Glucocorticoid receptor inhibitors

Abstract

To provide a novel glucocorticoid receptor inhibitor.SOLUTION: A glucocorticoid receptor inhibitor contains 3α-bufalin, a 3α-bufalin derivative, or a pharmaceutically acceptable salt of a 3α-bufalin derivative as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to glucocorticoid receptor inhibitors. [Background technology]

[0002] Glucocorticoids are a type of hormone released from the adrenal cortex, and are known to suppress inflammatory responses and deal with various stresses. Substances that act as agonists on glucocorticoid receptors have strong anti-inflammatory and immunosuppressive effects, and are widely used to treat chronic adrenal insufficiency (Addison's disease), collagen diseases, allergies, autoimmune diseases, blood diseases, etc. However, because these substances act systemically, it is known that large doses or long-term administration can cause various side effects such as elevated blood sugar, high blood pressure, osteoporosis, peptic ulcers, and susceptibility to infection.

[0003] On the other hand, it has been revealed in recent years that excessive glucocorticoids cause damage to the central nervous system and are associated with the worsening of pathology in patients with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, chronic inflammatory reactions, and treatment-resistant depression, and therefore drugs that have an inhibitory effect on glucocorticoid receptors are one of the drug discovery targets for the treatment of these diseases (Non-Patent Document 1).

[0004] Specifically, it has been confirmed that glucocorticoid receptor antagonists improve behavioral disorders in genetically modified mice, which are considered to be model animals for Alzheimer's disease (Non-Patent Document 2), and that glucocorticoid receptor inhibitors reduce anxiety behavior in chronic stress model rats. It has been reported that glucocorticoid receptor inhibitors may contribute to the prevention and treatment of these diseases (Non-Patent Documents 3 and 4).

[0005] Mifepristone (RU-486) ​​is known as a glucocorticoid receptor inhibitor (Non-Patent Document 5). This mifepristone also has an anti-luteinizing hormone effect, and is clinically used as an abortion drug in the early stages of pregnancy in the United States and Germany. In addition, Relacorilant, which is currently under development, is also known to have an anti-luteinizing hormone effect (Non-Patent Document 6), and the development of a substance with higher receptor selectivity is desired.

[0006] In recent years, the number of patients with central nervous system degenerative diseases such as Alzheimer's disease and Parkinson's disease has been increasing, but there is no cure for the disease. For this reason, the invention of drugs that contribute to their prevention and treatment is one of the most urgent issues. In addition, depression, insomnia, cardiovascular disorders such as high blood pressure, and autonomic nervous system dysregulation disorders caused by chronic stress have led to reduced productivity in social activities and have become social problems. It has been reported that patients suffering from such chronic stress suffer from organic disorders of the central nervous system.

[0007] Research is being conducted into substances that can prevent or repair organic damage to the central nervous system, as well as into regenerative medicine treatments. However, there is no specific drug that can effectively improve central nervous system disorders, and regenerative medicine has not yet been widely put to practical use due to cost and safety issues. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Alejandro F. De Nicola et al., 2020. Insights into thetherapeutic potential of glucocorticoid receptor modulators for neurodegenerative diseases Int J. mol. Sci. 21: 2137. [Non-Patent Document 2] M. Pedrazzoli et al., 2019. Glucocorticoid receptors modulated endric spine plasticity and microglia activity in and animal model of Alzheimer's disease. Neurobiology of Disease 131: 104568. [Non-Patent Document 3] J. Ding et al., 2019. Late glucocorticoid receptor antagonism changes the outcome of adult life stress. Psychoneuroendocrinology 107:169-178. [Non-Patent Document 4] Yukio Ago et al., 2009. Depression and adrenal cortical hormone receptors. Japanese Journal of Pharmacology 134:304-308. [Non-Patent Document 5] Sergiu Dalm, Adriaan M. Karssen, Onno C. Meijer1, Joseph K.Belanoff, E. Ronald de Kloet1:Resetting the Stress System with a Mifepristone Challenge. Cellular and Molecular Neurobiology(2019) 39:503-522. [Non-Patent Document 6] Sherice Williams and Chaitali Ghosh: Neurovascular glucocorticoid receptors and glucocorticoids: implications in health, neurological disorders and drugtherapy. Drug Discovery Today (2020) 25:89-106. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel glucocorticoid receptor inhibitor. [Means for solving the problem]

