Glutaminase inhibitor

Abstract

To provide a glutaminase inhibitor effective for prophylaxis, amelioration or therapy of cancer, inflammatory disease, nervous system disease, aging and obesity.SOLUTION: The present invention pertains to a glutaminase inhibitor comprising, as an active component, at least one substance selected from the group consisting of peptides, L-carnitine, N2-(1-oxopropyl)-L-glutamine, N2-(1-oxobutyl)-L-glutamine, sialic acid, eucomic acid, and 4-imidazoleacetic acid hydrochloride or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Glutaminase (glutaminase I, L-glutaminase, glutamine aminohydrolase) is an amidohydrolase that converts glutamine into glutamic acid. It has been reported that there are tissue-specific isozymes of glutaminase.

[0003] Mammalian tissues have two major isozymes of glutaminase, liver-type glutaminase (LGA) and kidney-type glutaminase (KGA) (Non-Patent Document 1). Non-Patent Document 1 suggests that expression of the gene encoding LGA is observed in the liver, brain, pancreas, breast cancer cells, etc., and that it is expressed in highly proliferative cells. In addition, expression of the gene encoding KGA is observed in all tissues except the liver, and is particularly strongly expressed in the kidney and brain, suggesting that expression increases in differentiated cells and non-proliferative cells.

[0004] Glutaminase inhibitors are a general term for compounds that have the function of inhibiting glutaminase activity. Glutamine is an important energy source for cancer cells, and glutaminase plays an important role in the metabolism of glutamine, so glutaminase inhibitors have been developed primarily as anticancer drugs (Patent Document 1).

[0005] In recent years, it has been revealed that glutaminase inhibitors have various functions other than anti-cancer effects, and various functions have been reported, such as a glutaminase inhibitor that has the effect of suppressing the increase in visceral fat and subcutaneous fat (Patent Document 2), an anti-inflammatory agent that has the effect of improving diseases caused by inflammation (Patent Document 3), an inhibitor of neuronal cell death that has the effect of inhibiting the production of glutamate in microglia (Patent Document 4), and an agent for removing senescent cells (Patent Document 5).

[0006] Examples of glutaminase inhibitors known to date include 6-diazo-5-oxo-L-norleucine [(S)-2-amino-6-diazo-5-oxocaproic acid or a salt thereof (DON)], CB-839, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), Ebselen, Compound 968, GlutaDON (registered trademark) (PEG-PGA+DON) (manufactured by New Medical Enzymes AG), and GlutaChemo (PEG-PGA+ideal candidate) (manufactured by New Medical Enzymes AG) (Patent Documents 2 and 3). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2019-163290 A [Patent Document 2] JP 2020-28239 A [Patent Document 3] JP 2020-29412 A [Patent Document 4] International Publication No. 2007 / 088712 [Patent Document 5] International Publication No. 2020 / 095971 [Non-patent literature]

[0008] [Non-Patent Document 1] Neurochem Int., 2009 Jul-Aug; 55(1-3):71-5. doi: 10.1016 / j.neuint.2009.01.008. Epub 2009 Feb Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, glutaminase inhibitors have been developed, but there is a demand for glutaminase inhibitors that exhibit excellent glutaminase inhibitory activity for the prevention, amelioration and / or treatment of diseases and symptoms related to cancer, inflammatory diseases, nervous system diseases, aging and obesity.

[0010] Therefore, an object of the present invention is to provide a glutaminase inhibitor that exhibits excellent glutaminase inhibitory activity. [Means for solving the problem]

[0011] The present inventors have discovered a substance that exhibits excellent glutaminase inhibitory activity and have completed the present invention.

[0012] The present invention is as follows. 1. Peptides, L-carnitine, N 2 -(1-oxopropyl)-L-glutamine, N 2 A glutaminase inhibitor comprising, as an active ingredient, at least one selected from the group consisting of -(1-oxobutyl)-L-glutamine, sialic acid, eucomic acid, and 4-imidazoleacetic acid hydrochloride, or a pharma- ceutically acceptable salt thereof. 2. The glutaminase inhibitor described in 1 above, wherein the peptide is an oligopeptide. 3. The glutaminase inhibitor according to 1 or 2 above, wherein the peptide is a tripeptide or a dipeptide. 4. The glutaminase inhibitor according to any one of 1 to 3 above, wherein the peptide is a peptide containing glutamic acid and cysteine. 5. The glutaminase inhibitor according to any one of 1 to 4 above, wherein the peptide is a peptide containing γ-glutamylcysteine. 6. The glutaminase inhibitor according to any one of 1 to 5 above, wherein the peptide is at least one peptide selected from the group consisting of homoglutathione, N-acetyl-oxidized glutathione, reduced glutathione, and oxidized glutathione. 7. The glutaminase inhibitor according to any one of 1 to 6 above, wherein the glutaminase is at least one of kidney-type glutaminase and liver-type glutaminase. 8. The glutaminase inhibitor according to any one of 1 to 7 above, wherein the glutaminase is kidney-type glutaminase. Effect of the Invention

[0013] The glutaminase inhibitor of the present invention exhibits excellent glutaminase inhibitory activity and is useful in the prevention, amelioration and / or treatment of diseases, disorders or symptoms associated with glutaminase. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the glutamine conversion rate (%) of each compound for evaluation of glutaminase inhibitory activity shown in Table 2, for evaluating renal glutaminase inhibitory activity. [Diagram 2] FIG. 2 shows the glutamine conversion rate (%) of each compound for evaluation of glutaminase inhibitory activity shown in Table 2, for evaluating renal glutaminase inhibitory activity. [Diagram 3] FIG. 3 shows the glutamine conversion rate (%) of each compound for evaluation of glutaminase inhibitory activity shown in Table 2, for evaluating hepatic glutaminase inhibitory activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] <Active ingredient> The glutaminase inhibitor of the present invention contains an active ingredient having a glutaminase inhibitory effect. The active ingredient may be a peptide, L-carnitine, N 2 -(1-oxopropyl)-L-glutamine, N 2 At least one selected from the group consisting of -(1-oxobutyl)-L-glutamine, sialic acid, eucomic acid, and 4-imidazoleacetic acid hydrochloride, or a pharma- ceutically acceptable salt thereof. These components may be contained alone or in combination.

