Therapeutic or prophylactic agent for polycystic ovarian syndrome

Abstract

Provided is a therapeutic or prophylactic agent for PCOS, which is for treating or ameliorating PCOS.　A 2-oxapregnane compound represented by formula (1) or a pharmaceutically acceptable salt thereof is used as an active ingredient for the therapeutic or prophylactic agent for PCOS. (In the formula, R1 to R3 each independently represent an alkyl group, R4 represents a hydrogen atom or an alkylcarbonyl group, X represents a halogen atom, and Y represents a hydroxyl or oxo group which is bonded to position-11, position-15 or position-16 in the steroid skeleton.)

Description

Treatment or prevention of polycystic ovary syndrome

[0001] The present invention relates to a therapeutic or preventive agent (ameliorating or alleviating agent) for polycystic ovary syndrome (PCOS), which comprises a pregnane compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0002] PCOS is the most common hormonal disorder affecting women of reproductive age, affecting 5-10% of women of reproductive age. Women with PCOS typically have high androgen levels, which can lead to the development of numerous fluid-filled sacs (cysts) in the ovaries (polycystic ovaries), enlargement and swelling of the ovaries, irregular menstrual periods, facial and body hair growth, infrequent ovulation, and reduced fertility.

[0003] The most common signs and symptoms of PCOS include irregular or amenorrhea, enlarged or polycystic ovaries, acne, infertility, hirsutism, obesity, male pattern baldness, and acanthosis nigricans (thick, dark patches of skin on the back of the neck, under the arms, or under the breasts).

[0004] The exact cause of PCOS is not fully understood, but factors that may be associated with its development include androgen excess, insulin resistance, genetic influences, and inflammation. Treatment is based on symptoms, medical history, other health conditions, and desire to become pregnant, and involves a combination of lifestyle modifications, medications, and surgery.

[0005] The diagnostic criteria for PCOS set by the Japan Society of Obstetrics and Gynecology in Japan (revised in 2024) state that all three of the following conditions must be met: (1) menstrual cycle abnormalities, (2) polycystic ovaries or elevated levels of anti-Müllerian hormone (AMH), and (3) hyperandrogenism or elevated levels of luteinizing hormone (LH). Overseas, the criteria are based on the Rotterdam Diagnostic Criteria 2003, and state that at least two of the three conditions - ovulation disorder, hyperandrogenism, and polycystic ovaries - must be present.

[0006] Currently, because the underlying cause of PCOS is unknown, there is no treatment that can improve all symptoms, and treatment is primarily symptomatic, focusing on the primary complaint. For example, in cases of infertility due to PCOS, treatment focuses on improving ovulation rate using ovulation-inducing drugs rather than improving symptoms. In cases of menstrual irregularities, hormone treatment is performed using oral contraceptives (OCs) / low-dose estrogen-progestin combination drugs (LEPs). In cases of virilization, treatment involves improving hormone balance or suppressing androgen levels with antiandrogens. In cases of insulin resistance, treatment is performed with metformin. However, symptoms often recur when medication is discontinued, or no improvement is seen in many cases, and a complete cure is not currently possible.

[0007] Japanese Patent No. 2591640 (Patent Document 1) describes that 17α-acetoxy-6-chloro-15β-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione has excellent antiandrogenic activity and is useful as a therapeutic agent for benign prostatic hyperplasia; a therapeutic agent for prostate cancer; a therapeutic agent for juvenile, middle-aged, senile, areata, seborrheic, and pityriasis alopecia; a hair care product; a therapeutic agent for acne vulgaris, etc.

[0008] Patent No. 2591640

[0009] However, Patent Document 1 does not describe PCOS.

[0010] Therefore, an object of the present invention is to provide a therapeutic or prophylactic agent for treating or preventing PCOS.

[0011] As a result of intensive research to achieve the above-mentioned object, the present inventors have found that 2-oxapregnane compounds having an oxo group at the 2-position of the steroid skeleton are effective in treating or preventing PCOS, and have completed the present invention.

[0012] That is, the present invention includes the following aspects.

[0013] Aspect [1]: A therapeutic or preventive agent for PCOS, comprising, as an active ingredient, a 2-oxapregnane compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, wherein the steroid skeleton in the following formula (1) is numbered by a position number:

[0014]

[0015] (In the formula, R １ ~R ３ each independently represents an alkyl group, and R ４ represents a hydrogen atom or an alkylcarbonyl group, X represents a halogen atom, and Y represents a hydroxyl group or an oxo group bonded to the 11th, 15th, or 16th position of the steroid skeleton.

[0016] Aspect [2]: In the formula (1), R １ ~R ３ represents a methyl group, and R ４ The therapeutic or preventive agent for PCOS according to the above aspect [1], wherein X represents an acetyl group, X represents a chlorine atom, and Y represents a hydroxyl group or an oxo group bonded to the 15-position of the steroid skeleton.

[0017] Aspect [3]: The agent for treating or preventing PCOS according to Aspect [1] or [2], wherein the 2-oxapregnane compound represented by Formula (1) is 17α-acetoxy-6-chloro-15β-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione.

[0018] Aspect [4]: ​​The therapeutic or preventive agent for PCOS according to any of Aspects [1] to [3], which improves at least one symptom selected from the group consisting of menstrual cycle abnormalities, ovarian hypertrophy, polycystic ovary syndrome, high levels of anti-Müllerian hormone, hyperandrogenism, high levels of luteinizing hormone, high levels of luteinizing hormone / follicle-stimulating hormone ratio, acne, infertility, hirsutism, obesity, male pattern baldness, and acanthosis nigricans.

[0019] Aspect [5]: The therapeutic or preventive agent for PCOS according to any one of Aspects [1] to [4], which improves at least two symptoms selected from the group consisting of menstrual cycle disorders, polycystic ovary syndrome, and hyperandrogenism.

[0020] Aspect [6]: The therapeutic or preventive agent for PCOS according to any one of Aspects [1] to [5], which improves menstrual cycle disorders, polycystic ovary syndrome, and hyperandrogenism.

[0021] The present invention also includes a therapeutic or preventive agent for PCOS according to any one of the above aspects [1] to [6], which has at least one feature selected from the following (1) to (5):

[0022] (1) reducing at least one selected from the number of cysts, testosterone concentration, luteinizing hormone (LH) concentration, and the ratio of the luteinizing hormone (LH) concentration to the follicle-stimulating hormone (FSH) concentration [LH / FSH]; (2) increasing progesterone concentration; (3) restoring estrus and improving the periodicity of the estrous cycle; (4) restoring the number of ovulations; and (5) restoring the formation of new corpora lutea.

[0023] Aspect [7]: The agent for treating or preventing PCOS according to any one of Aspects [1] to [6], which has at least one feature selected from the following (1) to (4): (1) reducing at least one selected from the number of cysts, testosterone concentration, luteinizing hormone (LH) concentration, and the ratio of the luteinizing hormone (LH) concentration to the follicle-stimulating hormone (FSH) concentration [LH / FSH], (2) increasing progesterone concentration, (3) restoring estrus and improving the periodicity of the estrous cycle, and (4) restoring the number of ovulations.

[0024] Aspect [8]: The agent for treating or preventing PCOS according to any one of Aspects [1] to [7], which treats or prevents PCOS in patients with obesity and / or insulin resistance.

[0025] Aspect [9]: The PCOS therapeutic or preventive agent according to any one of Aspects [1] to [8], which treats or prevents PCOS in women of reproductive age.

[0026] Aspect

[10] : The agent for treating or preventing PCOS according to any one of Aspects [1] to [9], which is a solid formulation, a semi-solid formulation, or a liquid formulation.

[0027] Aspect

[11] : The agent for treating or preventing PCOS according to any one of Aspects [1] to

[10] , which is an orally administered formulation.

[0028] Aspect

[12] : The therapeutic or preventive agent for PCOS according to any one of Aspects [1] to

[11] , which is in the form of a tablet.

[0029] Aspect

[13] : A method for treating or preventing PCOS using, as an active ingredient, a 2-oxapregnane compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

[0030] Aspect

[14] : Use of the 2-oxapregnane compound represented by formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient for treating or preventing PCOS.

[0031] Aspect

[15] : Use of the 2-oxapregnane compound represented by formula (1) or a pharmaceutically acceptable salt thereof for the manufacture of the agent for treating or preventing PCOS according to any of Aspects [1] to

[12] .

