Promoters or improvers of endometrial decidualization

Abstract

This invention provides a formulation capable of promoting normal decidualization of the endometrium. An endometrial decidualization promoter or improver containing an effective amount of pyrroloquinoline quinone or its salt is prepared. Furthermore, a cell fusion promoter or improver containing an effective amount of pyrroloquinoline quinone or its salt is also prepared.

Description

Technical Field

[0001] This invention relates to an agent that promotes or improves endometrial decidualization. Background Technology

[0002] According to statistics from the Japan Society of Obstetrics and Gynecology, the number of infertility treatment cases each year increased dramatically from about 10,000 in 2004 to more than 400,000 in 2015, and has remained at a high level since then, at about 450,000. This indicates that a large number of couples are suffering from infertility problems.

[0003] In Japan, since 2022, infertility treatments such as artificial insemination ("general infertility treatment") and in-vitro fertilization (IVF / IVT) have been covered by insurance, aiming to alleviate the economic burden of many infertility problems.

[0004] However, insurance coverage for infertility treatments applicable to women has age and frequency restrictions. Therefore, even if treatment is covered by insurance, it cannot be said that a woman's concerns about infertility have been fully resolved.

[0005] Female infertility is classified into ovarian factors, fallopian tube factors, and uterine factors based on its causes. However, the ovaries, fallopian tubes, and uterus influence each other. Without elucidating their mechanisms, it is difficult to understand the complex physiological phenomenon of ovulation-fertilization-implantation-embryonic development, thus making it difficult to fundamentally solve infertility.

[0006] In this complex physiological phenomenon, the inventors focused on the decidualization of endometrial cells, believing that if normal decidualization cannot occur, it may lead to implantation failure or miscarriage. They established an in vitro experimental system to simulate the decidualization of human endometrial cells and studied "normal decidualization" (Non-Patent Literature 1).

[0007] On the other hand, pyrroloquinoline quinone (PQQ) is an organic molecule found in bacteria as a redox coenzyme, and is a highly versatile and safe food ingredient. Based on existing research on PQQ and infertility, it is known to improve sperm motility (Patent Document 1) and enhance the normality of eggs or ovaries (Patent Document 2).

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: International Publication No. 2020 / 013086

[0011] Patent Document 2: International Publication No. 2020 / 262325

[0012] Non-patent literature

[0013] Non-patent literature 1: Kazuya Kusama et al. Biochemical and Biophysical Research Communications 571 (2021) 174-180 Summary of the Invention

[0014] According to the aforementioned patent documents, PQQ inhibits DNA damage caused by oxidation of sperm or eggs through its antioxidant effect, thereby producing sperm with high motility or eggs with high fertilization capacity.

[0015] However, even if normal sperm and egg successfully fertilize, if the aforementioned "normal decidualization of the endometrium" does not occur, the implantation window (an extremely precise timing) will be missed, resulting in implantation failure. Furthermore, without "normal decidualization of the endometrium," excessive decidualization may lead to endometrial cell senescence. In other words, promoting endometrial decidualization while simultaneously inhibiting abnormal cell senescence is "promoting normal decidualization of the endometrium," a necessary condition for implantation.

[0016] Therefore, the inventors conducted in-depth research on whether PQQ can "promote normal decidualization of the endometrium." The results showed that while significantly promoting decidualization, PQQ also inhibited the expression of metabolic waste products and aging genes, which are indicators of cellular aging. Furthermore, the inventors discovered that PQQ promotes implantation by acting on the "normal decidualization of the endometrium." In addition, the inventors found that PQQ can promote the fusion of placental trophoblast cells, which is crucial for successful pregnancy. Then, focusing on the connection between PQQ and improved fertility, the present invention was completed.

[0017] That is, the preferred embodiment of the present invention provides the following oral composition.

[0018] [1] An agent that promotes or improves endometrial decidualization, containing an effective amount of pyrroloquinoline quinone or its salt.

[0019] [2] An anti-aging agent for the endometrium, containing an effective amount of pyrroloquinoline quinone or its salt.

[0020] [3] According to the medicine described in [2], the anti-aging of the endometrium is to inhibit the transformation of endometrial cells into senescent cells.

[0021] [4] According to the medicine described in [2], the anti-aging of the endometrium is to remove senescent cells in the endometrium.

[0022] [5] A promoter or improver of placental trophoblast cell fusion, comprising an effective amount of pyrroloquinoline quinone or its salt.

[0023] [6] A fertility enhancer containing an effective amount of pyrroloquinoline quinone or its salt, which enhances fertility by promoting or improving endometrial decidualization and / or promoting or improving placental trophoblast cell fusion.

[0024] [7] According to the medicine described in [6], the improvement in fertility is an improvement in implantation or a continuation of the implantation state.

[0025] [8] The medicine according to any one of [1] to [7] is to be ingested for at least 7 days to 21 days after the first day of menstruation.

[0026] [9] The medicine according to any one of [1] to [7] is to be ingested for at least 14 days to 35 days after the first day of menstruation.

[0027] According to the present invention, since PQQ can significantly promote decidualization while also inhibiting the expression of metabolic waste and senescence genes, which are indicators of cellular senescence, it can provide an agent that promotes or improves endometrial decidualization. Furthermore, agents that promote or improve the fusion of placental trophoblast cells can be provided. Attached Figure Description

[0028] Figure 1 The results are from the cytotoxicity test in Example 1.

[0029] Figure 2 The results of the decidualization promotion assay of primary endometrial stromal cells (pESC) in Experiment Example 2-1 are shown.