[0010] In order to achieve the above object, the present inventors have conducted extensive research and found that 3α-bufalin has an inhibitory effect on the glucocorticoid receptor. That is, the present invention is a glucocorticoid receptor inhibitor containing 3α-bufalin, a 3α-bufalin derivative, or a pharma- ceutically acceptable salt of a 3α-bufalin derivative as an active ingredient. Effect of the Invention

[0011] According to the present invention, a novel glucocorticoid receptor inhibitor can be provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the antagonist activity of 3α-bufalin against the glucocorticoid receptor. [Diagram 2] FIG. 1 shows the antagonist activity of 3α-bufalin against vitamin D receptors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The glucocorticoid receptor inhibitor of the present invention contains 3α-bufalin, a 3α-bufalin derivative, or a pharma- ceutically acceptable salt of a 3α-bufalin derivative as an active ingredient. 3α-bufalin is a compound represented by the following chemical formula, and is the main metabolite of bufalin, which is one of the bufadienolides that are the main active ingredients of toad venom.

[0014] [ka]

[0015] Senso is a collection of ear gland secretions from the Asian toad (Bufogargarizans CANTOR) or the common toad (Bufo melanostictus SCHNEIDER), which is a member of the Bufonidae family, and is known to contain cardiac steroid bufadienolides and indole alkaloids as its main active ingredients (17th Revised Japanese Pharmacopoeia). Senso has long been known to have cardiotonic, anti-inflammatory, respiratory stimulant, and local anesthetic effects, and is therefore used in Japan as an ingredient or raw material for medicines that are effective in treating palpitations, shortness of breath, and alertness (17th Revised Japanese Pharmacopoeia Commentary (Hirokawa Shoten)).

[0016] The cardiac stimulant bufadienolide in Senso has a mechanism of action similar to that of cardiac glycosides derived from digitalis, but it is known that it is metabolized early in the body and the metabolites have weakened pharmacological effects. 3α-bufalin is the target molecule of the cardiac stimulant in Senso, Na + ,K + It is known that the inhibitory effect of bufalin on -ATPase is significantly weaker than that of bufalin (Tohma Shoji et al., 1991, In vivo kinetics of Senso components Bufalin and Cinobufagin. Pharmaceutical Journal 111:687-694.).

[0017] The present inventors investigated the effects of bufadienolides and their major metabolites on nuclear receptors and found that 3α-bufalin antagonizes the effects of glucocorticoid receptor agonists, thereby completing the present invention.

[0018] 3α-bufalin in the glucocorticoid receptor inhibitor of the present invention can be obtained by any method. For example, it can be synthesized by known organic chemical methods. Alternatively, bufalin can be obtained by extracting and purifying herbal medicine such as Senso or the ear gland secretion of a toad as a raw material by a conventional method, and the compound can be obtained by organic chemical or biological methods.

[0019] Examples of 3α-bufalin derivatives include the following: The hydroxyl group at the 3α-position is substituted with a methoxy group, acetoxy group, carboxyl group, acetate ester, succinate ester, glutarate ester, adipate ester, pimelate ester, or suberate ester The hydrogen atom at the 1β position is replaced by a hydroxyl group The hydrogen atom at the 5β position is replaced by a hydroxyl group The methyl group at the 10th position is replaced with a hydroxymethyl group. The hydrogen atom at the 11th position is replaced by an oxygen atom, the hydrogen atom at the 11α position is replaced by a hydroxyl group, or the hydrogen atom at the 11β position is replaced by a hydroxyl group The hydrogen atom at the 12-position is replaced by an oxygen atom, the hydrogen atom at the 12α-position is replaced by a hydroxyl group, or the hydrogen atom at the 12β-position is replaced by a hydroxyl group The hydrogen atom at the 16β position is replaced with an acetoxy group or a hydroxyl group.