[0016] Pharmaceutically acceptable salts are not particularly limited as long as they are pharma- ceutically acceptable, and examples thereof include halide salts, metal salts, ammonium salts, organic acid salts, inorganic acid salts, etc. More specific examples thereof include chloride salts, metal salts such as sodium salts, potassium salts, calcium salts, magnesium salts, zinc salts, etc., ammonium salts, organic acid salts such as acetates, propionates, lactates, tartrates, citrates, succinates, maleates, fumarates, etc., and inorganic acid salts such as hydrochlorides, sulfates, phosphates, etc. Each component will be described in detail below.

[0017] <<Peptide>> In the present invention, "peptide" includes pharma- ceutically acceptable peptide derivatives. Peptide derivatives refer to peptides whose amino groups have been chemically modified. In other words, peptides whose amino groups have had their hydrogen atoms replaced with functional groups include, for example, acylated peptides, glycerylated peptides, quaternary ammonium peptides, silylated peptides, alkylglycerylated peptides, and 2-hydroxyalkylated peptides.

[0018] Examples of amino acid residues constituting the peptide include glutamic acid, cysteine, alanine, valine, leucine, isoleucine, arginine, glutamine, lysine, aspartic acid, proline, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, and serine. From the viewpoint of improving glutaminase inhibitory activity, glutamic acid, cysteine, and alanine are preferred, and glutamic acid and cysteine ​​are more preferred.

[0019] The peptide preferably contains amino acid residues selected from glutamic acid, cysteine, alanine, and glycine at a content of 1% by mass or more, more preferably 10% by mass or more, and even more preferably 30% by mass or more.

[0020] Examples of peptides include polypeptide chains (also called protein hydrolysates) obtained by enzymatically or chemically decomposing proteins (polypeptides), and artificially synthesized polypeptide chains. Protein hydrolysates are obtained by partially hydrolyzing proteins with acids, alkalis, enzymes, or a combination of these. Protein sources for protein hydrolysates include, for example, animal proteins, vegetable proteins, and proteins derived from microorganisms.

[0021] Examples of animal proteins include collagen (including its denatured product, gelatin), keratin, fibroin, sericin, casein, conchiolin, elastin, protamine, and egg yolk protein and egg white protein from chickens.

[0022] Examples of vegetable proteins include proteins contained in soybeans, wheat, rice (rice bran), sesame, peas, corn, potatoes, etc. Examples of proteins derived from microorganisms include yeast proteins isolated from yeasts of the genera Saccharomyces, Candida, and Endomycopsis, yeasts known as brewer's yeast and sake yeast, proteins isolated from mushrooms (basidiomycetes) and Chlorella, and spirulina proteins derived from seaweed.

[0023] The peptide is preferably an oligopeptide. An oligopeptide is a peptide consisting of a relatively small number of amino acids, about 2 to 10. Examples of oligopeptides include dipeptides, tripeptides, and tetrapeptides, and dipeptides or tripeptides are preferred from the viewpoints of ease of solubility in water and ease of production.

[0024] Among dipeptides or tripeptides, peptides containing gamma-glutamyl-cysteine ​​are preferred.

[0025] Examples of peptides containing γ-glutamylcysteine ​​include homoglutathione (gamma-L-Glutamyl-L-Cysteinyl-beta-Alanine, CAS number 18710-27-5), N-acetyl-oxidized glutathione (CAS number 59524-81-1), reduced glutathione (CAS number 70-18-8), and oxidized glutathione (CAS number 27025-41-8).

[0026] Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine. CAS numbers are compound numbers used in Chemical Abstracts, a publication of the American Chemical Society, to uniquely identify compounds.

[0027] The peptide may be modified in various ways depending on the application, so long as the effects of the present invention are achieved. Examples of peptide modifications include amino group modification (e.g., biotinylation, myristoylation, palmitoylation, acetylation, maleimidation, etc.), carboxyl group modification (e.g., amidation, esterification, etc.), thiol group modification (e.g., farnesylation, geranylation, methylation, palmitoylation, etc.), hydroxyl group modification (e.g., phosphorylation, sulfation, octanoylation, palmitoylation, palmitoleoylation, etc.), various fluorescent labels (e.g., FITC, FAM, rhodamine, BODIPY, NBD, MCA, etc.), PEGylation, introduction of unnatural amino acids, D-amino acids, etc.

[0028] The glutaminase inhibitory activity can be evaluated, for example, by mixing a crude extract obtained by homogenizing kidney or liver, etc. with a component to be evaluated for inhibitory activity, incubating the mixture, adding glutamine as a reaction substrate to the mixture, and measuring the glutamic acid concentration in the solution after the reaction. The glutamic acid concentration in the solution can be measured by a known glutamic acid colorimetric method, sensor, etc.

