Agents for promoting or improving decidualization of endometrium

Abstract

Provided is a preparation with which it is possible to promote normal decidualization of the endometrium. An agent for promoting or improving decidualization of the endometrium, the agent containing an effective amount of pyrroloquinoline quinone or a salt thereof, is prepared. Furthermore, an agent for promoting or improving cell fusion, the agent containing an effective amount of pyrroloquinoline quinone or a salt thereof, is prepared.

Description

Agents for promoting or improving endometrial decidualization

[0001] The present invention relates to an agent for promoting or improving endometrial decidualization.

[0002] According to the Japan Society of Obstetrics and Gynecology, the number of annual infertility treatments has risen sharply from approximately 10,000 in 2004 to over 400,000 in 2015, and has remained at around 450,000 since then, indicating that a large number of couples are struggling with infertility.

[0003] In Japan, from 2022, infertility treatments will be covered by insurance for "general infertility treatments" such as artificial insemination, and "assisted reproductive technology" such as in vitro fertilization and intracytoplasmic sperm injection, helping to ease the financial burden that comes with the many worries associated with infertility.

[0004] However, infertility treatments covered by insurance for women have age restrictions and limitations on the number of treatments that can be given, and just because insurance is available does not mean that women's worries about infertility have been fully resolved.

[0005] Female infertility is classified according to its causes into ovarian factors, fallopian tube factors, and uterine factors. However, the ovaries, fallopian tubes, and uterus all crosstalk with each other, and unless this mechanism is elucidated, it will be difficult to understand the complex physiological phenomena of ovulation, fertilization, implantation, and embryo development, and to find a fundamental solution to infertility.

[0006] Among these complex physiological phenomena, the present inventors have focused on the decidualization of endometrial cells, and have hypothesized that failure to undergo normal decidualization may lead to implantation disorders and miscarriage. They have therefore established a test system that simulates decidualization in human endometrial cells in vitro and have been studying "normal decidualization" (Non-Patent Document 1).

[0007] On the other hand, pyrroloquinoline quinone (PQQ) is an organic molecule found in bacteria as a redox coenzyme, and is a highly safe food material with extremely diverse functions. Previous studies on PQQ's infertility have shown that it has the effect of increasing sperm activity (Patent Document 1) and improving the normality of eggs and ovaries (Patent Document 2).

[0008] International Publication No. WO 2020 / 013086 International Publication No. WO 2020 / 262325

[0009] Kazuya Kusama et al. Biochemical and Biophysical Research Communications 571 (2021) 174-180

[0010] According to the above patent documents, it has been revealed that the antioxidant effect of PQQ suppresses damage to DNA caused by oxidation in sperm and eggs, thereby resulting in sperm with high motility and eggs with high fertilization ability.

[0011] However, even if a normal sperm and egg are successfully fertilized, unless the aforementioned "normal endometrial decidualization" occurs, the implantation period (which is extremely precise) will not be synchronized, and implantation will not occur. Furthermore, if "normal endometrial decidualization" does not occur, excessive decidualization may cause endometrial cells to become senescent cells. In other words, promoting endometrial decidualization while minimizing abnormal senescence is "promoting normal endometrial decidualization," and is an essential condition for implantation.

[0012] Therefore, the present inventors have intensively investigated whether PQQ can "promote normal endometrial decidualization." As a result, they have found that PQQ can significantly promote decidualization while suppressing the expression of waste products and aging genes, which are indicators of senescent cells, and have discovered that PQQ acts to "promote normal endometrial decidualization" and promotes implantation. Furthermore, the present inventors have found that PQQ promotes the fusion of placental trophoblast cells, which is important for the completion of pregnancy. Then, they have focused on the fact that PQQ leads to improved fertility and have completed the present invention.

[0013] That is, a preferred embodiment of the present invention provides the following oral compositions: [1] An agent for promoting or improving endometrial decidualization, which comprises an effective amount of pyrroloquinoline quinone or a salt thereof.

[0014] [2] An endometrial anti-aging agent containing an effective amount of pyrroloquinoline quinone or a salt thereof.

[0015] [3] The agent according to [2], wherein the anti-aging of the endometrium is inhibition of transformation of endometrial cells into senescent cells.

[0016] [4] The agent according to [2], wherein the anti-aging of the endometrium is removal of senescent cells in the endometrium.

[0017] [5] An agent for promoting or improving placental trophoblast cell fusion, comprising an effective amount of pyrroloquinoline quinone or a salt thereof.

[0018] [6] A fertility enhancer comprising an effective amount of pyrroloquinoline quinone or a salt thereof, which promotes or improves endometrial decidualization and / or promotes or improves placental trophoblast cell fusion.

[0019] [7] The agent according to [6], wherein the improvement in fertility is improvement in implantation or continuation of the implantation state.

[0020] [8] The agent according to any one of [1] to [7], which is to be taken at least 7 to 21 days after the first day of menstruation.

[0021] [9] The agent according to any one of [1] to [7], which is to be taken at least during the period from 14 to 35 days after the first day of menstruation.

[0022] According to the present invention, PQQ can significantly promote decidualization while suppressing the expression of waste products and senescence genes, which are indicators of senescence, and therefore it is possible to provide an agent for promoting or improving endometrial decidualization and an agent for promoting or improving placental trophoblast cell fusion.