[0032] In the present invention, the therapeutic or preventive agent for PCOS contains, as an active ingredient, a 2-oxapregnane compound having an oxo group at the 2-position of the steroid skeleton, and therefore can treat or prevent PCOS.

[0033] FIG. 1 is a graph showing the relationship between the test substance and the estrous period in Test Example 1. FIG. 2 is a graph showing the relationship between the test substance and the number of ovulation eggs in Test Example 1. FIG. 3 is a graph showing the relationship between the test substance and the estrous period in Test Example 2. FIG. 4 is a graph showing the relationship between the test substance and the number of ovulation eggs in Test Example 2. FIG. 5 is a graph showing the relationship between the test substance and the estrous period in Test Example 3. FIG. 6 is a graph showing the relationship between the test substance and the change in body weight in Test Example 3. FIG. 7 is a graph showing the relationship between the test substance and the number of ovulation eggs in Test Example 3.

[0034] [Agent for treating or preventing PCOS] The agent for treating or preventing PCOS of the present invention comprises, as an active ingredient (pharmacologically or physiologically active ingredient), a 2-oxapregnane compound represented by formula (1) or a pharmaceutically acceptable salt thereof (hereinafter referred to as "pregnane compound (1)"). In particular, in the present invention, pregnane compound (1) acts as an androgen receptor modulator and directly inhibits peripheral androgens, thereby improving ovulation disorders and virilization symptoms associated with high androgen levels and normalizing hormone balance in the body, and is expected to be able to treat (improve or alleviate) or prevent various symptoms of PCOS.

[0035] In the formula (1), R １ ~R ３ Examples of the alkyl group (linear or branched alkyl group) represented by the formula (I) include C groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a s-butyl group, and a t-butyl group. １－６ Examples of R include alkyl groups. １ ~R ３ The alkyl groups represented by the formula (I) may be different from each other or may all be the same alkyl group, but it is preferable that all be the same alkyl group. １－４ Alkyl groups are preferred, and C １－３ Alkyl groups are more preferred, and C １－２ Alkyl groups are more preferred, and methyl groups are most preferred.

[0036] R ４ Examples of the alkylcarbonyl group (linear or branched alkyl group) represented by the formula (I) include C groups such as an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a t-butyryl group, a pentanoyl group (valeryl group), and a hexanoyl group. １－１０ Examples include alkyl-carbonyl groups. ４ may be either a hydrogen atom or an alkylcarbonyl group, and is usually an alkylcarbonyl group (alkanoyl group or acyl group). １－６ Alkyl-carbonyl groups are preferred, C １－４ Alkyl-carbonyl groups are more preferred, C １－３ An alkyl-carbonyl group is more preferred, and an acetyl group is most preferred. ４ The alkylcarbonyl group represented by R ４ The hydrogen atom represented by the formula (I) may be converted to an alkylcarbonyl group by an acylation enzyme.

[0037] The halogen atom represented by X includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Of these, a fluorine atom, a chlorine atom, and a bromine atom are preferred, and a chlorine atom is particularly preferred.

[0038] The substitution position of the hydroxyl group or oxo group represented by Y is either the 11th, 15th or 16th position of the steroid skeleton, with the 15th position being preferred.

[0039] The oxo group and hydroxyl group of the steroid skeleton may be mutually convertible by oxidoreductases in vivo. Therefore, Y may be either a hydroxyl group or an oxo group. A preferred Y is usually a hydroxyl group. When Y is a hydroxyl group, this hydroxyl group may have the α- or β-configuration represented by the following formula. For example, the hydroxyl group bonded to the 15-position of the steroid skeleton may have the β-configuration.

[0040]

[0041] The pregnane compound (1) includes, for example, 17α-C １－４ Alkyl-carbonyloxy-6-halo-15β-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione, 17α-C １－４ Examples include alkyl-carbonyloxy-6-halo-2-oxa-4,6-pregnadiene-3,15,20-trione, etc. A preferred pregnane compound (1) has a hydroxyl group represented by Y at the 11th, 15th, or 16th position of the steroid skeleton, and can be represented by the following formula (1a):

[0042]

[0043] Representative compounds represented by the formula (1a) or pharmaceutically acceptable salts thereof include 17α-acetoxy-6-chloro-15β-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione or pharmaceutically acceptable salts thereof.

[0044] The pregnane compound (1) may be a racemate or an optically active substance (or optical isomer). The pregnane compound (1) may be a metabolite.

[0045] Examples of salts include salts with inorganic acids (such as hydrochloric acid and sulfuric acid), organic acids (such as acetic acid), inorganic bases (such as alkali metals such as ammonia, sodium, and potassium, and alkaline earth metals such as calcium), and organic bases (such as amines such as triethylamine).

[0046] The pregnane compound (1) can be prepared by a conventional method, for example, in accordance with the method described in Patent Document 1.

[0047] The pregnane compound (1) may have antiandrogenic activity and may act as an androgen receptor modulator.

[0048] The agent for treating or preventing PCOS of the present invention only needs to contain at least the pregnane compound (1) (first active ingredient) as an active ingredient, and may also contain other active ingredients (biologically active ingredients or pharmacologically active ingredients, second active ingredients) as necessary. Examples of other active ingredients include antiandrogens (e.g., non-steroidal antiandrogens (antiandrogens not containing a steroid skeleton) such as flutamide, bicalutamide, and enzalutamide, and steroidal antiandrogens (antiandrogens containing a steroid skeleton) such as spironolactone and cyproterone acetate), oral contraceptives (OCs) or low-dose estrogen-progestin combination drugs (LEPs) (e.g., norethisterone, ethinyl estradiol, etc.), gonadotropins (e.g., luteinizing hormone, follicle-stimulating hormone, etc.), adrenocortical hormones (e.g., prednisolone, etc.), antidiabetic drugs (e.g., metformin, etc.), anti-Parkinson's drugs (e.g., cabergoline, etc.), and herbal medicines (e.g., Tokishakuyakusan, Saireito, Unkeito, etc.).

[0049] In general, non-steroidal antiandrogens such as flutamide and bicalutamide are known to have stronger antiandrogen activity than steroidal antiandrogens, and are expected to be more effective in treating or preventing PCOS.

[0050] However, as will be apparent from the examples below, even when flutamide is administered at a commonly used dose, no improvement is observed in the number of ovulations or luteinizing hormone (LH) levels [or the LH / follicle-stimulating hormone (FSH) ratio], which are indicators of ovulation disorders, nor is there a decrease in the number of polycystic follicles. These results suggest that there is an essential difference between flutamide and the pregnane compound (1) of the present invention in terms of their effect on improving ovulation disorders. One possible cause of this difference is the influence of the difference in their ability to be transported to the hypothalamus. That is, flutamide transports to the hypothalamus, where it exhibits antagonism against the androgen receptor (AR), thereby inhibiting the negative feedback mechanism and preventing the suppression of LH secretion, resulting in an increase in blood LH and testosterone levels. On the other hand, pregnane compound (1) has a different ability to penetrate the hypothalamus and exhibits a similar AR antagonistic effect, yet maintains LH secretion suppression, which is presumably why it exhibits therapeutic effects such as improved ovulation rate, normalized testosterone and LH levels, and reduced polycystic cysts. This suggests that not only are both compounds antiandrogenic and structurally distinct, but also that factors or mechanisms other than antiandrogenic activity, such as pharmacokinetics and mechanism of action at the hypothalamic level, are involved in improving ovulation disorders.

[0051] Furthermore, as will be apparent from the Examples below, even when spironolactone, a steroidal antiandrogen like the pregnane compound (1), is used in a commonly used amount, no improvement is observed in the number of ovulations or LH levels (or LH / FSH ratio), which are indicators of ovulation disorders, nor is there a decrease in the number of polycystic cysts. Spironolactone is a steroidal antiandrogen like the pregnane compound (1), and both share a steroid skeleton. However, their effects and behaviors in the treatment or prevention of PCOS are significantly different. This suggests that structures other than the steroid skeleton contribute to the effects of the present invention, and that factors or mechanisms of action other than antiandrogenic activity are also significantly involved.

[0052] Although antiandrogens such as spironolactone have been used to treat virilization symptoms, they have not been effective against the diagnostic criteria for PCOS, which are "menstrual cycle abnormalities," "polycystic ovary syndrome or elevated anti-Müllerian hormone (AMH) levels," and "hyperandrogenism or elevated luteinizing hormone (LH) levels." On the other hand, the pregnane compound (1) of the present invention can significantly improve at least two (preferably three) of the above diagnostic criteria for PCOS, and can be said to be effective in treating or preventing PCOS.

