Nestorone® for preventing and treating preterm birth

Abstract

Provided is a method of preventing and treating an onset of preterm labor and preterm birth, promoting fetal lung development, and reversing inflammation and reducing stress in the uterine environment by administering a pharmaceutically effective amount of Nestorone® (NES) to a pregnant individual in need thereof.

Description

VASTEB-066NESTORONE® FOR PREVENTING AND TREATING PRETERM BIRTHCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims the benefit of the filing date of United States Provisional Patent Application No. 63 / 698,781, filed on September 25, 2024, the disclosure of which is hereby incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] Preterm birth (PTB) has significant impact on infant mortality and morbidity. The World Health Organization (WHO) reported about 13.4 million infants were born preterm in 2020, and approximately 900,000 deaths in 2019 were due to PTB complications. The rates of PTB in the United States, Africa, and Southeast Asia are significant, with 1 / 9 babies born prematurely in the United States, and at least 12.5% of births and likely significantly more in Africa and Southeast Asia being preterm- according to the WHO. There is also significantly elevated risk for women who have previously delivered a premature baby; they are found to be 2.5 times more likely to deliver another baby prematurely.

[0003] During pregnancy, progesterone is a crucial hormone in maternal reproductive tissues and immune cells. Specifically, progesterone maintains myometrial quiescence during pregnancy by suppressing genes expressing contraction-associated proteins that induce labor by increasing myometrial contractility and excitability. In other words, progesterone prevents the uterus from contracting too soon. In most viviparous species, labor is triggered by a decrease in peripheral levels of progesterone, and blocking progesterone action induces preterm labor. Therefore, human parturition is likely triggered by a functional withdrawal of progesterone. Accordingly, progesterone is used in clinical interventions to increase the chances of embryo implantation and reduce the risk of miscarriage and premature labor, and vaginal progesterone hormone treatment is recommended for preventing PTB in patients with a short cervix. 17-hydroxyprogesterone caproate (17-OHPC), also known as Makena®, a synthetic progesterone derivative, had been used in pregnant women for about six decades to prevent recurrent PTB until March 2023 when the United States Food and Drug Administration (FDA) withdrew its approval due to a lack of efficacy shown from a confirmation trial called “PROLONG.”

[0004] Other than 17-OHPC, ritodrine, a tocolytic drug used to stop premature labor, was withdrawn from the U.S. market in 1998 due to severe maternal cardiopulmonary sideVASTEB-066 effects. Thus, there are currently no FDA-endorsed and evidence- supported pharmaceuticals available for preventing and treating PTB.

[0005] Premature infants are often bom with lungs that are not sufficiently developed since lungs are one of the last organs to fully mature in-utero. Surfactant production in lungs, which is vital for the structural integrity of lungs and gas transfer across alveolar membranes, does not begin until about 24 weeks, and is not sufficient for independent breathing until about 32 weeks of gestation. As a result, premature infants are at risk for short-term health problems, including respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and respiratory syncytial virus infection. RDS in particular, is one of the leading causes of premature infant mortality because underdeveloped lungs that lack a sufficient amount of surfactant cannot keep themselves properly inflated with air. Also, BPD results in extreme respiratory function patterns, resulting in premature lungs being particularly sensitive to injury and impaired healing. Premature infants are also at risk for long-term health problems, such as a risk of reduced exercise capacity, allergic asthma, general reduced lung function, and airway obstruction problems.

[0006] Evidence suggests that antenatal maternal inflammation is linked to PTB, and an increase in recurrent wheezing and asthma in infants bom prematurely. Corticosteroids, antiinflammation medicines, have been used in promoting fetal lung maturation and reducing the risk of respiratory distress syndrome in premature infants. They are usually given to women at risk of PTB, typically as two injections.

[0007] Therefore, there is an urgent need to develop new methods for preventing and treating PTB, promoting fetal lung development, and reducing the risk of respiratory distress syndrome in premature infants.BRIEF SUMMARY OF THE INVENTION

[0008] In one aspect, the disclosure is directed to a method for preventing and treating an onset of preterm labor and preterm birth by administering a pharmaceutically effective amount of Nestorone® (NES) to a pregnant individual in need thereof.

[0009] In one embodiment, the NES may be administered vaginally.

[0010] In one embodiment, the NES may be administered vaginally through an intravaginal ring, a gel or a suppository.VASTEB-066

[0011] In one embodiment, the NES may be administered beginning about the 18th to 22nd week of gestation until about the 37th week of gestation.

[0012] In one embodiment, the NES may be administered in an amount of about 50 |jg to about 20 mg per day.

[0013] In one embodiment, the NES may be administered in combination with a second active agent for preventing and treating preterm labor and preterm birth.

[0014] In one aspect, the disclosure is directed to a method of promoting fetal lung development by administering a pharmaceutically effective amount of NES to a pregnant individual in need thereof.

[0015] In one embodiment, the NES may be administered vaginally.

[0016] In one embodiment, the NES may be administered vaginally through an intravaginal ring, a gel or a suppository.

[0017] In one embodiment, the NES may be administered beginning about the 18th to 22nd week of gestation until about the 37th week of gestation.

[0018] In one embodiment, the NES may be administered in an amount of about 50 pg to about 20 mg per day.

[0019] In one embodiment, the NES may be administered in combination with a second active agent for preventing and treating preterm labor and preterm birth.

[0020] In one aspect, the disclosure is directed to a composition for preventing and treating an onset of preterm labor and preterm birth, comprising a pharmaceutically effective amount of NES and a pharmaceutically acceptable excipient.

[0021] In one embodiment, the composition may further comprise a second active agent for preventing and treating preterm labor and preterm birth.

[0022] In one embodiment, the second active agent comprises at least one of a tocolytic agent, or an anti-inflammatory agent selected from alpha lipoic acid, amitriptyline or interferons.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows in vitro effects of NES on inflammasome activity measured by Caspase- 1 induced luminosity in LPS stimulated human PBMCs.

[0024] FIGs. 2A and 2B shows in vitro effects of NES on LPS stimulated human PBMCs using Luminex assays.VASTEB-066

[0025] FIG. 3 shows in vitro effects of NES on LPS stimulated human PBMCs using qPCR assays.

[0026] FIG. 4 shows in vitro effects of NES on LPS stimulated MCF-7 cells using qPCR assays.

[0027] FIG. 5 shows in vitro effects of NES on LPS stimulated USMC cells using qPCR assays.

[0028] FIG. 6 shows in vitro effects of NES on LPS stimulated BEAS-2B cells using qPCR assays.

[0029] FIG. 7 shows in vivo effects of NES on cytokine levels in mouse uteri extracted from LPS-treated C57BL / 6 pregnant mice using qPCR assays.

[0030] FIG. 8 shows in vivo effects of NES on cytokine levels in mouse spleens extracted from LPS-treated C57BL / 6 pregnant mice using qPCR assays.