[0029] <<L-カルニチン> > Carnitine is a vitamin-like substance synthesized from lysine and methionine. L-carnitine is a substance identified by CAS number 541-15-1. L-carnitine may be a pharma- ceutical acceptable derivative thereof. These are commercially available products.

[0030] L-carnitine derivatives are not particularly limited as long as they are pharma- ceutically acceptable, and examples thereof include L-carnitine esters such as acetyl-L-carnitine, butyryl-L-carnitine, valeryl-L-carnitine, isovaleryl-L-carnitine, propyl-L-carnitine, and proprionyl-L-carnitine.

[0031] < <N 2 -(1-oxopropyl)-L-glutamine, N 2 -(1-Oxobutyl)-L-glutamine>> N 2 -(1-Oxopropyl)-L-glutamine is N-(1-oxopropyl)-L-glutamine according to the CAS number 111726-88-6. 2 -(1-Oxobutyl)-L-glutamine is a substance identified by the CAS number 1308962-52-8. These are commercially available products.

[0032] <<Sialic acid>> Sialic acid is a family name that collectively refers to substances in which the amino group or hydroxyl group of neuraminic acid, which has an amino group or a carboxyl group, has been substituted. Examples of sialic acid include N-acetylneuraminic acid, N-glycolylneuraminic acid, and hydrates thereof, with N-acetylneuraminic acid or N-acetylneuraminic acid hydrate (CAS number 131-48-6) being preferred, and N-acetylneuraminic acid being particularly preferred. These can be commercially available products.

[0033] <<Eucominic acid>> Eucominic acid ((R)-2-Hydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid) is a compound found in plants such as Lotus japonicus. Eucominic acid is identified by the CAS number 60449-48-1, and commercially available products can be used.

[0034] <<4-Imidazoleacetic acid hydrochloride>> Imidazole-4-acetic Acid Hydrochloride is a substance identified by the CAS number 3251-69-2, and commercially available products can be used.

[0035] <Other ingredients> The glutaminase inhibitor of the present invention may contain other components such as pharma- ceutically acceptable excipients within the scope of the present invention, depending on the form, etc. Examples of other components include additives such as water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, water-soluble polymers, surfactants, metal soaps, alcohols, polyhydric alcohols, pH adjusters, antioxidants, UV inhibitors, preservatives, fragrances, powders, thickeners, pigments, and chelating agents.

[0036] The glutaminase which the glutaminase inhibitor of the present invention has an inhibitory effect on is preferably at least one of kidney-type glutaminase and liver-type glutaminase, more preferably kidney-type glutaminase.

[0037] In one embodiment of the glutaminase inhibitor of the present invention, when the glutaminase inhibitor is a renal-type glutaminase inhibitor, the inhibitor may be a peptide, L-carnitine, N 2 -(1-oxopropyl)-L-glutamine, N 2It is preferable that the composition contains at least one selected from the group consisting of -(1-oxobutyl)-L-glutamine, sialic acid, eucomic acid, and 4-imidazoleacetic acid hydrochloride, or a pharma- ceutically acceptable salt thereof, as an active ingredient. As the peptide, homoglutathione, N-acetyl-oxidized glutathione, reduced glutathione, and oxidized glutathione are preferable.

[0038] In one embodiment of the glutaminase inhibitor of the present invention, when it is a liver-type glutaminase inhibitor, it is preferable that the active ingredient contains at least one selected from the group consisting of peptides, 4-imidazole acetate hydrochloride, and eucomic acid, or a pharma- ceutically acceptable salt thereof. As the peptide, homoglutathione, reduced glutathione, N-acetyl-oxidized glutathione, and oxidized glutathione are preferable, and N-acetyl-oxidized glutathione and oxidized glutathione are more preferable.

[0039] <Usage form> The glutaminase inhibitor of the present invention can be used as an active ingredient of various agents or compositions for inhibiting glutaminase [e.g., foods and drinks, medicines, health promotion agents, nutritional supplements (e.g., supplements, etc.), food additives, etc.]. In addition, the glutaminase inhibitor of the present invention can be applied (e.g., administered, ingested, inoculated, etc.) to non-human animals and humans either as it is or in combination with conventional components to form a glutaminase inhibitor.

[0040] The application form of the glutaminase inhibitor of the present invention is not particularly limited, and examples thereof include oral, transdermal, enteral, mucosal, intravenous, intraarterial, subcutaneous, and intramuscular. The glutaminase inhibitor of the present invention can be used in any application form to exert a glutaminase inhibitory effect, and can be applied to various products such as food and beverages, medicines, feed, and pet food. The glutaminase inhibitor of the present invention may be directly ingested (administered) as a supplement or the like, or may be used as an additive to impart a glutaminase inhibitory effect to a composition such as food and beverages.

[0041] The dosage form of the glutaminase inhibitor of the present invention can be appropriately determined depending on the type and use of the product, and may be, for example, a solid, semi-solid, or liquid form.