[0030] Figure 3 The results of the epithelial differentiation promotion experiment of Ishikawa cells and EM1 cells in Experiment Example 2-2 are shown.

[0031] Figure 4 The results are from the inhibition assay of aging-related β-galactosidase associated with pESC decidualization in Experiment Example 3-1.

[0032] Figure 5 The results are from the experiment in Example 3-2 on the inhibition of the aging gene p53 associated with pESC decidualization and the promotion of anti-aging genes.

[0033] Figure 6 The results are from the gene expression experiment in the endometrial cells of mice that drank PQQ in Experiment 4-1.

[0034] Figure 7 To show photographs and charts of immunostaining in the endometrium of mice that drank PQQ in Experiment 4-2.

[0035] Figure 8The results of the implantation rate evaluation experiment for mice that drank PQQ in Experiment Example 5.

[0036] Figure 9 The results are from the gene expression assay used as an indicator of cell fusion in Experiment Example 6.

[0037] Figure 10 A photograph showing the cell fusion state of Experiment 7-1.

[0038] Figure 11 A graph showing the changes in the number of fused cells in Experiment 7-2. Detailed Implementation

[0039] [Agents that promote or improve endometrial decidualization]

[0040] The formulation prepared in this manner contains an effective amount of pyrroloquinoline quinone or its salt.

[0041] (pyrroloquinoline quinone or its salt)

[0042] Pyrroloquinoline quinone is a known compound represented by Formula 1 below.

[0043]

[0044] Examples of salts of pyrroloquinoline quinone include, for example, alkali metal salts, alkaline earth metal salts, and ammonium salts. Examples of alkali metal salts include, for example, sodium salts, potassium salts, and lithium salts. Examples of alkaline earth metal salts include, for example, calcium salts and magnesium salts.

[0045] As a salt of pyrroloquinoline quinone or 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.

[0046] Pyrroloquinoline quinone or its salts can also be used commercially available products. Furthermore, pyrroloquinoline quinone or its salts can be used alone or in combination of two or more.

[0047] From the viewpoint of significantly maximizing the effects of the present invention, the content of pyrroloquinoline quinone or its salt relative to the total amount of the formulation can 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.

[0048] In addition, relative to the total amount of the formulation, the content of pyrroloquinoline quinone or its salt can be, for example, 30% or less by mass, 25% or less by mass, 20% or less by mass, 15% or less by mass, 10% or less by mass, 5% or less by mass, 1% or less by mass, 0.5% or less by mass, 0.2% or less by mass, etc.

[0049] In addition, the content of pyrroloquinoline quinone or its salt relative to the total amount of the formulation can be, for example, 0.001–30% by mass, 0.001–25% by mass, 0.001–20% by mass, 0.001–15% by mass, 0.001–10% by mass, 0.001–5% by mass, 0.001–1% by mass, 0.001–0.5% by mass, 0.001–0.2% by mass, 0.005–30% by mass, 0.005–25% by mass, 0.005–20% by mass, 0.005–15% by mass, 0.005–10% by mass, 0.005–5% by mass, 0.005–1% by mass, 0.0 0.5–0.5% by mass, 0.005–0.2% by mass, 0.01–30% by mass, 0.01–25% by mass, 0.01–20% by mass, 0.01–15% by mass, 0.01–10% by mass, 0.01–5% by mass, 0.01–1% by mass, 0.01–0.5% by 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.

[0050] The daily intake of pyrroloquinoline quinone or its salts may vary depending on the individual's condition (weight, age, sex, etc.), the form of the preparation, etc., but from the viewpoint of significantly exerting the effects of the present invention, for example, 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.

[0051] In addition, the daily intake of pyrroloquinoline quinone or its salts can be, for example, less than 100 mg, less than 80 mg, less than 60 mg, less than 50 mg, less than 40 mg, less than 30 mg, less than 25 mg, etc.

[0052] In addition, the daily intake of pyrroloquinoline quinone or its salts can be listed as follows: 5–100 mg, 5–80 mg, 5–60 mg, 5–50 mg, 5–40 mg, 5–30 mg, 5–25 mg, 10–100 mg, 10–80 mg, 10–60 mg, 10–50 mg, 10–40 mg, 10–30 mg, 10–25 mg, 15–100 mg, 15–80 mg, 15–60 mg, 15–50 mg, 15–40 mg, 15–30 mg, 15–25 mg, 20–100 mg, 20–80 mg, 20–60 mg, 20–50 mg, 20–40 mg, 20–30 mg, 20–25 mg, etc.

[0053] Provided that the effects of the present invention are not compromised, the formulations of this embodiment may contain pharmacologically or physiologically active ingredients other than pyrroloquinoline quinone or its salts.

[0054] Specific examples of such pharmacologically or physiologically active ingredients include, for instance, 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, and ornithine. Carnitine, glutamic acid, glutamine, creatine, luteolin, quercetin, genistein, anthocyanins, 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, placental extract, pycnogenol (proanthocyanidins), or equol, etc. From the viewpoint of further improving the efficacy of the present invention, the pharmacologically or physiologically active ingredient is preferably selected from one or more of ubiquinone (coenzyme Q10), vitamin B12, vitamin C, vitamin E, astaxanthin, zinc, carnitine, resveratrol, isoflavones, equol, pycnogenol (proanthocyanidins), vitamin D, iron, and folic acid. More preferably, it is selected from one or more of ubiquinone (coenzyme Q10), astaxanthin, zinc, resveratrol, isoflavones, equol, pycnogenol (proanthocyanidins), vitamin D, iron, and folic acid. Even more preferably, it is selected from one or more of zinc, vitamin D, iron, and folic acid. The pharmacologically or physiologically active ingredient may be used alone or in combination of two or more.