[0053] These second active ingredients can be used alone or in combination of two or more. Among these, oral contraceptives (OCs) such as norethisterone and ethinylestradiol, or low-dose estrogen-progestin combination drugs (LEPs) are preferred.

[0054] The total proportion of these second active ingredients is, for example, 100 parts by mass or less (e.g., 0.1 to 100 parts by mass), preferably 50 parts by mass or less (e.g., 0.5 to 50 parts by mass), and more preferably 30 parts by mass or less (e.g., 1 to 30 parts by mass) relative to 100 parts by mass of the pregnane compound (1).

[0055] The pregnane compound (1) may account for 50% by mass or more of the active ingredients (the total amount of the first active ingredient and the second active ingredient), preferably 70% by mass or more, even more preferably 80% by mass or more, even more preferably 90% by mass or more, and most preferably 100% by mass.

[0056] The total proportion of the active ingredient in the PCOS therapeutic or preventive agent may be 0.1% by mass or more, for example, 1% by mass or more, preferably 5% by mass or more, and more preferably 10% by mass or more.

[0057] The therapeutic or preventive agent for PCOS of the present invention is effective in improving and / or alleviating various symptoms of PCOS. Therefore, the therapeutic or preventive agent for PCOS can also be referred to as an improving agent and / or alleviating agent for improving and / or alleviating PCOS.

[0058] Furthermore, the therapeutic or preventive agent for PCOS of the present invention is effective in preventing various symptoms of PCOS.

[0059] Symptoms of PCOS include, for example, menstrual cycle abnormalities (or ovulation disorders), ovarian hypertrophy (or ovarian enlargement), polycystic ovaries, high levels of anti-Mullerian hormone (AMH), hyperandrogenism, high levels of luteinizing hormone (LH), high levels of luteinizing hormone (LH) / follicle-stimulating hormone (FSH) ratio, acne (also known as acne vulgaris, a typical symptom of PCOS that corresponds to the cardinal symptom of virilization in human females (particularly women of reproductive age)), infertility, hirsutism, obesity, male pattern baldness, and acanthosis nigricans (thick black patches of skin on the back of the neck, under the armpits, and under the chest).

[0060] Examples of the menstrual cycle abnormality include menstrual irregularities such as amenorrhea, oligomenorrhea, polymenorrhea, anovulatory cycle, etc. Among these, the agent for treating or preventing PCOS of the present invention is effective in improving (or alleviating) amenorrhea, oligomenorrhea, and anovulatory cycle.

[0061] The therapeutic or preventive agent for PCOS of the present invention can improve (or alleviate) one or more of these symptoms, preferably two or more, and more preferably three or more.

[0062] Although the onset or mechanism of action of PCOS has not been fully elucidated, it is presumed that a high level of androgen, together with hormone imbalances such as high LH levels (high LH / FSH ratio), insulin resistance, etc., causes the ovaries to become polycystic, and the aforementioned characteristic clinical symptoms (acne, signs of virilization such as hirsutism and male pattern baldness, menstrual irregularities or ovulation disorders, infertility, obesity, etc.) are exhibited. Based on this presumed mechanism, the international standard, the Rotterdam Diagnostic Criteria 2003, and the latest domestic standard, the diagnostic criteria for PCOS by the Japan Society of Obstetrics and Gynecology (revised in 2024), have established diagnostic criteria focusing on menstrual cycle abnormalities, polycystic ovaries, and hyperandrogenism.

[0063] Therefore, the therapeutic agent for PCOS of the present invention may be a therapeutic agent that improves at least two symptoms selected from the group consisting of menstrual cycle disorders, polycystic ovary syndrome, and hyperandrogenism, and the improvement of these two or more symptoms satisfies the Rotterdam Diagnostic Criteria 2003, an international standard.

[0064] Menstrual cycle abnormalities may be evaluated based on the state of ovulation disorder, and may be evaluated using, for example, the presence or absence of ovulation, the length (25 to 38 days) and regularity of the menstrual cycle, and hormone balance (e.g., blood androgen concentration, LH concentration, LH / FSH ratio, etc.) as indicators. Note that in animals (e.g., rats, etc.), the presence or absence of ovulation may be evaluated based on whether or not a new corpus luteum is observed.

[0065] Polycystic ovary syndrome may be evaluated by high AMH levels instead of confirming the number and condition of cysts. However, it is preferable to confirm the number and condition of cysts using ultrasound tomography or the like, as this allows for accurate evaluation of cyst findings.

[0066] Hyperandrogenism can be confirmed by hormone balance, typically evaluated by androgen concentration, but can also be evaluated by high LH levels (high basal LH levels) or high LH / FSH ratios. Hyperandrogenism may also be evaluated by hirsutism. Among these, androgen concentration is preferred because it can be evaluated simply and accurately. Androgen concentration may be evaluated by total testosterone concentration, free testosterone concentration, androstenedione concentration, etc., and is preferably evaluated by total testosterone concentration (blood testosterone concentration).

[0067] In particular, the therapeutic or preventive agent for PCOS of the present invention may be a therapeutic or preventive agent that improves (or alleviates) menstrual cycle abnormalities and hyperandrogenism, and is most preferably a therapeutic or preventive agent that improves (or alleviates) menstrual cycle abnormalities, polycystic ovary syndrome, and hyperandrogenism. Improvement (or alleviation) of all three symptoms, i.e., menstrual cycle abnormalities, polycystic ovary syndrome, and hyperandrogenism, meets the diagnostic criteria for PCOS (revised in 2024) of the Japan Society of Obstetrics and Gynecology, which is the domestic standard.

[0068] The agent for treating or preventing PCOS of the present invention can also be effectively used in patients with obesity and / or insulin resistance (pre-diabetes).

[0069] The agent for treating or preventing PCOS of the present invention may be the pregnane compound (1) alone as a therapeutic or preventive agent, or may be used as a pharmaceutical composition or a physiologically active composition in combination with a carrier (e.g., a pharmacologically or physiologically acceptable carrier). The carrier for the agent for treating or preventing PCOS (pharmaceutical composition, physiologically active composition) can be selected depending on the formulation (i.e., dosage form), administration mode, and intended use. The dosage form is not particularly limited and may be a solid preparation (powders, powders, granules (e.g., granules, fine granules), pills, tablets, capsules (e.g., soft capsules, hard capsules), dry syrup, suppositories, film or sheet preparations), semi-solid preparations (e.g., creams, ointments, gels, gummies), or liquid preparations (e.g., injections, syrups). Of these, solid preparations are preferred.

[0070] The powder formulation also includes sprays and aerosols. Capsules may be either soft or hard capsules, liquid-filled capsules, or capsules filled with a solid agent such as granules. The formulation may also be a lyophilized formulation. Furthermore, the PCOS therapeutic or preventive agent (or formulation) of the present invention may be a formulation with a controlled drug release rate (sustained-release formulation, immediate-release formulation). The route of administration of the PCOS therapeutic or preventive agent (or formulation) of the present invention is not particularly limited, and may be an oral formulation (granules, powders, tablets (e.g., sublingual tablets, orally disintegrating tablets), capsules, film formulations, etc.) or a parenteral formulation (e.g., inhalants, transdermal formulations, nasal formulations, suppositories, injections, etc.). Furthermore, the formulation may be a topical formulation (e.g., ointments, patches, or poultices). Among these, oral formulations (particularly tablets) are preferred.

[0071] The agent (or preparation) for treating or preventing PCOS of the present invention is preferably a solid preparation (e.g., a solid preparation for oral administration), particularly preferably a tablet. Therefore, the following explanation will focus on the components of the solid preparation.

[0072] The carrier can be selected from ingredients (e.g., excipients, binders, disintegrants, lubricants, coating agents, etc.) listed in, for example, the 19th Revised Japanese Pharmacopoeia (JP), (1) Pharmaceutical Additives Handbook, Japan Pharmaceutical Additives Association (2007), (2) "Dictionary of Pharmaceutical Additives 2021" (Yakuji Nipposha, February 2021), (3) Pharmaceutics, Revised 5th Edition, Nankodo Co., Ltd. (1997), and (4) Pharmaceutical Additives Standards 2018 (Yakuji Nipposha, July 2018), depending on the administration route and formulation purpose. As the carrier for solid formulations, at least one carrier selected from excipients, binders, and disintegrants is often used, and it is preferable to use at least one carrier selected from excipients and disintegrants. For solid formulations in tablet form, it is preferable to use a lubricant. Furthermore, the PCOS treatment or prevention agent (or formulation) may contain a lipid.