[0023] Figure 1 shows the results of a cytotoxicity test in Test Example 1. Figure 2 shows the results of a test to promote decidualization of primary endometrial stromal cells (pESCs) in Test Example 2-1. Figure 3 shows the results of a test to promote epithelial differentiation of Ishikawa cells and EM1 cells in Test Example 2-2. Figure 4 shows the results of a test to inhibit senescence-related β-galactosidase associated with decidualization of pESCs in Test Example 3-1. Figure 5 shows the results of a test to inhibit the senescence gene p53 and promote anti-senescence genes associated with decidualization of pESCs in Test Example 3-2. Figure 6 shows the results of a gene expression test in endometrial cells of PQQ-drinking mice in Test Example 4-1. Figure 7 shows photographs and graphs illustrating immunostaining in the endometrium of PQQ-drinking mice in Test Example 4-2. Figure 8 shows the results of an implantation rate evaluation test in PQQ-drinking mice in Test Example 5. Figure 9 shows the results of a gene expression test serving as an indicator of cell fusion in Test Example 6. Figure 10 shows photographs illustrating the state of cell fusion in Test Example 7-1. Figure 11 is a graph showing the change in the number of fused cells in Test Example 7-2.

[0024] [Agent for Promoting or Improving Endometrial Decidualization] The preparation of this embodiment contains an effective amount of pyrroloquinoline quinone or a salt thereof.

[0025] (Pyrroloquinoline quinone or a salt thereof) Pyrroloquinoline quinone is a compound represented by the following formula 1: It is a known compound represented by the formula:

[0026] Examples of salts of pyrroloquinoline quinone include alkali metal salts, alkaline earth metal salts, and ammonium salts. Examples of alkali metal salts include sodium salts, potassium salts, and lithium salts. Examples of alkaline earth metal salts include calcium salts and magnesium salts.

[0027] As pyrroloquinoline quinone or a salt thereof, an alkali metal salt of pyrroloquinoline quinone is preferred, a sodium salt of pyrroloquinoline quinone is more preferred, and a disodium salt of pyrroloquinoline quinone is even more preferred.

[0028] Commercially available pyrroloquinoline quinone or a salt thereof may be used. One type of pyrroloquinoline quinone or a salt thereof may be used alone, or two or more types may be used in combination.

[0029] From the viewpoint of significantly exhibiting the effects of the present invention, the content of pyrroloquinoline quinone or a salt thereof may be, for example, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, etc., based on the total amount of the formulation. Furthermore, the content of pyrroloquinoline quinone or a salt thereof may be, for example, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.5% by mass or less, 0.2% by mass or less, etc., based on the total amount of the formulation. The content of pyrroloquinoline quinone or a salt thereof may be, for example, 0.001 to 30 mass%, 0.001 to 25 mass%, 0.001 to 20 mass%, 0.001 to 15 mass%, 0.001 to 10 mass%, 0.001 to 5 mass%, 0.001 to 1 mass%, 0.001 to 0.5 mass%, 0.001 to 0.2 mass%, 0.005 to 30 mass%, 0.005 to 25 mass%, 0.005 to 20 mass%, 0.005 to 15 mass%, 0.005 to 10 mass%, 0.005 to 5 mass%, 0.005 to 1 mass%, 0.005 to 0.5 mass%, 0.005 to 0.2 mass%, 0.01 to 30 mass%, 0.01 to 25 mass%, 0.01 to 20 mass%, 0.01 to 15 mass%, 0.01 to 10 mass%, 0.01 to 5 mass%, 0.01 to 1 mass%, 0.01 to 0.5 mass%, 0. 01-0.2% by mass, 0.05-30% by mass, 0.05-25% by mass, 0.05-20% by mass, 0.05-15% by mass, 0.05-10% by mass, 0.05-5% by mass, 0.05-1% by mass, 0.05-0.5% by mass, 0.05-0.2% by mass, etc.

[0030] The daily intake of pyrroloquinoline quinone or a salt thereof may vary depending on the condition of the individual taking it (body weight, age, sex, etc.), the formulation form, etc., but from the viewpoint of significantly exhibiting the effects of the present invention, examples include 1 mg or more, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 6 mg or more, 7 mg or more, 8 mg or more, 9 mg or more, 10 mg or more, 15 mg or more, 20 mg or more, etc. Furthermore, examples of the daily intake of pyrroloquinoline quinone or a salt thereof include 100 mg or less, 80 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 25 mg or less, etc. Examples of the daily intake of pyrroloquinoline quinone or a salt thereof include 5 to 100 mg, 5 to 80 mg, 5 to 60 mg, 5 to 50 mg, 5 to 40 mg, 5 to 30 mg, 5 to 25 mg, 10 to 100 mg, 10 to 80 mg, 10 to 60 mg, 10 to 50 mg, 10 to 40 mg, 10 to 30 mg, 10 to 25 mg, 15 to 100 mg, 15 to 80 mg, 15 to 60 mg, 15 to 50 mg, 15 to 40 mg, 15 to 30 mg, 15 to 25 mg, 20 to 100 mg, 20 to 80 mg, 20 to 60 mg, 20 to 50 mg, 20 to 40 mg, 20 to 30 mg, and 20 to 25 mg.

[0031] The preparation according to this embodiment may contain pharmacologically active ingredients or physiologically active ingredients other than pyrroloquinoline quinone or a salt thereof, as long as the effects of the present invention are not impaired.