[0055] [use]

[0056] The endometrium is essential for the establishment and maintenance of pregnancy. During the menstrual cycle, it proliferates and thickens in response to ovarian steroid hormones, transforming into a state called decidualization, which allows a fertilized egg to implant. During the proliferative phase (from the first day of menstruation to approximately 14 days after menstruation), 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 a fertilized egg; during menstruation, the endometrium sheds and is expelled from the body. The endometrium is mainly composed of stromal cells and glandular epithelial cells. The formation of the decidual membrane through the proliferation of stromal cells is called decidualization.

[0057] As mentioned above, even if a normal sperm and egg successfully fertilize, if "normal decidualization of the endometrium" does not occur, the implantation window (an extremely precise timing) will be missed, resulting in implantation failure. Furthermore, the absence of "normal decidualization of the endometrium" can lead to excessive decidualization and endometrial cell senescence. In other words, promoting endometrial decidualization while simultaneously inhibiting abnormal cell senescence is "promoting normal decidualization of the endometrium," a necessary condition for implantation.

[0058] In the following examples, using primary endometrial stromal cells (pESCs) that maintained the phenotype of actual human endometrial stromal cells, pyrroloquinoline quinone or its salt (hereinafter also referred to as PQQ) was found to be a component that could promote endometrial decidualization.

[0059] In Experiment 1, no toxicity of PQQ to pESCs was observed. Furthermore, in Experiment 2-1, when maturation-stimulating components (Db: dibutyryl-cAMP, and / or MPA: medroxyprogesterone acetate) simulating stimulation from ovarian steroid hormones were applied to pESCs together with PQQ, even the lowest concentration (1 nM) of PQQ significantly promoted decidualization of pESCs.

[0060] In the simulated in vitro decidualization test system of the later embodiments, although Db or MPA is used to simulate the stimulation brought about by ovarian steroid hormones, in vivo, the stimulation of progesterone begins to rise from the late proliferative phase and rises sharply during the secretory phase (approximately 14 days after the first day of menstruation). Therefore, based on the results of the later embodiments, PQQ is preferably taken starting, for example, at least 7 days after the first day of menstruation, and preferably continued after 14 days from the first day of menstruation. Furthermore, in another embodiment, it is preferable to take it until the period when normal decidualization of the endometrium is completed and the endometrium matures, i.e., 21 days after the first day of menstruation, but it is also possible, for example, to take it up to 35 days later, and is not limited thereto.

[0061] Furthermore, in subsequent embodiments, when maturation-stimulating components (Db and / or MPA) were applied to pESCs together with PQQ, aging-related β-galactosidase was significantly inhibited, thus confirming the inhibition of the aging gene p53 and the overactivation of the anti-aging gene SIRT1. This indicates that PQQ has an inhibitory function (anti-aging) during the maturation of endometrial tissue. Therefore, it is confirmed that PQQ has the effect of inhibiting endometrial cell senescence and clearing senescent cells.

[0062] Furthermore, in the following examples, PQQ was confirmed not only in vitro but also in animal models to promote endometrial epithelial differentiation and assist and promote endometrial implantation. It is strongly speculated that this is because oral intake of PQQ promotes normal decidualization of the endometrium, inhibits cell senescence, and facilitates a better maturation and implantation period, thereby improving fertility, implantation, and maintaining implantation.

[0063] (Additives, etc.)

[0064] In addition to the above-mentioned ingredients, the formulation of this embodiment may also contain various additives, provided that the effect of the present invention is not compromised.

[0065] Specific examples of such additives include excipients, binders, disintegrants, lubricants, colorants, flavoring agents, odorants, sugars, sugar alcohols / polyols, high-sweetness sweeteners, oils, emulsifiers, thickeners, acidulants, and fruit juices.

[0066] Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, gelatin, calcium carbonate, kaolin, crystalline cellulose, and silicic acid.

[0067] Examples of adhesives 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.

[0068] Examples of disintegrants include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, glyceryl monostearate, and lactose.

[0069] Examples of lubricants include talc, stearates, borax, and polyethylene glycol.

[0070] Examples of flavoring agents include sucrose, orange peel, citric acid, and tartaric acid.

[0071] Examples of sugars include sucrose, isomerized sugars, glucose, fructose, palaginose, trehalose, lactose, xylose, and so on.

[0072] Examples of sugar alcohols / polyols include sorbitol, xylitol, erythritol, lactitol, palaginitol, reduced maltose syrup, reduced maltose syrup, glycerol, and propylene glycol.

[0073] Examples of high-sweetness sweeteners include aspartame, stevia, acesulfame potassium, and sucralose.

[0074] Examples of oils include safflower oil, grapeseed oil, sunflower seed oil, olive oil, corn oil, sesame oil, soybean oil, rapeseed oil, and perilla oil.

[0075] Examples of emulsifiers include sucrose fatty acid esters, glycerol fatty acid esters, and lecithin.

[0076] Examples of thickeners include carrageenan, xanthan gum, guar gum, pectin, and locust bean gum.

[0077] Examples of acidulants include citric acid, lactic acid, and malic acid. Examples of fruit juices include lemon juice, orange juice, and berry juice.

[0078] One additive can be used alone, or two or more additives can be used in combination.

