JP2022056936A - Glucocorticoid receptor inhibitor - Google Patents

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

The present invention relates to glucocorticoid receptor inhibitors.

Glucocorticoid is a kind of hormone released from the adrenal cortex, and is known to have an action of suppressing an inflammatory reaction and coping with various stresses. Substances that act as agonists for glucocorticoid receptors have strong anti-inflammatory and immunosuppressive effects, such as chronic hypocorticosteroid (Azison's disease), collagen disease, allergies, autoimmune diseases, and blood diseases. Widely used in. However, since these substances act systemically, it is known that high-dose administration or long-term administration causes various side effects such as increased blood glucose, hypertension, osteoporosis, digestive ulcer, and easy infection.

On the other hand, drugs that have an inhibitory effect on glucocorticoid receptors have an excess of glucocorticoid in the central nervous system in patients with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, chronic inflammatory reactions, and refractory depression. In recent years, it has become clear that it causes damage to the disease and is associated with the worsening of its condition. Therefore, it has become one of the drug discovery targets for the treatment of these diseases (Non-Patent Document 1).

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

Mifepristone (RU-486) is known as a glucocorticoid receptor inhibitor (Non-Patent Document 5). This mifepristone also has anti-luteinizing hormone action and is clinically used as an abortion drug in the early stages of pregnancy in the United States and Germany. Further, it is known that relacolirant, which is currently under development, also has an antiluteinizing hormone action (Non-Patent Document 6), and development of a substance having more receptor selectivity is desired.

In recent years, the number of patients with central neurodegenerative diseases such as Alzheimer-type dementia and Parkinson's disease has been increasing, but there is no radical therapeutic agent. Therefore, the invention of a drug or the like that contributes to the prevention or treatment thereof is one of the urgent issues. In addition, depression and insomnia due to chronic stress, cardiovascular disorders such as hypertension, and autonomic dysregulation have become social problems due to a decrease in productivity in social activities. It has been reported that these chronically stressed patients have organic disorders of the central nervous system.

For diseases that cause organic disorders of the central nervous system, research on substances having a disorder prevention or tissue repair effect and research on treatment by regenerative medicine are underway. However, there is no effective silver bullet to improve central nervous system disorders, and regenerative medicine has not been widely put into practical use due to cost and safety issues.

Alejandro F. De Nicola et al., 2020. Insights into thetherapeutic potential of glucocorticoid receptor modulators for neurodegenerative diseases Int J. mol. Sci. 21: 2137. M. Pedrazzoli et al., 2019. Glucocorticoid receptors modulatedendric spine plasticity and microglia activity in and animal model of Alzheimer ’s disease. Neurobiology of Disease 131: 104568. J. Ding et al., 2019. Late glucocorticoid receptor antagonismchanges the outcome of adult life stress. Psychoneuroendocrinology 107: 169-178. Yukio Ago et al., 2009. Depression and corticosteroid receptors. Journal of Pharmacology 134: 304-308. Sergiu Dalm, Adriaan M. Karssen, Onno C. Meijer1, Joseph K.Belanoff, E. Ronald de Kloet1: Resetting the StressSystem with a Mifepristone Challenge. Cellular and Molecular Neurobiology (2019) 39: 503-522. Sherice Williams and Chaitali Ghosh: Neurovascular glucocorticoid receptorsand glucocorticoids: implications in health, neurological disorders and drugtherapy. Drug Discovery Today (2020) 25:89-106.

An object of the present invention is to provide a novel glucocorticoid receptor inhibitor.

As a result of intensive studies to achieve the above object, the present inventors have found that 3α-bufalin has an action of inhibiting a glucocorticoid receptor. That is, the present invention is a glucocorticoid receptor inhibitor containing a pharmaceutically acceptable salt of 3α-bufalin, 3α-bufalin derivative, or 3α-bufalin derivative as an active ingredient.

According to the present invention, it is possible to provide a novel glucocorticoid receptor inhibitor.

It is a figure which shows the antagonist activity of 3α-bufalin to a glucocorticoid receptor. It is a figure which shows the antagonist activity of 3α-bufalin to the vitamin D receptor.

The glucocorticoid receptor inhibitor of the present invention contains, as an active ingredient, a pharmaceutically acceptable salt of 3α-bufalin, a 3α-bufalin derivative, or a 3α-bufalin derivative. 3α-bufalin is a compound represented by the following chemical formula and is a main metabolite of bufalin, which is one of the main medicinal components of Bufoto.