[0042] Furthermore, the glutaminase inhibitor of the present invention may be blended with other components having glutaminase inhibitory activity depending on the form, use, etc., within a range that does not impair the effects of the present invention. Examples of such ingredients include nicotinic acid or its derivatives, cholesterol synthesis inhibitors, probucol, various plant extracts such as swelling dietary fiber, appetite suppressants such as sibutramine, central feeding regulators such as Sanorex (generic name: mazindol), digestive and absorption inhibitors such as cetilistat, lipase inhibitors such as orlistat, thermogenic enhancers such as capsaicin, 6-diazo-5-oxo-L-norleucine (CAS No: 51481-10-8) [(S)-2-amino-6-diazo-5-oxocaproic acid or its salt (DON)], CB-839 (CAS No: 1439399-58-2), bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) (CAS No: 314045-39-1), Ebselen, Compound 968 (CAS No: 311795-38-7), GlutaDON (registered trademark) (PEG-PGA+DON) (manufactured by New Medical Enzymes AG), GlutaChemo (PEG-PGA+ideal candidate) (manufactured by New Medical Enzymes AG), etc. In addition to these, other examples include proteins or peptides such as neutralizing antibodies against KGA or fragments thereof, and nucleic acids such as siRNA and miRNA for knocking out the gene (GLS1) that codes for KGA. The amounts of these to be added are not limited as long as they do not impair the effects of the present invention.

[0043] When the glutaminase inhibitor of the present invention is used in foods and beverages, the glutaminase inhibitor is prepared into a desired form either as is or in combination with other food materials or additives, and provided as a food or beverage exhibiting the desired effect.

[0044] The foods and beverages include, in addition to general foods and beverages, for example, health foods, functional foods, nutritional supplements, and supplements, as well as health functional foods such as foods labeled to reduce the risk of disease (e.g., specific health foods, nutritional functional foods, and functional food, etc.), and foods for the sick.

[0045] The form of the food and drink is not particularly limited, but specific examples thereof include liquid foods such as drinks, soups, non-alcoholic beverages, alcoholic beverages, jelly-like beverages, and functional beverages; semi-solid foods such as jellies and yogurt; fermented foods such as miso and fermented beverages; Western confectioneries such as cookies and cakes, Japanese confectioneries such as buns and yokan, candies, gums, gummies, frozen desserts, and ice desserts; oil-containing products such as edible oils, dressings, mayonnaise, and margarine; carbohydrate-containing foods such as rice, rice cakes, noodles, breads, and pastas; processed livestock foods such as ham and sausages; processed seafood foods such as kamaboko, dried fish, and shiokara; processed vegetable foods such as pickles; retort products such as curry, thickened sauce, and Chinese soup; instant foods such as instant soup and instant miso soup; microwaveable foods; processed products using eggs, and processed seafood or meat products; seasonings; and the like. Further examples include health foods prepared in the form of powder, granules, tablets, capsules, liquid, paste, or jelly. These can be used for the above-mentioned purposes. The medical foods are provided for patients who require prevention, amelioration, and / or treatment of diseases and / or symptoms involving glutaminase.

[0046] When the glutaminase inhibitor of the present invention is used in a food or drink, the amount of the glutaminase inhibitor to be added to the food or drink varies depending on the form of the food or drink, etc., but a person skilled in the art can appropriately set the amount according to the glutaminase inhibitor to be used while predicting the effect based on known information, animal tests, etc. The amount of the active ingredient having glutaminase inhibitory activity that can be set in this way is, for example, preferably 0.00001 to 100 mass%, more preferably 0.0001 to 100 mass%, even more preferably 0.001 to 100 mass%, and particularly preferably 0.01 to 100 mass%.

[0047] Furthermore, when the glutaminase inhibitor of the present invention is used in food or beverage, the glutaminase inhibitor of the present invention can be provided as a food additive alone or in combination with other ingredients. When the glutaminase inhibitor of the present invention is used as a food additive, the content of the active ingredient having glutaminase inhibitory activity in the food additive, the amount of the food additive added to the food or beverage, etc. can be appropriately set so that the glutaminase inhibitor satisfies the above-mentioned content in the food or beverage to which it is added.

[0048] Furthermore, when the glutaminase inhibitor of the present invention is used as a medicine, the glutaminase inhibitor of the present invention is adjusted to a desired form alone or in combination with other pharmacologically active ingredients, pharma- ceutically acceptable bases, additives, etc., and provided as a pharmaceutical composition that exerts the desired effect.

[0049] The form of such a pharmaceutical composition is not particularly limited, but specific examples include oral administration preparations such as tablets, granules, powders, capsules, soft capsules, and syrups; transdermal or transmucosal administration preparations such as liquids, ointments, creams, gels, sprays, patches, inhalants, and suppositories; and injections.

[0050] When the glutaminase inhibitor of the present invention is used in a pharmaceutical composition, the blending ratio of the glutaminase inhibitor to the pharmaceutical composition varies depending on the form of the pharmaceutical composition, etc., but a person skilled in the art can appropriately set the blending ratio according to the glutaminase inhibitor used while predicting the effect based on known information, animal tests, etc. The blending amount of the active ingredient having glutaminase inhibitory activity that can be set in this way is, for example, preferably 0.00001 to 100 mass%, more preferably 0.0001 to 100 mass%, even more preferably 0.001 to 100 mass%, and particularly preferably 0.01 to 100 mass%.

[0051] The application (e.g., administration, ingestion, inoculation, etc.) of the glutaminase inhibitor of the present invention is not particularly limited as long as it is an effective amount that exerts its effect, and the mass of the active ingredient having glutaminase inhibitory activity is generally preferably 10 mg to 30 g, more preferably 50 mg to 10 g, and even more preferably 200 mg to 5 g per day. The above application amount is preferably administered once or more times a day (e.g., 1 to 3 times a day) in divided doses, and can be appropriately increased or decreased depending on age, pathological condition, and symptoms.