[0079] From the viewpoint of the stability of pyrroloquinoline quinone or its salts during formulation, the additive preferably contains oils.

[0080] (Dosage form, etc.)

[0081] The dosage form of the formulation in this embodiment is not particularly limited, and examples include: oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, lozenges, etc.), granules, powders, soft capsules, hard capsules, lozenges, gels, or liquids (including suspensions, emulsions, syrups, etc.); external preparations such as ointments, suppositories, patches, and sprays; and injections. From the viewpoint of ease of processing and further improving the effect of the present invention, oral preparations are preferred, and tablets, granules, soft capsules, and hard capsules are more preferred.

[0082] The formulations of this embodiment can be used, for example, as ingredients in pharmaceuticals, quasi-pharmaceuticals, cosmetics, and food and beverage products (beverages, foods). Furthermore, the formulations of this embodiment can also be used, for example, as pharmaceutical preparations, quasi-pharmaceutical preparations, foods for specific health purposes, nutritional functional foods, foods for the elderly, foods for special purposes, functionally labeled foods, health supplements, food preparations (e.g., candy tablets), and other foods. In addition, the pharmaceuticals of this embodiment can also be used, for example, as veterinary pharmaceuticals and feed additives.

[0083] When the formulation of this embodiment is used as a food, the food may be a food incorporating pyrroloquinoline quinone or its salt, and other ingredients as needed, into a general food. Examples of such foods include solid foods such as cookies, biscuits, snacks, jellies, gummies, chocolates, chewing gum, candies, and cheese; and liquid foods such as nutritional drinks, fruit juices, tea drinks, coffee drinks, and milk drinks.

[0084] The formulation of this embodiment is suitable for use in subjects requiring normal decidualization of the endometrium (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 improving the effects of the present invention, humans are preferred as the target. Furthermore, from the viewpoint of further improving the effects of the present invention, among non-human animals, cattle, pigs, and horses are preferred, with pigs being more preferred.

[0085] [Manufacturing Method]

[0086] The method for manufacturing the formulation in this embodiment is not particularly limited, and known methods can be used. Specifically, a method can be given as follows: heating and stirring the components as needed to dissolve or disperse them, mixing them, cooling and stirring simultaneously, and then allowing them to stand at room temperature, but this method is not particularly limited.

[0087] [Route of administration]

[0088] The formulation described in this embodiment is not limited and can be stored and / or sold in known containers in dosages of 1 week or more, 10 days or more, 14 days or more, 20 days or more, or 30 days or more.

[0089] The formulation of this invention is not limited, and the administration period may be at least 3 days, or 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.

[0090] [Methods to promote or improve endometrial decidualization, methods for anti-aging of the endometrium, and methods to improve fertility due to endometrial anti-aging]

[0091] Pyrroloquinoline quinone or its salts significantly promote decidualization while also inhibiting the expression of metabolic waste products and senescence genes, which are indicators of cellular senescence. Therefore, as an embodiment of the present invention, methods for promoting or improving endometrial decidualization, methods for anti-aging of the endometrium, and methods for improving fertility caused by promoting endometrial decidualization are provided. These methods include the following steps: ingesting a preparation containing pyrroloquinoline quinone or its salts. The endometrium can be human endometrium or endometrium from 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 improving the effects of the present invention, human endometrium is preferred. It should be noted that the types, contents, and daily intake of pyrroloquinoline quinone or its salts in the preparations of these embodiments, the types, contents, and daily intake of other components, and the form of the preparation are as described above.

[0092] [Agents to promote or improve placental trophoblast cell fusion]

[0093] The formulation prepared in this manner contains an effective amount of pyrroloquinoline quinone or its salt.

[0094] The definition, content, and daily intake of pyrroloquinoline quinone or its salts are based on the information recorded in [Promoters or Improvers of Endometrial Decidualization].

[0095] Provided that the effects of the present invention are not compromised, the formulations of this embodiment may also contain pharmacologically or physiologically active ingredients other than pyrroloquinoline quinone or its salts.

[0096] Specific examples of such pharmacologically or physiologically active ingredients are provided in the section on [Promoters or Improvers of Endometrial Decidualization].

[0097] [use]

[0098] The implantation process for establishing pregnancy can be broadly divided into three stages: attachment, adhesion, and invasion of the fertilized egg into the uterine lining. It also involves complex processes such as maternal immune regulation. During invasion, the trophoblast cells of the fertilized egg that have migrated into the uterine lining continue to proliferate, differentiate, and fuse, continuously forming syncytiotrophoblast cells (cell fusion). In other words, after fertilization, the fusion of trophoblast cells in the uterus is indispensable for the establishment and maintenance of normal implantation. Therefore, promoting or improving placental trophoblast cell fusion helps assist in the establishment of fertilized egg implantation, thereby aiding in the completion of pregnancy.

[0099] In later examples, pyrroloquinoline quinone or its salts were found to be components that can promote the fusion of placental trophoblast cells.

[0100] In Experiment 6, it was confirmed that PQQ contributes to the increased expression of syncytin 2 and PAPPA, which are indicators, during the process of inducing placental trophoblast cells to fuse with syncytiotrophoblast cells (fused trophoblast cells).

[0101] In the simulated in vitro placental trophoblast cell fusion experimental system described in later embodiments, although forskolin was used to stimulate cell fusion, in vivo cell fusion occurs near the completion of implantation. It is strongly hypothesized that by promoting cell fusion that occurs from the beginning to the completion of the implantation process, thus facilitating successful implantation, fertility is further improved, involving the establishment and maintenance of the implantation state.

