Method for preventing nitration of tyrosine residues in hepatocyte growth factor using trisulfide compounds

Abstract

The present invention provides a trisulfide-compound-containing agent for inhibiting nitration of tyrosine residues in hepatocyte growth factor, the trisulfide compound being: glutathione trisulfide or a pharmaceutically acceptable salt thereof; or a compound represented by formula (1) (wherein X represents -OR1 or -NR2R3, R1 represents a hydrogen atom or an alkyl group containing 1 to 6 carbon atoms, R2 and R3 independently represent a hydrogen atom or an alkyl group containing 1 to 6 carbon atoms, and the alkyl group may have one or more substituents selected from the group consisting of amino groups and carboxy groups), a pharmaceutically acceptable salt thereof, or a cyclodextrin clathrate thereof.

Description

[Technical field]

[0001] The present invention relates to a method for preventing the nitration of tyrosine residues in hepatocyte growth factor using trisulfide compounds. [Background technology]

[0002] Hepatocyte growth factor (hereinafter also referred to as "HGF") was discovered as an in vivo protein that promotes the proliferation of mature hepatocytes. Subsequent research has revealed that in addition to cell proliferation, HGF has a variety of physiological activities, such as promoting cell motility, inhibiting cell death, inducing morphogenesis, anti-fibrosis, and angiogenesis, and is involved in the regeneration and protection of various tissues and organs, not only the liver, but also the gastrointestinal mucosa, nerves, lungs, kidneys, heart, and skin (Non-Patent Documents 5 to 7).

[0003] It has further been reported that HGF is an activator of muscle stem cells (satellite cells), and satellite cells are known to contribute to the regeneration of muscle fibers and the like (Non-Patent Document 1).

[0004] In recent years, it has been reported that when tyrosine residues in the HGF protein are nitrated, the physiological activity of HGF disappears (Non-Patent Documents 2, 8) (Ryuichi Tatsumi, "Basic Science of Muscle Stem Cells and Their Health and Medical Applications," Kyushu University Industry-Academia-Government Exchange Promotion Seeds Presentation in Tokyo 2019, March 14, 2019) (A. Elgaabari et al., "Insight linking between nitration and myogenic dysfunction of HGF / NK1 domain," 128th Annual Meeting of the Japanese Society of Animal Science). The report suggests that HGF dysfunction causes inhibition of satellite cell activation and reduced proliferation. Therefore, it is believed that nitration of tyrosine residues in the HGF protein inhibits muscle growth, hypertrophy, regeneration, or maintenance, resulting in muscle atrophy, muscle regeneration failure, muscle growth inhibition, etc.

[0005] Trisulfide compounds such as glutathione trisulfide are converted to active sulfur molecular species such as glutathione persulfide in vivo. It has been reported that active sulfur molecular species have a strong antioxidant effect and may have physiological functions such as anti-aging (e.g., Non-Patent Documents 3 and 4). In addition, a method described in Patent Document 1 is known as a method for producing a trisulfide compound. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 117186 [Non-patent literature]

[0007] [Non-Patent Document 1] R.Tatsumi, “Mechano-biology of skeletal muscle hypertrophy and regeneration: possible mechanism of stretch-induced activation of resident myogenic stem cells”, Animal Science Journal 81(1),11-20(2010). [Non-Patent Document 2] Nana Imatomi et al., "Physiological significance of inactivation of muscle stem cell activating factor HGF by nitration: A breakthrough in the main cause of age-related muscle atrophy and regeneration failure," Abstract of the 128th Annual Meeting of the Japanese Society of Animal Science [Non-Patent Document 3] T.Ida,et al., “Reactive cysteine ​​persulfides and S-polythiolation regulate oxidative stress and redox signaling”,PNAS,May27,2014,111(21)7606-7611 [Non-Patent Document 4] T.Akaike,et al., “Cysteinyl-tRNA synthetase governs cysteine ​​polysulfidation and mitochondrial bioenergetics”, Nature Communications,2017,Oct27;8(1):1177 [Non-Patent Document 5] Hirohito Tsubouchi, "Discovery of HGF and its Clinical Application," Journal of the Japanese Society of Gastroenterology, Vol. 110, No. 12, pp. 2033-2041 (2013) [Non-Patent Document 6] Shinya Mizuno and Toshikazu Nakamura, "Clinical application of regenerative factor HGF," Drug Delivery System, 16-6 (2001) [Non-Patent Document 7] C. Desole, et al., “HGF and MET: From Brain Development to Neurological Disorders”, Frontiers in Cell and Developmental Biology, Vol. 9(published 09 June 2021, Article 683609). [Non-Patent Document 8] A. Elgaabari, et. al., “A pilot study on nitration / dysfunction of NK1 segment of myogenic stem cell activator HGF”, Biochemistry and Biophysics Reports Vol. 31, 101295 (2022) (published June 11, 2022) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide an agent that suppresses the nitration of tyrosine residues in HGF. [Means for solving the problem]

[0009] The present inventors found that only HGF undergoes nitration of tyrosine residues among cell growth factors, and considered that this nitration of tyrosine residues in HGF has physiologically important significance.The present inventors then found that glutathione trisulfide and lipoic acid trisulfide (compounds represented by formula (4) described below) concentration-dependently suppress the nitration of tyrosine residues in HGF, which is considered to be important, and completed the present invention.