[0052] <effect> The glutaminase inhibitor of the present invention has an excellent glutaminase inhibitory effect and is useful in the prevention, amelioration and / or treatment of diseases, disorders or symptoms involved in glutaminase. The subject to which the glutaminase inhibitor of the present invention can be applied is preferably a mammal, more preferably a human.

[0053] The subjects specifically include, for example, farm animals, including cows, sheep, pigs, horses, and goats; pets, such as dogs and cats; exotic and / or zoo animals; laboratory animals, including mice, rats, rabbits, guinea pigs, and hamsters; and poultry, such as chickens, turkeys, ducks, and geese.

[0054] Diseases and / or conditions in which glutaminase is involved include, for example, cancer, diseases characterized by abnormal cell proliferation, inflammatory or autoimmune diseases, dermatitis, respiratory diseases, hyperproliferative disorders, diseases associated with vasculogenesis or angiogenesis, diseases associated with the accumulation of visceral fat or subcutaneous fat, nervous system diseases associated with neuronal cell death due to excitatory neuropathy, and aging or aging-related diseases caused by the accumulation of senescent cells and / or symptoms associated with these diseases. Each action will be explained below.

[0055] (1) Anti-cancer and anti-inflammatory effects The anti-cancer and anti-inflammatory effects of the glutaminase inhibitors of the present invention include the prevention, amelioration and / or treatment of diseases such as cancer, diseases characterized by abnormal cell proliferation, inflammatory or autoimmune diseases, dermatitis, respiratory diseases, hyperproliferative disorders, diseases associated with vasculogenesis or angiogenesis, and symptoms associated with these diseases.

[0056] Cancers include, for example, bladder cancer, such as transitional cell carcinoma, urothelial carcinoma (transitional cell carcinoma) in the bladder, tumors in the urothelial cells lining the bladder, squamous cell carcinoma, adenocarcinoma, and small cell carcinoma; breast cancer, such as ductal carcinoma, medullary carcinoma, colloid carcinoma, tubular carcinoma, and inflammatory breast cancer in the ductal tissue of the breast; colon cancer; kidney cancer; liver cancer; lung cancer, such as non-small cell lung cancer (NSCLC), which is classified as squamous cell carcinoma, adenocarcinoma, and large cell undifferentiated carcinoma, and small cell lung cancer; esophageal cancer; gallbladder cancer; adenocarcinoma of the ovaries and glandular carcinoma that has spread from the ovaries to the peritoneal cavity. ovarian cancer, including epithelial ovarian tumors such as cancer; pancreatic cancer, such as epitheliod carcinoma in the pancreatic duct tissue and adenocarcinoma in the pancreatic duct; lymphoma; gastric cancer, such as gastric stromal tumors and carcinoid tumors; cervical cancer, such as adenocarcinoma in the cervical epithelium, including squamous cell carcinoma and adenocarcinoma, cervical cancer caused by human papillomavirus (HPV); thyroid cancer, such as papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; prostate cancer, such as adenocarcinoma or adenocarinoma that has spread to the bone; basal cell Skin cancers, including carcinoma, melanoma, squamous cell carcinoma and actinic keratosis; lymphoid hematopoietic malignancies, including leukemia, acute and chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mastocytosis, hairy cell leukemia, hairy cell lymphoma and Burkett's lymphoma; hematopoietic malignancies of the myeloid lineage, including acute and chronic myelogenous leukemia, multiple myeloma, myelodysplasia, myeloproliferative disorders, myelodysplastic syndromes and myeloid leukemia; human lymphotropic virus type 1 (HTLV-I) and adult T-cell leukemia / lymphoma; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; central nervous system (CNS) cancers, such as primary brain tumors, including glioma (astrocytoma, anaplastic astrocytoma, or glioblastoma multiforme), oligodendroglioma, ependymoma, meningioma, lymphoma, schwannoma, and medulloblastoma; peripheral nervous system (PNS) cancers, such as malignant peripheral nerve sheath tumors (MPNST), including acoustic neuroma, neurofibroma, and schwannoma, malignant fibrocytoma, malignant fibrous histiocytoma, malignant meningioma, malignant mesothelioma, and malignant mixed Mullerian tumor; melanoma; seminoma; teratoma; osteosarcoma;keratoctanthoma; follicular thyroid carcinoma; Kaposi's sarcoma; primary liver cancer; testicular cancer, such as germ cell tumors (GCTs), including seminomas and non-seminomas, gonadal stromal tumors, including Leydig cell tumors and Sertoli cell tumors; thymic carcinomas, such as thymoma, thymic carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, carcinoid or carcinoid tumors; rectal carcinoma, and other tumors;

[0057] Diseases characterized by abnormal cell proliferation include, for example, benign prostatic hyperplasia, familial adenomatous polyposis, neurofibromatosis, atherosclerosis, pulmonary fibrosis, arthritic diseases (e.g., arthritis), psoriasis, glomerulonephritis, restenosis after angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplant rejection, endotoxic shock, and fungal infections.

[0058] Inflammatory or autoimmune diseases include inflammatory diseases, autoimmune diseases, or inflammatory reactions associated with diseases associated with undesired immune responses, such as asthma, emphysema, allergies, dermatitis, rheumatoid arthritis, psoriasis, lupus erythematosus, graft-versus-host disease, type 2 diabetes, hyperglycemia, insulin resistance, hypertension, atherosclerosis, NAFLD, fatty liver, dyslipidemia, arthritis, Alzheimer's, inflammatory bowel disease, and heart disease.