[0073] The carrier may be, for example, a carrier not in the form of a metal salt (first carrier), a carrier in the form of a polyvalent metal salt of an inorganic acid (second carrier), or a carrier in the form of a metal salt of an organic acid (third carrier). Examples of metal salts include inorganic acid salts such as sulfates, hydrochlorides, phosphates, carbonates, and silicates; and organic acid salts such as carboxylates and sulfonates.

[0074] When the carrier is a first carrier that is not in the form of a metal salt and / or a second carrier that is in the form of a polyvalent metal salt of an inorganic acid, the therapeutic or preventive agent for PCOS of the present invention (pharmaceutical composition or formulation, particularly a solid formulation) is effectively prevented from discoloration or coloration over a long period of time and has excellent stability.

[0075] Examples of the carrier (first carrier) that is not in the form of a metal salt include sugars or sugar alcohols such as lactose (or lactose hydrate), glucose, sucrose, maltose (maltose or maltose hydrate), maltitol, mannitol, sorbitol, xylitol, and isomaltose hydrate; starches such as corn starch and potato starch; soluble starches such as pregelatinized starch and partially pregelatinized starch; polysaccharides such as crystalline cellulose (including microcrystalline cellulose), carboxymethylcellulose (carmellose, CMC), dextrin, gum arabic, pectin, agar, and gelatin; polyvinylpyrrolidone-based polymers (polyvinylpyrrolidone (PVP, povidone), copolymers such as vinylpyrrolidone-vinyl acetate copolymer (copolyvidone), cross-linked polyvinylpyrrolidone, and the like. Examples of the first carrier include synthetic polymers such as poly(meth)acrylic acid polymers (methacrylic acid copolymers such as Eudragit L, S, L30D-55, and aminoalkyl methacrylic acid copolymers such as Eudragit E and RS), polylactic acid, and polyethylene glycol (polyethylene glycol 6000, and the like); cellulose ethers such as methylcellulose (MC), ethylcellulose (EC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), low-substituted hydroxypropylcellulose, and hydroxypropylmethylcellulose (HPMC); silicon oxides such as light anhydrous silicic acid, silicon dioxide, and hydrous silicon dioxide; and waxes such as beeswax. These first carriers can be used alone or in combination of two or more.

[0076] Examples of the carrier (second carrier) in the form of a polyvalent metal salt of an inorganic acid include polyvalent metal salts of silicic acid such as divalent silicates (or alkaline earth metal salts of silicic acid) such as calcium silicate, magnesium silicate, and magnesium aluminosilicate, and trivalent silicates such as kaolin (or aluminum silicate); polyvalent metal salts of phosphoric acid (particularly calcium phosphates) such as divalent phosphates (or alkaline earth metal salts of phosphoric acid) such as calcium monohydrogen phosphate, calcium dihydrogen phosphate, and anhydrous calcium hydrogen phosphate; and carbonates such as magnesium carbonate. These second carriers can be used alone or in combination of two or more.

[0077] Examples of the carrier in the form of a metal salt of an organic acid (third carrier) include polysaccharides such as sodium alginate, metal salts of carboxymethylcellulose (or crosslinked products thereof) such as carmellose sodium, carmellose calcium, and croscarmellose sodium, sodium carboxymethyl starch (sodium starch glycolate), low-substituted sodium carboxymethyl starch, etc. These third carriers can be used alone or in combination of two or more.

[0078] These carriers can be used alone or in combination of two or more. It should be noted that the same component may perform multiple functions or roles, to varying degrees, and therefore the same component may be classified as multiple carrier components. For example, excipients and / or disintegrants may be classified as binders, or excipients may be classified as disintegrants. For example, depending on the degree of pregelatinization, pregelatinized starch may function as a binder or a disintegrant, and if the degree of pregelatinization is low or if it is partially pregelatinized, it may function as a disintegrant. Therefore, the present specification and claims are not limited to the types of carriers classified, and may be replaced with other types of carriers depending on their functions or roles.

[0079] Representative excipients include the components exemplified as the first carrier, for example, sugars or sugar alcohols such as lactose, glucose, sucrose, mannitol, sorbitol, and xylitol; starches such as corn starch; polysaccharides such as crystalline cellulose (including microcrystalline cellulose); silicon oxides or silicates such as light anhydrous silicic acid; and the like.

[0080] Representative binders include the components exemplified as the first carrier, such as soluble starches such as pregelatinized starch and partially pregelatinized starch; polysaccharides such as gum arabic and dextrin; synthetic polymers such as polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), carboxyvinyl polymer, polyacrylic acid polymer, polylactic acid, and polyethylene glycol; cellulose ethers such as methylcellulose (MC), ethylcellulose (EC), carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and hydroxypropylmethylcellulose (HPMC); and the components exemplified as the third carrier, such as sodium alginate and sodium carboxymethylcellulose.

[0081] Representative disintegrants include the components exemplified as the first carrier, such as carmellose, crospovidone, low-substituted hydroxypropyl cellulose, etc.; and the components exemplified as the third carrier, such as carboxymethyl starch sodium, carmellose sodium, carmellose calcium, croscarmellose sodium, etc. As mentioned above, pregelatinized starch is also a representative disintegrant. These carriers can be used alone or in combination of two or more.

[0082] The proportion of the excipient may be, for example, about 50% by mass or more (e.g., 55 to 99.5% by mass), preferably 60% by mass or more (e.g., 65 to 99% by mass), and more preferably 70% by mass or more (e.g., 75 to 90% by mass) per unit dosage form (e.g., one plain tablet (uncoated tablet) or uncoated tablet).

[0083] The proportion of the binder may be, for example, about 50% by mass or less (e.g., 0.01 to 50% by mass), preferably 40% by mass or less (e.g., 0.05 to 40% by mass), more preferably 0.1 to 30% by mass, and more preferably about 0.5 to 20% by mass per unit dosage form (e.g., one plain tablet (plain tablet) or uncoated tablet).

[0084] The proportion of the disintegrant may be, for example, about 0.01 to 50% by mass, preferably about 0.05 to 40% by mass, more preferably about 0.1 to 30% by mass, and more preferably about 0.5 to 20% by mass (e.g., 1 to 15% by mass) per unit dosage form (e.g., one plain tablet (uncoated tablet) or uncoated tablet).

[0085] Examples of the lubricant include higher unsaturated fatty acids or metal salts thereof such as sodium stearyl fumarate, higher saturated fatty acids or metal salts thereof (polyvalent metal salts) such as stearic acid, magnesium stearate, aluminum stearate, and calcium stearate, and minerals such as talc. The proportion of the lubricant per unit dosage form (e.g., one plain tablet (uncoated tablet) or uncoated tablet) may be, for example, about 0.01 to 20% by mass, preferably about 0.05 to 10% by mass, and more preferably about 0.1 to 5% by mass.

[0086] Examples of the coating agent include sugars, cellulose derivatives such as ethyl cellulose and hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, methyl methacrylate-(meth)acrylic acid copolymer, and Eudragit (methacrylic acid-acrylic acid copolymer). The coating agent may be an enteric component such as cellulose phthalate, hydroxypropyl methylcellulose phthalate, or methyl methacrylate-(meth)acrylic acid copolymer, or a gastric component composed of a polymer (such as Eudragit) containing a basic component such as a dialkylaminoalkyl (meth)acrylate. The formulation may also be a capsule containing such an enteric component or gastric component in its coating.

[0087] In the formulation, known additives can be used appropriately depending on the administration route, dosage form, etc. Examples of such additives include disintegration aids, antioxidants, stabilizers, preservatives, bactericides, antibacterial agents, antistatic agents, flavoring agents, masking agents, colorants, odorants, fragrances, refreshing agents, antifoaming agents, etc. These additives can be used alone or in combination of two or more.

[0088] In a pharmaceutical composition or a physiologically active composition combined with a carrier (e.g., a pharmacologically or physiologically acceptable carrier), the content of the pregnane compound (1) can be appropriately selected depending on the species, age, weight, and condition (general condition, pathology, presence or absence of complications, etc.) of the subject to be administered, the administration time, dosage form, administration method, etc., and can be selected, for example, from a range of about 0.01 to 30% by mass per unit preparation, for example, 0.1 to 20% by mass, preferably 0.5 to 15% by mass, and more preferably 1 to 10% by mass.

