Compositions and methods for providing estrogen

Abstract

The invention provides a novel methods and pharmaceutical compositions of testosterone and aromatase in providing estrogen or treating postmenopausal symptoms and other estrogen deficiency-related disease or condition.

Description

Patent Application Atty. Docket No. YPK-009PCTCOMPOSITIONS AND METHODS FOR PROVIDING ESTROGENPriority Claims and Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No.63 / 741,877, filed January 4, 2025, the entire content of which is incorporated herein by reference for all purposes.Technical Field of the Invention

[0002] The invention generally relates to novel therapeutic methods and pharmaceutical compositions. More particularly, the invention relates to novel methods and pharmaceutical compositions of testosterone and aromatase in providing estrogen or treating postmenopausal symptoms and other estrogen deficiency-related diseases or conditions.Background of the Invention

[0003] A sharp decline in estrogen levels during menopause can cause a number of symptoms and health issues in postmenopausal women, significantly affecting their health and quality of life. These postmenopausal symptoms include deterioration of bone and muscle health, hot flashes and night sweats, mood swings, insomnia, headaches, losing sexual desire and weight gain, among others. Studies have also shown that the risk of cardiovascular and atherosclerotic disease increases dramatically after menopause, which has been attributed to declining levels of estrogens in postmenopausal women. (Mendelsohn et al. 1999 N Engl J Med 340: 1801-1811; Blaustein 2008 Endocrinology 149:2697-2698; Schreckenberg et al. 2009 Endocrinology 150:3735-3741; https: / / my.clevelandclinic.org / health / diseases / 22354-low-estrogen.)

[0004] Estrogen is a category of sex hormones responsible for the development and regulation of the female reproductive system and secondary sex characteristics. There are three major forms of estrogens: estrone (El), estradiol (E2), and estriol (E3). Another estrogen, estetrol (E4), is produced only during pregnancy. Estradiol is the most potent and prevalent form of estrogens. (Huether et al. 2019 Understanding Pathophysiology, Elsevier Health Sciences p.767; Satoskar et al. 2017 Pharmacology and Pharmacotherapeutics, Elsevier Health Sciences p.Patent Application Attv. Docket No. YPK-009PCT 943; Delgado etal. 2021 "Estrogen". StatPearls [Internet], StatPearls Publishing PMID 30855848.)

[0005] Estrogen replacement therapy (ERT) is a treatment method that administers a patient with the hormone estrogen to increase the amount of estrogen in the body. It is typically given to women who have gone through menopause or who have early menopause caused by other conditions such as surgical removal of ovaries. ERT may help relieve symptoms of menopause, such as hot flashes, night sweats, vaginal dryness, osteoporosis and sleep problems. There are, however, health risks associated with ERT, including increased risks of certain serious conditions, such as breast cancer, heart disease, stroke, and blood clots. (Chen et al. l^UAMA 287(6):734-741; Judd et al. 1983 Ann Intern Med 98(2): 195-205; Hormone therapy: Is it right for you? https: / / www.mayoclinic.org / diseases-conditions / menopause / in-depth / hormone-therapy / art-20046372.)

[0006] Based on results of studies carried out by the Women’s Health Initiative program, ERT in younger women might not have significantly higher risk of breast cancer as had been previously believed. In older women especially those 59 or older, however, increased breast cancer risks remain a major concern when considering ERT. (Chlebowski et al. 2020 Cancer 126:2956-2964; Prentice etal. 2021 Am J Epidemiology 190(3):365- 375.)

[0007] The therapeutics and methods currently available for treating postmenopausal symptoms and other estrogen deficiency-related diseases and conditions are inadequate and / or with significant side effects. There remains an urgent and ongoing need for novel and improved therapeutics.Summary of the Invention

[0008] The invention is based in part on the unexpected discovery of therapeutic methods and pharmaceutical compositions, as demonstrated herein, that can be used to treat postmenopausal symptoms and other estrogen deficiency-related diseases and conditions. In particular, disclosed herein are methods using testosterone and aromatase together in specifically selected dosages in treatment and / or prevention of estrogen deficiency-associated diseases and conditions.Patent Application Attv. Docket No. YPK-009PCT

[0009] The novel strategy disclosed herein employs testosterone in combination with aromatase such that testosterone is converted into estradiol in the presence of the enzyme aromatase.