[0102] (Additives, etc.)

[0103] Provided that the effects of the present invention are not compromised, the formulation of this embodiment may contain various additives in addition to the above-mentioned components.

[0104] Specific examples and conditions of such additives are based on the information described in [Promoters or Improvers of Endometrial Decidualization].

[0105] The dosage form of the formulation in this embodiment is also based on the information described in [Promoters or Improvers of Endometrial Decidualization].

[0106] The formulations of this embodiment can be used, for example, as ingredients in pharmaceuticals, quasi-pharmaceuticals, cosmetics, and food and beverage products (beverages, foods). Furthermore, the formulations of this embodiment can also be used, for example, as pharmaceutical preparations, quasi-pharmaceutical preparations, foods for specific health purposes, nutritional functional foods, foods for the elderly, foods for special purposes, functionally labeled foods, health supplements, food preparations (e.g., candy tablets), and other foods. In addition, the pharmaceuticals of this embodiment can also be used, for example, as veterinary pharmaceuticals and feed additives.

[0107] When the formulation of this embodiment is used as a food, the food may be a food incorporating pyrroloquinoline quinone or its salt, and other ingredients as needed, into a general food. Examples of such foods include solid foods such as cookies, biscuits, snacks, jellies, gummies, chocolates, chewing gum, candies, and cheese; and liquid foods such as nutritional drinks, fruit juices, tea drinks, coffee drinks, and milk drinks.

[0108] The formulation of this embodiment is suitable for use in subjects who benefit from promoting or improving 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 the target. Furthermore, 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.

[0109] [Manufacturing Method]

[0110] The method for manufacturing the formulation in this embodiment is not particularly limited, and known methods can be used. Specifically, a method can be given as follows: heating and stirring the components as needed to dissolve or disperse them, mixing them, cooling and stirring simultaneously, and then allowing them to stand at room temperature, but this method is not particularly limited.

[0111] [Route of administration]

[0112] The formulation described in this embodiment is not limited and can be stored and / or sold in known containers in dosages of 1 week or more, 10 days or more, 14 days or more, 20 days or more, 30 days or more, or 35 days or more.

[0113] The formulation used in this embodiment is not limited, and the administration period can be, for example, at least 3 days, or 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.

[0114] [Methods to promote or improve placental trophoblast cell fusion, and methods to enhance fertility resulting from promoting or improving placental trophoblast cell fusion]

[0115] Pyrroloquinoline quinone or its salts can significantly promote the transformation of undifferentiated placental trophoblast cells into fused trophoblast cells. This can be verified, for example, by detecting increased expression of Syncytin 2 or PAPPA, which are markers of placental trophoblast cell fusion. Therefore, as an embodiment of the present invention, methods for promoting or improving placental trophoblast cell fusion and methods for improving fertility caused by promoting placental trophoblast cell fusion are provided, these methods comprising the step of ingesting a preparation containing pyrroloquinoline quinone or its salts. Placental trophoblast cells can be human cells or cells from 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 improving the effects of the present invention, human cells are preferred. It should be noted that the type, content, and daily intake of pyrroloquinoline quinone or its salts in the preparations of these embodiments, the type, content, and daily intake of other components, and the form of the preparation are as described above.

[0116] The present invention includes the following methods.

[0117] [1] A method for promoting or improving endometrial decidualization, comprising administering an effective amount of pyrroloquinoline quinone or a salt thereof.

[0118] [2] A method for preventing endometrial aging, comprising administering an effective amount of pyrroloquinoline quinone or its salt.

[0119] [3] According to the method described in [2], the anti-aging of the endometrium is to inhibit the transformation of endometrial cells into senescent cells.

[0120] [4] According to the method described in [2], the anti-aging of the endometrium is to remove senescent cells from the endometrium.

[0121] [5] A method for promoting or improving placental trophoblast cell fusion by administering an effective amount of pyrroloquinoline quinone or its salt.

[0122] [6] A method for improving fertility, comprising administering an effective amount of pyrroloquinoline quinone or its salt to improve fertility by promoting or improving endometrial decidualization and / or promoting or improving placental trophoblast cell fusion.

[0123] [7] According to the method of [6], wherein the improvement in fertility is an improvement in implantation or a continuation of the implantation state.

[0124] [8] The method according to any one of [1] to [7], wherein the administration is performed for at least 7 to 21 days after the first day of menstruation.

[0125] [9] The method according to any one of [1] to [7], wherein the administration is performed at least 14 days after the first day of menstruation and up to 35 days after the first day of menstruation.

[0126]

[10] Use of pyrroloquinoline quinone or its salts in the manufacture of agents that promote or improve decidualization of the endometrium.

[0127]

[11] Use of pyrroloquinoline quinone or its salts in the manufacture of anti-aging agents for the endometrium.

[0128]

[12] According to the use described in

[11] , the anti-aging of the endometrium is to inhibit the transformation of endometrial cells into senescent cells.

[0129]

[13] According to the use described in

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

[0130]

[14] Use of pyrroloquinoline quinone or its salts in the production of placental trophoblast cell fusion promoters or improvers.

[0131]

[15] The use of pyrroloquinoline quinone or its salts in the manufacture of fertility enhancers improves fertility by promoting or improving endometrial decidualization and / or placental trophoblast cell fusion.

[0132]

[16] According to the use described in

[15] , the improvement in fertility is either improved implantation or continued implantation status.