[0010] That is, the present invention provides the following [1] to

[30] . [1] An agent for suppressing the nitration of a tyrosine residue in hepatocyte growth factor, comprising a trisulfide compound, wherein the trisulfide compound is glutathione trisulfide or a pharma- ceutical acceptable salt thereof, or a compound represented by the formula (1): [ka]

[0033] In the formula, X is -OR 1 or -NR 2 R 3 R 1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R 2 and R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and the alkyl group may have one or more substituents selected from the group consisting of an amino group and a carboxy group.] (hereinafter also referred to as "compound (1)"), a pharma- ceutically acceptable salt thereof, or a cyclodextrin clathrate thereof. [2] The nitration inhibitor according to [1], wherein the trisulfide compound is glutathione trisulfide or a pharma- ceutically acceptable salt thereof. [3] The nitration inhibitor described in [2], wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof includes at least one selected from the group consisting of glutathione trisulfide, amino acid salts of glutathione trisulfide, and alkali metal salts of glutathione trisulfide. [4] The nitration inhibitor described in [2], wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof includes at least one selected from the group consisting of glutathione trisulfide, an arginine salt of glutathione trisulfide, and a sodium salt of glutathione trisulfide. [5] The nitration inhibitor according to [1], wherein the trisulfide compound is lipoic acid trisulfide or a pharma- ceutically acceptable salt thereof. [6] A preventive or therapeutic agent for muscle atrophy and / or muscle regeneration failure, comprising the nitration inhibitor according to any one of [1] to [5]. [7] A method for preventing or treating muscle atrophy and / or muscle regeneration failure, comprising administering the nitration inhibitor according to any one of [1] to [5] to a subject in need thereof. [8] A method for preventing or treating muscle atrophy and / or muscle regeneration failure in a companion animal, comprising administering to the companion animal a nitration inhibitor according to any one of [1] to [5]. [9] The nitration inhibitor according to any one of [1] to [5] for use in the prevention or treatment of muscle atrophy and / or muscle regeneration failure.

[10] Use of the nitration inhibitor according to any one of [1] to [5] for the manufacture of an agent for the prevention or treatment of muscle atrophy and / or muscle regeneration failure.

[11] An agent for suppressing or ameliorating muscle growth inhibition (reduced meat productivity) caused by heat stress in livestock or poultry, comprising the nitration inhibitor according to any one of [1] to [5].

[12] A method for suppressing or ameliorating muscle growth inhibition (reduced meat productivity) caused by heat stress, comprising administering the nitration inhibitor according to any one of [1] to [5] to livestock or poultry in need thereof.

[13] A method for suppressing or ameliorating muscle growth inhibition (reduced meat productivity) caused by heat stress in livestock or poultry, comprising administering to livestock or poultry a nitration inhibitor according to any one of [1] to [5].

[14] The nitration inhibitor according to any one of [1] to [5] for use in inhibiting or improving muscle growth inhibition (reduced meat productivity) caused by heat stress in livestock or poultry.

[15] Use of the nitration inhibitor according to any one of [1] to [5] for the manufacture of an agent for suppressing or improving muscle growth inhibition (reduced meat productivity) caused by heat stress in livestock or poultry.

[16] A method for suppressing nitration of tyrosine residues in hepatocyte growth factor, comprising administering a trisulfide compound to a subject in need thereof, wherein the trisulfide compound is glutathione trisulfide or a pharmaceutically acceptable salt thereof, or compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex of ...).

[17] The method according to

[16] , wherein the trisulfide compound is glutathione trisulfide or a pharma-ceutically acceptable salt thereof.

[18] The method according to

[17] , wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof comprises at least one selected from the group consisting of glutathione trisulfide, amino acid salts of glutathione trisulfide, and alkali metal salts of glutathione trisulfide.

[19] The method according to

[17] , wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof comprises at least one selected from the group consisting of glutathione trisulfide, an arginine salt of glutathione trisulfide, and a sodium salt of glutathione trisulfide.

[20] The method according to

[16] , wherein the trisulfide compound is lipoic acid trisulfide or a pharma-ceutically acceptable salt thereof.

[21] A trisulfide compound for use in inhibiting the nitration of tyrosine residues in hepatocyte growth factor, the trisulfide compound being glutathione trisulfide or a pharmaceutically acceptable salt thereof, or compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex thereof, or a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex thereof.

[22] The trisulfide compound for use according to

[21] , wherein the trisulfide compound is glutathione trisulfide or a pharma- ceutically acceptable salt thereof.

[23] The trisulfide compound for use according to

[22] , wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof comprises at least one selected from the group consisting of glutathione trisulfide, an amino acid salt of glutathione trisulfide, and an alkali metal salt of glutathione trisulfide.

[24] The trisulfide compound for use according to

[22] , wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof comprises at least one selected from the group consisting of glutathione trisulfide, an arginine salt of glutathione trisulfide, and a sodium salt of glutathione trisulfide.

[25] The trisulfide compound for use according to

[21] , wherein the trisulfide compound is lipoic acid trisulfide or a pharma-ceutically acceptable salt thereof.