Senso is a collection of parotid gland secretions of the Asian toad (Bufogargarizans CANTOR) or the Asian common toad (Bufo melanostictus SCHNEIDER) of the toad family. (17th revised Japanese Pharmacy). Since ancient times, Senso has been known to have cardiotonic, anti-inflammatory, respiratory excitatory, and local anesthetic effects, so it is also used as an ingredient or raw material for pharmaceuticals with palpitation, shortness of breath, and care. (17th revised Japanese Pharmacopoeia manual (Hirokawa Shoten)).

Bufadienolide, the cardiotonic component of senso, has a mechanism of action similar to that of digitalis-derived cardiac glycosides, but is known to be metabolized early in vivo and its metabolites have weakened pharmacological action. It is known that 3α-bufalin has a significantly attenuated inhibitory effect on Na ⁺ , K ⁺ -ATPase, which is a target molecule of the cardiotonic component contained in senso, as compared with bufalin (Shoji Toma et al.,. 1991, In vivo dynamics of the senso components Bufalin and Cinobufagin. Journal of Pharmaceutical Sciences 111: 687-694.).

As a result of investigating the action of bufadienolide and its main metabolite on the nuclear receptor, the present inventors have found that 3α-bufalin antagonizes the action of a glucocorticoid receptor agonist, and make the present invention. It has reached.

The 3α-bufalin in the glucocorticoid receptor inhibitor of the present invention can be obtained by any method. For example, it can be synthesized by a known organic chemical method. Alternatively, a crude drug such as senso or a parotid gland secretion of a toad can be used as a raw material and extracted and purified by a conventional method to obtain bufalin, which can be obtained from this compound by an organic chemical or biological method.

Examples of the 3α-bufalin derivative include the following.
-The hydroxyl group at the 3α position is replaced with a methoxy group, an acetoxy group, a carboxyl group, an acetic acid ester, a succinic acid ester, a glutalylic acid ester, an adipic acid ester, a pimeric acid ester, or a suberic acid ester. Is replaced with a hydroxyl group ・ The hydrogen atom at the 5β position is replaced with a hydroxyl group ・ The methyl group at the 10th position is replaced with a hydroxymethyl group ・ The hydrogen atom at the 11th position is an oxygen atom and the hydrogen atom at the 11α position Is a hydroxyl group or a hydrogen atom at the 11β position is replaced with a hydroxyl group ・ A hydrogen atom at the 12th position is replaced with an oxygen atom, a hydrogen atom at the 12α position is replaced with a hydroxyl group, or a hydrogen atom at the 12β position is replaced with a hydroxyl group ・ 16β position Hydrogen atom is replaced with an acetoxy group or a hydroxyl group

Further, in the present specification, the pharmaceutically acceptable salt includes various salts such as an acid addition salt, a metal salt, an ammonium salt, an organic amine addition salt and an amino acid addition salt. Examples thereof include inorganic acid salts such as hydrochlorides, sulfates and phosphates, and organic acid salts such as acetates, trifluoroacetates, citrates, maleates, fumarates, lactates and tartrates. It can, but is not limited to these.

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 salts such as ammonium and tetramethylammonium. Examples of the organic amine addition salt include a morpholine addition salt and a piperidine addition salt. Examples of amino acid addition salts include glycine addition salt, phenylalanine addition salt, lysine addition salt, aspartic acid addition salt, glutamic acid addition salt, and arginine addition salt.

The glucocorticoid receptor inhibitor of the present invention is useful for prevention, progression inhibition, and / or treatment of disorders of the central nervous system due to 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 for improving central nervous system disorders. As long as the effect of the present invention is not impaired, it may be dissolved in a solvent generally used with pharmaceuticals such as water and ethanol, foods and drinks, and used.

Alternatively, the glucocorticoid receptor inhibitor of the present invention can be further blended with an active ingredient and used as a composition for improving central nervous system disorders. Such active ingredients include, for example, other substances having a glucocorticoid receptor regulating action, neurodegenerative diseases such as dementia and Parkinson's disease, and substances having a preventive or therapeutic effect on chronic stress disorders. Can be mentioned. Specifically, it is a crude drug saffron or an extract thereof, a crude drug Senso or an extract thereof, and the like. The daily amount of saffron may be about 4 to 100 mg, and the daily amount of senso may be about 1 to 5 mg, but the amount is not particularly limited.

The composition for improving central nervous system disorder of the present invention includes calculus bovis, carrot, ginseng horn, pearl, agarwood, dragon brain, animal ginseng, and so on. Crude drugs such as musk, red ginseng, buffalo horn and licorice can also be added as further active ingredients.