[0031] FIG. 9 shows in vivo effects of NES on cytokine levels in mouse brains extracted from LPS-treated C57BL / 6 pregnant mice using qPCR assays.

[0032] FIG. 10 shows in vivo effects of NES on cytokine levels in mouse lungs extracted from LPS-treated C57BL / 6 pregnant mice using qPCR assays.DETAILED DESCRIPTION

[0033] Before describing various embodiments of the invention in detail, it is to be understood that the invention is not limited in the present application to the details set forth in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure. It is also to be understood that the phrasing and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0034] All percentages and ratios used herein are by weight of the total composition unless otherwise indicated herein. All measurements made herein are at room temperature (25°C ± 5°C) and normal pressure unless otherwise designated. All temperatures used herein are in degrees Celsius unless specified otherwise.

[0035] The present disclosure can comprise (open ended) or consist essentially of the components as well as other ingredients or elements described herein. As used herein, the termVASTEB-066“comprising” means having the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” used herein are also to be construed as open ended unless the context suggests otherwise. The terms “comprising,” “having” and “including” encompass the terms “consisting of’ and “consisting essentially of.” The term “consisting essentially of’ herein means that the composition or method may include additional ingredients and / or steps, but only if the additional ingredients and / or steps do not materially alter the characteristics of the claimed composition or method.

[0036] All ranges recited herein include the endpoints, and those falling between. Terms such as “about,” “generally,” “substantially” and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms are defined by the circumstances and the terms that they modify as those are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error, and instrument error for a given technique used to measure a value. Unless otherwise indicated, the term “about” as used herein includes all values in a range of the specific value indicated ±10%.

[0037] Unless otherwise indicated, as used herein, the terms “a” and “an” include the plural, such that, e.g., “an excipient” can mean at least one excipient, as well as a plurality of excipients including but not limited to, excipients of different types.

[0038] Where used herein, the term “and / or” when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition is described as comprising agents A, B, and / or C, the composition can comprise A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0039] As used herein, the term “preterm birth” or “PTB” generally describes babies bom alive before the 37th week of gestation. Preterm births may occur, for example, before the 28th week of gestation (referred to as extremely preterm), between the 28th to the 32nd week of gestation (referred to as very preterm), or between the 32nd to the 37th week of gestation (referred to as moderate to late preterm).

[0040] As used herein, the term “preterm labor” refers to dilation and / or effacement of the cervix, detected by digital examination and associated with persistent uterine contractionsVASTEB-066 before 37 weeks of gestation. Specifically, preterm labor may refer to six or more uterine contractions per hour accompanied by documented cervical change, cervical dilation greater than 2 cm, cervical effacement greater than 80%, or documented change in cervical effacement greater than 50%.

[0041] As used herein, the term “short cervix” refers to a cervical length greater than 1.0 cm, and preferably ranging between 1.0 cm to 8.0 cm. In other examples, the term “short cervix” refers to a cervical length less than or equal to 3.0 cm and more than 1.0 cm. In other examples, the term “short cervix” refers to a cervical length less than or equal to 2.5 cm and more than 1.0 cm. A cervix that is less than 1.0 cm is referred to as an “ultra-short cervix” and is clinically distinguishable from “short cervix.” Pregnant individuals having a short cervix may be identified and clinically diagnosed as understood by one skilled in the art. For example, a diagnosis may be made following sonographic and clinical examination.

[0042] As used herein, the term “active,” “pharmacologically active,” “therapeutically active,” “pharmaceutically active” or “physiologically active” to describe “ingredient” or “agent” means any chemical material or compound used alone or in combination suitable for administration, for example, vaginal administration, which induces a desired systemic effect.

[0043] In one embodiment, the term “active agent” used herein refers to a medication administered to prevent or treat PTB, and / or promote fetal lung development.

[0044] As used herein, the term “treat” or “treatment” refers to any treatment of a disease or disorder, including, not limited to, arresting or suppressing the development of clinical symptoms, ameliorating clinical symptoms, and eliminating the causes of regression of clinical symptoms. The term “prevent” or “prevention” refers to reduction of the likelihood of occurrence or development of a disease or disorder. It is understood by one of the ordinary skill in the art that it is not always possible to distinguish between “treating” and “preventing” since the ultimate inductive event or events may be unknown, latent, or the patient is not evaluated until well after the occurrence of the event or events.

[0045] As used herein, the term “effective,” “pharmacologically effective,” “therapeutically effective,” “pharmaceutically effective” or “physiologically effective” amount of an active agent means a non-toxic but sufficient amount of a compound used alone or in combination with one or more other compounds to provide the desired therapeutic effect.

[0046] As understood by one of skill in the art, the effective amount of an active agent, or the actual weight percentage of such agent used in the composition, can vary dependingVASTEB-066 upon a variety of factors, including for example, the formulation of the composition, the actual dosage form, and the manner of delivery of the composition, e.g., whether the dosage form is intended for extended release. Such activity can be determined according to conventional methods, and the amounts of active agents may be formulated accordingly.

[0047] As used herein, the term “unit dose form” refers to a composition in a form that is administered to a subject in a single dose, and the term “daily dosage” refers to the daily dosage of any active agents used alone or in combination in the composition to provide the desired therapeutic effect.

[0048] As used herein, the term “pharmaceutically acceptable excipient” refers to carriers or vehicles, gel forming agents or thickening agents, suspending agents, emollients, moisturizers, solubilizers, stabilizers, pH adjusters, penetration enhancers, or preservatives, which do not cause significant irritation to an organism and do not abrogate the biological activity and properties of the applied active agent.

[0049] As used herein, the term “carriers” or “vehicles” refers to carrier materials suitable for administration and includes any such material known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer or the like, which is non-toxic and which does not interact with other components of the composition in a deleterious manner. Examples of suitable carriers include water, alcohols, mineral oil, silicone, liquid sugars, waxes, petroleum jelly, and a variety of other oils and polymeric materials.

[0050] As used herein, the term “release enhancers” refers to compounds that enhance the rate of release of the agent(s) from the delivery device, for example, an intravaginal ring, vaginal gels, or suppositories. Such compounds include, but are not limited to, polyvinylpyrrollidone (PVP or povidone), modified cellulose ethers (e.g., hydroxyethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose), microcrystalline cellulose, polyacrylic acid, carbomer, alginic acid, carrageenan, cyclodextrins, 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).

[0051] As used herein, the term “penetration enhancer” refers to a material that helps increase the permeability of a pharmacologically active agent to skin, so as to increase the rate at which the pharmacologically active agent permeates through the skin and enters the bloodstream.VASTEB-066

[0052] According to one aspect of the present disclosure, provided is a method of preventing and treating an onset of preterm labor and preterm birth (PTB) by administering a pharmaceutically effective amount of Nestorone® (NES) to a pregnant individual in need thereof.

[0053] In another aspect, the present disclosure is directed to a method of promoting fetal lung development by administering a pharmaceutically effective amount of NES to a pregnant individual in need thereof.