[0059] Dermatitis includes contact or atopic dermatitis. Contact dermatitis includes irritant dermatitis, phototoxic dermatitis, allergic dermatitis, photoallergic dermatitis, contact urticaria, and generalized contact dermatitis. Irritant dermatitis can occur when too many substances are used on the skin when the skin is sensitive to a substance. Atopic dermatitis, sometimes called eczema, is a type of dermatitis that is an atopic skin disease.

[0060] Respiratory diseases include, for example, diseases that affect lung lobes, pleural cavity, bronchi, trachea, upper airway, or nerves and muscles for breathing.Specific examples include obstructive pulmonary diseases, especially chronic obstructive pulmonary disease (COPD).Chronic obstructive pulmonary disease (COPD) is a general term for airway diseases characterized by airflow obstruction or restriction, and specifically includes chronic bronchitis, emphysema, and bronchiectasis.

[0061] Hyperproliferative disorders include, for example, acute myeloid leukemia; thymoma / thymic carcinoma; brain tumors; lung cancer; squamous cell carcinoma; skin cancer; eye tumors; retinoblastoma; intraocular melanoma; oral and oropharyngeal cancers, such as hypopharyngeal, laryngeal, nasopharyngeal, and oropharyngeal cancer; bladder cancer; gastric and stomach cancer; pancreatic cancer; bladder cancer; breast cancer; head and neck cancer; renal and kidney cancer; liver cancer; ovarian cancer; prostate cancer; colorectal cancer, such as colon and rectal cancer; esophageal cancer; testicular cancer; gynecological cancer; cancers such as thyroid cancer; cancers of the central nervous system (CNS); cancers of the peripheral nervous system (PNS); cancers related to AIDS, such as diffuse large B-cell lymphoma, B-cell immunoblastic lymphoma, and small noncleaved cell lymphoma (e.g., lymphoma and Kaposi's sarcoma); virus-induced cancers, such as hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatocellular carcinoma, and noncancerous hyperproliferative disorders, such as benign hyperplasia of the skin (e.g., psoriasis), restenosis, or prostate [e.g., benign prostatic hyperplasia (BPH)].

[0062] Diseases associated with vasculogenesis or angiogenesis include, for example, tumor angiogenesis, chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, skin diseases such as psoriasis, eczema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangiomas, gliomas, melanomas, Kaposi's sarcoma, and ovarian, breast, lung, pancreatic, prostate, colon, and epidermoid cancers.

[0063] Other diseases include endotoxemia, sepsis and xenoderma pigmentosum.

[0064] The glutaminase inhibitors according to the present invention have the effect of interfering with the function of leukocytes or interfering with the function of osteoclasts. Inflammatory responses are particularly associated with the influx of leukocytes and / or leukocyte (e.g., neutrophil) chemotaxis.

[0065] The glutaminase inhibitor according to the present invention has effects such as suppressing the expression of TNFα, suppressing fat inflammation, and suppressing macrophages (e.g., macrophage-1, macrophage-2). Examples of the effects of the glutaminase inhibitor according to the present invention include suppressing, preventing, or improving at least one selected from elevated blood glucose level, elevated blood pressure, elevated blood neutral fat, arteriosclerosis, arthritis, chronic knee joint pain, and dementia (e.g., dementia accompanied by accumulation of foreign bodies or waste products in the brain), improving lipid metabolism, and enhancing or improving at least one of memory learning function and cognitive function. Examples of the improving effect of lipid metabolism include lowering blood neutral fat level, lowering small dense LDL cholesterol level, and lowering the proportion of small dense LDL cholesterol in total LDL.

[0066] (2) Anti-obesity effect The anti-obesity effects of the glutaminase inhibitor according to the present invention include, for example, an effect of suppressing weight gain, an effect of reducing weight, an effect of suppressing the accumulation of visceral fat and subcutaneous fat, an effect of reducing visceral fat and subcutaneous fat, etc. In particular, an effect of suppressing the accumulation of visceral fat and subcutaneous fat, an effect of reducing visceral fat and subcutaneous fat, etc. are included.

[0067] The visceral fat is not particularly limited, but examples thereof include visceral fat around abdominal organs, preferably visceral fat around organs (e.g., intestines, kidneys, etc.) that are partially or entirely present in the abdomen around the navel, etc. In addition, the subcutaneous fat is not particularly limited, but examples thereof include fat present subcutaneously in the abdomen, waist, buttocks, thighs, etc.

[0068] The glutaminase inhibitor according to the present invention can be used in foods, beverages, and medicines that are effective for preventing, improving, or treating the accumulation of visceral fat or subcutaneous fat. Examples of diseases and symptoms accompanied by visceral fat accumulation include hypertension, glucose metabolism disorder, and lipid metabolism disorder. Examples of diseases and symptoms accompanied by subcutaneous fat accumulation include obesity, cellulite, sagging (e.g., skin aging and loss of elasticity), and edema (swelling). The glutaminase inhibitor according to the present invention can prevent, improve, or treat the onset of such diseases or symptoms by suppressing the accumulation of visceral fat or subcutaneous fat.