[26] Use of a trisulfide compound for the manufacture of an inhibitor of nitration of tyrosine residues in hepatocyte growth factor, wherein the trisulfide compound is glutathione trisulfide or a pharmaceutically acceptable salt thereof, or compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex of compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex of compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex of compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex of compound (1).

[27] The use according to

[26] , wherein the trisulfide compound is glutathione trisulfide or a pharma-ceutically acceptable salt thereof.

[28] The use according to

[27] , wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof comprises at least one selected from the group consisting of glutathione trisulfide, amino acid salts of glutathione trisulfide, and alkali metal salts of glutathione trisulfide.

[29] The use described in

[27] , wherein the glutathione trisulfide or a pharma- ceutically acceptable salt thereof includes at least one selected from the group consisting of glutathione trisulfide, an arginine salt of glutathione trisulfide, and a sodium salt of glutathione trisulfide.

[30] The use according to

[26] , wherein the trisulfide compound is lipoic acid trisulfide or a pharma-ceutically acceptable salt thereof. Effect of the Invention

[0011] According to the present invention, an agent for suppressing the nitration of tyrosine residues in HGF can be provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of Western blot analysis of the inhibitory effect of glutathione trisulfide on the nitration of recombinant HGF. [Diagram 2] FIG. 2 shows the results of Western blot analysis of the inhibitory effect of lipoic acid trisulfide on the nitration of recombinant HGF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will be described in detail below, but is not limited to the following embodiments. In this specification, "inhibition of muscle growth and / or reduction in meat productivity" may be expressed as "inhibition of muscle growth (reduction in meat productivity)".

[0014] The nitration inhibitor, the agent for preventing or treating muscle atrophy and / or muscle regeneration failure, and the method for preventing or treating the same of the present invention can be administered or applied to humans or pet animals. The nitration inhibitor, the agent for suppressing or improving muscle growth inhibition (reduction in meat productivity) caused by heat stress, and the method for suppressing or improving the same of the present invention can be administered or applied to livestock or poultry.

[0015] In human HGF, the 198th and 250th tyrosine residues may be nitrated. In mammalian HGF among the pets and livestock described below, the tyrosine residues corresponding to the 198th and 250th tyrosine residues of human HGF may be nitrated. In avian HGF among the pets and livestock or poultry described below, the tyrosine residue corresponding to the 250th tyrosine residue of human HGF may be nitrated. Nitration of the 198th and / or 250th tyrosine residues in human HGF and the tyrosine residues in non-human HGF at the corresponding positions eliminates the activity of HGF. It is believed that dysfunction of HGF leads to inhibition of muscle growth, hypertrophy, regeneration or maintenance, and causes muscle atrophy, muscle regeneration failure, etc. The nitration inhibitor of the present invention can inhibit the nitration of tyrosine residues at the specific positions described above.

[0016] The trisulfide compound used in the nitration inhibitor of the present invention is glutathione trisulfide or a pharma- ceutical acceptable salt thereof, or compound (1), a pharma-ceutical acceptable salt thereof, or a cyclodextrin inclusion complex thereof. Glutathione trisulfide is represented by formula (2). [ka]

[0017] In the present invention, examples of pharma- ceutically acceptable salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with amino acids such as arginine.

[0018] The pharmaceutically acceptable salt of glutathione trisulfide is preferably an amino acid salt or an alkali metal salt, more preferably an arginine salt or a sodium salt. The pharmaceutically acceptable salt of compound (1) is preferably a salt with an alkali metal, more preferably a sodium salt.

[0019] Glutathione trisulfide or its pharmaceutically acceptable salt, or compound (1) or its pharmaceutically acceptable salt may have crystalline polymorphism, but is not limited to any crystalline form, and may be a single or mixture of any crystalline form. Glutathione trisulfide or its pharmaceutically acceptable salt, or compound (1) or its pharmaceutically acceptable salt also includes amorphous forms. Glutathione trisulfide or its pharmaceutically acceptable salt, or compound (1) or its pharmaceutically acceptable salt includes anhydrides and solvates (particularly hydrates).

[0020] In one embodiment, compound (1) has the formula (3): [ka] A compound represented by the formula: 1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. 1 may be, for example, a hydrogen atom, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a hexyl group. 1 is a hydrogen atom, and in this case, the compound represented by formula (3) is lipoic acid trisulfide represented by formula (4). [ka]

[0021] In another embodiment, compound (1) is represented by formula (5): [ka] A compound represented by the formula: 2 and R3 R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and the alkyl group may have one or more substituents selected from the group consisting of an amino group and a carboxy group. 2 and R 3 R may be a hydrogen atom or an alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a hexyl group. These alkyl groups may have one or both of an amino group and a carboxy group as substituents. 2 and R 3 may be, for example, a group represented by formula (6) (wherein * represents a bond). Specific examples of the compound represented by formula (5) include, for example, 2 and R 3 are both hydrogen atoms, R 2 is a hydrogen atom and R 3 is a group represented by formula (6). [ka]

[0022] The compound represented by formula (5) can be produced by a step (step 1) of oxidizing a compound represented by formula (5a) with an oxidizing agent to obtain a sulfoxide compound, and a step (step 2) of reacting the obtained sulfoxide compound with a sulfur source. [ka] [In the formula, R 2 and R 3 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and the alkyl group may have one or more substituents selected from the group consisting of an amino group and a carboxy group.]