[0089] The agent for treating or preventing PCOS (pharmaceutical composition or formulation) of the present invention can be prepared using, in addition to the active ingredient, carrier components, and, if necessary, additives, etc., by a conventional formulation method, for example, the manufacturing method described in the 19th Edition of the Japanese Pharmacopoeia or a method similar to this manufacturing method.

[0090] As mentioned above, the pregnane compound (1) is a known compound that has been used for many years and is highly stable and safe.

[0091] The PCOS therapeutic or preventive agent (pharmaceutical composition or preparation) of the present invention can be administered to humans and non-human animals, and can be safely administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.). Of these, administration to humans is preferred, and administration to obese humans accompanied by hyperandrogenemia is particularly preferred because the PCOS therapeutic agent of the present invention acts effectively.

[0092] The dosage can be selected depending on the species, age, body weight, and condition (general condition, symptoms, presence or absence of complications, etc.) of the subject, the administration time, dosage form, administration method, etc. For example, the dosage (daily dose) for humans may be, for example, about 0.1 to 300 mg / day, preferably about 0.5 to 200 mg / day, and more preferably about 1 to 100 mg / day, calculated as the compound represented by formula (1). Note that the compound represented by formula (1) of the present invention is effective even at small doses, so the dosage may be, for example, about 0.01 to 100 mg / day, preferably about 0.05 to 75 mg / day, and more preferably about 0.1 to 50 mg / day, calculated as the compound represented by formula (1).

[0093] The administration method may be oral, topical, or parenteral (for example, subcutaneous, intramuscular, rectal, or vaginal administration). In a preferred embodiment, the PCOS therapeutic agent (pharmaceutical composition or formulation) of the present invention is orally administered. The number of administrations is not particularly limited and can be selected from, for example, about 1 to 5 times per day (for example, 1 to 3 times per day), and may be, for example, once per day, or multiple times per day (for example, 2 to 3 times per day) as needed.

[0094] In addition to the features described in the section "Means for Solving the Problems," preferred aspects of the therapeutic or preventive agent for PCOS of the present invention are as follows.

[0095] (A) Treating or ameliorating at least one symptom selected from menstrual cycle abnormalities (or ovulation disorders), ovarian hypertrophy (or ovarian enlargement), polycystic ovary syndrome, high levels of anti-Mullerian hormone (AMH), hyperandrogenism, high levels of luteinizing hormone (LH), high levels of luteinizing hormone (LH) / follicle-stimulating hormone (FSH) ratio, acne (also known as acne vulgaris, a typical symptom of PCOS that corresponds to the cardinal symptom of virilization in human females (particularly women of reproductive age)), infertility, hirsutism, obesity, male pattern baldness, and acanthosis nigricans (thick, dark patches of skin on the back of the neck, under the armpits, or under the chest).

[0096] (B) In (A), the menstrual cycle abnormality is irregular menstruation such as amenorrhea, oligomenorrhea, and anovulatory cycle.

[0097] (C) In the above (A) or (B), at least one symptom selected from menstrual cycle abnormalities (or ovulation disorders), polycystic ovary syndrome, and hyperandrogenism is treated or improved.

[0098] (D) In ​​any one of (A) to (C), at least two symptoms selected from menstrual cycle abnormalities (or ovulation disorders), polycystic ovary syndrome, and hyperandrogenism are treated or improved.

[0099] (E) In any of (A) to (D), menstrual cycle abnormalities (or ovulation disorders), polycystic ovary syndrome, and hyperandrogenism are treated or improved.

[0100] (F) In any of (A) to (E) above, the product has at least one feature selected from the following (1) to (5):

[0101] (1) reducing at least one selected from the number of cysts, testosterone concentration, luteinizing hormone (LH) concentration, and the ratio of the luteinizing hormone (LH) concentration to the follicle-stimulating hormone (FSH) concentration [LH / FSH]; (2) increasing progesterone concentration; (3) restoring estrus and improving the periodicity of the estrous cycle; (4) restoring the number of ovulations; and (5) restoring the formation of new corpora lutea.

[0102] (G) In (F), the product has at least one feature selected from (1) to (4).

[0103] (H) In (F) or (G), the product has at least two features selected from (1) to (4).

[0104] (I) In any of (F) to (H) above, the present invention has at least the features of (1), (2), (3) and (4) above.

[0105] (J) In any of (A) to (E) above, it has at least one feature selected from the following (1-1) to (5-1).

[0106] (1-1) Decrease the number of cysts, testosterone concentration, luteinizing hormone (LH) concentration, and the ratio of luteinizing hormone (LH) concentration to follicle-stimulating hormone (FSH) concentration [LH / FSH]. (2-1) Increase progesterone concentration. (3-1) Restore estrus and improve the periodicity of the estrus cycle. (4-1) Restore the number of ovulations. (5-1) Restore the formation of new corpora lutea.

[0107] (K) In (J), the product has at least one feature selected from (1-1) to (4-1).

[0108] (L) In (J) or (K), the product has at least two features selected from (1-1) to (4-1).

[0109] (M) In any of (J) to (L), the present invention has at least the features of (1-1), (2-1), (3-1) and (4-1).

[0110] The therapeutic or preventive agent for PCOS of the present invention may be specified by combining the matters described in this specification with at least one embodiment selected from these embodiments (A) to (M).

[0111] In addition to the agent for treating or preventing PCOS, the present invention also includes a method for treating (ameliorating or alleviating) or preventing PCOS by administering the pregnane compound (1) or a pharmaceutically acceptable salt thereof as an active ingredient to a subject suffering from PCOS, or a method for treating (ameliorating or alleviating) or preventing PCOS by administering the agent for treating or preventing PCOS (pharmaceutical composition or preparation).

[0112] The present invention also encompasses use of the pregnane compound (1) or a pharmaceutically acceptable salt thereof as an active ingredient for treating (improving or alleviating) or preventing PCOS, or use of the therapeutic or preventive agent for PCOS (pharmaceutical composition or preparation) for treating (improving or alleviating) or preventing PCOS; and use of the pregnane compound (1) or a pharmaceutically acceptable salt thereof for producing the therapeutic or preventive agent for PCOS (drug for treating or preventing PCOS).

[0113] Furthermore, the present invention also encompasses the pregnane compound (1) or a pharmaceutically acceptable salt thereof for treating (ameliorating or alleviating) or preventing PCOS.

[0114] The present invention also encompasses a pharmaceutical composition (or preparation) comprising at least the pregnane compound (1) or a pharmaceutically acceptable salt thereof; a pharmaceutical composition (or preparation) comprising at least the pregnane compound (1) or a pharmaceutically acceptable salt thereof and a carrier; and a pharmaceutical composition (or preparation) for treating (ameliorating or alleviating) or preventing PCOS, which comprises the pregnane compound (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0115] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. The materials and evaluation methods used in the following examples are shown below. The masses of the materials used are all solid content masses.

[0116] [Materials used] Letrozole: manufactured by Tokyo Chemical Industry Co., Ltd. Pregnane compound: 17α-acetoxy-6-chloro-15β-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione Flutamide: manufactured by Sigma-Aldrich Co., Ltd. Metformin: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Spironolactone: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. CE2: general feed, manufactured by CLEA Japan, Inc. HFD32: high-fat diet, manufactured by CLEA Japan, Inc.

[0117] Test Example 1 Using letrozole-induced rats in which the estrus phase had been abolished by letrozole, the effect on a PCOS model was examined.

[0118] Specifically, letrozole was administered repeatedly at a dose of 0.1 mg / kg to rats (female, Wistar-Imamichi, 8 weeks old; manufactured by Japan SLC Co., Ltd.) once daily, and smear observations were initiated one week later. Two weeks after letrozole administration, the rats were assigned to univariate block allocations based on body weight, with eight rats per group. Each test substance was administered for 3 to 5 weeks, and smear observations were continued. Vaginal smear samples were collected from the rats, and the estrous cycle was evaluated by observing smear images under a microscope. The proportions of each estrous cycle [proestrus, estrus, metestrus, and diestrus (interphase)] are shown in Figure 1. The composition of the test groups is shown in Table 1.

[0119]

[0120] In the test system, a 0.5 w / v % aqueous solution of methylcellulose and a 0.5 w / v % aqueous solution of polysorbate 80 were used as vehicles for administering the treatment reagent and test substance.