[0133]

[17] Use according to any one of

[10] to

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

[0134]

[18] Use according to any one of

[10] to

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

[0135] Example

[0136] The present invention will be specifically described below through examples and test cases, but the present invention is not limited to the following examples and test cases. Unless otherwise specified, the units of the components in the table are mass%.

[0137] [Endometrial lining removal and isolation of human primary endometrial stromal cells (pESCs)]

[0138] To simulate endometrial maturation in vitro, primary cultured cells that maintain the phenotype of actual human endometrial stromal cells were used.

[0139] Human endometrial tissue was collected from three women under 45 years of age with endometriosis and regular menstrual cycles. The collected endometrial tissue was then treated with a Ca²⁺-free solution. + / Mg² + The cells were washed with Hanks' balanced salt solution (HBSS, manufactured by Fujifilm and Kojun Pharmaceutical Co., Ltd.), cut into small pieces, and digested for 2 hours at 37°C with type I collagenase (2.5 mg / ml, manufactured by Sigma-Aldrich Japan) and DNase I (25 mg / ml, manufactured by Nippon Gene Co., Ltd.) in the presence of PSN (100 mg / ml penicillin, 100 mg / ml streptomycin, and 200 mg / ml neomycin; manufactured by Thermo Fisher Scientific). Undigested tissue was removed by passing the digest through a 250 mm pore size filter, followed by filtration through a 38 mm filter to separate epithelial cells from stromal cells. The cells obtained in this way were used as primary endometrial stromal cells (pESCs).

[0140] [Culture of endometrial cell populations (human primary endometrial stromal cells, human endometrial epithelial cell lines, and human endometrial glandular epithelial cell lines)]

[0141] The above-mentioned pECS, human endometrial epithelial cell line (Ishikawa 3-H-12, JCRB cell bank, hereinafter referred to as "Ishikawa cells"), and human endometrial glandular epithelial cell line (EM-E6 / E7 / TERT-1, hereinafter referred to as "EM1 cells") were resuspended in Dulbecco modified Eagle medium / F12 (DMEM / F12) (1:1) medium (manufactured by Fujifilm and Koichi Pure Chemical Industries Co., Ltd.) supplemented with 10% fetal bovine serum (FBS), antibiotics and antifungal drugs. The cells were then inoculated into gelatin-coated culture dishes at 37°C in humidified air containing 5% CO2.

[0142] [Stimulation of maturation of endometrial cell populations]

[0143] To evaluate the maturity of endometrial cell populations (pESC, 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 pESC, dibutyryl-cAMP (500 mM, manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter referred to as "Db") and progesterone (1 mM, manufactured by Sigma Aldrich Japan, medroxyprogesterone acetate, hereinafter referred to as "MPA") were added to the culture medium. For Ishikawa cells and EM1 cells, dibutyryl-cAMP (500 mM) was added to the culture medium, along with various concentrations of PQQ, and the cells were cultured for 48 hours.

[0144] [Cytotoxicity assay of pESC]

[0145] To evaluate the cytotoxicity of PQQ against pESC, the cell proliferation assay kit (Cell Counting Kit-8, manufactured by Dojin Chemical Research Institute Co., Ltd.) and the cytotoxicity assay kit (Cytotoxicity LDH Assay Kit-WST, manufactured by Dojin Chemical Research Institute Co., Ltd.) were used in the cultured cells described above, and the assays were performed according to the instructions.

[0146] [Staining of pESCs with senescence-associated β-galactosidase 1]

[0147] To assess the senescence of pESCs, the cultured pESCs were fixed with 4% paraformaldehyde for 10 minutes and then washed twice with PBS. Staining was performed overnight at 37°C in phosphate-buffered saline adjusted to pH 6.0 with 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mM MgCl2, and 1 mg / ml X-gal. The number of SA-β-Gal positive cells was determined in five randomly selected fields of view and calculated as a ratio to a control without added maturation stimulants.

[0148] [RNA extraction and RT-PCR-based quantification]

[0149] For endometrial cell populations (pESC, Ishikawa cells, EM1 cells) and cells obtained from removed uteruses, genes were extracted from cultured cells using Isogen II (manufactured by Nippon Gene Co., Ltd.). mRNA was reverse transcribed using the ReverTra AceqPCR RT Kit (manufactured by Toyobo Co., Ltd.), and the resulting cDNA was amplified by qPCR using PowerUP SYBR Green MasterMix (manufactured by Thermo Fisher Scientific). The Ct value of each gene was used as the expression level, and the expression ratio was calculated using the expression level without PQQ as a control.

[0150] [Evaluation of gene expression in mouse endometrial epithelium]

[0151] One week prior to the experiment, 10-week-old female C57BL / 6 mice were administered PQQ (2 mg / day) via drinking water. In these mice, day 0 was designated as the day of estrus, and pseudopregnancy was induced (adult female mice were mated with vasectomized male mice at a 3:1 ratio; vaginal plugs were confirmed the following morning, and mice with vaginal plugs were used). The uterus was removed 4-5 days later as a specimen. For this specimen, uterine tissue was homogenized and lysed with lysis buffer to evaluate gene expression (Example 4-1). Additionally, paraffin sections were prepared directly from the uterus, and FOXO1-positive cells were immunostained (Example 4-2).

