The vaginal ring according to claim 1, wherein the non-hormonal active substance comprises at least one selected from copper, zinc, or lactide components.

3. The vaginal ring according to claim 1, wherein the non-hormonal active substance comprises a lactide component and at least one selected from a copper component or a zinc component.

4. The vaginal ring according to claim 1, wherein the non-hormonal active substance comprises a lactide component, a copper component, and a zinc component.

5. The vaginal ring according to claim 2, wherein the copper component is metallic copper, copper oxide, copper salt, or copper ion-ligand complex.

6. The vaginal ring according to claim 2, wherein the zinc component is metallic zinc, zinc oxide, or a zinc salt.

7. The vaginal ring according to claim 2, wherein the lactide component is D-lactide, L-lactide, DL-lactide, or lactic acid.

8. The vaginal ring according to claim 1, wherein the elastomer is selected from silicone, polyethylene vinyl acetate copolymer (EVA), styrene-butadiene-styrene block copolymer, polyphosphazene, poly(isoprene), poly(isobutylene), polybutadiene, polyurethane, nitrile rubber, neoprene rubber, or a combination thereof.

9. The vaginal ring according to claim 1, wherein the elastomer is present in an amount of about 50% to about 99% of the total weight of the vaginal ring.

10. The vaginal ring according to claim 1, wherein the vaginal ring is in the form of a matrix, and the elastomer is silicone.

11. The vaginal ring according to claim 1, wherein the vaginal ring is in the form of an exposed core, and the elastomer is polyethylene vinyl acetate or polyurethane.

12. The vaginal ring according to claim 5, wherein the copper salt is anhydrous copper sulfate or copper sulfate hydrate.

13. The vaginal ring according to claim 6, wherein the zinc salt is zinc acetate, zinc formate, zinc lactate, zinc chloride, zinc sulfate, zinc iodide, zinc citrate, or zinc orotate, each in the form of anhydrous or hydrate.

14. The vaginal ring according to claim 1, wherein the non-hormonal active substance is contained in an amount of about 5% to about 50% by weight relative to the total amount of the vaginal ring.

15. The vaginal ring according to claim 4, wherein copper ions, zinc ions, and lactic acid are released in a molar ratio of 1:1:1 to 1:6:6 after the first day period.

16. The vaginal ring according to claim 4, wherein copper ions are released at a rate of 2 mg / day to 31 mg / day during a 30-day period of use.

17. The vaginal ring according to claim 4, wherein zinc ions are released at a rate of 1 mg / day to 17 mg / day during a 30-day period of use.

18. The vaginal ring according to claim 4, wherein lactic acid is released at a rate of 12 mg / day to 105 mg / day during a 30-day period of use.

Images