[0032] Specific examples of such pharmacologically active ingredients or physiologically active ingredients include ubiquinone (coenzyme Q10), vitamin B1, vitamin B2, vitamin B6, folic acid, vitamin B12, vitamin C, vitamin D, vitamin A, vitamin E, niacin, calcium pantothenate, taurine, carnosine, anserine, balenine, citrulline, γ-aminobutyric acid, valine, leucine, isoleucine, glycine, arginine, ornithine, carnitine, glutamic acid, and glutamic acid. Examples of suitable antioxidants include amine, creatine, carnitine, luteolin, quercetin, genistin, cyanidin, resveratrol, diosgenin, isoflavone aglycones, isoflavones, lipoic acid, zinc, iron, calcium, selenium, astaxanthin, zeaxanthin, beta-carotene, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, placenta extract, pycnogenol (proanthocyanidin), and equol. From the viewpoint of further enhancing the effects of the present invention, the pharmacologically active ingredient or physiologically active ingredient is preferably one or more selected from the group consisting of ubiquinone (coenzyme Q10), vitamin B12, vitamin C, vitamin E, astaxanthin, zinc, carnitine, resveratrol, isoflavone, equol, pycnogenol (proanthocyanidin), vitamin D, iron, and folic acid, more preferably one or more selected from the group consisting of ubiquinone (coenzyme Q10), astaxanthin, zinc, resveratrol, isoflavone, equol, pycnogenol (proanthocyanidin), vitamin D, iron, and folic acid, and even more preferably one or more selected from the group consisting of zinc, vitamin D, iron, and folic acid. One pharmacologically active ingredient or physiologically active ingredient may be used alone, or two or more may be used in combination.

[0033] [Uses] The endometrium is an essential tissue for the establishment and maintenance of pregnancy. It proliferates and thickens in response to ovarian steroid hormones during the menstrual cycle, transforming into a state known as the decidua, which allows for the implantation of a fertilized egg. During the proliferative phase (after menstruation, from the first day of menstruation to approximately 14 days later), the endometrium proliferates and thickens in preparation for the next ovulation. During the secretory phase (approximately 14 days after the first day of menstruation), the endometrium matures and awaits the fertilized egg. During menstruation, the endometrium is shed and expelled from the body. The endometrium is primarily composed of stromal cells and glandular epithelial cells, and the formation of the decidua through the proliferation of stromal cells is called decidualization.

[0034] As mentioned above, even if a normal sperm and egg are successfully fertilized, if "normal endometrial decidualization" does not occur, the implantation period (which is extremely precise) will not be synchronized and implantation will not occur. Furthermore, if "normal endometrial decidualization" does not occur, excessive decidualization may cause endometrial cells to become senescent cells. In other words, promoting endometrial decidualization while minimizing abnormal senescence is "promoting normal endometrial decidualization" and is an essential condition for implantation.

[0035] In the Examples described below, pyrroloquinoline quinone or a salt thereof (hereinafter simply referred to as PQQ) has been found to be a component capable of promoting endometrial decidualization using primary endometrial stromal cells (pESCs) that maintain the phenotype of actual human endometrial stromal cells.

[0036] PQQ was not confirmed to be toxic to pESCs in Test Example 1. Furthermore, in Test Example 2-1, it was confirmed that when maturation-stimulating components (Db: dibutyryl-cAMP and / or MPA: medroxyprogesterone acetate) that mimic stimulation by ovarian steroid hormones were allowed to act on pESCs together with PQQ, decidualization of pESCs was significantly promoted even at the minimum concentration of PQQ (1 nM).

[0037] In the test system simulating in vitro decidualization in the examples described below, Db and MPA are used to simulate the stimulation by ovarian steroid hormones, but in living organisms, the stimulation of progesterone begins to increase in the late proliferative phase, and the stimulation rises rapidly in the secretory phase (about 14 days after the first day of menstruation).Therefore, from the results of the examples described below, it is preferable to start taking PQQ at least 7 days after the first day of menstruation, and it is preferable to continue taking it after 14 days after the first day of menstruation.In addition, in another embodiment, it is preferable to take PQQ from the first day of menstruation until 21 days after, which is the time when normal decidualization of endometrium is completed and endometrium matures, but it can also be taken up to 35 days after, and is not limited thereto.

[0038] Furthermore, in the Examples described below, when maturation-stimulating components (Db and / or MPA) were applied to pESCs together with PQQ, senescence-associated β-galactosidase was significantly suppressed, and further, suppression of the senescence gene p53 and enhancement of the anti-senescence gene SIRT1 were confirmed, indicating that PQQ has the function of suppressing senescence (anti-senescence) in the maturation of endometrial tissue. This confirmed that PQQ has the effect of suppressing the transformation of endometrial cells into senescent cells and removing senescent cells.

[0039] Furthermore, in the examples described below, it has been confirmed that PQQ promotes endometrial epithelial differentiation and aids and promotes endometrial implantation not only in vitro but also in animal models. It is strongly speculated that oral intake of PQQ promotes normal endometrial decidualization, suppresses senescent cells, and allows the maturation or implantation period to occur in a better state, thereby improving fertility, improving implantation, and maintaining the implantation state.

[0040] (Additives, etc.) The preparation according to this embodiment can contain various additives in addition to the above components, as long as the effects of the present invention are not impaired.

[0041] Specific examples of such additives include excipients, binders, disintegrants, lubricants, colorants, flavoring agents, odorants, sugars, sugar alcohols / polyalcohols, high-intensity sweeteners, oils and fats, emulsifiers, thickeners, acidulants, and fruit juices.

[0042] Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, gelatin, calcium carbonate, kaolin, crystalline cellulose, and silicic acid.

[0043] Examples of binders include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, calcium phosphate, and polyvinylpyrrolidone.

[0044] Disintegrants include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose and the like.

[0045] Lubricants include talc, stearates, borax, polyethylene glycol, and the like.

[0046] Flavoring agents include sucrose, orange peel, citric acid, tartaric acid, etc.

[0047] Examples of sugars include sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, and xylose.

[0048] Examples of sugar alcohols and polyhydric alcohols include sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose starch syrup, glycerin, and propylene glycol.

[0049] High-intensity sweeteners include aspartame, stevia, acesulfame potassium, and sucralose.

[0050] Examples of oils include safflower oil, grape seed oil, sunflower oil, olive oil, corn oil, sesame oil, soybean oil, rapeseed oil, and perilla oil.