[0010] In one aspect, the invention generally relates to a method for providing estrogen to women, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

[0011] In another aspect, the invention generally relates to a method for treating a disease or condition associated with estrogen deficiency, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

[0012] In yet another aspect, the invention generally relates to a method for treating one or more postmenopausal symptoms, comprising administering to a postmenopausal female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

[0013] In yet another aspect, the invention generally relates to a method for increasing estrogen level in a postmenopausal woman without increasing risk of breast cancer, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof.

[0014] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.Patent Application Attv. Docket No. YPK-009PCT

[0015] In yet another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

[0016] In yet another aspect, the invention generally relates to a kit comprising (a) a unit dosage form of testosterone, or a pharmaceutically acceptable form thereof, (b) a unit dosage form of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, and (c) instructions for administration thereof.

[0017] In yet another aspect, the invention generally relates to use of (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, for the manufacture of a medicament for the treatment of a disease or condition associated with estrogen deficiency.

[0018] In yet another aspect, the invention generally relates to use of (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, for the manufacture of a medicament for the treatment of one or more postmenopausal symptoms.Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following terms, unless indicated otherwise according to the context wherein the terms are found, are intended to have the following meanings.

[0020] When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.

[0021] Ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 14 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0022] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference, unless the context clearly dictates otherwise.Patent Application Attv. Docket No. YPK-009PCT

[0023] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0024] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0025] The term “comprising”, when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. The term “consisting essentially of’, when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of’ refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of’, when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0026] As used herein, the terms “disease”, “disorder” or “condition” are used interchangeably herein to refer to a pathological condition, for example, one that can be identified by symptoms or other identifying factors as diverging from a healthy or a normal state. The term “disease” includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[0027] As used herein, a "pharmaceutically acceptable form" of a disclosed compound includes, but is not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, isomers, prodrugs, and isotopically labeled derivatives of disclosed compounds. In one embodiment, a "pharmaceutically acceptable form" includes, but is not limited to, pharmaceutically acceptable salts, esters, isomers, prodrugs and isotopically labeled derivatives of disclosed compounds. In some embodiments, a "pharmaceutically acceptable form" includes,Patent Application Attv. Docket No. YPK-009PCT but is not limited to, pharmaceutically acceptable salts and isotopically labeled derivatives of disclosed compounds.

[0028] In certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, lactic acid, trifluoracetic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0029] The salts can be prepared in situ during the isolation and purification of the disclosed compounds, or separately, such as by reacting the free base or free acid of a parent compound with a suitable base or acid, respectively.Patent Application Attv. Docket No. YPK-009PCT

[0030] In certain embodiments, the pharmaceutically acceptable form is a "solvate" (e.g., a hydrate). As used herein, the term "solvate" refers to compounds that further include a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. The solvate can be of a disclosed compound or a pharmaceutically acceptable salt thereof. Where the solvent is water, the solvate is a "hydrate". Pharmaceutically acceptable solvates and hydrates are complexes that, for example, can include 1 to about 100, or 1 to about 10, or 1 to about 2, about 3 or about 4, solvent or water molecules. It will be understood that the term "compound" as used herein encompasses the compound and solvates of the compound, as well as mixtures thereof.

[0031] In certain embodiments, the pharmaceutically acceptable form is a prodrug. As used herein, the term "prodrug" (or “pro-drug”) refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable form of the compound. A prodrug can be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug has improved physical and / or delivery properties over the parent compound. Prodrugs can increase the bioavailability of the compound when administered to a subject (e.g., by permitting enhanced absorption into the blood following oral administration) or which enhance delivery to a biological compartment of interest (e.g., the brain or lymphatic system) relative to the parent compound. Exemplary prodrugs include derivatives of a disclosed compound with enhanced aqueous solubility or active transport through the gut membrane, relative to the parent compound.

[0032] The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7- 9, 21-24 (Elsevier, Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein. Exemplary advantages of a prodrug can include, but are not limited to, its physical properties, such as enhanced water solubility for parenteral administration at physiological pH compared to the parent compound, or it can enhance absorption from the digestive tract, or it can enhance drug stability for long-term storage.Patent Application Attv. Docket No. YPK-009PCT

[0033] As used herein, the term “pharmaceutically acceptable” excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0034] As used herein, the terms “prevent”, “preventing”, or “prevention” refer to a method for precluding, delaying, averting, or stopping the onset, incidence, severity, or recurrence of a disease or condition. For example, a method is considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of a disease or condition or one or more symptoms thereof in a subject susceptible to the disease or condition as compared to a subject not receiving the method. The disclosed method is also considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of one or more symptoms of a disease or condition in a subject susceptible to the disease or condition after receiving the method as compared to the subject's progression prior to receiving treatment. The reduction or delay in onset, incidence, severity, or recurrence of cancer can be about a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.Patent Application Attv. Docket No. YPK-009PCT

[0035] Prevention and the like do not mean preventing a subject from ever getting the specific disease or disorder. Prevention may require the administration of multiple doses.Prevention can include the prevention of a recurrence of a disease in a subject for whom all disease symptoms were eliminated, or prevention of recurrence in a relapsing-remitting disease.