[0023] In the above production method, the reactions in steps 1 and 2 may be carried out in one pot without isolating the sulfoxide compound.

[0024] The solvent used in step 1 is not particularly limited as long as it dissolves the compound represented by formula (5a) and the oxidizing agent and does not inhibit the oxidation reaction. Examples of such a solvent include water, an aqueous sulfuric acid solution, an aqueous ethanol solution, and an aqueous acetonitrile solution, and water is preferred. The amount of the solvent used in step 1 can be 1 mL to 500 mL, and is preferably 10 mL to 20 mL, per 1 g of the compound represented by formula (5a).

[0025] The oxidizing agent used in step 1 includes potassium peroxymonosulfate (sold under the trade name Oxone (registered trademark), etc.), peracetic acid, hydrogen peroxide, and sodium periodate. Hydrogen peroxide may be used together with a catalytic amount of methyltrioxorhenium. From the viewpoint of safety and cost, potassium peroxymonosulfate is a preferred oxidizing agent. The amount of the oxidizing agent used may be 0.8 to 2.0 equivalents relative to 1 equivalent of the compound represented by formula (5a), and is preferably 1.0 to 1.3 equivalents.

[0026] The reaction temperature in step 1 can be -20°C to 30°C, and is preferably -5°C to 5°C.

[0027] The reaction time in step 1 can be from 5 minutes to 24 hours, and is preferably from 0.5 hours to 2 hours.

[0028] The solvent used in step 2 is not particularly limited as long as it dissolves the sulfoxide compound and the sulfur source and does not inhibit the subsequent reaction. Examples of such a solvent include water, an aqueous sulfuric acid solution, an aqueous ethanol solution, and an aqueous acetonitrile solution, and water is preferred. The amount of the solvent used in step 2 can be 1 mL to 500 mL, and is preferably 10 mL to 20 mL, per 1 g of the sulfoxide compound.

[0029] Examples of the sulfur source used in step 2 include sodium sulfide, potassium sulfide, sodium hydrogen sulfide, potassium hydrogen sulfide, and hydrogen sulfide. The amount of the sulfur source used can be 0.5 to 4.0 equivalents, and preferably 0.9 to 1.2 equivalents, relative to 1 equivalent of the sulfoxide compound.

[0030] The reaction temperature in step 2 can be -20°C to 30°C, and is preferably -5°C to 25°C.

[0031] The reaction time in step 2 can be from 10 minutes to 2 days, and is preferably from 0.5 hours to 2 hours.

[0032] When step 1 and step 2 are carried out in one pot, examples of the reaction solvent include water, an aqueous sulfuric acid solution, an aqueous ethanol solution, and an aqueous acetonitrile solution, and preferably water. The amount of the solvent can be 1 mL to 500 mL, and preferably 10 mL to 20 mL, per 1 g of the compound represented by formula (5a). Examples of the oxidizing agent used include potassium peroxymonosulfate, peracetic acid, hydrogen peroxide (which may be used together with a catalytic amount of methyltrioxorhenium), and sodium periodate, and preferably potassium peroxymonosulfate. The amount of the oxidizing agent used can be 0.8 equivalents to 2.0 equivalents, and preferably 1.0 equivalents to 1.3 equivalents, per equivalent of the compound represented by formula (5a). Examples of the sulfur source used include sodium sulfide, potassium sulfide, sodium hydrogen sulfide, potassium hydrogen sulfide, and hydrogen sulfide. The amount of the sulfur source used can be 0.5 to 4.0 equivalents relative to 1 equivalent of the compound represented by formula (5a), and is preferably 0.9 to 1.2 equivalents. The reaction temperature can be -20°C to 30°C, and is preferably -5°C to 25°C. The reaction time can be 15 minutes to 2 days, and is preferably 1 to 4 hours.

[0033] In addition to steps 1 and 2, a step of protecting a functional group such as a hydroxy group, a carbonyl group, an amino group, a carboxy group, etc. and a step of deprotecting the protected functional group may be included, if necessary. Protective groups and protection / deprotection reactions for these functional groups are well known to those skilled in the art, and appropriate protecting groups and protection / deprotection reactions can be selected by referring to "Greene's Protective Groups in Organic Synthesis" or the like.

[0034] The compound represented by formula (5a) is a combination of lipoic acid and NHR 2 R 3 The compound represented by formula (5a) can be produced by condensing the above. Examples of the solvent for the condensation reaction include dichloromethane, chloroform, and tetrahydrofuran, and preferably tetrahydrofuran. The amount of the solvent can be 1 mL to 200 mL, and preferably 3 mL to 35 mL, per 1 g of the compound represented by formula (5a). Examples of the condensation agent used include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and its salts, N,N'-dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) (N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt) may be used as an additive). The amount of the condensation agent used can be 0.8 equivalents to 2.0 equivalents, and preferably 1.0 equivalents to 1.5 equivalents, per 1 equivalent of the compound represented by formula (5a). The reaction temperature can be -10°C to 40°C, and preferably 15°C to 25°C. The reaction time can be from 1 hour to 3 days, and is preferably from 1 hour to 24 hours.

[0035] The compound represented by formula (5) is a mixture of lipoic acid trisulfide and NHR 2 R 3 The condensation conditions are the same as above.