[0051] Examples of emulsifiers include sucrose fatty acid esters, glycerin fatty acid esters, and lecithin.

[0052] Examples of thickeners include carrageenan, xanthan gum, guar gum, pectin, and locust bean gum.

[0053] Examples of acidulants include citric acid, lactic acid, malic acid, etc. Examples of fruit juices include lemon juice, orange juice, berry juice, etc.

[0054] The additives may be used alone or in combination of two or more.

[0055] From the viewpoint of the stability of pyrroloquinoline quinone or a salt thereof during formulation, it is preferable to include an oil or fat as an additive.

[0056] (Dosage Form, etc.) The dosage form of the preparation according to this embodiment is not particularly limited, and examples thereof include oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, troche tablets, etc.), granules, powders, soft capsules, hard capsules, troches, jellies, or liquid preparations (including suspensions, emulsions, syrups, etc.); external preparations such as ointments, suppositories, patches, and sprays; injections, etc. Among these, from the viewpoint of ease of handling and further enhancing the effects of the present invention, oral preparations are preferred, and tablets, granules, soft capsules, and hard capsules are more preferred.

[0057] The preparation according to this embodiment can be used as an ingredient of, for example, medicines, quasi-drugs, cosmetics, and food and drink (drinks, foods).In addition, the preparation according to this embodiment can also be used as, for example, pharmaceutical preparations, quasi-drug preparations, specified health foods, nutritional functional foods, foods for the elderly, special dietary foods, functional food, health supplements (supplements), food preparations (e.g., confectionery tablets), and clear foods.In addition, the agent according to this embodiment can also be used as, for example, veterinary medicines and feed additives.

[0058] When the preparation according to the present embodiment is used as a food, the food may be prepared by blending pyrroloquinoline quinone or a salt thereof, and optionally other ingredients, into a general food, such as solid foods like cookies, biscuits, snacks, jellies, gummies, chocolates, chewing gum, candies, and cheese, and liquid foods like nutritional drinks, juices, tea drinks, coffee drinks, and dairy drinks.

[0059] The preparation according to this embodiment can be suitably used in subjects requiring normal endometrial decidualization (e.g., humans; non-human animals such as cattle, pigs, horses, sheep, goats, dogs, rabbits, mice, rats, guinea pigs, gerbils, hamsters, and ferrets). Humans are preferred as subjects from the viewpoint of further enhancing the effects of the present invention. Among non-human animals, cattle, pigs, and horses are preferred, with pigs being more preferred, from the viewpoint of further enhancing the effects of the present invention.

[0060] [Manufacturing Method] The manufacturing method of the formulation according to the present embodiment is not particularly limited, and any known method can be used. Specifically, a method in which the components are stirred while being heated as necessary to dissolve or disperse them, mixed, stirred while being cooled, and then allowed to stand at room temperature can be mentioned, but the manufacturing method is not particularly limited thereto.

[0061] [Administration Mode] The formulation according to this embodiment may be stored and / or sold in a known container in an amount to be administered for, but not limited to, one week or more, 10 days or more, 14 days or more, 20 days or more, or 30 days or more.

[0062] The administration period of the formulation according to this embodiment can be, for example, but not limited to, at least 3 days or more, and may be 5 days or more, 7 days or more, 10 days or more, 14 days or more, 20 days or more, 30 days or more, or 35 days or more.

[0063] [Method for Promoting or Improving Endometrial Decidualization, Method for Antiaging of Endometrium, and Method for Improving Fertility Due to Antiaging of Endometrium] Pyrroloquinoline quinone or a salt thereof significantly promotes decidualization while suppressing the expression of waste products and aging genes, which are indicators of senescent cell formation. Thus, one embodiment of the present invention provides a method for promoting or improving endometrial decidualization, a method for antiaging of endometrium, and a method for improving fertility due to the promotion of endometrial decidualization, which comprise ingesting a formulation containing pyrroloquinoline quinone or a salt thereof. The endometrium may be human endometrium, or may be endometrium of a non-human animal such as a cow, pig, horse, sheep, goat, dog, rabbit, mouse, rat, guinea pig, gerbil, hamster, or ferret. From the viewpoint of further enhancing the effects of the present invention, human endometrium is preferred. In these embodiments, the type, content, and daily intake of pyrroloquinoline quinone or a salt thereof in the formulation, the type, content, and daily intake of other components, and the formulation form are as described above.

[0064] [Agent for Promoting or Improving Placental Trophoblast Cell Fusion] The preparation of this embodiment contains an effective amount of pyrroloquinoline quinone or a salt thereof.

[0065] The definition, content, daily intake, and other conditions of pyrroloquinoline quinone or its salts are the same as those described in [Agents for promoting or improving endometrial decidualization].

[0066] The preparation according to this embodiment may also contain pharmacologically active ingredients or physiologically active ingredients other than pyrroloquinoline quinone or a salt thereof, as long as the effects of the present invention are not impaired.

[0067] Specific examples of such pharmacologically active ingredients or physiologically active ingredients are the same as those described in [Agents for promoting or improving endometrial decidualization].

[0068] [Uses] The implantation process, which leads to the establishment of pregnancy, can be roughly divided into three stages: attachment, adhesion, and invasion of the fertilized egg to the endometrium. Complex processes such as maternal immune control are also involved. During the invasion process, the trophoblast cells of the fertilized egg that have migrated into the endometrium continue to proliferate, differentiate, and fuse, leading to syncytialization (cell fusion), in which they become syncytiotrophoblast cells. In other words, fusion of trophoblast cells in the uterus after fertilized egg formation is essential for the establishment and maintenance of normal implantation. Therefore, promoting or improving placental trophoblast cell fusion will aid in the establishment of fertilized egg implantation and ultimately the completion of pregnancy.