[0054] In a further aspect, the present disclosure is directed to a method of reversing inflammation and reducing stress in the uterine environment by administering a pharmaceutically effective amount of NES to a pregnant individual in need thereof.

[0055] Preferably, NES may be administered to a pregnant individual who has a risk of preterm labor, for example, individuals who have previously delivered a premature baby or who have a short or effaced cervix in mid-pregnancy.

[0056] NESTORONE®, referred to as NES throughout the present, is a progestin, a synthetic 19-nor-progesterone derivative. It is 16-methylene- 17 a- acetoxy- 19-norpregn-4-ene- 3, 20-dione, having a structure close to the physiological hormone progesterone, also referred to as Prog herein. NES has strong antiovulatory, anti-gonadotropic and progestational properties, and does not carry androgenic, or estrogenic actions at therapeutic levels. NES has been studied extensively in non-oral forms, including transdermal gels, implants, and vaginal rings for hormone replacement therapy and contraception. It was approved by the FDA in 2018 in a first formulation combined with an estrogen as a vaginal system marketed as Annovera® for female contraception.

[0057] NES may be administered parenterally, by injection, orally, topically, intravenously, intraperitoneally, subcutaneously, transcutaneously, intradermally, subdermally, intra- articularly, intraventricularly, intrathecally, intravaginally, or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, through a buccal, sublingual, intraperitoneal, intravaginal, anal or intracranial route. In preferred embodiments, NES may be administered through vaginal delivery, i.e., delivery by passage of active agents, by means of intravaginal devices such as intravaginal rings, suppositories, gels, creams, foams, lotions, and the like, through vaginal tissue. Preferably, NES may be delivered vaginally through intravaginal rings, gels or suppositories. More preferably, NES may be delivered vaginally through intravaginalVASTEB-066 rings. The vaginal route of administration may be chosen due to its potential for greater patient satisfaction compared with intramuscular dosing, and for improved efficacy through enhanced drug delivery to target tissues. Vaginal administration also avoids first-pass metabolism problems, e.g., provides a first uterine pass effect, and direct delivery to the uterus allows for lower systemic drug concentrations.

[0058] In one embodiment, NES may be administered in a unit dose including, but not limited to, from every four weeks, every three weeks, every two weeks, once a week, once a day, twice a day, three times a day, and up to four times a day, depending on factors including the type of formulation, the concentration of NES, and the administration route.

[0059] Preferably, it may be administered beginning about the 18thto 22ndweek of gestation until about the 37thweek of gestation, or for approximately 14 to 19 weeks, depending on the gestational age at the beginning of treatment and the date of delivery. In another embodiment, NES may be administered beginning about the 16thweek of gestation until about the 37thweek of gestation, or for approximately 21 weeks. In still other embodiments, NES may be administered beginning about the time of a positive pregnancy test until about the 37thweek of gestation, or beginning about the 2ndto 4thweek of gestation, for approximately 33 to 35 weeks.

[0060] NES may be administered in an amount including, but not limited to, about 50 pg to 20 mg, 100 pg to 18 mg, 500 pg to 15 mg, 800 pg to 12 mg, 1 mg to 10 mg, 1.25 mg to 5 mg, and 2 mg to 4 mg per day, depending on factors including the type of formulation, and the route of administration. NES may be administered in a vaginal ring in an amount including, but not limited to, about 50 pg to 2 mg, 100 pg to 1.8 mg, 500 pg to 1.5 mg, and 800 pg to 1.2 mg per day. NES may be administered in a form of gels (including vaginal gels and transdermal gels) or suppositories in an amount up to 10 times higher than NES administered in a vaginal ring.

[0061] For some embodiments, the amount and concentration of NES needs to be sufficient to remain in the subject to provide prevention or treatment, such as for about 1 hour or more, preferably greater than about 2 hours, even more preferably greater than 6 hours, still more preferably greater than about 12 hours, yet more preferably greater than about 24 hours, and most preferably greater than about 36 hours.

[0062] NES can be administered in combination with other treatment options for preterm birth or for promoting fetal lung development. For example, NES may be administeredVASTEB-066 in combination with other active agents for treating or preventing PTB, or may be administered in addition to cervical cerclage, a surgical procedure to close the cervix in women with an incompetent cervix, or in addition to bed rest.

[0063] The other active agents for treating or preventing PTB may include, but not limited to, tocolytics including oxytocin receptor antagonists (OTR antagonists) such as atosiban, nolasiban, retosiban, nifedipine, magnesium sulfate, and betamethasone; antibiotics such as coamoxiclay, erythromycin, beta-lactams, macrolides, clindamycin, metronidazole, and azithromycin; and anti-inflammatory medications including nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin, celecoxib, aspirin, rofecoxib, nimesulide and sulindac, prostaglandin inhibitors such as TGH113, AS604872, and ebopiprant, cytokinesuppressive anti-inflammatory drugs (CSAIDs) such as NF-KB inhibitor sulfasalazine, IKKB inhibitors parthenolide and TPCA-1, MAPK inhibitors U0126, SB202190, SP600125 and SB239063, chemokine inhibitors somatotaxin, and FX125L, IL-6 antibody tocilizumab, IL-ip receptor antagonist rytvela, immune response modulators, for example, resveratrol, N-N- dimethylacetamide, N-acetylcysteine, melatonin, folic acid, 15d-PGJ2, naloxone, rosiglitazone, naltrexone, endothelin-1 inhibitors, Sphk inhibitors, BQ- 123, omega-3 fatty acid, lipoxin A4, and simvastatin, and other anti-inflammatory agents such as alpha lipoic acid, amitriptyline and interferons.

[0064] According to one aspect of the present disclosure, provided is a composition for preventing and treating PTB, promoting fetal lung development, and reversing inflammation and reducing stress in the uterine environment disclosed above, where the composition comprises a pharmaceutically effective amount of NES and a pharmaceutically acceptable excipient.

[0065] Such a composition may be in forms of intravaginal rings, suppositories, gels, creams, foams, lotions, or the like, preferably in the form of intravaginal rings, gels or suppositories preferably administered vaginally through means of a delivery device inserted into the vagina, and more preferably, in the form of intravaginal rings.

[0066] The composition may comprise NES in a weight amount from about 0.01% to about 50%, preferably from about 1% to about 40%, more preferably from about 2.5% to about 30%, even more preferably from about 5% to about 20%, and still more preferably from about 6% to about 15%, based on a total weight of the composition.VASTEB-066

[0067] In particular, the composition, preferably in a form of an intravaginal ring, a gel, or a suppository may further comprise a pharmaceutically acceptable excipient including, but not limited to, a carrier, an emollient, a moisturizer, and a pH adjuster.