[0069] In addition, when the patient has symptoms of visceral fat obesity or subcutaneous fat obesity, the condition may progress to various diseases or syndromes ranging from mild to severe. Examples of diseases or syndromes caused by visceral fat obesity include arteriosclerosis, myocardial infarction, angina pectoris, cerebral infarction, and obliterative arteriosclerosis. Examples of diseases or syndromes caused by subcutaneous fat obesity include sleep apnea syndrome, frequent urination, alopecia, menstrual abnormalities, growth retardation due to hormone deficiency, anemia, ovarian cancer, uterine cancer, breast cancer, infertility, liver cirrhosis, hemorrhoids, deep vein thrombosis, pulmonary embolism, and venous thromboembolism. The glutaminase inhibitor according to the present invention can also prevent, improve, or treat the onset of such diseases or syndromes.

[0070] (3) Cell death inhibition effect The cell death inhibitory effect of the glutaminase inhibitor according to the present invention can be, for example, the prevention / treatment of nervous system diseases associated with neuronal cell death due to excitatory nerve damage, such as ischemic damage, inflammatory nerve diseases, and neurodegenerative diseases.

[0071] Examples of ischemic disorders include stroke, cerebral hemorrhage, cerebral infarction, and cerebrovascular dementia. Examples of inflammatory neurological disorders include central nervous system inflammatory neurological disorders such as encephalitis sequelae, acute disseminated encephalomyelitis, bacterial meningitis, tuberculous meningitis, fungal meningitis, viral meningitis, and vaccine-induced meningitis. Examples of neurodegenerative disorders include Alzheimer's disease, head trauma, cerebral palsy, Huntington's disease, Pick's disease, Down's syndrome, Parkinson's disease, AIDS encephalopathy, multiple system atrophy, multiple sclerosis, amyotrophic lateral sclerosis, and spinocerebellar ataxia.

[0072] (4) Senescent cell removal effect The senescent cell-eliminating effect of the glutaminase inhibitor according to the present invention includes, for example, the effect of inducing cell death in senescent cells in vivo or in vitro, and selectively removing senescent cells from a cell population containing senescent cells.

[0073] As used herein, senescent cells refer to cells whose cell proliferation or cell cycle has irreversibly stopped. Whether a cell is a senescent cell can be evaluated by using the characteristics of cellular senescence as an indicator. The characteristics of cellular senescence include increased expression of p16 (CDKN2A) protein, activation of senescence-specific β-galactosidase (SA-β-gal), increased expression of p21 (CDKN1A) protein, increased expression of p19 protein, senescence-specific heterochromatin formation (Senescence-associated heterochromatic foci (SAHF)), DNA damage response (DDR) and senescence-associated secretory phenotype (SASP) (for details on the characteristics of cellular senescence, see, for example, Kuilman et al., Genes Dev 24:2463-2479, 2010).

[0074] The senescent cell-eliminating effect of the glutaminase inhibitor according to the present invention can be effective in preventing or treating aging or aging-related diseases caused by the accumulation of senescent cells, such as arteriosclerosis, osteoporosis, cataracts, glaucoma, dementia, Parkinson's disease, pulmonary fibrosis, chronic obstructive pulmonary disease, cancer, type 2 diabetes, chronic renal failure, cardiac hypertrophy, liver cirrhosis, sarcopenia, and emaciation. EXAMPLES

[0075] The following examples are given, but the present invention is not limited to these examples.

[0076] [Example 1] Evaluation of renal glutaminase inhibitory activity (Preparation of crude kidney extract for evaluation) Kidneys were removed from 8-week-old male C57BL / 6J mice (Charles River Biosciences Japan and CLEA Japan) and stored frozen at -80°C until use in the study. On the day of the study, the kidneys were thawed and lightly washed with PBS buffer, after which homogenization buffer was added and the mice were homogenized using a BioMasher III (Nippi Biosciences).

[0077] The protein concentration of the obtained crude extract was measured using Pierce™ 660 nm Protein Assay Reagent (Thermo Fisher Scientific). The extract was diluted with PBS buffer to a total protein concentration of approximately 25 mg / mL to prepare a kidney crude extract for evaluation. The composition of the homogenate buffer is shown in Table 1.

[0078] [Table 1]

[0079] (Preparation of evaluation samples) 27 types of compounds for evaluating glutaminase inhibitory activity were dissolved or suspended in PBS buffer at a concentration of 200 mM to prepare samples for evaluation. The compounds for evaluating glutaminase inhibitory activity used and their CAS numbers are shown in Table 2.

[0080] [Table 2]

[0081] (Kidney-type glutaminase inhibitory activity evaluation) A mixture of 20 μL of the above-mentioned crude kidney extract for evaluation, 5 μL of the sample for evaluating glutaminase inhibitory activity, and 55 μL of kidney assay buffer was incubated for 20 minutes at 37° C. The composition of the kidney assay buffer is shown in Table 3.

[0082] [Table 3]

[0083] 20 μL of glutamine (final concentration 40 mM) was added as a reaction substrate to the mixture, and the mixture was further incubated at 37° C. for 10 minutes to carry out a conversion reaction from glutamine to glutamic acid by glutaminase. After the reaction was completed, 10 μL of 3M HCl was added to stop the enzyme reaction, and the glutamic acid in the solution after the reaction was quantified using the L-glutamic acid measurement kit "Yamasa" NEO (manufactured by Yamasa Shoyu Co., Ltd.). Each sample was measured three times, and the average value was used as the glutamic acid concentration. 20 mM DON was used as a positive control for glutaminase inhibitors, and PBS buffer was used as a negative control.

[0084] The glutamine conversion activity was calculated by [glutamic acid concentration of the reaction solution (g / L)] / 147.13 / 0.04. Furthermore, the glutamine conversion rate (%) was calculated by [glutamine conversion activity of each evaluation sample] / [glutamine conversion activity of the negative control (PBS buffer)]×100. The inhibitory activity evaluation of each evaluation sample was performed twice.