[0069] In the Examples described below, pyrroloquinoline quinone or a salt thereof has been found to be a component capable of promoting placental trophoblast cell fusion.

[0070] In Test Example 6, it was confirmed that PQQ contributes to the increase in the expression of syncityn2 and PAPPA, which are indicators of the induction of fusion of placental trophoblast cells into syncytiotrophoblast cells (fused trophoblast cells).

[0071] In the test system simulating in vitro placental trophoblast cell fusion described in the Examples below, cell fusion is stimulated with forskolin, but in vivo cell fusion is a phenomenon observed near the completion of implantation. It is strongly speculated that promoting cell fusion, which occurs from the start of the implantation process to its completion, will facilitate the completion of implantation, further improve fertility, and lead to the establishment and continuation of the implantation state.

[0072] (Additives, etc.) The preparation according to this embodiment can contain various additives in addition to the above components, as long as the effects of the present invention are not impaired.

[0073] Specific examples of such additives and other conditions are the same as those described in [Agents for promoting or improving endometrial decidualization].

[0074] The dosage form of the preparation according to this embodiment is also in accordance with the content described in [Agent for promoting or improving endometrial decidualization].

[0075] The preparation according to this embodiment can be used as an ingredient of, for example, medicines, quasi-drugs, cosmetics, and food and drink (drinks, foods).In addition, the preparation according to this embodiment can also be used as, for example, pharmaceutical preparations, quasi-drug preparations, specified health foods, nutritional functional foods, foods for the elderly, special dietary foods, functional food, health supplements (supplements), food preparations (e.g., confectionery tablets), and clear foods.In addition, the agent according to this embodiment can also be used as, for example, veterinary medicines and feed additives.

[0076] When the preparation according to the present embodiment is used as a food, the food may be prepared by blending pyrroloquinoline quinone or a salt thereof, and optionally other ingredients, into a general food, such as solid foods like cookies, biscuits, snacks, jellies, gummies, chocolates, chewing gum, candies, and cheese, and liquid foods like nutritional drinks, juices, tea drinks, coffee drinks, and dairy drinks.

[0077] The preparation according to this embodiment can be suitably used for subjects who can benefit from the promotion or improvement of placental trophoblast cell fusion (e.g., humans; non-human animals such as cattle, pigs, horses, sheep, goats, dogs, rabbits, mice, rats, guinea pigs, gerbils, hamsters, and ferrets). From the viewpoint of further enhancing the effects of the present invention, humans are preferred as subjects. Furthermore, from the viewpoint of further enhancing the effects of the present invention, among non-human animals, cattle, pigs, and horses are preferred, and pigs are more preferred.

[0078] [Manufacturing Method] The manufacturing method of the formulation according to the present embodiment is not particularly limited, and any known method can be used. Specifically, a method in which the components are stirred while being heated as necessary to dissolve or disperse them, mixed, stirred while being cooled, and then allowed to stand at room temperature can be mentioned, but the manufacturing method is not particularly limited thereto.

[0079] [Administration Mode] The formulation according to this embodiment may be stored and / or sold in a known container in an amount for administration for, but not limited to, one week or more, 10 days or more, 14 days or more, 20 days or more, 30 days or more, or 35 days or more.

[0080] The administration period of the formulation according to this embodiment can be, for example, but not limited to, at least 3 days or more, and may be 5 days or more, 7 days or more, 10 days or more, 14 days or more, 20 days or more, 30 days or more, or 35 days or more.

[0081] [Method for Promoting or Improving Placental Trophoblast Cell Fusion, and Method for Improving Fertility Due to Promotion or Improvement of Placental Trophoblast Cell Fusion] Pyrroloquinoline quinone or a salt thereof can significantly promote the transition from undifferentiated placental trophoblast cells to fused trophoblast cells. This can be verified, for example, by detecting increased expression of Syncytin 2 or PAPPA, which are fusion markers of placental trophoblast cells. Therefore, as one embodiment of the present invention, there is provided a method for promoting or improving placental trophoblast cell fusion, and a method for improving fertility due to promotion of placental trophoblast cell fusion, which comprises ingesting a formulation containing pyrroloquinoline quinone or a salt thereof. Placental trophoblast cells may be human cells or cells from non-human animals such as cows, pigs, horses, sheep, goats, dogs, rabbits, mice, rats, guinea pigs, gerbils, hamsters, and ferrets. From the viewpoint of further enhancing the effects of the present invention, human cells are preferred. In these embodiments, the type, content, and daily intake of pyrroloquinoline quinone or a salt thereof in the formulation, the type, content, and daily intake of other components, and the formulation form are as described above.

[0082] The present invention includes the following aspects: [1] A method for promoting or improving endometrial decidualization, which comprises administering an effective amount of pyrroloquinoline quinone or a salt thereof.

[0083] [2] A method for preventing endometrial aging, comprising administering an effective amount of pyrroloquinoline quinone or a salt thereof.

[0084] [3] The method according to [2], wherein the anti-aging of the endometrium is suppression of senescence of endometrial cells.

[0085] [4] The method according to [2], wherein the anti-aging of the endometrium is removal of senescent cells in the endometrium.

[0086] [5] A method for promoting or improving placental trophoblast cell fusion, comprising administering an effective amount of pyrroloquinoline quinone or a salt thereof.

[0087] [6] A method for improving fertility by promoting or improving endometrial decidualization and / or promoting or improving placental trophoblast cell fusion, comprising administering an effective amount of pyrroloquinoline quinone or a salt thereof.

[0088] [7] The method according to [6], wherein the improvement in fertility is improvement in implantation or continuation of the implantation state.

[0089] [8] The method according to any one of [1] to [7], wherein the administration is performed at least during the period from 7 days to 21 days after the first day of menstruation.