[0068] A carrier, or alternatively a vehicle, in the composition may include, but is not limited to, water, alcohols, mineral oil, silicone, liquid sugars, waxes, petroleum jelly, and mixtures thereof. The carrier may be present from about 90% to 99% wt / wt, preferably from about 94% to 98% wt / wt, or from about 95% to 97% wt / wt, and more preferably from about 96% to 96.5% of the composition depending on the type of carriers used. Preferably, the carrier in the present transdermal composition may include water and a C2 to C4 alcohol. Water may be present from about 20% to 40% w / w, preferably from about 25% to 32% wt / wt, or from about 27% to 29%, and more preferably from about 28.0% to 28.5% w / w of the composition. The C2 to C4 alcohol may also function here as a penetration enhancer, and preferably is ethanol, isopropanol, n propanol, or mixtures thereof, and more preferably is ethanol. The C2 to C4 alcohol may be present from about 50% to 85% wt / wt, or 52% to 83% wt / wt, preferably from about 62% to 73% wt / wt, from about 65% to 71% wt / wt, from about 66.5% to 69.5% wt / wt, or from about 67.5% to 68.5 wt / wt, and more preferably around about 67.9% wt / wt of the composition. The C2 to C4 alcohol is preferably ethanol. Ethanol may be present from about 50% to 85% wt / wt, preferably from about 65% to 71% wt / wt, and more preferably at about 67.93% wt / wt of the composition.

[0069] An emollient, and / or a moisturizer, in the composition may be used to enhance penetration (thus functioning as a penetration enhancer), soften and smooth the skin, or to hold and retain moisture. It includes, but not limited to, isopropyl myristate, cetyl alcohol, cetearyl alcohol, cocoa butter, isopropyl palmitate, lanolin, liquid paraffin, polyethylene glycols, shea butter, silicone oils, stearic acid, stearyl alcohol, castor oil, and mixtures thereof, as well as other oils. The emollient / moisturizer may be present from about 0.2% to 4.0% wt / wt, preferably from about 0.5% to 3.0% wt / wt of the composition depending on the type of emollient / moisturizer used. Preferably, the emollient / moisturizer in the present transdermal composition may include isopropyl myristate, which may be present from about 0.2% to 3.0% wt / wt, preferably from about 0.5% to 2.0% wt / wt, and more preferably around 1.0% wt / wt of the composition.

[0070] A pH adjuster, or alternatively a pH regulator, in the composition may be used to maintain the desired pH for the proper chemical and physical properties. It may include, butVASTEB-066 not limited to, an acid and a solution thereof such as a hydroxy acid such as lactic acid, and tartric acid, a base and a solution thereof such as sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonium hydroxide, and a ternary amine and a solution thereof such as triethanolamine, tromethamine, and tetrahydroxypropylethylendiamine, and a buffer and a solution thereof such as carbonate buffers, citrate buffers, phosphate buffers, and acetate buffers, tris buffers, or a combination thereof.

[0071] The composition is preferably formulated so as to have a pH value of about 3 to 9, preferably a pH value of about 4 to 8, or about 5 to 7, and more preferably a pH value of about 5.7. Sodium hydroxide is preferably used in the present transdermal composition as a pH adjuster and may be present from about 0.01% to 0.4% wt / wt, preferably from about 0.015% to 0.03% wt / wt, and more preferably around 0.028% wt / wt of the composition.

[0072] The composition may further comprise an additional penetration enhancer. The additional penetration enhancer may include, but not limited to, a glycol, a monoalkyl ether of diethylene glycol, a fatty alcohol, acetone, acyl lactylates, acyl peptides, acylsarcosinates, alkanolamine salts of fatty acids, alkyl benzene sulphonates, alkyl ether sulphates, alkyl sulphates, anionic surfatactive agents, benzyl benzoate, benzyl salicylate, butan-l,4diol, butyl benzoate, butyl laurate, butyl myristate, butyl stearate, cationic surface-active agents, citric acid, cocoamidopropylbetaine, decyl methyl sulfoxide, decyl oleate, dibutyl azelate, dibutyl phthalate, dibenzyl sebacate, dibutyl sebacate, dibutyl suberate, dibutyl succinate, dicapryl adipate, didecyl phthalate, diethylene glycol, diethyl sebacate, diethyl-m-toluamide, di(2- hydroxypropyl) ether, diisopropyl adipate, diisopropyl sebacate, N,N-dimethyl acetamide, dimethyl azelate, N,N-dimethyl formamide, l,5-dimethyl-2-pyrrolidone, dimethyl sebacate, dimethyl sulphoxide, dioctyl adipate, dioctyl azelate, dioctyl sebacate, 1,4 dioxane, 1- dodecylazacyloheptan-2one, dodecyl dimethyl amine oxides, ethyl caprate, ethyl caproate, ethyl caprylate, 2-dehyl-hexyl pelargonate, ethyl-2-hydroxypropanoate, ethyl laurate, ethyl myristate, l-ethyl-2-pyrrolidone, ethyl salicylate, hexyl laurte, 2-hydroxyoctanoic acid, 2- hydroxypropanoic acid, 2-hydroxypropionic acid, isethionates, isopropyl isostearate, isopropyl palritate, guar hydroxypropyltrimonium chloride, hexan-2,5-diol, kheliin, lamepons, lauryl alcohol, maypons, metal salts of fatty acids, methyl nicotinate, 2-methyl propan-2-ol, 1-methyl- 2-pyrrolidone, 5-methyl-2-pyrrolidone, methyl taurides, miranol, nonionic surface-active agents, octyl alcohol, octylphenoxy polyethoxyethanol, oleic ethanolamide, pleyl alcohol, pentan-2, 4-diol, phenoxyethanol, phosphatidyl choline, phosphine oxides, polyalkoxylatedVASTEB-066 ether glycollates, poly(diallylpiperidinium chloride), poly(dipropyldiallylammonium chloride), polyglycerol esters, polyoxyethylene lauryl ether, polyoxy:polyoxy ethylene stearate, polyoxypropylene 15 stearyl ether, poly (vinyl pyridinium chloride), propan- l-ol, propan-2-ol, propylene glycol dipelargonate, pyroglutamic acids, 2-pyrrolidone, pyruvic acids, Quatemium 5, Quatemium 18, Quatemium 19, Quaternium 23, Quaternium 31, Quatemium 40, Quatemium 57, quartenary amine salts, quaternised poly (dimethylaminoethylmethacrylate), quatemised poly (vinyl alcohol), sapamin hydrohloride, sodium cocaminopropionate, sodium dioctyl sulphonsuccinate, sodium laurate, sodium lauryl ether sulphate, sodium lauryl sulphate, sugar esters, sulphosuccinate, tetrahydrofuran, tetrahydrofurfural alcohol, transcutol, triethanolamine dodecyl benzene sulphonate, triethanolamine oleate, urea and a derivative thereof, and mixtures thereof. In addition to a C2-C4 alcohol, preferably ethanol, an emollient and / or a moisturizer as a penetration enhancer mentioned above, the additional penetration enhancers may be optionally present in the composition from about 0.2% to 3.0% wt / wt, preferably from about 0.5% to 2.0% wt / wt of the composition depending on the type of penetration enhancer used.