[0036] R 1The compound represented by formula (3), in which is an alkyl group having 1 to 6 carbon atoms, can be produced by a step (step 1) of oxidizing a compound represented by formula (3a) with an oxidizing agent to obtain a sulfoxide compound, and a step (step 2) of reacting the obtained sulfoxide compound with a sulfur source. The reaction conditions are the same as those described above. [ka] [In the formula, R 1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.]

[0037] The compound represented by formula (3a) is a mixture of lipoic acid and R 1 It can be produced by condensing OH as described above.

[0038] R 1 The compound represented by formula (3) in which R is an alkyl group having 1 to 6 carbon atoms is prepared by combining lipoic acid trisulfide and R 1 It can also be produced by condensing OH. The condensation conditions are the same as above.

[0039] The cyclodextrin may be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a derivative thereof, where the term “derivative” means that the hydrogen atom of at least one hydroxyl group of each cyclodextrin is replaced by an alkyl group or a sugar which may have a substituent. Examples of cyclodextrin derivatives that can be used include methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, dimethyl-α-cyclodextrin, dimethyl-β-cyclodextrin, dimethyl-γ-cyclodextrin, hydroxyethyl-α-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxyethyl-γ-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, glucosyl-γ-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, and sulfobutylether-β-cyclodextrin.

[0040] The cyclodextrin inclusion complex can be produced by the steps of dissolving cyclodextrin in a solvent (step a), adding compound (1) or a pharma- ceutically acceptable salt thereof to the resulting solution and stirring (step b), filtering the solution after stirring, washing with the same solvent as that used in step a, freezing the filtrate, and freeze-drying (step c). Note that the filtering and washing steps in step c may be omitted.

[0041] The solvent used in step a is preferably water.

[0042] The amount of the solvent used in step a can be 1 to 350 ml, and preferably 1 to 80 ml, per 1 g of cyclodextrin.

[0043] In the step b, the mass ratio of cyclodextrin to compound (1) or a pharma- ceutically acceptable salt thereof can be 2 to 20, and is preferably 5 to 16.5.

[0044] The stirring temperature in step b can be 20 to 50° C., and may be room temperature.

[0045] The stirring time in step b can be from 0.25 to 40 hours, and is preferably from 2 to 35 hours.

[0046] In step b, after compound (1) or a pharma- ceutically acceptable salt thereof is added, the same solvent as that used in step a may be added before stirring. In this case, the amount of the solvent may be 0 to 30 ml, and preferably 0 to 20 ml, per 1 g of cyclodextrin.

[0047] The amount of the solvent used in step c can be 0 to 150 ml, and is preferably 0 to 20 ml, per 1 g of cyclodextrin.

[0048] The freezing temperature in step c can be -30 to -20°C, and is preferably -20°C.

[0049] The freezing time in step c can be 10 to 50 hours.

[0050] The freeze-drying in step c can be performed at an absolute pressure of 20 to 100 Pa and an external temperature of 10 to 40°C, preferably 20°C.

[0051] The freeze-drying period in step c can be 1 to 5 days.

[0052] The preventive or therapeutic agent for muscle atrophy and / or muscle regeneration failure of the present invention contains the above-mentioned nitration inhibitor.

[0053] The muscle atrophy in the present invention includes muscle atrophy caused by long-term non-use of muscles or reduced frequency or strength of use due to aging, bedriddenness, trauma, postoperative immobilization, weightlessness, contraction of some disease, etc. The muscle atrophy or muscle regeneration failure in the present invention is related to disorders of muscle growth, hypertrophy, regeneration, or maintenance triggered by inhibition of activation or reduced proliferation of muscle stem cells (satellite cells), and representative examples include age-related sarcopenia, amyotrophic lateral sclerosis, spinal muscular atrophy, spinal-bulbar muscular atrophy, and muscular dystrophy.

[0054] Examples of pet animals include dogs, cats, hamsters, rabbits, ferrets, parakeets, Java sparrows, parrots, and the like.

[0055] The dosage of the nitration inhibitor, the preventive or therapeutic agent for muscle atrophy and muscle regeneration failure of the present invention is a therapeutically effective amount, and varies depending on symptoms, age, administration method, dosage form, etc. In general, glutathione trisulfide or its pharmaceutically acceptable salt, or compound (1), its pharmaceutically acceptable salt, or their cyclodextrin inclusion complexes can be administered to an adult at 0.5 to 2000 mg per day, and it is preferable to administer 1 to 800 mg once or in several divided doses per day every day. It is considered that when the dosage of the above compound is within this range, a preventive or therapeutic effect against muscle atrophy and muscle regeneration failure is exhibited.

[0056] The dosage of the nitration inhibitor, the preventive or therapeutic agent for muscle atrophy and muscle regeneration failure of the present invention is a therapeutically effective amount, and varies depending on symptoms, age, administration method, dosage form, etc. In general, glutathione trisulfide or its pharmaceutically acceptable salt, or compound (1), its pharmaceutically acceptable salt, or their cyclodextrin inclusion complexes can be administered to pet animals at 0.1 to 400 mg / kg per day, and it is preferable to administer 0.1 to 400 mg / kg once or several times per day for consecutive days. It is considered that when the dosage of the above compound is within this range, a preventive or therapeutic effect against muscle atrophy and muscle regeneration failure is exhibited.