[0152] [Implantation evaluation in mouse uterus]

[0153] Starting one week prior to the experiment, 10-week-old female C57 / BL6 mice were administered PQQ (2 mg / day) via drinking water. In these mice, day 0 was designated as the day of estrus, and a pseudopregnancy treatment was initiated (adult female mice were mated with vasectomized male mice at a ratio of 3:1; vaginal plugs were confirmed the following morning, and mice with vaginal plugs were used). The following day, two-cell stage embryos were transferred into the fallopian tubes. Ten days later, the uterus was retrieved, and the number of implanted embryos was counted. Mice that did not receive PQQ served as controls, and the implantation rate was calculated.

[0154] [Experimental Example 1. Cytotoxicity Assay]

[0155] Add 1 pM to 1 mM (common ratio 10) of PQQ to pESCs. Use NADH as an indicator to evaluate the number of viable cells and LDH as an indicator to evaluate the number of dead cells. Results are shown in... Figure 1 .

[0156] As a result, no significant increase in dead cells or decrease in live cells was observed in any of the evaluated concentration regions. Therefore, the effects of cell death on the reduced expression of various factors were deemed negligible in the following experimental cases.

[0157] [Experimental Example 2-1. Decidualization Promotion Test of pESC]

[0158] PQQ was added to pESC at a concentration of 1 nM to 100,000 nM (common ratio 10) simultaneously with the maturation stimulant (MPA+Db). The group without the maturation stimulant was designated as the control (CM), the group with only the maturation stimulant added was designated as (MPA+Db), and the group with PQQ added simultaneously with the maturation stimulant was designated as "PQQ+DM".

[0159] RNA was extracted and quantified by RT-PCR in each group to evaluate gene expression associated with decidualization. Results are shown in... Figure 2 .

[0160] As a result, at any concentration of PQQ, the representative genes of decidualization, RPL, IGFBP1, and FOXO1, were all significantly upregulated.

[0161] In summary, PQQ significantly promoted decidualization of human primary endometrial stromal cells even at the lowest concentration of 1 nM.

[0162] [Experiment 2-2. Epithelial Differentiation Promotion Assay of Ishikawa Cells and EM1 Cells]

[0163] PQQ was added to Ishikawa cells and EM1 cells at concentrations ranging from 1 nM to 100,000 nM (common ratio 10) along with the maturation stimulant (Db). The group without the maturation stimulant (CM) was designated as (Db), and the group with PQQ added along with the maturation stimulant was designated as "PQQ+D".

[0164] RNA was extracted and quantified by RT-PCR in each group to evaluate the expression of genes associated with epithelial differentiation. Results are shown in... Figure 3 .

[0165] As a result, in Ishikawa cells and EM1 cells, at any concentration of PQQ, the representative genes of epithelial differentiation, LIF and COX2, were significantly upregulated.

[0166] In summary, PQQ significantly promoted epithelial differentiation of human endometrial epithelial cell lines and human endometrial glandular epithelial stromal cell lines, even at the lowest concentration of 1 nM.

[0167] As shown in Experiments 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 endometrial tissue maturation and assisting implantation.

[0168] [Experimental Example 3-1. Inhibition of senescence-related β-galactosidase associated with pESC decidualization]

[0169] PQQ of 1 nM to 100 nM (common ratio 10) was added to pESC simultaneously with the maturation stimulant (MPA+Db). The group without the maturation stimulant was used as the control (Ctrl). The group with only the maturation stimulant added was denoted as (DM), the group containing only PQQ was denoted as (PQQ), and the group with PQQ added simultaneously with the maturation stimulant was denoted as "DM+PQQ".

[0170] In each group, senescence-related β-galactosidases were stained, and the stained cell percentage (number of stained cells / total number of cells) was calculated. Results are shown in... Figure 4 .

[0171] As a result, maturation stimulation led to a significant increase in aging-related β-galactosidase, but at any concentration of PQQ, aging-related β-galactosidase was significantly inhibited.

[0172] [Experiment 3-2. Inhibition of the aging gene p53 and promotion of anti-aging genes accompanying pESC decidualization]

[0173] PQQ of 1 nM to 1000 nM (common ratio 10) was added to pESC simultaneously with the maturation stimulant (MPA+Db). The group without the maturation stimulant was designated as the control (CM), the group with only the maturation stimulant added was designated as (DM), and the group with PQQ added simultaneously with the maturation stimulant was designated as "DM+PQQ".

[0174] The expression levels of the aging gene p53 and the anti-aging gene SIRT1 in each group were measured, and their relative expression ratios compared to the control were calculated. The results are shown in... Figure 5 .

[0175] The results showed that maturation stimulation significantly enhanced the expression of the aging gene p53, but at any concentration of PQQ, p53 expression was significantly suppressed to the same level as the control. Additionally, maturation stimulation suppressed the expression of the anti-aging gene SIRT1, but at any concentration of PQQ, SIRT1 expression was significantly enhanced to the same level as the control.

[0176] As shown in the results of experiments 3-1 and 3-2, in primary endometrial stromal cells, PQQ inhibited aging factors (aging-related β-galactosidase, aging gene p53) caused by decidualization stimulation and enhanced anti-aging factors (anti-aging gene SIRT1). Therefore, it was determined that PQQ has the function of inhibiting aging during the maturation process of endometrial tissue.

[0177] In summary, this indicates that PQQ can promote the maturation of endometrial tissues (stromal, epithelium, and glandular epithelium) in vitro while also inhibiting cell senescence. Therefore, it is concluded that PQQ promotes and assists normal endometrial maturation, which helps to promote and assist implantation.