[0073] The composition may further include one or more additives including, but not limited to, humectants, preservatives, antioxidants, emulsifiers, occlusive agents, solubilizing agents, and surfactants. The type and amount of the additive can be determined by one of skill in the art using conventional methods.

[0074] Suitable humectants may include, without limitation, glycerin, propylene, glycol, sorbitol, and triacetin. Suitable preservatives may include, without limitation, one or more alkanols, disodium EDTA (ethylenediamine tetraacetate), EDTA salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycols, sorbates, urea derivatives such as diazolindinyl urea, or any combinations thereof. Suitable antioxidants may include, without limitation, tocopherol and derivatives, ascorbic acid and derivatives, butylated hydroxy anisole, butylated hydroxy toluene, fumaric acid, malic acid, propyl gallate, metabisulfates and derivatives, and mixtures thereof. Suitable emulsifiers that can be used in the context of the present composition include, without limitation, one or more sorbitans, alkoxylated fatty alcohols, alkylpolyglycosides, soaps, alkyl sulfates, monoalkyl and dialkyl phosphates, alkyl sulphonates, acyl isothionates, or any combinations thereof. Suitable occlusive agents that can be used in the context of the present composition include, without limitation, petrolatum, mineral oil, beeswax, silicone oil, lanolin and oil-soluble lanolinVASTEB-066 derivatives, saturated and unsaturated fatty alcohols such as behenyl alcohol, hydrocarbons such as squalane, and various animal and vegetable oils such as almond oil, peanut oil, wheat germ oil, linseed oil, jojoba oil, oil of apricot pits, walnuts, palm nuts, pistachio nuts, sesame seeds, rapeseed, cade oil, com oil, peach pit oil, poppyseed oil, pine oil, castor oil, soybean oil, avocado oil, safflower oil, coconut oil, hazelnut oil, olive oil, grape seed oil and sunflower seed oil. Representative examples of solubilizing agents may include, without limitation, complexforming solubilizers such as citric acid, ethylenediamine-tetraacetate, sodium meta-phosphate, succinic acid, urea, cyclodextrin, polyvinylpyrrolidone, diethylammonium-ortho-benzoate, and micelle-forming solubilizers such as tweens and spans e.g., TWEEN 80. Other solubilizers that are usable for the compositions of the present disclosure are, for example, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene n-alkyl ethers, n-alkyl amine n-oxides, poloxamers, organic solvents, phospholipids and cyclodextrines. The composition may further include anionic, non-ionic or cationic surfactants.

[0075] The composition may further comprise other active agents for treating or preventing PTB described above.

[0076] The composition may further comprise other pharmaceutically compatible agents in a suitable amount. Examples of pharmacologically active agents include, but are not limited to, any suitable antimicrobial agents including antibacterial, antifungal, antiprotozal and antiviral agents known in the art.

[0077] When employed, the other active agents described above may be present in an amount of about 0.5 to about 40 w / w % and preferably about 2.5 to about 15 w / w % of the composition.

[0078] The composition may be in a form of an intravaginal ring, and NES can diffuse out of the vaginal system with release rates that vary over time. In some embodiments, the intravaginal ring may provide a gradual release of NES through a polymeric structure over extended periods of time.

[0079] The intravaginal ring used here is commonly known in the art. In some embodiments, the intravaginal ring may comprise of at least a silicone elastomer. The elastomer may be a methyl siloxane-based polymer. In some embodiments, the intravaginal ring may further comprise inactive ingredients, including titanium dioxide, dibutyltin dilaurate, and silicone medical adhesive.VASTEB-066

[0080] The composition may be in a form of a gel, and may further comprise a gel forming agent.

[0081] A gel forming agent, or alternatively a thickening agent, may be present in an amount sufficient to alter the viscosity of the composition. A gel forming agent can be selected from the group including: carbomer; carboxyethylene or polyacrylic acid such as Carbopol 980 USP / NF / EP, or 940 NF, 981 or 941 NF, 1382 or 1342 NF, 5984 or 934 NF, ETD 2020, 2050, 934P NF, 97 IP NF, 974P NF, and Noveon AA-1 USP; cellulose derivatives such as ethylcellulose, hydroxypropylmethylcellulose (HPMC), ethylhydroxyethylcellulose (EHEC), carboxymethylcellulose (CMC), hydroxypropylcellulose (HPC) (Klucel different grades), hydroxyethylcellulose (HEC) (Natrosol grades), HPMCP 55, and Methocel grades; natural gums such as arabic, xanthan, guar gums, and alginates; polyvinylpyrrolidone derivatives such as Kollidon grades; polyoxyethylene polyoxypropylene copolymers such as Lutrol F grades 68, and 127; and mixtures thereof. Other gel forming agents may include chitosan, polyvinyl alcohols, pectins, and veegum grades. A tertiary amine, such as triethanolamine or trolamine, may also be included to thicken and neutralize the system.

[0082] A polymer or copolymer of acrylic acid, such as a carbomer acts as a gel forming agent and facilitates the release of lipophilic active agent and penetration enhancer. Preferably, the gel forming agent is Lutrol F grades and Carbopol grades.

[0083] The amount and the type of the gel forming agent in the composition may be selected to provide the desired product consistency and / or viscosity to facilitate application to the skin. The gel forming agent may be present from about 0.2% to 3.0% wt / wt of the composition depending on the type of gel forming agent used. For example, the gel forming agent may be preferably present in an amount between about 0.5% and 2% wt / wt and more preferably at about 1.0% wt / wt for polyacrylic acids.

[0084] The composition may be in a form of a suppository. For suppositories, any traditional binder and / or carrier may be used, for example, one or more polyalkalene glycols or triglycerides.EXAMPLESMATERIALS:Nestorone® (NES), Progesterone and Lipopolysaccharide (LPS)VASTEB-066

[0085] NES was sourced from Crystal Pharma. For the in vivo experiments, the dose of NES used was 0.02 mg / dam in 100 pL of sesame oil / ethanol.

[0086] Progesterone was sourced from Sigma. For the in vivo experiments, the dose of progesterone used was 2 mg / dam in 100 pl of sesame oil / ethanol.

[0087] LPS extracted from E. coli was sourced from Sigma- Aldrich (LPS 0111: B4) For the in vivo experiments, the dose of EPS used was 1 pg / gram body weight / dam in lOOpE of saline.Cell Cultures

[0088] Peripheral Blood Mononuclear Cells (PBMCs) were purified from whole blood enriched for leukocytes via leukapheresis obtained from healthy volunteers at the New York Blood Bank. The blood was diluted 1:1 in PBS (Gibco-BRL) and then layered on top of Ficoll Paque PLUS (GE Healthcare Biosciences AB) in specially designed Sepmate tubes (StemCell Technologies) for density dependent separation. Cells were then centrifuged at 1200 xg for 10 minutes at room temperature, z.e., a temperature ranging from 68 to 77°F, to obtain the purified mononuclear fraction, which was carefully collected and transferred to a new tube. The cells were washed up to 6 times to remove platelet contamination by resuspending in PBS and centrifuging at 300g for 10 minutes, and the pellet was resuspended in RPMI-1640 media for subsequent analyses. For treatment assays in vitro, the PBMCs from healthy volunteers were quantified and transferred to a 24-well or 96-well culture plate for immunological assays.