[0090] [9] The method according to any one of [1] to [7], wherein the administration is performed during a period of at least 14 to 35 days after the first day of menstruation.

[10] Use of pyrroloquinoline quinone or a salt thereof in the manufacture of an agent for promoting or improving endometrial decidualization.

[0091]

[11] Use of pyrroloquinoline quinone or a salt thereof in the production of an endometrial anti-aging agent.

[0092]

[12] The use according to

[11] , wherein the anti-aging of the endometrium is suppression of senescence of endometrial cells.

[0093]

[13] The use according to

[11] , wherein the anti-aging of the endometrium is removal of senescent cells in the endometrium.

[0094]

[14] Use of pyrroloquinoline quinone or a salt thereof in the manufacture of an agent for promoting or improving placental trophoblast cell fusion.

[0095]

[15] Use of pyrroloquinoline quinone or a salt thereof in a fertility enhancer resulting from promotion or improvement of endometrial decidualization and / or promotion or improvement of placental trophoblast cell fusion.

[0096]

[16] The use according to

[15] , wherein the improvement in fertility is improvement of implantation or continuation of the implantation state.

[0097]

[17] The use according to any one of

[10] to

[16] , wherein the agent is administered at least during the period of 7 to 21 days after the first day of menstruation.

[0098]

[18] The use according to any one of

[10] to

[16] , wherein the agent is administered during a period of at least 14 to 35 days after the first day of menstruation.

[0099] Next, the present invention will be specifically described with reference to Examples and Test Examples, but the present invention is not limited to the following Examples and Test Examples. The unit of the amount of each component in the tables is % by mass unless otherwise specified.

[0100] [Excision of the endometrium and isolation of primary human endometrial stromal cells (pESCs)] To simulate endometrial maturation in vitro, we used primary cultured cells that maintain the phenotype of actual human endometrial stromal cells.

[0101] Human endometrial tissues were collected from female endometriosis patients (n=3) under the age of 45 who had regular menstrual cycles. ２＋ / Mg ２＋ The tissue was washed with Hank's balanced salt solution (HBSS, Fujifilm Wako Pure Chemical Industries, Ltd.), cut into small pieces, and digested with type I collagenase (2.5 mg / ml, Sigma-Aldrich Japan) and DNase I (25 mg / ml, Nippon Gene Co., Ltd.) in the presence of PSN (100 mg / ml penicillin, 100 mg / ml streptomycin, and 200 mg / ml neomycin; Thermo Fisher Scientific) at 37°C for 2 hours. The resulting digestion product was passed through a 250 mm pore size strainer to remove undigested tissue, and then the epithelial cells were separated from the stromal cells by filtration through a 38 mm strainer. The resulting cells were designated primary endometrial stromal cells (pESCs).

[0102] [Culturing of Endometrial Cell Groups (Human Primary Endometrial Stromal Cells, Human Endometrial Epithelial Cell Line, Human Endometrial Glandular Epithelial Cell Line)] The aforementioned pECS, human endometrial epithelial cell line (Ishikawa 3-H-12, JCRB Cell Bank, hereinafter abbreviated as "Ishikawa cells"), and human endometrial glandular epithelial cell line (EM-E6 / E7 / TERT-1, hereinafter abbreviated as "EM1 cells") were resuspended in Dulbecco's Modified Eagle Medium / F12 (DMEM / F12) (1:1) medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, and cultured at 5% CO. ２ The cells were seeded onto gelatin-coated culture dishes at 37°C in humidified air containing HCl.

[0103] [Maturation Stimulation of Endometrial Cells] To evaluate the maturation of endometrial cells (pESCs, Ishikawa cells, and EM1 cells) in vitro, the following maturation stimulating components were added to the cultured cells to promote endometrial differentiation in preparation for implantation. For pESCs, dibutyryl-cAMP (500 mM, manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter abbreviated as "Db") and progesterone (1 mM, manufactured by Sigma-Aldrich Japan, medroxyprogesterone acetate, hereinafter abbreviated as "MPA") were added to the medium. For Ishikawa cells and EM1 cells, dibutyryl-cAMP (500 mM) was added to the medium, and various concentrations of PQQ were also added to each. The cells were cultured for 48 hours.

[0104] [Cytotoxicity Test in pESCs] To evaluate the cytotoxicity of PQQ to pESCs, cell proliferation (Cell Counting Kit-8, Dojindo Laboratories) and cytotoxicity (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories) were measured in the above cultured cells according to the respective instructions.

[0105] [Sensation-associated β-galactosidase 1 staining in pESCs] To assess the degree of senescence in pESCs, the cultured pESCs were fixed with 4% paraformaldehyde for 10 minutes and then washed twice with PBS. Staining was performed using 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mM MgCl in phosphate-buffered saline (PBS) adjusted to pH 6.0. ２ and 1 mg / ml X-gal overnight at 37°C. The number of SA-β-Gal-positive cells was measured in five randomly selected fields and calculated as a ratio to the control in which no maturation-stimulating components were added.

[0106] [RNA extraction and quantification by RT-PCR] Genes were extracted from cultured endometrial cells (pESC, Ishikawa cells, EM1 cells) and cells obtained from excised uterus using Isogen II (Nippon Gene Co., Ltd.). mRNA was reverse transcribed using ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd.), and the resulting cDNA was subjected to qPCR amplification using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific). The Ct value of each gene was used as the expression level, and the expression level without PQQ was used as a control to calculate the expression ratio.