[0036] As used herein, the terms “subject” and “patient” are used interchangeably herein to refer to a living animal (human or non-human). The subject may be a mammal. The terms “mammal” or “mammalian” refer to any animal within the taxonomic classification mammalia. A mammal may be a human or a non-human mammal, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. The term "subject" does not preclude individuals that are entirely normal with respect to a disease or condition, or normal in all respects.

[0037] As used herein, the term “therapeutically effective amount” refers to the dose of a therapeutic agent or agents sufficient to achieve the intended therapeutic effect with minimal or no undesirable side effects. A therapeutically effective amount can be determined by a skilled physician, e.g., by first administering a low dose of the pharmacological agent(s) and then incrementally increasing the dose until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0038] As used herein, the terms “treatment” or “treating” a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition, or one or more symptoms of such disease or condition, before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and / or the underlying pathology. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

[0039] Any compositions or methods disclosed herein can be combined with one or more of any of the other compositions and methods provided herein.

[0040] Isotopically-labeled compounds are also within the scope of the present disclosure. As used herein, an "isotopically-labeled compound" refers to a presently disclosed compound including pharmaceutical salts thereof, each as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated intoPatent Application Attv. Docket No. YPK-009PCT compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,170,31P,32P,35S,18F, and36C1, respectively.

[0041] By isotopically-labeling the presently disclosed compounds, the compounds may be useful in drug and / or substrate tissue distribution assays. Tritiated (3H) and carbon- 14 (14C) labeled compounds are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds presently disclosed, including pharmaceutical salts, esters, and prodrugs thereof, can be prepared by any means known in the art.

[0042] Further, substitution of normally abundant hydrogen (1H) with heavier isotopes such as deuterium can afford certain therapeutic advantages, e.g., resulting from improved absorption, distribution, metabolism and / or excretion (ADME) properties, creating drugs with improved efficacy, safety, and / or tolerability. Benefits may also be obtained from replacement of normally abundant12C with13C. (See, WO 2007 / 005643, WO 2007 / 005644, WO 2007 / 016361, and WO 2007 / 016431.)

[0043] Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 95% (“substantially pure”), which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are more than 99% pure.

[0044] Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the present invention include, for example, hydrates.

[0045] Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intraventricular, intracorporeal, intraperitoneal, rectal, or oral administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease or condition being treated or the nature of the therapy being used and on the nature of the active compound.

[0046] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof are admixed with at least one inert customary excipient (or carrier) such as sodium citrate orPatent Application Attv. Docket No. YPK-009PCT dicalcium phosphate or (i) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (ii) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (iii) humectants, as for example, glycerol, (iv) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (v) solution retarders, as for example, paraffin, (vi) absorption accelerators, as for example, quaternary ammonium compounds, (vii) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (viii) adsorbents, as for example, kaolin and bentonite, and (ix) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art.

[0047] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, such as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like. Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

[0048] Materials, compositions, and components disclosed herein can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed andPatent Application Attv. Docket No. YPK-009PCT discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.Detailed Description of the Invention

[0049] The invention provides a novel approach to treatment of postmenopausal symptoms and estrogen deficiency-related diseases and conditions The therapeutic methods and pharmaceutical compositions disclosed herein can benefit patients in terms of a novel treatment option, improved treatment outcome, less safety risks, and fewer side effects.

[0050] A key advantage of the invention is that, unlike in ERT, the herein disclosed treatment does not directly administer estrogen, thereby reducing or minimizing the health risks associated with ERT, in particular the increased risk of breast cancer in older women. Instead, the method of the invention allows a pre-selected and controlled pace of providing estrogen from in situ conversion of testosterone to estradiol via aromatase.