[0057] According to another embodiment of the present invention, an agent for suppressing or ameliorating muscle growth inhibition (reduction in meat productivity) caused by heat stress in livestock or poultry contains the above-mentioned nitration inhibitor.

[0058] In the present invention, causes of muscle growth inhibition (reduced meat productivity) due to heat stress in livestock or poultry include an increase in the outside temperature of the livestock or poultry rearing environment due to the heat in summer, and abnormal or deviation in temperature management in the livestock or poultry rearing management area. These causes are thought to cause respiratory alkalosis, reduced thyroid hormones, increased corticosterone, changes in immune response, and excessive production of mitochondrial reactive oxygen species (ROS) in the livestock or poultry. As a result, inhibition of activation and reduced proliferation of muscle stem cells (satellite cells) leads to disorders in muscle growth, hypertrophy, regeneration, or maintenance, and reduced meat productivity.

[0059] Examples of livestock or poultry include cows, horses, pigs, donkeys, sheep, goats, chickens, ducks, geese, ostriches, turkeys, ducks, quails, and the like.

[0060] The dosage of the nitration inhibitor of the present invention, the agent for inhibiting or improving muscle growth inhibition (reduction in meat productivity) in livestock or poultry, is a therapeutically effective amount, and varies depending on symptoms, administration method, etc. In general, glutathione trisulfide or a pharmaceutically acceptable salt thereof, or compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex thereof can be administered to livestock or poultry at 0.1 to 400 mg / kg per day, and it is preferable to administer 0.1 to 400 mg / kg once or in several divided doses per day for consecutive days. It is considered that when the dosage of the compound is within this range, the effect of inhibiting or improving muscle growth inhibition (reduction in meat productivity) in livestock or poultry is exhibited.

[0061] The nitration inhibitor, the preventive or therapeutic agent for muscle atrophy and / or muscle regeneration failure, and the agent for inhibiting or improving muscle growth inhibition (reduced meat productivity) of the present invention can be administered orally, for example, as a tablet, capsule, powder, granule, liquid, or syrup, or parenterally, as an injection, infusion, or suppository. These dosage forms can be formulated by known formulation techniques. In the case of a solid formulation, a pharmaceutically acceptable excipient, such as starch, lactose, refined white sugar, glucose, crystalline cellulose, carboxycellulose, carboxymethylcellulose, carboxyethylcellulose, calcium phosphate, magnesium stearate, gum arabic, etc., can be blended during formulation, and if necessary, a lubricant, a binder, a disintegrant, a coating agent, a colorant, etc. can be blended. In the case of a liquid formulation, a stabilizer, a dissolving agent, a suspending agent, an emulsifier, a buffer, a preservative, etc. can be blended. EXAMPLES

[0062] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Note that GSSSG stands for glutathione trisulfide.

[0063] Experimental Example 1 <Peroxynitrite (ONOO - Inhibitory effect of GSSSG on the nitration of recombinant HGF protein by Samples were prepared by adding and mixing GSSSG dihydrate produced by the method described in Patent Document 1 to a purified recombinant mouse HGF preparation (2207-HG-025 / CF, manufactured by R&D Systems, carrier protein not included) dissolved in PBS so that the molar ratio of HGF:GSSSG was 1:10, 1:50, or 1:400. Samples were also prepared in which no GSSSG dihydrate was added and the molar ratio of HGF:GSSSG was 1:0. Each sample was immediately treated with peroxynitrite (ONOO). - ) by HGF:ONOO - The molar ratio of was 1:500. The mixture was kept at pH 7.2 to 7.4 and 25 to 37° C. for 30 minutes (nitration treatment) to obtain a nitrated sample.

[0064] Western blotting was performed using the obtained nitrated sample according to the usual method. The nitrated sample was treated with a solution containing SDS and β-mercaptoethanol to prepare a sample for reduced SDS-PAGE. 10% polyacrylamide gel was used for electrophoresis. The transfer membrane used was made of nitrocellulose. HRP-labeled anti-nitrotyrosine monoclonal antibody (catalog number: sc-32757 HRP, manufactured by Santa Cruz Biotechnology) and HRP-labeled anti-HGF monoclonal antibody (catalog number: sc-374422 HRP, manufactured by Santa Cruz Biotechnology) were used. The immune reaction was recorded with an image capture device using ECL reagent. Nitrated bovine serum albumin (BSA) was used as a positive control sample. nitro ) was used. PL (Protein Ladder) and MW-STD (Molecular Weight-Standard) in Figure 1 are molecular weight markers. Material A in Figure 1 refers to GSSSG.

[0065] After confirming that the reaction intensity with anti-HGF antibody was roughly the same in each lane (lower panel of Fig. 1), we confirmed that the reaction intensity with anti-nitrotyrosine antibody decreased with increasing molar ratio of GSSSG added (upper panel of Fig. 1), thus visualizing the inhibitory effect of GSSSG on the nitration of tyrosine residues in HGF.