[0121] Furthermore, 5 to 7 weeks after the start of treatment reagent administration, estrus rats were euthanized by exsanguination under deep anesthesia, and the ovaries were removed. The oviducts were separated from the removed ovaries and immersed in a dish containing 450 μL of L-15 medium (Leibovitz's L-15 medium (containing L-glutamine, phenol red, and sodium pyruvate), Fujifilm Wako Pure Chemical Industries, Ltd.). Under a microscope, a perfusion needle was inserted through the ovarian inlet, and oocyte cumulus cell complexes (COCs) were expelled. 50 μL of hyaluronidase solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was left at room temperature for 1 hour. The oocytes were denuded and counted under a microscope. The removed ovaries were evaluated for intraovarian pathology (number of cysts and number of rats with newly formed corpora lutea). The results are shown in Table 2. Furthermore, the concentrations of various hormones in the collected blood were measured. Table 3 shows the results.

[0122] In Test Example 1, the methods for measuring the estrus period, the number of rats that ovulated, the concentrations of various hormones, and intraovarian pathology were as follows.

[0123] [Method for measuring estrus period] The estrus cycle after administration of the test substance was calculated using formula (1).

[0124] [Calculation formula (1)] Prophase: (Prophase smears observed from the start date of administration of the test substance, etc. to the date of autopsy / X) x 100 (%) Estrus: (Estrus smears observed from the start date of administration of the test substance, etc. to the date of autopsy / X) x 100 (%) Late: (Late smears observed from the start date of administration of the test substance, etc. to the date of autopsy / X) x 100 (%) Interphase: (Interphase smears observed from the start date of administration of the test substance, etc. to the date of autopsy / X) x 100 (%) *X means the number of days from the start date of administration of the test substance, etc. to the date of autopsy, and will differ for each individual.

[0125] [Number of rats that ovulated (number of ovulating rats)] After oviductal irrigation under a microscope, rats in which ova were confirmed were determined as ovulating rats, and the number of ova ovulated by each rat was counted. The average value is shown in FIG.

[0126] [Hormones Concentration] Testosterone (T), Progesterone (P4): Collected blood was centrifuged at 3000 rpm and 4°C for 10 minutes to separate the plasma. 50 μL of acetonitrile was added to 50 μL of the separated plasma, followed by mixing. 50 μL of internal standard solution was then added and stirred. 50 μL of 5 mol / L ammonium acetate was then added and stirred. The mixture was then centrifuged at 3500 rpm and 4°C for 5 minutes to separate the plasma. 200 μL of 50% acetonitrile was added to 50 μL of the upper layer and stirred to prepare a pretreated sample. The sample was analyzed using a liquid chromatograph mass spectrometer (LC / MS / MS) to measure the concentrations of each hormone in the plasma (calibration curve range: T: 0.01-10 ng / mL, P4: 0.1-100 ng / mL).

[0127] Luteinizing hormone (LH), follicle-stimulating hormone (FSH): Using 25 μL of plasma, measurements were performed by MILLIPLEX® multiplex assay using MILLIPLEX MAP Rat Pituitary Magnetic Bead Panel - Endocrine Multiplex Assay (Merck KGaA) (calibration curve range: LH: 3.2 to 10,000 pg / mL, FSH: 32 to 100,000 pg / mL).

[0128] Ovarian pathology (number of cysts and rats with neo-luteal formation) Ovaries were fixed in 10% by volume neutral buffered formalin, embedded in paraffin (Sakura Finetech Japan Co., Ltd.), and thinly sliced ​​to a thickness of 4 μm using a microtome. The resulting thin sections were stained with hematoxylin (Sakura Finetech Japan Co., Ltd.) and eosin (Sakura Finetech Japan Co., Ltd.). The number of cysts and the number of rats with neo-luteal formation were counted using an optical microscope for the hematoxylin-eosin-stained ovarian tissue. Cystic atretic follicles were counted as cysts, and basophilic, small, spindle-shaped luteal bodies with neovascularization in the granulosa cell layer were counted as neo-luteal formation.

[0129] [Statistical Analysis] A two-tailed Student's t-test was performed between the normal group and the control group. A risk of less than 5% was considered to be statistically significant, and significant differences were indicated by "*" (p<0.05). A Dunnett's multiple comparison test was performed between the control group and the A3, A10, A30, and B30 groups. A risk of less than 5% was considered to be statistically significant, and significant differences were indicated by "#" (p<0.05). A two-tailed Student's t-test was performed between the B30 group and the A3, A10, and A30 groups. A risk of less than 5% was considered to be statistically significant, and significant differences were indicated by "$" (p<0.05).

[0130] As is clear from Figure 1, compared to the normal group, the disappearance of estrus was confirmed in all cases in the control group, whereas when the pregnane compound was administered, the recovery of estrus that had disappeared in the control group was confirmed (groups A3, A10, and A30).

[0131]

[0132]

[0133] As is clear from Table 2 and Figure 2, when a pregnane compound was administered to treat ovulation disorders caused by letrozole treatment, the number of rats that ovulated increased in a dose-dependent manner, and in the A30 group, which received 30 mg / kg of a pregnane compound, the average number of ovulations was 6.3 ± 6.5, a significant increase.

[0134] Furthermore, as is clear from Table 2, the number of polycystic ovaries observed in the control group was significantly reduced by administering 30 mg / kg of the pregnane compound, and polycystic ovaries were improved. In contrast, only a slight reduction was observed with flutamide.

[0135] Furthermore, as is clear from Table 2, neo-luteal formation (corpora lutea within one sexual cycle after ovulation), which was no longer observed in the control group, was observed in 1 / 8 or 5 / 8 cases after administration of 10 mg / kg or 30 mg / kg of a pregnane compound. Neo-luteal formation was also observed in 2 / 8 cases with flutamide, but the number was lower than that of the pregnane compound at the same dose.

[0136] As is clear from Table 3, letrozole treatment in the control group increased testosterone and LH levels and decreased progesterone levels, while administration of the pregnane compound improved the balance of these hormone levels. In particular, administration of 10 mg / kg of the pregnane compound significantly decreased testosterone and LH levels, and administration of 30 mg / kg significantly increased progesterone levels, restoring them to the same level as the normal group. In contrast, flutamide had a weak effect on improving hormone levels.

[0137] Test Example 2 By extending the administration period of the pregnane compound to a longer period than in Test Example 1, the effect on the PCOS model was examined using the following letrozole-induced rats.

[0138] Specifically, rats (female, Wistar-Imamichi, 8 weeks old; Animal Reproduction Research Institute) were assigned to univariate block allocation based on body weight, with 10 rats in each group. Starting the day after group assignment, all groups except the control group were repeatedly administered letrozole at a dose of 0.1 mg / kg once daily, and smear observations began six weeks later. Two weeks after letrozole administration, each test substance was administered for six to eight weeks, and smear observations were continued. Vaginal smears were collected from the rats, and the estrous cycle was evaluated by observing smear images under a microscope. The proportions of each estrous cycle [proestrus, estrus, metestrus, and diestrus (interphase)] are shown in Figure 3. The composition of the test groups is shown in Table 4.

[0139]

[0140] In the test system, a 0.5 w / v % aqueous solution of methylcellulose and a 0.5 w / v % aqueous solution of polysorbate 80 were used as vehicles for administering the treatment reagent and test substance.

[0141] Furthermore, 8 to 10 weeks after the start of treatment reagent administration, estrus rats were euthanized by exsanguination under deep anesthesia, and the ovaries were removed. The oviducts were separated from the removed ovaries and placed in a dish containing 450 μL of L-15 medium (Leibovitz's L-15 medium (containing L-glutamine, phenol red, and sodium pyruvate), Fujifilm Wako Pure Chemical Industries, Ltd.) and 50 μL of hyaluronidase (Fujifilm Wako Pure Chemical Industries, Ltd.) + L-15 medium solution (a solution of hyaluronidase dissolved in L-15 medium at a concentration of 0.2%). Under a microscope, a perfusion needle was inserted into the oviduct from the ovarian entrance, and oocyte cumulus cell complexes (COCs) were expelled. The oocytes were denuded by immersion for several minutes, and counted under a microscope. The results of evaluation of intraovarian pathology (cyst count) from the removed ovaries are shown in Table 5. Furthermore, the results of measuring the concentrations of various hormones in the collected blood are shown in Table 6. In Table 5, the parameter for the number of ovulating rats in group A10 differs from the number of specimens in Table 4. This is because there was one case where the number of ovulations deviated when confirming the number of ovulations, and other parameters of the rats (estrous cycle, hormone concentrations, ovarian histopathological evaluation) were measured.