[0178] [Experiment 4-1. Gene expression in endometrial epithelium of mice drinking PQQ (RT-PCR)]

[0179] The expression of genes related to decidualization and epithelial differentiation in the entire uterus of mice that consumed PQQ was evaluated. The results showed that the expression of genes related to decidualization and epithelial differentiation, including LIF1, ITGB3, FOXO1, BMP2, and COX2, was significantly enhanced, consistent with in vitro results. Figure 6 ).

[0180] [Experimental Example 4-2. Gene expression and immunostaining of endometrial epithelium in mice treated with PQQ]

[0181] The expression of FOXO1, a representative gene associated with epithelial differentiation, in the uterus of mice fed PQQ was evaluated. The results showed a significant increase in FOXO1-positive cells, indicating that epithelial differentiation occurred even at the cellular level in the uterus. Figure 7 ).

[0182] As shown in the results of Experiments 4-1 and 4-2, in the uterus of mice that drank PQQ, the characteristic gene for epithelial differentiation was highly expressed, and the number of epithelial differentiated cells increased. Therefore, it was determined that PQQ can promote epithelial differentiation of the endometrium not only in vitro, but also in animal models, and assist and promote the implantation of the endometrium.

[0183] [Experiment 5. Implantation rate evaluation test in mice that drank PQQ]

[0184] The implantation rate of embryos transferred in the uterus of mice that drank PQQ was evaluated, and the results showed a significantly increased implantation rate compared with mice that did not drink PQQ. Figure 8 ).

[0185] Based on the results of experiments 1-5, it is shown that PQQ has the effect of maturing endometrial stromal cells, epithelial cells, and glandular epithelial cells that play an important role in implantation. At the same time, it also has the effect of inhibiting cell senescence caused by overactivation, which is ideal for promoting and assisting implantation. As a result of this effect, the embryo implantation rate is improved.

[0186] [Experiment 6. Gene Expression Assay for Cell Fusion]

[0187] For placental trophoblast cells, with and without the addition of Foskerlin (0.5 μM, Caymanchemical, hereinafter referred to as "FSK") as a fusion stimulus, 10 nM of PQQ was simultaneously added to the culture medium [containing 4% knockout serum substitute (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (Fujifilm and Kojin Chemical Co., Ltd.), 0.3% bovine serum albumin (Fujifilm and Kojin Chemical Co., Ltd.), 1% ITS-X supplement (Fujifilm and Kojin Chemical Co., Ltd.), 2.5 μM Y27632 (Fujifilm and Kojin Chemical Co., Ltd.), 50 ng / mL epidermal growth factor (Fujifilm and Kojin Chemical Co., Ltd.), and Dulbecco modified Eagle medium / F12 (DMEM / F12) (1:1) medium containing antibiotics and antifungal drugs (Fujifilm and Kojin Chemical Co., Ltd.)]. The fusion results were compared between the state without PQQ and the state with PQQ.

[0188] RNA extraction and RT-PCR quantification were performed under various conditions to evaluate gene expression of Syncytin 2 and PAPPA as fusion markers. Results are shown in... Figure 9 .

[0189] Figure 9 The diagram shows the state of no fusion stimulation (Ctrl), only PQQ added (PQQ), the state of becoming a fused trophoblast cell through fusion stimulation (FSK), and the state of adding PQQ on the basis of fusion stimulation (FSK+PQQ).

[0190] The results showed that adding PQQ significantly increased the gene expression of Syncytin 2 and PAPPA, which are fusion markers.

[0191] In summary, PQQ significantly promotes cell fusion.

[0192] [Experiment Example 7. Cell Fusion Promotion Assay]

[0193] Subsequently, human trophoblast stem cells treated with fusion-stimulated FSK were fixed with methanol and incubated with anti-E-cadherin antibody (1:200, #3195, CST) and Alexa Fluor 488-labeled goat anti-mouse antibody (Thermo Fisher Scientific) to recognize the cell surface. The staining images at this stage are shown below. Figure 10 (Experimental Example 7-1)

[0194] Cell nuclei were counterstained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI). The number of nuclei and the total number of nuclei within syncytiotrophoblast cells (fused cells) were counted in five randomly selected microscopic fields for each sample and calculated as a fusion index [(number of nuclei in fused cells / total number of nuclei) × 100] in three independent experiments. The resulting increase in the number of fused cells is illustrated in the figure. Figure 11 It was confirmed that the use of PQQ increased the number of fused cells.

Claims

1. An agent that promotes or improves endometrial decidualization, comprising an effective amount of pyrroloquinoline quinone or its salt.

2. An anti-aging agent for the endometrium, comprising an effective amount of pyrroloquinoline quinone or its salt.

3. The medicament according to claim 2, wherein, The anti-aging effect of the endometrium is to inhibit the transformation of endometrial cells into senescent cells.

4. The pharmaceutical preparation according to claim 2, wherein, The anti-aging process of the endometrium involves removing senescent cells from the endometrium.

5. A placental trophoblast cell fusion promoter or improver, comprising an effective amount of pyrroloquinoline quinone or its salt.

6. A fertility enhancer containing an effective amount of pyrroloquinoline quinone or its salt, which enhances fertility by promoting or improving endometrial decidualization and / or promoting or improving placental trophoblast cell fusion.

7. The pharmaceutical agent according to claim 6, wherein, The improvement in fertility refers to improved implantation or continued implantation.

8. The medicine according to any one of claims 1 to 7, wherein it is ingested for at least 7 days to 21 days after the first day of menstruation.

9. The medicine according to any one of claims 1 to 7, wherein it is ingested for at least 14 days to 35 days after the first day of menstruation.

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