[0066] Experimental Example 2 <Peroxynitrite (ONOO - Inhibitory effect of lipoic acid trisulfide on the nitration of recombinant HGF protein by The use of lipoic acid trisulfide instead of GSSSG, HGF:ONOO - Nitration treatment and Western blotting were performed in the same manner as in Experimental Example 1, except that the molar ratio of was 1:4000. Note that material B in Figure 1 means lipoic acid trisulfide.

[0067] As in Experimental Example 1, after confirming that the reaction intensity with anti-HGF antibody was roughly the same in each lane (lower frame in FIG. 2), it was confirmed that the reaction intensity with anti-nitrotyrosine antibody decreased with increasing molar ratio of added lipoic acid trisulfide (upper frame in FIG. 2). In other words, the inhibitory effect of lipoic acid trisulfide on the nitration of tyrosine residues in HGF was visualized.

[0068] GSSSG or a pharma- ceutically acceptable salt thereof, or compound (1), a pharma- ceutically acceptable salt thereof, or a cyclodextrin inclusion complex thereof is peroxynitrite (ONOO - ), it is expected that it will be possible to suppress the nitration of tyrosine residues in HGF caused by various oxidative stresses occurring in vivo, and therefore it is expected that it will also suppress HGF dysfunction caused by various oxidative stresses occurring in vivo. GSSSG or a pharmaceutically acceptable salt thereof, or compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex thereof is expected to have a preventive or therapeutic effect against muscle atrophy and muscle regeneration failure caused by the nitration of tyrosine residues in HGF. In addition, GSSSG or a pharmaceutically acceptable salt thereof, or compound (1), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion complex thereof is expected to be able to suppress or improve muscle growth inhibition (reduced meat productivity) caused by the nitration of tyrosine residues in HGF caused by heat stress.

[0069] Reference example 1 <Production of (R)-lipoic acid trisulfide> [ka] In a 200 mL four-neck flask, 24.38 g (118.17 mmol) of (R)-α-lipoic acid and 488 mL (20.0 v / w) of 75% aqueous ethanol solution were charged. After confirming that the contents of the flask were dissolved, the internal temperature was cooled to 0°C. Oxone (registered trademark) (41.40 g, 124.20 mmol, 1.05 equivalents) was added in two portions and reacted for about 50 minutes. Insoluble matter in the reaction solution was removed by filtration and washed with 65 mL (2.67 v / w) of ethanol. To the filtrate and washings, 400mL (206.93mmol, 1.75 equivalents) of Na2S aqueous solution (Na2S·9H2O70.70g dissolved in water569mL) was added dropwise over about 2.5 hours at an internal temperature of 2-6℃ (pH was controlled to 6-7 using 3mol / L sulfuric acid aqueous solution during the addition and reaction, total amount used was 14mL). After reacting for about 50 minutes at an internal temperature of 3℃ and pH7, 41mL (1.7v / w) of 3mol / L sulfuric acid aqueous solution was added dropwise to adjust the pH to 1.3. Next, 320mL (13.1v / w) of water and 320mL (13.1v / w) of ethyl acetate were added, and extraction was performed with ethyl acetate. The aqueous layer was extracted four times with 160mL (6.6v / w) of ethyl acetate, and the organic layers were combined and concentrated under reduced pressure at an external temperature of 30℃. Ethanol was added to the concentrate to dissolve it, and then column purification was performed with ODS. The fraction was concentrated under reduced pressure at an external temperature of 30° C. and then dried using an oil pump to obtain 10.69 g of (R)-lipoic acid trisulfide (44.84 mmol, yield 38%, HPLC purity 99.7%, white solid).

[0070] <Production of (R)-Lipoamide Trisulfide> [ka] In a 200mL four-neck flask, 2.00g (8.39mmol) of (R)-lipoic acid trisulfide and 65mL (32.5v / w) of methylene chloride were charged. After confirming that the contents of the flask were dissolved, 2.07g (10.77mmol, 1.28 equivalents) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1.42g (12.33mmol, 1.47 equivalents) of N-hydroxysuccinimide (NHS) were added. After replacing the air in the flask with nitrogen, the reaction was carried out at room temperature for about 8 hours. Next, 2.28mL (33.74mmol, 4.02 equivalents) of 28% ammonia water was added dropwise at room temperature over 5 minutes, and the reaction was carried out overnight. Then, 60 mL (30.0 v / w) of water was added at room temperature and the mixture was separated. The organic layer was washed three times with 60 mL (30.0 v / w) of 2.5% aqueous sodium hydrogen carbonate solution, and then washed four times with 60 mL (30.0 v / w) of water. The washed organic layer was then concentrated under reduced pressure at an external temperature of 25°C, and dried with an oil pump to obtain 1.93 g (8.13 mmol, yield 97%, HPLC purity 99.6%, white solid) of (R)-lipoamide trisulfide. 1 H-NMR: (CDCl3,400MHz)δ(ppm)=5.36(bs,2H),3.33(m,1H),3.13(m,2H),2.22(m,3H),1.89(m,1H),1.74-1.42(m,6H). HR-ESI-TOF-MS:m / z 236.0238 ([MH] - ),calcd for[C8H 14 NOS3]-236.0243.