[0107] [Evaluation of gene expression in mouse endometrial epithelium] 10-week-old C57BL / 6 female mice were administered PQQ (2 mg / day) in drinking water starting one week before the test. The mice were subjected to pseudopregnancy treatment (adult female mice were mated with vasectomized male mice in a 3:1 ratio, and the next morning vaginal plugs were confirmed. The mice with vaginal plugs were used). Four or five days later, the uteri were excised and used as samples. The uterine tissues were homogenized and lysed in a lysis buffer to evaluate gene expression (Test Example 4-1). Paraffin sections were prepared from the intact uteri and immunostained for FOXO1-positive cells (Test Example 4-2).

[0108] [Evaluation of Implantation in Mouse Uteri] PQQ (2 mg / day) was administered to 10-week-old C57 / B16 female mice in drinking water starting one week before the test. The mice were subjected to pseudopregnancy treatment (adult female mice were mated with vasectomized male mice in a 3:1 ratio, and the next morning vaginal plugs were confirmed and used), with the day of estrus being considered as day 0. The next day, two-cell stage embryos were transferred into the oviducts, and 10 days later, the uteri were recovered and the number of embryos implanted was counted. The implantation rate was calculated using mice that did not drink PQQ as a control.

[0109] Test Example 1: Cytotoxicity Test PQQ was added to the medium of pESCs at 1 pM to 1 mM (common ratio 10), and the number of viable cells was evaluated using NADH as an indicator, and the number of dead cells was evaluated using LDH as an indicator. The results are shown in Figure 1.

[0110] As a result, no significant increase in dead cells or decrease in live cells was observed across the entire concentration range evaluated. In other words, it was determined that the effects of cell death on the decreased expression of each factor in the following test examples were negligible.

[0111] Test Example 2-1. Test for promoting decidualization of pESCs PQQ was added to pESCs at 1 nM to 100,000 nM (common ratio 10) simultaneously with the maturation-stimulating components (MPA+Db). A control (CM) was used without the maturation-stimulating components, while a control (MPA+Db) was used with only the maturation-stimulating components, and a control (PQQ+DM) was used with PQQ added simultaneously with the maturation-stimulating components.

[0112] RNA extraction and RT-PCR quantification were performed for each sample to evaluate the expression of genes related to decidualization. The results are shown in Figure 2.

[0113] As a result, at all concentrations of PQQ, the expression of all of the representative genes involved in decidualization, RPL, IGFBP1, and FOXO1, was significantly and remarkably increased.

[0114] From the above, PQQ significantly promoted decidualization of human primary endometrial stromal cells even at the minimum concentration of 1 nM.

[0115] Test Example 2-2. Epithelial differentiation promotion test in Ishikawa cells and EM1 cells. Ishikawa cells and EM1 cells were added with a maturation-stimulating component (Db) and 1 nM to 100,000 nM (common ratio 10) of PQQ at the same time. A control (CM) was added without the maturation-stimulating component, a maturation-stimulating component alone was added (Db), and a control to which PQQ was added simultaneously with the maturation-stimulating component was added was called "PQQ+D."

[0116] RNA extraction and RT-PCR quantification were performed for each sample to evaluate the expression of genes related to epithelial differentiation. The results are shown in Figure 3.

[0117] As a result, in both Ishikawa cells and EM1 cells, the expression of both LIF and COX2, which are representative genes involved in epithelial differentiation, was significantly increased at all concentrations of PQQ.

[0118] From the above, it was found that PQQ significantly promoted epithelial differentiation of human endometrial epithelial cell lines and human endometrial glandular epithelial stromal cell lines even at the minimum concentration of 1 nM.

[0119] As shown in Test Examples 2-1 and 2-2, PQQ significantly promoted decidualization in primary endometrial stromal cells, and promoted differentiation in endometrial epithelial cell lines and endometrial glandular epithelial cell lines. Therefore, it was determined that PQQ has the function of promoting the maturation of endometrial tissue and assisting implantation.

[0120] Test Example 3-1. Test for suppression of senescence-associated β-galactosidase associated with decidualization of pESCs. PQQ at 1 nM to 100 nM (common ratio 10) was added to pESCs simultaneously with the maturation-stimulating components (MPA+Db). A control (Ctrl) was used without the maturation-stimulating components, a maturation-stimulating component alone was used (DM), PQQ alone was used as "PQQ", and a maturation-stimulating component and PQQ added simultaneously were used as "DM+PQQ".

[0121] Each cell was stained for senescence-associated β-galactosidase, and the stained cell rate (number of stained cells / total number of cells) was calculated. The results are shown in Figure 4.

[0122] As a result, a significant increase in senescence-associated β-galactosidase was observed due to maturation stimulation, but PQQ significantly inhibited senescence-associated β-galactosidase at all concentrations.

[0123] Test Example 3-2. Test for suppression of aging gene p53 and promotion of anti-aging genes associated with decidualization of pESCs. PQQ at 1 nM to 1000 nM (common ratio 10) was added to pESCs simultaneously with the maturation stimulating components (MPA+Db). A control (CM) was used without the maturation stimulating components, a maturation stimulating component alone was used (DM), and a control (DM+PQQ) was used in which PQQ was added simultaneously with the maturation stimulating components.

[0124] The expression levels of the senescence gene p53 and the anti-senescence gene SIRT1 were measured in each group, and the relative expression ratios to the control were calculated. The results are shown in Figure 5.

[0125] As a result, maturation stimulation significantly increased the expression of the aging gene p53, but PQQ at all concentrations significantly suppressed p53 expression to the same level as the control.Furthermore, maturation stimulation suppressed the expression of the anti-aging gene SIRT1, but PQQ at all concentrations significantly increased SIRT1 expression to the same level as the control.

[0126] As shown in the results of Test Examples 3-1 and 3-2, PQQ suppressed aging factors (senescence-associated β-galactosidase, aging gene p53) induced by decidualization stimulation in primary endometrial stromal cells and enhanced anti-aging factors (pro-aging gene SIRT1). Therefore, it was determined that PQQ has the function of suppressing aging in the maturation of endometrial tissue.