[0089] Michigan Cancer Foundation-7 (MCF-7), Bronchial Epithelium transformed with Adl2-SV40 2B (BEAS-2B), and Uterine Smooth Muscle Cells (USMCs) were obtained from American Type Culture Collection (ATCC), where the cell lines were routinely cultured in RPMI-1640 medium (Gibco) with high glucose containing 10% fetal bovine serum (FBS) (Gibco), 1% penicillin / streptomycin, 1% Glutamax(Gibco) and 2g / L sodium bicarbonate.INDUCTION OF INFLAMMATION IN PREGNANT MICE:

[0090] Pregnant mice were treated with LPS obtained above in a dose of 1 mg / kg, which did not cause any maternal loss while still inducing preterm birth, and may lead to fetal death due to a lack of properly formed organs from insufficient fetal development. According to Hudalla el al., LPS-induced maternal inflammation promotes fetal leukocyte recruitment and prenatal organ infiltration in mice (Hudalla H, Karenberg K, Kuon RJ, Pbschl J, Tschada R, Frommhold D. LPS-induced maternal inflammation promotes fetal leukocyte recruitmentVASTEB-066 and prenatal organ infiltration in mice. Pediatr Res. 2018; 84:757-764). Pregnant mice were then monitored with a digital video camera to record potential preterm birth, the number of pups, and their viability. Stillbirth was defined as death upon delivery with no pulse or respiration. Control animals were injected with equivalent volumes of saline or sesame oil / ethanol.IN VITRO EFFECTS OF NES ON LPS TREATED HUMAN PBMCs, MCF-7, USMCs, AND BEAS-2B CELLS:

[0091] To investigate the effects on NES on human PBMCs, MCF-7, USMCs, and BEAS-2B cells, mainly in regulating immune pathways involved in inflammation, the potential of NES in preventing PTB was explored in comparison to that of hydrocortisone (HC) and / or progesterone (Prog).Human PBMCs:Caspase- 1 Inflammasome Assay

[0092] Caspase- l is a key enzyme in the inflammatory response and programmed cell death (apoptosis) pathways. It plays a crucial role in the maturation and secretion of pro- inflammatory cytokines, such as interleukin- ip (IL- 1 P) and interleukin- 18 (IL- 18). The activity of caspase- 1 is tightly regulated, and dysregulation of its activity has been implicated in various inflammatory conditions. A caspase- 1 activity assay kit from Promega was used to explore the potential therapeutic effect of NES. 50,000-60,000 human PBMCs per well in a 96-well plate were treated with LPS, a known inflammasome inducer, to assess inflammasome activation and subsequent suppression by using NES. The assay was performed according to the manufacturer’s instructions. Human PBMCs with or without being primed with LPS were treated with NES at 5 pm and 10 pm, respectively. The cell culture supernatant was used to analyze the inhibition of inflammasome induction by NES.

[0093] As shown in FIG. 1, the results demonstrated significant caspase- 1 activity in PBMCs primed with LPS. NES treatments suppressed the inflammasome induction, suggesting its anti-inflammatory role. The inflammasome was robustly active during inflammation caused by LPS bacterial endotoxin and suppressed by NES in human PBMCs. The study supports that NES suppresses inflammasome activation, a marker of inflammation severity, showing its potential as a therapeutic agent for suppressing inflammation in-vivo.VASTEB-066Luminex Assay

[0094] During labor, the inflammatory response can significantly impact the activity of PBMCs in the body. PBMCs consist of lymphocytes, monocytes, natural killer (NK) cells, and dendritic cells (DCs). These cells were stimulated with LPS (100 ng / ml) for 30 minutes before NES or HC treatments. NES / HC was added at 5, 10, 20, or 50 pM concentrations at 30 minutes after LPS stimulation. The cells were then incubated for 24 hours at 37 °C. After incubation, the culture supernatants were collected and assessed for cytokine levels at the baseline and post-treatment as measured by the Invitrogen cytokine human magnetic 25-plex panel kit (Invitrogen, Frederick, MD) on the Luminex platform. The expression of pro-inflammatory cytokines such as IL-6, TNF-a, IL-ip, IL-12, IL-17a, and GM-CSF, as well as regulatory cytokines IL- 10 and IL-2Ra, were monitored using the filter plate method. The Luminex 200 (Luminex, Austin, TX) and Star Station software (Applied Cytometry Systems, Sacramento, CA) were used to analyze samples by using the Kruskall-Wallis test. The data were shown in FIGs. 2A and 2B.

[0095] As shown in FIG. 2B, the results showed that NES inhibited the secretion of pro-inflammatory cytokines such as IL-17a and GM-CSF in LPS -stimulated PBMCs, with no loss in cell viability detected at concentrations of 10, 20, and 50 pM (data not shown). The effective concentration of 20 pM was then used in the assay, and the data was shown in FIG. 2A. FIG. 2A demonstrated that LPS (100 ng / ml), which was used to trigger inflammation in the cells, increased the production of TNF-a, IL-6, IL-ip, and IL-12 (p < 0.01), confirming that an inflammatory response was induced. NES inhibited this effect while increasing the expression of the anti-inflammatory cytokines, such as IL- 10. NES was at least as effective in inducing an anti-inflammatory immune response as HC, used as a positive control.RNA Isolation and qPCR Assay

[0096] To investigate the effects of NES on human PBMCs, particularly in regulating immune pathways involved in inflammation, the relative changes in cytokine expression levels were explored using quantitative PCR (qPCR). PBMCs cells were stimulated with LPS (100 ng / ml) for 30 minutes before adding NES or Prog treatment. The concentration of NES / Prog was 20 pM for the treatment in this assay.

[0097] Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The cDNA was prepared from a 1-2 pg RNA sample pretreated with DNAse I (Invitrogen). For cDNA synthesis, High-Capacity cDNA Reverse Transcription KitVASTEB-066(applied biosystems) was used according to the manufacturer’s protocol. The primers were designed using the Primer Quest tool, and were selected from the Harvard Primer bank and manufactured by Integrated DNA Technologies (IDT) (htps: / / pga.nigh.harvard.edu / prmierbarrk / ). The primers were synthesized based on sequences provided therein.