[0142] In Test Example 2, the methods for measuring the estrus period, the number of rats that ovulated, the concentrations of various hormones, and intraovarian pathology were as follows.

[0143] [Method for measuring estrus period] The estrus cycle after administration of the test substance was calculated using formula (2).

[0144] [Calculation formula (2)] Percentage of each sexual cycle: A / B x 100 (%) *A means the number of days on which a smear was confirmed for each sexual cycle from day 43 to day 50 (8 days) after starting letrozole administration. B is 8 days (days 43 to 50 after starting letrozole administration).

[0145] [Number of rats that ovulated (number of ovulating rats)] After oviductal perfusion under a microscope, rats in which ova were confirmed were determined as ovulating rats, and the number of ova from each rat was counted. The average value is shown in FIG.

[0146] [Concentrations of Various Hormones] Testosterone (T), Progesterone (P4): After mixing the internal standard solution and methanol, the solvent was removed using a centrifugal evaporator. 0.2 mL of plasma was added to the residue to prepare the measurement sample. 0.3 mL of water was added to the measurement sample, followed by stirring. 2 mL of t-butyl methyl ether was then added and stirred again. After centrifugation, the separated organic layer was distilled off, and 0.5 mL of methanol and 1 mL of water were added to the residue and stirred. The sample was loaded onto a solid-phase extraction column previously conditioned with methanol and water. After sequential washing with water, a methanol / water / acetic acid mixture (45:55:1 (volume ratio)), and a water / ammonia water mixture (100:1 (volume ratio)), testosterone (T) and progesterone (P4) were eluted with 1 mL of methanol. After removing the eluate, 0.1 mL of an acetonitrile / water mixture (4:6) was added to the residue and stirred to prepare the pretreated sample. The samples were analyzed using a liquid chromatograph mass spectrometer (LC / MS / MS) to measure the plasma concentrations of each hormone (calibration curve range T: 1 to 2500 pg / mL, P4: 0.25 to 100 ng / mL).

[0147] Luteinizing hormone (LH), follicle-stimulating hormone (FSH): Using 25 μL of plasma, measurements were performed by MILLIPLEX® multiplex assay using MILLIPLEX MAP Rat Pituitary Magnetic Bead Panel - Endocrine Multiplex Assay (Merck KGaA) (calibration curve range: LH: 3.2 to 10,000 pg / mL, FSH: 32 to 100,000 pg / mL).

[0148] [Intraovarian pathology (cyst count)] Ovaries were fixed in 10% by volume neutral buffered formalin, embedded in paraffin (Sakura Finetech Japan Co., Ltd.), and thinly sliced ​​to a thickness of 4 μm using a microtome. The obtained thin sections were stained with hematoxylin (Sakura Finetech Japan Co., Ltd.) and eosin (Sakura Finetech Japan Co., Ltd.). The number of cysts in the hematoxylin-eosin-stained ovarian tissue was counted using an optical microscope. Cystic atretic follicles were counted as cysts.

[0149] [Statistical Analysis] A two-tailed Student's t-test was performed between the normal group and the control group. A risk of less than 5% was considered to be statistically significant, and significant differences were indicated by a "*" (p<0.05). A Dunnett's multiple comparison test was performed between the control group and groups A1, A3, A10, and A30. A risk of less than 5% was considered to be statistically significant, and significant differences were indicated by a "#" (p<0.05). A two-tailed Student's t-test was performed between the control group and groups B30 and C5. A risk of less than 5% was considered to be statistically significant, and significant differences were indicated by a "†" (p<0.05). In addition, Dunnett's multiple comparison test was performed between group B30 or group C5 and groups A1, A3, A10, and A30, and a risk level of less than 5% was determined to be statistically significant, and significance was indicated by "$" or "‡" (p<0.05).

[0150] As is clear from Figure 3, compared to the normal group, the disappearance of estrus was confirmed in all cases in the control group, whereas when the pregnane compound was administered, the recovery of estrus that had disappeared in the control group was confirmed (Groups A1, A3, A10, and A30).

[0151]

[0152]

[0153] As is clear from Table 5 and Figure 4, when a pregnane compound was administered to treat ovulation disorders caused by letrozole treatment, the number of rats that ovulated increased in a dose-dependent manner, and in the A30 group, which received 30 mg / kg of a pregnane compound, the average number of ovulations was 5.0 ± 5.2, a significant increase.

[0154] Furthermore, as is clear from Table 5, the number of cysts observed in the control group was significantly reduced by the administration of 30 mg / kg of the pregnane compound, and an improvement in polycystic ovaries was observed. In contrast, no reduction was observed with flutamide or spironolactone.

[0155] As is clear from Table 6, letrozole treatment in the control group increased testosterone and LH levels and decreased progesterone levels, while administration of a pregnane compound improved the balance of these hormone levels. In particular, administration of 10 mg / kg of a pregnane compound significantly decreased testosterone and LH levels and significantly increased progesterone levels. Administration of 30 mg / kg of a pregnane compound also significantly decreased the LH / FSH ratio. Thus, by extending the administration period of the pregnane compound beyond that of Test Example 1, a statistically significant pharmacological activity was observed in the PCOS model, even when the dose of the pregnane compound was reduced. In contrast, flutamide and spironolactone did not show any effect of improving hormone levels.

[0156] Test Example 3 Using high-fat diet-loaded letrozole-induced rats that had been fed a high-fat diet and had their estrus phase abolished with letrozole (Tokyo Chemical Industry Co., Ltd.), the effect on insulin-resistant PCOS model rats (IR-PCOS model) was investigated.

[0157] Specifically, letrozole was repeatedly administered once daily at a dose of 0.1 mg / kg to rats (female, Iar: Wistar-Imamichi, 4 weeks old; Japan SLC Co., Ltd.), and the rats were allowed to eat a high-fat diet ad libitum. Smear observations were initiated one week later. After 12 weeks of letrozole administration, each test substance was administered for 3 to 5 weeks, and smear observations were continued. Vaginal smears were collected from the rats, and the estrous cycle was evaluated by observing smear images under a microscope. The proportions of each estrous cycle [proestrus, estrus, metestrus, and diestrus (interphase)] are shown in Figure 5. The composition of the test groups is shown in Table 7.

[0158]

[0159] In the test system, a 0.5 w / v % aqueous solution of methylcellulose and a 0.5 w / v % aqueous solution of polysorbate 80 were used as vehicles for administering the treatment reagent and test substance.

[0160] After 3 weeks of administration of the test substance, etc., the animals were fasted for 17 hours, and then euthanized by collecting all blood samples under deep anesthesia, and the ovaries were removed. The results of evaluating the pathology (number of cysts) of the removed ovaries are shown in Table 8. Furthermore, the results of measuring the concentrations of various hormones, fasting blood glucose levels, insulin concentrations, and HOMA-IR in the collected blood are shown in Table 9. Figure 6 also shows the changes in body weight between the day before the start of administration of the test substance, etc., and the day of dissection.

[0161] For animals used to evaluate ovulation numbers, estrus rats were euthanized under deep anesthesia 3 to 5 weeks after administration of the test substance, and the ovaries were removed. The oviducts were separated from the removed ovaries and immersed in a dish containing 450 μL of L-15 medium (Leibovitz's L-15 medium (containing L-glutamine, phenol red, and sodium pyruvate), Fujifilm Wako Pure Chemical Industries, Ltd.). Under a microscope, a perfusion needle was inserted through the ovarian inlet, and oocyte cumulus cell complexes (COCs) were expelled. 50 μL of hyaluronidase solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was left at room temperature for 1 hour. The oocytes were denuded and counted under a microscope. The average ovulation number for each individual is shown in Figure 7.

[0162] The methods for measuring the estrous cycle, intraovarian pathology, number of rats that ovulated, and various hormone concentrations were the same as in Test Example 1, and the methods for measuring body weight changes, fasting blood glucose levels, insulin concentrations, and HOMA-IR were as follows.

[0163] [Weight Fluctuations] The body weights of the animals were measured on the day before the start of administration of the test substance, etc. and on the day of dissection. The weight ratio (%) on the day of dissection, with the body weight on the day before the start of administration of the test substance, etc. being taken as 100%, was calculated as the weight fluctuation.