[0127] From the above, it was found that PQQ promotes the maturation of endometrial tissue (stroma, epithelium, glandular epithelium) in vitro while suppressing cellular senescence. Therefore, it was determined that PQQ promotes and supports the maturation of normal endometrium and contributes to the promotion and support of implantation.

[0128] Test Example 4-1. Gene expression (RT-PCR) test in endometrial epithelium in PQQ-drinking mice. When the expression of genes related to decidualization and epithelial differentiation was evaluated in the entire uterus of mice that had been given PQQ as drinking water, the expression of genes related to decidualization and epithelial differentiation, LIF1, ITGB3, FOXO1, BMP2, and COX2, was significantly increased, similar to the results of the in vitro test ( FIG. 6 ).

[0129] Test Example 4-2. Gene expression and immunostaining in endometrial epithelium in PQQ-drinking mice. When the expression of FOXO1, a representative gene involved in epithelial differentiation, was evaluated in the uterus of mice that had been given PQQ as drinking water, a significant increase in cells positive for the epithelial differentiation gene FOXO1 was found, indicating that epithelial differentiation was progressing even at the cellular level in the uterus ( FIG. 7 ).

[0130] As shown in the results of Test Examples 4-1 and 4-2, genes characteristic of epithelial differentiation were highly expressed and epithelial differentiated cells increased in the uterus of mice drinking PQQ. Therefore, it was determined that PQQ promotes epithelial differentiation of the endometrium and assists and promotes endometrial implantation not only in vitro but also in animal models.

[0131] [Test Example 5. Evaluation of implantation rate in PQQ-drinking mice] When the implantation rate of transplanted embryos was evaluated in the uterus of mice that had been given PQQ as drinking water, the implantation rate was significantly increased compared to mice that had not been given PQQ as drinking water (Figure 8).

[0132] The results of Test Examples 1 to 5 showed that PQQ has the effect of maturing endometrial stromal cells, epithelial cells, and glandular epithelial cells, which play an important role in implantation, while suppressing cell aging due to excessive activation, making it ideal for promoting and assisting implantation, and as a result, it was shown that the embryo implantation rate is improved.

[0133] Test Example 6: Gene expression test during cell fusion Placental trophoblast cells were cultured with or without forskolin (0.5 μM, manufactured by Cayman Chemical Co., hereinafter abbreviated as "FSK") as a fusion stimulus. 10 nM PQQ was simultaneously added to the culture medium [4% Knockout Serum Replacement (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.3% bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1% ITS-X Supplement (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 2.5 μM Y27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 50 ng / mL PQQ was added to Dulbecco's modified Eagle's medium / F12 (DMEM / F12) (1:1) medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with epidermal growth factor (Fujifilm Wako Pure Chemical Industries, Ltd.), antibiotics, and antimycotics. The fusion status was compared between the condition without PQQ and the condition with PQQ.

[0134] In each case, RNA extraction and RT-PCR quantification were performed to assess gene expression of the fusion markers Syncytin 2 and PAPPA. The results are shown in Figure 9.

[0135] FIG. 9 shows the results without fusion stimulation (Ctrl), with addition of PQQ only (PQQ), the state in which fused trophoblast cells were formed by fusion stimulation (FSK), and the state in which PPQ was added in addition to fusion stimulation (FSK+PQQ).

[0136] As a result, the addition of PQQ significantly increased the expression of the fusion markers Syncytin 2 and PAPPA genes.

[0137] From the above, it is clear that PQQ significantly promotes cell fusion.

[0138] Test Example 7. Cell Fusion Promotion Test Furthermore, human trophoblast stem cells that had undergone fusion-stimulating FSK treatment were fixed with methanol and incubated with an anti-E-cadherin antibody (1:200, #3195, CST) and an AlexaFluor 488-labeled goat anti-mouse antibody (Thermo Fisher Scientific) to identify the cell surface. A photograph of the stained cells is shown in Figure 10. (Test Example 7-1)

[0139] Nuclei were counterstained with 4',6-diamino-2-phenylindole 2HCl (DAPI). The number of nuclei within syncytiotrophoblast cells (fused cells) and the total number of nuclei were counted in five randomly selected microscopic fields for each sample, and the fusion index was calculated in three independent experiments using the fusion index [(number of nuclei within fused cells / total number of nuclei) x 100]. The increase in the number of fused cells measured in this way is shown in Figure 11. We were able to confirm the increase in the number of fused cells using PQQ.

Claims

1. An agent for promoting or improving endometrial decidualization, which contains an effective amount of pyrroloquinoline quinone or a salt thereof.

2. An endometrial anti-aging agent containing an effective amount of pyrroloquinoline quinone or a salt thereof.

3. The agent according to claim 2, wherein the anti-aging of the endometrium is inhibition of senescent transformation of endometrial cells.

4. The agent according to claim 2, wherein the anti-aging of the endometrium is removal of senescent cells in the endometrium.

5. An agent for promoting or improving placental trophoblast cell fusion, comprising an effective amount of pyrroloquinoline quinone or a salt thereof.

6. A fertility enhancer which contains an effective amount of pyrroloquinoline quinone or a salt thereof and which promotes or improves endometrial decidualization and / or promotes or improves placental trophoblast cell fusion.

7. The agent according to claim 6, wherein the improvement in fertility is improvement of implantation or continuation of the implantation state.

8. The agent according to any one of claims 1 to 7, which is to be taken during the period from at least 7 days to 21 days after the first day of menstruation.

9. The agent according to any one of claims 1 to 7, which is to be taken during the period from at least 14 to 35 days after the first day of menstruation.

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