[0085] (Evaluation Results) The renal-type glutaminase inhibitory activity of the compounds for evaluation of glutaminase inhibitory activity shown in Table 2 was measured by the above method. The glutamine conversion rate (%) of each compound is shown in Figures 1 and 2.

[0086] A decrease in glutamine conversion rate was observed in the evaluation samples No. 18 to 27. 2 -(1-oxopropyl)-L-glutamine, N 2 -(1-Oxobutyl)-L-glutamine, homoglutathione, N-acetyl-oxidized glutathione, reduced glutathione, oxidized glutathione, N-acetylneuraminic acid, eucomic acid, and 4-imidazoleacetic acid hydrochloride were found to exhibit inhibitory activity against kidney-type glutaminase.

[0087] Among these, the compounds No. 19 to No. 26 [N 2 -(1-oxopropyl)-L-glutamine, N 2 -(1-oxobutyl)-L-glutamine, homoglutathione, N-acetyl-oxidized glutathione, reduced glutathione, oxidized glutathione, N-acetylneuraminic acid, and eucominic acid] reduced the glutamine conversion rate to less than 50%, indicating that they exhibit significant renal glutaminase inhibitory activity.

[0088] [Example 2] Evaluation of hepatic glutaminase inhibitory activity (Preparation of crude liver extract for evaluation) The livers of 8-week-old male mice C57BL / 6J (Charles River Biosciences Japan and CLEA Japan) were excised, and a crude liver extract for evaluation was prepared in the same manner as in Example 1 for the preparation of the crude kidney extract for evaluation.

[0089] (Assessment of hepatic glutaminase inhibitory activity) The preparation of samples for evaluating glutaminase inhibitory activity was carried out as described in Example 1. Of the compounds listed in Table 2, Nos. 21 to 27 were subjected to evaluation of liver-type glutaminase inhibitory activity. A mixture obtained by mixing 20 μL of the crude liver extract for evaluation, 5 μL of the sample for evaluating glutaminase inhibitory activity, and 55 μL of liver assay buffer was incubated at 37° C. for 20 minutes. The composition of the liver assay buffer is shown in Table 4.

[0090] [Table 4]

[0091] 20 μL of glutamine (final concentration 40 mM) was added as a reaction substrate to the mixture, and the mixture was further incubated at 37° C. for 10 minutes to carry out a conversion reaction of glutamine to glutamic acid by glutaminase. After the reaction was completed, 10 μL of 3M HCl was added to stop the enzyme reaction, and the glutamic acid in the solution after the reaction was quantified using the L-glutamic acid measurement kit "Yamasa" NEO (manufactured by Yamasa Shoyu Co., Ltd.). Each sample was measured three times, and the average value was taken as the glutamic acid concentration. 20 mM DON was used as the positive control for the glutaminase inhibitor, and PBS buffer was used as the negative control. The glutamine conversion rate (%) was calculated by the method described in Example 1. The inhibitory activity evaluation of each evaluation sample was carried out twice.

[0092] (Evaluation Results) The hepatic glutaminase inhibitory activity of compounds No. 21 to No. 27 in Table 2 for evaluation of glutaminase inhibitory activity was measured by the above method. The glutamine conversion rate (%) of each compound is shown in FIG.

[0093] The glutamine conversion rate of DON used as a positive control was not reduced, indicating that DON does not exhibit hepatic glutaminase inhibitory activity. The glutamine conversion rate of homoglutathione (No. 21), reduced glutathione (No. 23), and 4-imidazole acetate hydrochloride (No. 27) was reduced to about 80%, suggesting that these compounds may exhibit hepatic glutaminase inhibitory activity.

[0094] Furthermore, the glutamine conversion rate was reduced to less than 40% in the cases of N-acetylglutathione (No. 22), oxidized glutathione (No. 24), and eucomic acid (No. 26), strongly suggesting that these compounds may exhibit hepatic glutaminase inhibitory activity.

Claims

1. Peptides, L-carnitine, N ２ -(1-oxopropyl)-L-glutamine, N ２ A glutaminase inhibitor comprising, as an active ingredient, at least one member selected from the group consisting of 1-(1-oxobutyl)-L-glutamine, sialic acid, eucomic acid, and 4-imidazoleacetic acid hydrochloride, or a pharma- ceutical acceptable salt thereof.

2. The glutaminase inhibitor of claim 1 , wherein the peptide is an oligopeptide.

3. The glutaminase inhibitor according to claim 1 or 2, wherein the peptide is a tripeptide or a dipeptide.

4. The glutaminase inhibitor according to any one of claims 1 to 3, wherein the peptide is a peptide containing glutamic acid and cysteine.

5. The glutaminase inhibitor according to any one of claims 1 to 4, wherein the peptide is a peptide containing γ-glutamylcysteine.

6. The glutaminase inhibitor according to any one of claims 1 to 5, wherein the peptide is at least one peptide selected from the group consisting of homoglutathione, N-acetyl-oxidized glutathione, reduced glutathione, and oxidized glutathione.

7. The glutaminase inhibitor according to any one of claims 1 to 6, wherein the glutaminase is at least one of kidney-type glutaminase and liver-type glutaminase.

8. The glutaminase inhibitor according to any one of claims 1 to 7, wherein the glutaminase is kidney-type glutaminase.

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