[0051] Another major advantage offered by the disclosed invention is due to the fact that testosterone has been shown to protect women from breast cancer by decreasing tissue proliferation. (Glaser etal. 2015 Maturitas 82(3): 291-295; Glaser et al. 2019 BMC Cancer 19: 1271.)

[0052] In one aspect, the invention generally relates to a method for providing estrogen to women, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.Patent Application Attv. Docket No. YPK-009PCT

[0053] In another aspect, the invention generally relates to a method for treating a disease or condition associated with estrogen deficiency, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

[0054] In yet another aspect, the invention generally relates to a method for treating one or more postmenopausal symptoms, comprising administering to a postmenopausal female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

[0055] In yet another aspect, the invention generally relates to a method for increasing estrogen level in a postmenopausal woman without increasing risk of breast cancer, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof.

[0056] Testosterone is the most common form of androgens, which are steroid hormones that regulate the development and maintenance of male characteristics in vertebrates by binding to androgen receptors. Androgens are synthesized in the testes, the ovaries, and the adrenal glands. (Moini 2015 Fundamental Pharmacology for Pharmacy Technicians, C engage Learning, p. 338; Gylys et al. 2017 Medical Terminology Systems: A Body Systems Approach. F.A. Davis, p. 82.)

[0057] Aromatase, also known as estrogen synthetase or estrogen synthase, is an enzyme responsible for a key step in the biosynthesis of estrogens. In humans, aromatase is encoded by the gene CYP19. Aromatase is responsible for the aromatization of androgens into estrogens. In particular, it catalyzes the last steps of estrogen biosynthesis from androgens. Specifically, it transforms androstenedione to estrone and testosterone to estradiol. (Ghosh et al. 2009 J Steroid Biochem Mol Biol. 118(4-5): 197-202; Subramanian et al. 2008 Breast Cancer Res & Treat. 111(2): 191-202; Chen 1998 F w / . inBiosc. 3:922-933.)Patent Application Atty. Docket No. YPK-009PCT

[0058] Testosterone

[0059] In certain embodiments of the methods, (a) is a testosterone ester (e.g., testosterone cypionate, testosterone enanthate, and testosterone undecanoate).Testosterone enanthatePatent Application Attv. Docket No. YPK-009PCTTestosterone undecanoate

[0060] In certain embodiments of the methods, (b) the aromatase is an aromatase-PEG.

[0061] PEGylation is a process that attaches PEG chains to molecules, such as proteins. PEG is a non-toxic, water-soluble polymer. PEGylation can improve one or more properties of aromatase, for example, solubility, stability and / or permeability. Aromatase-PEG is superior over aromatase in terms of improved pharmacokinetics, reduced immunogenicity, less degradation, and / or improved toxicity profde.

[0062] In certain embodiments of the methods, (b) the aromatase is an aromatase-PEG wherein the polyethylene glycol (PEG) has a molecular weight Mnin the range of about 200 to about 500,000 e.g., about 200 to about 10,000, about 10,000 to about 100,000, about 100,00 to about 250,000, about 250,000 to about 500,000, about 1,000 to about 50,000). In certain embodiments the PEG in aromatase-PEG has a Mnin the range of about 1,000 to about 50,000.

[0063] In certain embodiments of the methods, the subject is administered a weekly dose of about 5 mg to about 250 mg (e.g., about 5 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 250 mg, about 10 mg to about 100 mg) of (a) and about 10 mg to about 2,000 mg (e.g., about 10 mg to about 100 mg, about 100 mg to about 500 mg, about 500 mg to about 1,000 mg, about 1,000 mg to about 2,000 mg, about 10 mg to about 1,000 mg, about 25 mg to about 500 mg) of (b). In certain embodiments of the methods, the subject is administered a weekly dose of about 10 mg to about 100 mg of (a) and about 25 mg to about 500 mg of (b).

[0064] In certain embodiments of the methods, (a) and (b) are each administered every three days to once a month. In certain embodiments of the methods, (a) and (b) are each administered once weekly. In certain embodiments of the methods, (a) and (b) are each administered twice weekly.

[0065] In certain embodiments of the methods, (a) and (b) are administered subcutaneously for a time period of about 1 week to about 2 years (e.g., about 2 to about 4 weeks, about 4 to about 8 weeks, about 8 to about 12 weeks, about 12 to about 6 months, about 6 to 12 months).Patent Application Attv. Docket No. YPK-009PCT

[0066] In certain embodiments, the method further comprises administering to the subject a further therapy.

[0067] In certain embodiments, the method further comprises administering to the subject a further therapeutic agent.