In addition to the above-mentioned active ingredients, the composition for improving central nervous system of the present invention contains excipients, sweeteners, acidulants, thickeners, flavors, pigments, emulsifiers, and others as long as the effects of the present invention are not impaired. May contain additives and materials commonly used in pharmaceuticals, foods and drinks, and the like.

The central nervous system disorder improving composition of the present invention can be used as a pharmaceutical product or a quasi-drug. Pharmaceuticals or quasi-drugs can be obtained by formulating them by a method known to those skilled in the art together with appropriate excipients and the like, depending on the administration method. Examples of the administration method include oral administration, external administration, transrectal administration (suppository), parenteral administration such as eye drops. Of these, oral administration, external administration and the like are preferable. In the case of oral administration, the above-mentioned daily dose can be administered at one time or in several divided doses. The administration may be performed before, after, or between meals, and the administration period is not particularly limited and can be appropriately set.

The drug or quasi-drug can be in any dosage form for oral or parenteral administration. Examples thereof include pills, granules, powders, tablets, coated tablets, sugar-coated tablets, capsules (eg, hard or soft gelatin capsules), liquids, emulsions, suspensions, fine granules and chewables. .. These can be formulated by a conventional method. Examples of the form for injection include direct intravenous injection, infusion administration, and the like. If necessary, carriers such as lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin and gelatin, and usual additives such as solvents, solubilizing agents and isotonic agents are appropriately added. May be good.

Alternatively, the pharmaceutical product or quasi-drug of the present invention can be administered parenterally, for example, intravenously, intramuscularly, intraperitoneally, intraperitoneally, subcutaneously, or rectally in the form of a suppository. .. Furthermore, it is localized or transdermal in the form of external agents such as ointments, creams, liquids, lotions, emulsions, tinctures, ointments, aqueous gels, oily gels, aerosols, powders, shampoos and soaps. It may be administered locally to the eye in the form of an ointment or a lotion. The daily dose of the external preparation can be appropriately set according to the symptom, purpose, age, administration method and the like of the subject to be administered.

The dose of the drug or quasi-drug is not particularly limited, but can be appropriately selected depending on the dosage form, the age, weight and symptoms of the ingestor such as the user or the patient or the ingesting animal. For example, the amount of the active ingredient is 0.00001 to 0.1 g, preferably 0.00001 to 0.05 g, more preferably 0.0001 to 0. For oral administration per 1 kg of the body weight of the ingestor or the ingested animal per day. In the case of parenteral administration, 05 g may be administered in an amount of 0.000001 to 0.01 g, preferably 0.000001 to 0.005 g, and more preferably 0.00001 to 0.005 g.
The ingestion period can be arbitrarily determined according to the age, symptoms, administration route, etc. of the user or patient.

The central nervous system disorder improving composition of the present invention can be used as a food or drink. In that case, it may be in the form of a liquid, a powder, a granule, a capsule, or a tablet. Food and drink includes various foods and drinks such as general foods, functional foods, health foods, health supplements, nutritional supplements (supplements), health functional foods, specified health foods, nutritionally functional foods, beauty foods and foods with specific functional claims. It can be used as an item.

The forms of food and drink include, for example, beverages (soft beverages, carbonated beverages, nutritional beverages, powdered beverages, fruit beverages, dairy beverages, jelly beverages, etc.), confectionery (candy, chocolate, cookies, cakes, chewing gum, candies, tablets). , Gummy, bun, sheep drink, pudding, jelly, ice cream, sherbet, etc.), processed marine products (fish sausage, kamaboko, chikuwa, hampen, etc.), processed livestock products (hamburger, ham, sausage, wiener, cheese, butter, yogurt, etc.) , Fresh cream, cheese, margarine, fermented milk, etc.), soup (powdered soup, liquid soup, etc.), main foods (rice, noodles (dried noodles, raw noodles), bread, cereals, etc.), seasonings (mayonnaise, shortning) , Dressing, sauce, sauce, soy sauce, etc.), supplements, etc.

When the composition for improving central nervous system disorder of the present invention is contained in foods and drinks, an appropriate amount may be ingested so that a predetermined amount of the glucocorticoid receptor inhibitor is ingested. The intake amount of the food or drink can be appropriately selected depending on the content of the glucocorticoid receptor inhibitor in the food or drink.

Furthermore, the central nervous system disorder improving composition of the present invention can also be blended and used in animal feeds. As a form, it can be blended in dry dog food, dry cat food, wet dog food, wet cat food, semi-moist dock food, poultry feed, and livestock feed such as cattle and pigs.