[0164] [Fasting Blood Glucose Levels] Fasting blood glucose levels were measured using Laboassay® Glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the accompanying measurement instructions. Specifically, 300 μL of the accompanying color-developing reagent was added to 2 μL of plasma, and the reaction was allowed to proceed for 5 minutes to develop color. Absorbance at 505 nm was measured using a microplate reader, and blood glucose levels were calculated using a calibration curve (calibration curve range: 50-500 mg / dL).

[0165] [Insulin Concentration] Measurements were performed using an ultrasensitive rat insulin assay kit (manufactured by Morinaga Biochemical Research Institute, Inc.) according to the assay instructions provided with the kit. Specifically, 5 μL of plasma was added to the plate provided with the kit along with 95 μL of sample diluent, and the mixture was allowed to react for 2 hours. After washing, 100 μL of insulin antibody solution was added and the mixture was allowed to react for 30 minutes. After washing, 100 μL of enzyme substrate solution was added, and after 40 minutes of reaction, 100 μL of stop solution was added. Absorbance at 450 nm was measured using a microplate reader, and a calibration curve was prepared in the range of 0.1 to 6.4 ng / mL.

[0166] [HOMA-IR] HOMA-IR was calculated using the formula (3).

[0167] [Calculation formula (3)] HOMA-IR = fasting blood glucose × insulin concentration × 26 / 405

[0168] [Statistical Analysis] A two-tailed Student's t-test was performed between the normal group and the control group, and a risk of less than 5% was considered to be statistically significant, with significance indicated by "*" (p<0.05). A Dunnett's multiple comparison test was also performed between the vehicle group (control group) and groups A3, A10, A30, and B30, with a risk of less than 5% considered to be statistically significant, with significance indicated by "#" (p<0.05). A Dunnett's multiple comparison test was also performed between group B30 and groups A3, A10, and A30, with a risk of less than 5% considered to be statistically significant, with significance indicated by "$" (p<0.05).

[0169] As is clear from Figure 5, the recovery of the estrous cycle was confirmed in the IR-PCOS model at doses of 3 mg / kg or more for the pregnane compound. That is, the improvement of menstrual irregularities in PCOS accompanied by obesity and insulin resistance was confirmed with the pregnane compound.

[0170]

[0171]

[0172] As is clear from Table 8 and Figure 7, when a pregnane compound was administered at a dose of 30 mg / kg to treat ovulation disorders caused by letrozole treatment, the number of rats that ovulated increased compared to the control group, with the average number of ovulations being 2.0 ± 2.3, whereas the number of rats that ovulated did not increase when flutamide or metformin was administered.

[0173] Furthermore, a decrease in the number of cysts was observed with the pregnane compound, and neo-luteal formation was also observed at the dose of 30 mg / kg, where natural ovulation occurred. However, no decrease in the number of cysts was observed with flutamide or metformin, and no neo-luteal formation was observed in any individual.

[0174] As is clear from Table 9, the control group showed a significant increase in testosterone concentration and LH concentration and a significant decrease in progesterone concentration, as in Test Example 1, whereas administration of the pregnane compound decreased testosterone concentration and LH concentration and increased progesterone concentration depending on the dose.

[0175] Furthermore, administration of pregnane compounds had no effect on insulin resistance-related indices (fasting blood glucose, insulin concentration, HOMA-IR) that were increased in the control group. Flutamide also had no effect, while metformin significantly reduced insulin concentration and HOMA-IR.

[0176] As is clear from FIG. 6, when a pregnane compound was administered to the mice in response to weight gain caused by letrozole treatment and a high-fat diet, a dose-dependent suppression of weight gain was confirmed.

[0177] (Preparation of Formulations) Formulation examples are shown below, but as mentioned above, the same component of the carrier may perform multiple functions or roles, although to varying degrees. Therefore, the carriers used in the following formulation examples are not limited to the types of carriers classified, and can be replaced with other types of carriers depending on the function or role. For example, the following formulation example uses pregelatinized starch as a disintegrant, not as a binder.

[0178] Formulation Example 1 300 g of the active ingredient was mixed with excipients (lactose hydrate 2415 g, crystalline cellulose 1290 g, corn starch 450 g), a disintegrant (pregelatinized starch 450 g), and a lubricant (magnesium stearate 45 g), and the resulting mixture was compressed into tablets to produce tablets (20 mg tablets) containing the ingredients shown in Table 10 below per tablet.

[0179]

[0180] Formulation Example 2 100 g of the active ingredient was mixed with excipients (lactose hydrate 1810 g, crystalline cellulose 1000 g, corn starch 300 g), a disintegrant (pregelatinized starch 60 g), and a lubricant (magnesium stearate 30 g), and the resulting mixture was compressed into tablets to produce tablets (5 mg tablets) containing the ingredients shown in Table 11 below per tablet.

[0181]

[0182] Formulation Example 3 75 g of the active ingredient was mixed with excipients (lactose hydrate 2790 g, crystalline cellulose 1500 g, corn starch 450 g), a disintegrant (pregelatinized starch 90 g), and a lubricant (magnesium stearate 45 g), and the resulting mixture was compressed into tablets to produce tablets (2.5 mg tablets) containing the ingredients shown in Table 12 below per tablet.

[0183]

[0184] Formulation Example 4 300 g of the active ingredient was mixed with excipients (lactose hydrate 2415 g, crystalline cellulose 1290 g, corn starch 450 g), a disintegrant (pregelatinized starch 450 g), and a lubricant (magnesium stearate 45 g), and the resulting mixture was compressed into tablets to produce tablets (10 mg tablets) containing the ingredients shown in Table 13 below per tablet.

[0185]

[0186] Formulation Example 5 15 g of the active ingredient was mixed with excipients (lactose hydrate 2850 g, crystalline cellulose 1500 g, corn starch 450 g), a disintegrant (pregelatinized starch 90 g), and a lubricant (magnesium stearate 45 g), and the resulting mixture was compressed into tablets to produce tablets (0.5 mg tablets) containing the ingredients shown in Table 14 below per tablet.

[0187]

[0188] These tablets were stored in a thermostatic chamber at a temperature of 40° C. and a relative humidity of 75% for 4 and 8 weeks, but almost no discoloration of the tablets was observed.

[0189] The PCOS therapeutic or preventive agent of the present invention can be used for mammals, and can be effectively used as a therapeutic or preventive agent (therapeutic or prophylactic drug) for treating (improving or alleviating) or preventing the symptoms of PCOS in preferably human females (particularly females of reproductive age).

Claims

1. A therapeutic or prophylactic agent for PCOS comprising 17α-acetoxy-6-chloro-15β-hydroxy-2-oxa-4,6-pregnadiene-3,20-dione or a pharmaceutically acceptable salt thereof as an active ingredient.

2. A PCOS treatment or preventive agent according to claim 1, which improves at least one symptom selected from the group consisting of menstrual cycle abnormalities, ovarian hypertrophy, polycystic ovary syndrome, high anti-Müllerian hormone levels, androgen excess, high luteinizing hormone levels, high luteinizing hormone / follicle-stimulating hormone ratio, acne, infertility, hirsutism, obesity, male pattern baldness, and acanthosis nigricans.

3. A PCOS treatment or preventive agent according to claim 1 or 2, which improves at least two symptoms selected from the group consisting of menstrual cycle disorders, polycystic ovary syndrome, and androgen hypertension.

4. A treatment or preventive agent for PCOS according to claim 1 or 2, which improves menstrual cycle disorders, polycystic ovary syndrome, and androgen hypertension.

5. A PCOS treatment or preventive agent according to claim 1 or 2, having at least one feature selected from (1) to (4) below. (1) Reduce at least one selected from the number of cysts, testosterone concentration, luteinizing hormone concentration, and the ratio of luteinizing hormone concentration to follicle-stimulating hormone concentration. (2) Increase progesterone levels (3) To restore the estrus period and improve the periodicity of the estrus cycle. (4) Restore the number of ovulations

6. A PCOS treatment or prophylactic agent according to claim 1 or 2, for treating or preventing PCOS in patients with obesity and / or insulin resistance.

7. A PCOS treatment or preventive agent according to claim 1 or 2, for treating or preventing PCOS in women of reproductive age.

8. A PCOS treatment or preventive agent according to claim 1 or 2, which is a solid preparation, a semi-solid preparation, or a liquid preparation.

9. The PCOS treatment or prophylactic agent according to claim 1 or 2, which is an orally administered formulation.

10. A PCOS treatment or preventive agent according to claim 1 or 2, in the form of a tablet.