[0068] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0069] In yet another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

[0070] In yet another aspect, the invention generally relates to a kit comprising (a) a unit dosage form of testosterone, or a pharmaceutically acceptable form thereof, (b) a unit form of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, and (c) instructions for administration thereof.

[0071] In yet another aspect, the invention generally relates to use of (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, for the manufacture of a medicament for the treatment of a disease or condition associated with estrogen deficiency.

[0072] In yet another aspect, the invention generally relates to use of (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of aromatase (e.g., a pegylated aromatase or "aromatase-PEG"), or a therapeutically acceptable form thereof, for the manufacture of a medicament for the treatment of one or more postmenopausal symptoms.ExamplesMaterials

[0073] Aromatase- 503 amino acids, Mass 57,883 Da; MTOXCE19A1 (NC2980831, Fisher)

[0074] m-PEG-Succinimidyl Valerate, MW 20,000 (BP-25241, BroadPharm)Patent Application Attv. Docket No. YPK-009PCT

[0075] TESTOSTERONE (200 mg / mL, pre-formulated in oil from Padagis)Methods

[0076] 500 mg Aromatase in 10 mL PBS (50 mg / mL) was stored at -20°C. The frozen material was warmed up to ambient temperature and mixed well before use for animal treatment. O.lmL (5mg) SubQ injection to each of 3 rats daily (treatment Group 1)

[0077] Aromatase-PEG: Aromatase was pegylated to prolong its half-life. 500 mg aromatase and 500 mg PEG in 6.7 mL PBS (75 mg / mL) were mixed well at ambient temperature for 30 min to prepare the conjugated PEG-aromatase (PEG conjugates with aromatase through an amino group in lysine) and stored at -20°C. The frozen material was warmed up to ambient temperature and mixed well before use for animal treatment. 0.2 mL (15 mg each of aromatase and PEG) subcutaneous injection to each of 3 rats every 3-6 days (treatment Group 2).

[0078] Testosterone 0.1 ml (20 mg) was given per rat every 6 days, 2 times during the study period.Study Details

[0079] All 10 animals were female SD rats (Sprague Dawley 200-220 g at arrival) from Charles River Laboratories and underwent ovariectomy (ovary-removal) surgery at Day 2.

[0080] 200 uL of rat blood was collected from tail vein using 23 -Gauge needle on Days 1, 9, 19, 22. 200 uL blood was centrifuged to produce 100 uL serum (frozen at -20 °C for future tests).

[0081] Animals 1-4 for Control. Animals 5-10 were treated with testosterone solution (200 mg / mL, pre-formulated in oil) 0.1 mL (20 mg) subcutaneously at Days 16 and 24 (after blood draw).

[0082] Animals 5-7 for Treatment 1 : Starts treatment at Day 16 (after blood draw) to Day 30 with Aromatase solution [500 mg Aromatase in lOmL sterile PBS (50 mg / mL)] 0.1 mL (5 mg) via subcutaneous injection to each of 3 rats daily.

[0083] Animals 8-10 for Treatment 2: Starts treatment at Days 16 (after blood draw), 19, 22, 28 (dose on day 25 was skipped on purpose) with Pegylated Aromatase solution [500mg Aromatase and 500mg PEG in 6.7mL sterile PBS, (75mg / mL)] 0.2mL (15mg) via Subcutaneous injection to each of 3 rats daily.

[0084] Testosterone testing using Enzo Life Sciences Testosterone ELISA kit for rats (Cat# 50-658-123). 15 uL of serum were diluted in 85uL Sample Dilution Buffer with luLPatent Application Attv. Docket No. YPK-009PCT displacement reagent included in the kit and analyzed together with control (Testosterone 1000-500-125-31-7.8 pg / mL)

[0085] Estradiol testing using Rat Estrogen ELISA Kit (Biomatik, Cat# EKF60790). 15 uL of serum were diluted in 35uL Sample Dilution Buffer in the kit and analyzed together with control (Estrogen 200-100-50-25-12.5-6.25-3.125-0 pg / mL).Study ResultsTable 1. Testosterone level in the blood of rats before and after ovariectomy (ng / ml)

[0086] The result showed that the baseline testosterone level was 8.56; after ovariectomized, it dropped to 2.63 in 3 weeks and was corrected by this treatment to 15. The testosterone level is higher in this study because the hormone was displaced from protein before measured.Table 1. Estradiol level in the blood of rats before and after ovariectomy (pg / ml)Patent Application Attv. Docket No. YPK-009PCT

[0087] The results showed that the baseline estradiol was 139 pg / mL, after ovariectomy it dropped to 76 pg / mL at day 19 and 10 pg / mL at 3 weeks. After the treatment, the estradiol level recovered. At 3 weeks, the treatment group levels were 69 pg / mL in treatment 1 and 11 Ipg / mL in treatment 2. The one treated with testosterone and PEG conjugated aromatase is better than the one treated with testosterone and unconjugated aromatase.