Animals in animal feeds include all animals in need of prevention or treatment of the disease, including pet animals, livestock animals, or animals kept in zoos and the like. The composition for improving central nervous system disorders of the present invention comprises animals other than humans, for example, domestic mammals such as cows, horses, pigs and sheep, poultry such as chickens, quails and ostriches, and pets such as insects, birds or small mammals. It can also be used as appropriate for species and the like.

Hereinafter, the glucocorticoid receptor inhibitor according to the present invention will be specifically described, but these are not limited to the present invention.

[Sample preparation]
Bufalin was isolated and purified from senso, and chromium oxide was added to the bufalin and oxidized in glacial acetic acid to give 3-ketobufalin. 3-Ketobfarin was purified by a conventional method and then dissolved in methanol. Sodium borohydride was added to the obtained solution, the mixture was stirred, and the mixture was concentrated after 30 minutes. 3α-bufalin was isolated and purified by scraping the portion corresponding to 3α-bufalin by silica gel thin layer chromatography, extracting and recrystallizing.
The 3α-bufalin thus obtained was dissolved in ethanol to prepare a test drug solution having a concentration of ^10-3 M. The test drug solution is a ^10-3 M 3α-bufarin ethanol solution.

[Evaluation of antagonist activity]
For the activity evaluation, a dual reporter assay system using 293T cells of human fetal-derived renal epithelial cell line was used. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% charcoal-treated fetal bovine serum at a typical 37 ° C. and 5% CO ₂ atmosphere. The cells were diluted approximately 10-fold approximately every 4 days for passage maintenance. At the time of activity evaluation, a medium containing no phenol red, which is a pH indicator, was used.

<Glucocorticoid receptor antagonist activity>
First, 293T cells were seeded on a 12-well multi-well plate at a cell number of 15 × 10 ⁵ cells / mL / well, and cultured day and night before. Then, using Lipofectamine 2000, a plasmid encoding a glucocorticoid receptor (25 ng), a plasmid encoding firefly luciferase downstream of the glucocorticoid receptor binding region (250 ng), and a sea urchin luciferase downstream of the CMV promoter were encoded. The plasmid (1 ng) was dissolved in 0.4 mL of OptiMEM medium and transfected into each well.

After 6 hours of incubation, the supernatant was removed and 1 mL of medium was added to each well. Further, 1 μL of the above-mentioned test drug solution was added and the cells were cultured for 48 hours. To assess antagonist activity, 1 μL of ^10-3 M ethanol solution of dexamethasone as a known ligand was also added and cultured.
After culturing, the supernatant was removed, washed with PBS, and the cells were disrupted. For crushing, 300 μL of Lysis Buffer from the Dual-Luciferase Reporter Assay System (Promega) kit was used for each well. By adding 20 μL of the color-developing reagent of the kit to 50 μL of the crushed liquid, the luminescence amount (F) by firefly luciferase and the luminescence amount (R) by sea urchin luciferase were measured.

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

<Vitamin D receptor antagonist activity>
First, 293T cells were seeded on a 12-well multi-well plate at a cell number of 15 × 10 ⁵ cells / mL / well, and cultured day and night before. Then, using Lipofectamine 2000, a plasmid encoding a vitamin D receptor (25 ng), a plasmid encoding a firefly luciferase downstream of the vitamin D receptor binding region (250 ng), and a plasmid encoding a sea urchin cercipherase downstream of the CMV promoter. (1 ng) and a plasmid (25 ng) encoding the retinoid X receptor were dissolved in 0.4 mL of OptiMEM medium and transfected into each well.

After 6 hours of incubation, the supernatant was removed and 1 mL of medium was added to each well. Further, 1 μL of the above-mentioned test drug solution was added and the cells were cultured for 48 hours. To assess antagonist activity, 1 μL of ^10-3 M ethanol solution of vitamin D as a known ligand was also added and cultured.
After culturing, the supernatant was removed, washed with PBS, and the cells were disrupted. For crushing, 300 μL of Lysis Buffer from the Dual-Luciferase Reporter Assay System (Promega) kit was used for each well. By adding 20 μL of the color-developing reagent of the kit to 50 μL of the crushed liquid, the luminescence amount (F) by firefly luciferase and the luminescence amount (R) by sea urchin luciferase were measured.

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

Claims (4)

A glucocorticoid receptor inhibitor containing a pharmaceutically acceptable salt of 3α-bufalin, 3α-bufalin derivative, or 3α-bufalin derivative as an active ingredient. A composition for improving central nervous system disorders, which comprises the glucocorticoid receptor inhibitor according to claim 1. The central nervous system disorder improving composition according to claim 2, which is a drug or a quasi-drug. The central nervous system disorder improving composition according to claim 2, which is a food or drink.

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