[0020] In the present specification, pharma- ceutically acceptable salts include various salts such as acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, etc. Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic acid salts such as hydrochloride, sulfate, phosphate, etc., and organic acid salts such as acetate, trifluoroacetate, citrate, maleate, fumarate, lactate, tartrate, etc.

[0021] Examples of metal salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of ammonium salts include ammonium and tetramethylammonium salts. Examples of organic amine addition salts include morpholine addition salts and piperidine addition salts. Examples of amino acid addition salts include glycine addition salts, phenylalanine addition salts, lysine addition salts, aspartic acid addition salts, glutamic acid addition salts, and arginine addition salts.

[0022] The glucocorticoid receptor inhibitor of the present invention is useful for preventing, inhibiting the progression of, and / or treating central nervous system disorders caused by neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and chronic stress. Therefore, the glucocorticoid receptor inhibitor of the present invention can be used by itself to improve central nervous system disorders. As long as the effects of the present invention are not impaired, the glucocorticoid receptor inhibitor may be dissolved in a solvent generally used for pharmaceuticals, foods, and beverages, such as water or ethanol, before use.

[0023] Alternatively, the glucocorticoid receptor inhibitor of the present invention may be combined with an additional active ingredient to be used as a composition for improving central nervous system disorders. Examples of such active ingredients include other substances that have glucocorticoid receptor regulating effects, or substances that have preventive or therapeutic effects on neurodegenerative diseases such as dementia and Parkinson's disease, or chronic stress disorders. Specifically, the active ingredients include the herb saffron or its extract, and the herb Senso or its extract. The amount of saffron to be combined may be about 4 to 100 mg per day, and the amount of Senso to be combined may be about 1 to 5 mg per day, but is not particularly limited.

[0024] The composition for improving central nervous system disorders of the present invention can also contain herbal medicines such as bezoar, ginseng, antelope horn, pearl, agarwood, bornole, animal gall, musk, red ginseng, buffalo horn, and licorice as additional active ingredients.

[0025] In addition to the above-mentioned active ingredient, the composition for improving central nervous system disorders of the present invention may contain excipients, sweeteners, acidulants, thickeners, flavorings, colorants, emulsifiers, and other additives and materials commonly used in pharmaceuticals, foods, beverages, etc., as long as the effects of the present invention are not impaired.

[0026] The composition for improving central nervous system disorders of the present invention can be used as a pharmaceutical or quasi-drug. Pharmaceuticals or quasi-drugs can be obtained by formulating the composition with an appropriate excipient or the like by a method known to those skilled in the art according to the administration method. Examples of the administration method include oral administration, topical administration, rectal administration (suppository), and parenteral administration such as eye drops. Among these, oral administration and topical administration are preferred. When administered orally, the above-mentioned daily amount can be administered at once or in several divided doses. Administration may be performed before, after, or between meals, and the administration period is not particularly limited and can be set appropriately.

[0027] The pharmaceutical or quasi-drug product may be in any dosage form for oral or parenteral administration. Examples include pills, granules, powders, tablets, coated tablets, sugar-coated tablets, capsules (e.g., hard or soft gelatin capsules), liquids, emulsions, suspensions, fine granules, and chewable agents. These can be formulated by conventional methods. Examples of the form for injection include direct intravenous injection and drip administration. If necessary, carriers such as lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, and gelatin, and conventional additives such as solvents, solubilizers, and isotonicity agents may be appropriately blended.

[0028] Alternatively, the pharmaceutical or quasi-drug of the present invention can be administered parenterally, for example, intravenously, intramuscularly, intraperitoneally, or subcutaneously as an injection, or rectally in the form of a suppository.Furthermore, it may be administered locally or transdermally in the form of an external preparation such as an ointment, cream, liquid, lotion, emulsion, tincture, ointment, aqueous gel, oily gel, aerosol, powder, shampoo, or soap, or it may be administered locally to the eye in the form of eye drops or eyewash.The daily dose of the external preparation can be appropriately set depending on the symptoms, purpose, age, administration method, etc. of the subject to be administered.