[0088] Based on the experiment in ovariectomized rats, it is an ideal treatment for postmenopausal women to replace estrogen with one treatment weekly or biweekly with testosterone in combination with PEG conjugated aromatase.

[0089] Applicant’s disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0090] The described features, structures, or characteristics of Applicant’s disclosure may be combined in any suitable manner in one or more embodiments. In the description, herein, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that Applicant’s composition and / or method may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.Patent Application Attv. Docket No. YPK-009PCT Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.Incorporation by Reference

[0092] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.Equivalents

[0093] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Patent Application Attv. Docket No. YPK-009PCT What is claimed is:CLAIMS1. A method for providing estrogen to women, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

2. A method for treating a disease or condition associated with estrogen deficiency, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

3. A method for treating one or more postmenopausal symptoms, comprising administering to a postmenopausal female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof.

4. A method for increasing estrogen level in a postmenopausal woman without increasing risk of breast cancer, comprising administering to a female subject in need thereof (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase- PEG), or a therapeutically acceptable form thereof.

5. The method of any one of claims 1-4, wherein (a) is testosterone.

6. The method of any one of claims 1-4, wherein (a) is a testosterone ester.Patent Application Attv. Docket No. YPK-009PCT 7. The method of claim 6, wherein the testosterone ester is selected from testosterone cypionate, testosterone enanthate, and testosterone undecanoate.

8. The method of any one of claims 1-7, wherein (b) the aromatase-PEG comprises a polyethylene glycol (PEG) having a Mnin the range of about 200 to about 500,000.

9. The method of claim 8, wherein (b) the aromatase-PEG comprises a polyethylene glycol (PEG) having a Mnin the range of about 1,000 to about 50,000.

10. The method of any one of claims 1-9, wherein the subject is administered a weekly dose of about 5 mg to about 250 mg of (a) and about 10 mg to about 1,000 mg of (b).

11. The method of claim 10, wherein the subject is administered a weekly dose of about 10 mg to about 100 mg of (a) and about 25 mg to about 500 mg of (b).

12. The method of any one of claims 1-11, wherein (a) and (b) are each administered subcutaneously once or twice weekly.

13. The method of claim 12, wherein (a) and (b) are administered for a time period of about 1 week to about 2 years.

14. The method of claim 12, wherein (a) and (b) are administered for a time period of about 3 weeks to about 12 weeks.

15. The method of any one of claims 1-14, further comprising administering to the subject a further therapy.

16. The method of any one of claims 1-15, further comprising administering to the subject a further therapeutic agent.Patent Application Attv. Docket No. YPK-009PCT 17. A pharmaceutical composition comprising (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

18. The pharmaceutical composition of claim 17, wherein (a) is testosterone.

19. The pharmaceutical composition of claim 17, wherein (a) is a testosterone ester.

20. The pharmaceutical composition of claim 19, wherein the testosterone ester is selected from testosterone cypionate, testosterone enanthate, and testosterone undecanoate.

21. The pharmaceutical composition of any one of claims 17-20, wherein (b) the aromatase- PEG comprises a polyethylene glycol (PEG) having a Mnin the range of about 200 to about 500,000.

22. A unit dosage form comprising a pharmaceutical composition according to any one of claims 17-21.

23. A kit comprising (a) a unit dosage form of testosterone, or a pharmaceutically acceptable form thereof, (b) a unit dosage form of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof, and (c) instructions for administration thereof.

24. Use of (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof, for the manufacture of a medicament for the treatment of a disease or condition associated with estrogen deficiency.Patent Application Attv. Docket No. YPK-009PCT 25. Use of (a) a therapeutically effective amount of testosterone, or a pharmaceutically acceptable form thereof, and (b) a therapeutically effective amount of a pegylated aromatase (aromatase-PEG), or a therapeutically acceptable form thereof, for the manufacture of a medicament for the treatment of one or more postmenopausal symptoms.

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