[0098] The real-time detection of mRNA expression was performed through the relative (rRT-PCR) quantitation protocol using Power SYBR™ Green PCR Master Mix expression assay on QuantStudio3 real-time PCR System (Applied Biosystems). For the relative quantitation of NF-KB, IL-6, IL- 10, TNF-a, IL-2Ra, and TLR4 genes, triplicates of the total reaction mixture of 25 pl were composed of 2 pl of cDNA, 12.5 pl of Power SYBR™ Green PCR Master Mix, 0.25 pl (90pM) of both sense and antisense primers and 10 pl of nuclease free water. After the initial denaturation at 95 °C for 15 minutes, target genes were amplified through 40 cycles of universal cycling conditions (95°C / 15s, 55°C / lmin, 72°C / lmin) followed by melt curve analysis with 0.15 second increments between 60°C and 95°C (95°C / 15s, 60°C / lmin, 95°C / ls). All PCR reactions were normalized to P-actin mRNA for human samples and GAPDH for mouse samples.

[0099] As shown in FIG. 3, LPS (100 ng / ml) increased the production of TNF-a, IL-6, NF-KB, and TLR4 (p < 0.01), confirming that an inflammatory response was induced. NES inhibited this effect while increasing the expression of anti-inflammatory cytokines, such as IL- 10. NES was more effective in inducing an anti-inflammatory immune response than Prog, used as a positive control.

[0100] The activation of TLR4 by LPS triggers the MyD88-dependent signaling pathway, leading to the translocation of phosphorylated NF-KB transcription factor from the cytosol to the nucleus. This, in turn, results in the expression of pro-inflammatory cytokines like TLR4, TNF-a and anti-inflammatory mediators such as IL- 10. These mediators play a crucial role in maintaining the lung epithelial barrier and host defense, with IL- 10 assisting in the resolution of inflammation.MCF-7:RNA Isolation and qPCR Assay

[0100] To investigate the effects of NES on MCF-7 cells, particularly in regulating immune pathways involved in inflammation, the relative changes in cytokine expression levels were explored using quantitative PCR (qPCR) in the same way as the procedure for PBMCs.VASTEB-066MCF-7 cells were stimulated with LPS (100 ng / ml) for 30 minutes before adding NES or Prog treatment. No loss of cell viability was detected in these cells post-NES treatment. Cytokine differential mRNA expression was assessed after 24hrs of NES treatments using the qPCR. The concentration of NES / Prog was 20 pM for the treatment in this assay.

[0101] As shown in FIG. 4, NES attenuated the LPS-induced increase in TNF-a, IL-6, TLR4, and NF-KB (p<0.001 while increasing IL- 10 mRNA expression. NES was more effective in inducing an anti-inflammatory immune response than Prog, used as a positive control.USMCs:RNA Isolation and qPCR Assay

[0102] To investigate the effects of NES on USMCs, particularly in regulating immune pathways involved in uterine inflammation, the relative changes in cytokine expression levels were explored using quantitative PCR (qPCR) in the same way as the procedure for PBMCs. USMCs cells were stimulated with LPS (100 ng / ml) for 30 minutes before adding NES or Prog treatment. No loss of cell viability was detected in these cells post-NES treatment. Cytokine differential mRNA expression was assessed after 24hrs of NES treatments using the qPCR. The concentration of NES / Prog was 20 pM for the treatment in this assay.

[0103] As shown in FIG. 5, NES attenuated the LPS-induced increase in TNF-a, IL-6, NF-KB, and TLR4(p<0.001) while increasing the expression of anti-inflammatory cytokines, such as IL- 10.. NES was more effective in inducing an anti-inflammatory immune response than Prog, used as a positive control.BEAS-2B:RNA Isolation and qPCR Assay

[0104] To investigate the effects of NES on BEAS-2B cells, particularly in regulating immune pathways involved in lung inflammation, the relative changes in cytokine expression levels were explored using quantitative PCR (qPCR) in the same way as the procedure for PBMCs. BEAS-2B cells were stimulated with LPS (100 ng / ml) for 30 minutes before adding NES or Prog treatment. No loss of cell viability was detected in these cells post-NES treatment. Cytokine differential mRNA expression was assessed after 24hrs of NES treatments using qPCR. The concentration of NES / Prog was 20 pM for the treatment in this assay.

[0105] As shown in FIG. 6, BEAS-2B cells exposed to LPS exhibited a significant increase in IL-6, TLR4, TNF-a, and NF-KB levels compared to control cells. Treatment withVASTEB-066NES resulted in a decrease in IL-6, TLR4, TNF-a, and NF-KB levels and an increase in immunoregulatory IL- 10 level in the LPS-stimulated cells.

[0106] These observations suggest that NES may have therapeutic potential in reducing inflammation in lung epithelial cells and promoting fetal lung development.IN VIVO ASSESSMENT IN SYSTEMIC INFLAMMATION MURINE MODEL:

[0107] Timed-pregnant C57BL / 6 mice were obtained from Charles River Laboratories and housed under specific pathogen-free conditions at the Comparative Biosciences Center (CBC) at Rockefeller University. They were provided with ad libitum food and water. Pregnant C57BL / 6 mice (n=36) were randomly divided into 6 groups. One-third of the animals (n=12) were given a subcutaneous (SC) injection of NES (20 ug / dam, n=12), one-third received an SC injection of progesterone 2 mg / dam, n=12), and one-third received an SC injection of ethanol / sesame oil (Vehicle, n=12). NES / progesterone / vehicle was first administered to pregnant mice on gestation day (GD) 15. The average gestation period for a mouse is 18-21 days. Twenty-four hours later, on GD 16, mice received the second NES / progesterone / vehicle injection before the LPS or saline injection. On GD 16, half of the mice treated with NES / progesterone / vehicle received an intraperitoneal injection of either 1 pg / g b.w. of LPS in 100 pl of sterile saline (LPS group, n=6 per subgroup) or 100 pl of sterile saline (Control group, n=6 per subgroup). The animals were closely monitored until delivery, and the delivery time was recorded for each animal.

[0108] Tissue samples were collected from mice that underwent preterm labor after being injected with Ipg / g b.w. of LPS per mouse on GD 16, which resulted in preterm birth in 100% of the animals within 24 hours in the vehicle / LPS group. Mice that carried a pregnancy to full term (GD 21) were sacrificed on the day of labor, defined as the delivery of at least 1 pup (n=6 per group for progesterone-treated and NES-treated animals). Mice that did not deliver at term were sacrificed 24 hours post-term (GD 22).

[0109] Parturition events were monitored from GD 17 through GD 21 by observing mice in the morning, at noon, and in the evening. Preterm birth for a mouse was defined as birth occurring earlier than GD 19.

[0110] Neither NES nor progesterone had any effect on maternal mortality or morbidity. The effects of NES on delivery time, maternal weight gain, and litter size wereVASTEB-066 assessed and are shown in the following Table. Maternal spleen, lungs, uterus and brain, and fetal tissues were collected for cytokine analysis.

[0111] Table. NES Prevents LPS Induced Preterm Labor

[0112] As shown from the above Table, NES by itself did not affect gestation or parturition of the mice. All 6 mice treated with NES remained pregnant and had normal term parturition at GD20. Additionally, NES produced no harmful effects on fetal outcomes or health. Both the NES and progesterone groups had a comparable number of pups and fetal weights to the control group treated with the vehicle alone and showed no signs of abnormalities or distress.