[0029] The dosage of a pharmaceutical or quasi-drug is not particularly limited, but can be appropriately selected depending on the dosage form, age, weight, and symptoms of the ingesting person or animal such as a user or patient. For example, the amount of active ingredient may be 0.00001 to 0.1 g, preferably 0.00001 to 0.05 g, and more preferably 0.0001 to 0.05 g, per 1 kg of body weight of the ingesting person or animal per day in the case of oral administration, and 0.000001 to 0.01 g, preferably 0.000001 to 0.005 g, and more preferably 0.00001 to 0.005 g in the case of parenteral administration. The intake period can be determined arbitrarily depending on the age, symptoms, administration route, etc. of the user or patient.

[0030] The composition for improving central nervous system disorders of the present invention can be used as food and drink. In that case, it may be in any form of liquid, powder, granule, capsule, or tablet. The food and drink can be used as various foods and drinks such as general foods, functional foods, health foods, health supplements, nutritional supplements (supplements), health functional foods, specific health foods, nutritional functional foods, beauty foods, and specific functional foods.

[0031] Examples of the form of food and drink include beverages (soft drinks, carbonated drinks, nutritional drinks, powdered drinks, fruit drinks, dairy drinks, jelly drinks, etc.), sweets (candy, chocolate, cookies, cakes, chewing gum, candies, tablets, gummy candies, buns, yokan, puddings, jellies, ice cream, sorbets, etc.), processed seafood products (fish sausages, kamaboko, chikuwa, hanpen, etc.), processed livestock products (hamburger steaks, ham, sausages, wieners, cheese, butter, yogurt, fresh cream, cheese, margarine, fermented milk, etc.), soups (powdered soups, liquid soups, etc.), staple foods (rice, noodles (dried noodles, fresh noodles), bread, cereals, etc.), seasonings (mayonnaise, shortening, dressings, sauces, sauces, soy sauce, etc.), and supplements.

[0032] When the composition for improving central nervous system disorders of the present invention is contained in a food or drink, an appropriate amount may be ingested so that a predetermined amount of the glucocorticoid receptor inhibitor is ingested. The amount of the food or drink to be ingested can be appropriately selected depending on the content of the glucocorticoid receptor inhibitor in the food or drink.

[0033] Furthermore, the composition for improving central nervous system disorders of the present invention can be used by being added to animal feed, such as dry dog ​​food, dry cat food, wet dog food, wet cat food, semi-moist dog food, chicken feed, and feed for livestock such as cows and pigs.

[0034] The animals for animal feed include all animals that require disease prevention or treatment, including pet animals, livestock animals, and animals kept in zoos, etc. The composition for improving central nervous system disorders of the present invention can also be used appropriately for animals other than humans, for example, livestock mammals such as cows, horses, pigs, and sheep, poultry such as chickens, quails, and ostriches, and pets such as reptiles, birds, and small mammals. EXAMPLES

[0035] The glucocorticoid receptor inhibitors according to the present invention will be specifically described below, but the present invention is not limited thereto.

[0036] [Sample preparation] Bufalin was isolated and purified from Senso, and chromium oxide was added to it and oxidized in glacial acetic acid to obtain 3-ketobufalin. 3-Ketobufalin was purified by the usual method and then dissolved in methanol. Sodium borohydride was added to the resulting solution, which was stirred and concentrated after 30 minutes. The portion corresponding to 3α-bufalin was scraped off using silica gel thin layer chromatography, extracted, and recrystallized to isolate and purify 3α-bufalin. The 3α-bufalin thus obtained was dissolved in ethanol and diluted with 10 -3 The test drug solution was prepared at a concentration of 10 -3 M 3α-bufalin ethanol solution.

[0037] [Evaluation of antagonist activity] To evaluate the activity, a dual reporter assay system using human fetal kidney epithelial cell line 293T cells was used. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% charcoal-stripped fetal bovine serum at 37°C and 5% CO. 2 The cells were cultured under atmospheric conditions. The cells were subcultured by diluting about 10-fold approximately every 4 days. When evaluating activity, a medium that did not contain phenol red, a pH indicator, was used.

[0038] <Glucocorticoid receptor antagonist activity> First, 15 × 10 293T cells were plated in a 12-well multiwell plate. 5 The cells were seeded at 1000x1000 cells / mL / well and precultured overnight. Then, using Lipofectamine 2000, a plasmid encoding the glucocorticoid receptor (25 ng), a plasmid encoding firefly luciferase downstream of the glucocorticoid receptor binding region (250 ng), and a plasmid encoding Renilla luciferase downstream of the CMV promoter (1 ng) were dissolved in 0.4 mL of OptiMEM medium and transfected into each well.

[0039] After 6 hours of incubation, the supernatant was removed and 1 mL of medium was added to each well. In addition, 1 μL of the test drug solution was added and cultured for 48 hours. To evaluate antagonist activity, 10 μL of dexamethasone, a known ligand, was added to each well. -3 1 μL of M ethanol solution was also added and incubated. After incubation, the supernatant was removed, the cells were washed with PBS, and the cells were disrupted. For the disruption, 300 μL of the lysis buffer included in the Dual-Luciferase Reporter Assay System (Promega) kit was used for each well. The amount of luminescence due to firefly luciferase (F) and Renilla luciferase (R) was measured by adding 20 μL of the color development reagent included in the kit to 50 μL of the disrupted solution.

[0040] The results are shown in Figure 1. In Figure 1, the vertical axis is (F / R), and the data show the mean ± standard deviation (mean ± SD). In the case of dexamethasone, the value was "4.67 ± 0.52," and in the case of adding the test drug solution, the value was "1.80 ± 0.74." This confirmed that 3α-bufalin has antagonist activity against the glucocorticoid receptor.

[0041] <Vitamin D receptor antagonist activity> First, 15 × 10 293T cells were plated in a 12-well multiwell plate. 5 The cells were seeded at 1000x1000 cells / mL / well and precultured overnight. Then, using Lipofectamine 2000, a plasmid encoding the vitamin D receptor (25 ng), a plasmid encoding firefly luciferase downstream of the vitamin D receptor binding region (250 ng), a plasmid encoding Renilla luciferase downstream of the CMV promoter (1 ng), and a plasmid encoding the retinoid X receptor (25 ng) were dissolved in 0.4 mL of OptiMEM medium and transfected into each well.

[0042] After 6 hours of incubation, the supernatant was removed and 1 mL of medium was added to each well. In addition, 1 μL of the test drug solution was added and cultured for 48 hours. To evaluate antagonist activity, 10 μL of vitamin D as a known ligand was added. -3 1 μL of M ethanol solution was also added and incubated. After incubation, the supernatant was removed, the cells were washed with PBS, and the cells were disrupted. For the disruption, 300 μL of the lysis buffer included in the Dual-Luciferase Reporter Assay System (Promega) kit was used for each well. The amount of luminescence due to firefly luciferase (F) and Renilla luciferase (R) was measured by adding 20 μL of the color development reagent included in the kit to 50 μL of the disrupted solution.

[0043] The results are shown in Figure 2. In Figure 2, the vertical axis is (F / R), and the data show the mean ± standard deviation (mean ± SD). In the case of vitamin D, the value was "1.24 ± 0.15," and in the case where the test drug solution was added, the value was "0.85 ± 0.24." This confirmed that 3α-bufalin has antagonist activity against the vitamin D receptor.

Claims

1. A glucocorticoid receptor inhibitor containing 3α-bufalin as an active ingredient.

2. A composition for improving central nervous system disorders, comprising the glucocorticoid receptor inhibitor according to claim 1.

3. The composition for improving central nervous system disorders according to claim 2, which is a drug or quasi-drug.

4. The composition for improving a central nervous system disorder according to claim 2, which is a food or drink.

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