[0113] The table also shows that LPS was fully effective at inducing PTB in pregnant mice. All 6 mice treated solely with an IP injection of 25 pg LPS / mouse on GDI 6 delivered prematurely between 12-24 hours post-treatment. All pups delivered this way were non-viable and many were malformed. However, there was no obvious effect on maternal health asides from the induction of PTB, and the mothers appeared healthy and active after parturition.

[0114] When LPS treated mice were also given NES, NES delayed parturition and improved fetal outcomes compared to the LPS only group. Despite the 100% incidence of PTB in the LPS only group, no mice delivered before term in the progesterone+LPS or NES+LPS treated groups. 3 / 6 NES+LPS-treated pregnant mice and 1 / 6 progesterone+LPS treated pregnant mice delivered <24 hours post-term, but those that did not deliver by this time were sacrificed on post-term GD 22.

[0115] In terms of fetal outcomes, NES appeared more effective than progesterone in improving fetal outcomes as 3 NES+LPS-treated animals delivered a total of 14 pups in contrast to that only 1 progesterone+LPS-treated mouse delivered 5 pups (P < 0.01). These pups that were delivered were all robust and healthy and did not have any apparent morbidities.VASTEB-066This stands in stark contrast to the LPS only-treated group that had no viable births. All the sacrificed mice who had not given birth by GD22 showed signs of having at least partially resorbed some fetuses, indicating that while NES and progesterone are fully capable of preventing LPS induced labor and PTB, they do not appear sufficient to prevent all the LPS induced damage to developing fetuses. In the control groups, NES or progesterone treatment had no apparent effect on maternal health, and there were no obvious differences in the mothers of any control groups observed prior to sacrifice or found during necropsy.

[0116] Pre-treating pregnant mice with NES (0.2mg / dam / day) 24 hours before LPS injection completely blocked preterm birth (100%). NES can completely suppress LPS-induced preterm parturition and rescue pups from spontaneous abortion without toxic effects on the mother, while it delays the parturition to post-term. NES performed significantly (p<0.005) better than progesterone in reducing the signs of resorption, delaying parturition, and improving delivery outcome. This supports its use as a stalling mechanism against preterm labor triggered by LPS induced inflammation.

[0117] Comprehensive cytokine profile analysis on harvested mice organs was also performed. The uteruses were examined for initial fetal loss or absorption. The spleens, uteruses, brains, and lungs were harvested for cytokine profile analysis (both pro-inflammatory and anti-inflammatory). The investigation revealed considerable differential gene expression related to cytokines.

[0118] As shown in FIGs. 7 to 10, NES inhibited the LPS-induced expression of proinflammatory cytokines in mouse uterus, spleen, brain and lung, specifically IL-6, IL- la, I- CAM, NAMPT, TNFa, NF-KB, IL-8, TLR4, and IL-ip, and increased anti-inflammatory cytokine IL- 10, to a higher degree compared to progesterone. In particular, NES inhibited the expression of LPS induced cytokines in mouse lung tissue, specifically TNF-a and TLR4, and increased IL- 10 levels to a higher degree, compared to progesterone. Thus, NES can both effectively reduce the expression of proinflammatory mediators, and increase antiinflammatory gene expression in an inflammation-induced preterm labor mouse model.

[0119] LPS administration led to an upsurge in maternal proinflammatory cytokines in the uterus, spleen, brain, and lung, explicitly IL-6, IL- la, IL-8, TLR4, IL-1 p, LCAM, NAMPT, NF-KB and TNF-a, which contribute to the onset of preterm labor during infection. With NES and progesterone, the immune response pivoted from inflammatory to antiinflammatory in the maternal spleen with a 4.27 -fold increase in IL- 10 and a 4 to 6-foldVASTEB-066 decrease (p<0.05) in IL-6, IL-la, IL-8, TLR4, IL-ip, LCAM and TNF-a cytokine expression levels, suggesting the inherent anti-inflammatory potential of NES. The pro-inflammatory cytokines were at basal levels and comparable in all control groups (NES, progesterone, and vehicle alone). Pretreatment with NES significantly inhibited the expression of all proinflammatory cytokines quantified. IL- 10 mRNA levels significantly increased in the uterus with NES treatment in the LPS- stimulated group. NES performed significantly (p<0.05) better than progesterone in inducing an anti-inflammatory cytokine profile.

[0120] The in vitro and in vivo LPS-induced PTB mouse model studies demonstrate the use of NES can effectively reduce the expression of proinflammatory mediators while increasing the expression of anti-inflammatory cytokines without toxicity. Further, the studies show that NES is capable of more robust suppression of proinflammatory markers compared to progesterone as well as more effectively increasing anti-inflammatory IL- 10 levels, suggesting that NES can be used as a potent anti-inflammatory treatment for preventing PTB, and improving fetal health and viability.

Claims

VASTEB-066CLAIMS1. A method for preventing and treating an onset of preterm labor and preterm birth by administering a pharmaceutically effective amount of Nestorone® to a pregnant individual in need thereof.

2. The method of claim 1, wherein the Nestorone® is administered vaginally.

3. The method of claim 1, wherein the Nestorone® is administered vaginally through an intravaginal ring, a gel or a suppository.

4. The method of claim 1, wherein the Nestorone® is administered beginning about the 18thto 22ndweek of gestation until about the 37thweek of gestation.

5. The method of claim 1, wherein the Nestorone® is administered in an amount of about 50 pg to about 20 mg per day.

6. The method of claim 1, wherein the Nestorone® is administered in combination with a second active agent for preventing and treating preterm labor and preterm birth.

7. The method of claim 6, wherein the second active agent comprises at least one of a tocolytic agent, or an anti-inflammatory agent selected from alpha lipoic acid, amitriptyline or interferons.

8. A method of promoting fetal lung development by administering a pharmaceutically effective amount of Nestorone® to a pregnant individual in need thereof.

9. The method of claim 8, wherein the Nestorone® is administered vaginally.

10. The method of claim 8, wherein the Nestorone® is administered vaginally through an intravaginal ring, a gel or a suppository.VASTEB-06611. The method of claim 8, wherein the Nestorone® is administered beginning about the 18thto 22ndweek of gestation until about the 37thweek of gestation.

12. The method of claim 8, wherein the Nestorone® is administered in an amount of about 50 pg to about 20 mg per day.

13. The method of claim 8, wherein the Nestorone® is administered in combination with a second active agent for preventing and treating preterm labor and preterm birth.

14. A composition for preventing and treating an onset of preterm labor and preterm birth, comprising a pharmaceutically effective amount of Nestorone® and a pharmaceutically acceptable excipient.

15. The composition of claim 14, further comprising a second active agent for preventing and treating preterm labor and preterm birth.

16. The composition of claim 15, wherein the second active agent comprises at least one of a tocolytic agent, or an anti-inflammatory agent selected from alpha lipoic acid, amitriptyline or interferons.

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