[0071] Reference example 2 <Production of Lipoamide Trisulfide (Racemate)> [ka] In a 500 mL four-neck flask, 1.00 g (4.87 mmol) of lipoamide (racemic form) and 182 mL (182.0 v / w) of 85% dimethylformamide aqueous solution were charged. After confirming that the contents of the flask were dissolved, the flask was cooled to an internal temperature of 4° C. In the same flask, 1.63 g (4.89 mmol, 1.00 equivalent) of Oxone (registered trademark) was added in three portions every 10 minutes, and the reaction was allowed to proceed for about 1 hour. While controlling the pH of the reaction solution to 5-11 using a 3 mol / L aqueous sulfuric acid solution, 1.24 g (5.16 mmol, 1.06 equivalent) of sodium sulfide nonahydrate was charged in portions at an internal temperature of 5° C., and the reaction was allowed to proceed for about 1.5 hours. 180mL (180.0v / w) of water and 50mL (50.0v / w) of methylene chloride were added, and the mixture was extracted with methylene chloride. The aqueous layer was then extracted twice with 50mL (50.0v / w) of methylene chloride. The organic layers were combined and concentrated under reduced pressure at an external temperature of 30°C or less. 80mL (80.0v / w) of water was added dropwise to the concentrated residue at room temperature for 30 minutes to crystallize the mixture. The slurry was filtered and then washed with 50mL (50.0v / w) of water. The wet crystals were dried under reduced pressure at 25°C to obtain 510mg (2.15mmol, yield 44%, HPLC purity 92%, white solid) of lipoamide trisulfide.

[0072] <Purity test of lipoamide trisulfide (HPLC)> Detector: Ultraviolet spectrophotometer (measurement wavelength: 220 nm) Column: LiChrosorb RP-18 (Kanto Chemical, 4.0 mm I.D. x 250 mm, 5 μm) Column temperature: constant temperature around 40℃ Mobile phase A: Phosphoric acid aqueous solution (pH 3) Mobile phase B: Methanol Delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as follows to control the concentration gradient.

[0073] [Table 1] Flow rate: 1mL / min Injection volume: 10μL Area measurement range: 35 minutes after injection of sample solution Retention time: Lipoamide sulfoxide (12-13 min), Lipoamide (approximately 17 min), Lipoamide trisulfide (approx. 19 min)

[0074] Reference examples 3~9 In the following, "HP" is an abbreviation for "hydroxypropyl," "Me" is an abbreviation for "methyl," and "Mal" is an abbreviation for "maltosyl."

[0075] <Production of lipoic acid trisulfide> [ka]

[0076] 2.0g (9.02mmol) of lipoic acid and 40mL of 75% ethanol aqueous solution were charged into a reaction vessel and cooled to an internal temperature of 0°C. 3.4g (10.20mmol) of Oxone (registered trademark) was added thereto and reacted for about 2 hours. Inorganic salts in the reaction solution were filtered and washed with 7mL of ethanol. 5.8g (24.1mmol) of sodium sulfide nonahydrate was added to the filtrate and reacted for about 1 hour. 7mL of 3mol / L sulfuric acid aqueous solution was dropped into this reaction solution, followed by adding 20mL of water and 45mL of ethyl acetate (AcOEt), and extraction with AcOEt was performed. The aqueous layer was extracted twice with 20mL of AcOEt, and the organic layers were combined and concentrated under reduced pressure. The concentrate was dissolved by adding 3 mL of ethanol, and the solution was purified using an ODS column (YMC Dispo PackAT, mobile phase: aqueous acetonitrile solution) to obtain 0.7 g (2.39 mmol, HPLC purity: 100%) of lipoic acid trisulfide.

[0077] <Production of CD inclusion complex of lipoic acid trisulfide> Reference Example 3: β-CD inclusion complex of lipoic acid trisulfide (racemic body) In a 100 mL eggplant flask, 20.0 mg (0.899 mmol) of β-CD10 and 80 mL of water were charged. After confirming that the contents of the flask were dissolved, 99.8 mg (0.419 mmol) of lipoic acid trisulfide was added, and the flask was washed with 20 mL of water. After stirring at 45°C for 15 minutes, the mixture was filtered, and the flask and the crystals were washed with 10 mL of water. The obtained filtrate was frozen in a freezer at -20°C for 23 hours. The mixture was freeze-dried at an external temperature of 20°C for about 4.5 days, and 980.0 mg of the inclusion compound (white solid) was obtained.

[0078] Reference Example 4: HP-β-CD inclusion complex of lipoic acid trisulfide (racemic form) In a 50 mL recovery flask, 1291.0 mg of HP-β-CD and 16 mL of water were charged. After confirming that the contents of the flask were dissolved, 100.0 mg (0.419 mmol) of lipoic acid trisulfide was added. After stirring at room temperature for about 28 hours, the mixture was filtered, and the flask and the crystals were washed with 10 mL of water. The obtained filtrate was frozen in a freezer at -20°C for about 2 days. The mixture was freeze-dried at an external temperature of 20°C for about 2 days to obtain 1330.0 mg of the inclusion compound (white solid).

[0079] Reference Example 5: (R)-Lipoic acid trisulfide inclusion complex with HP-β-CD In a 50 mL recovery flask, 969.9 mg of HP-β-CD and 10 mL of water were charged. After confirming that the contents of the flask had dissolved, 100.3 mg (0.421 mmol) of (R)-lipoic acid trisulfide was added, and the flask was washed with 4 mL of water. After stirring at room temperature for about 25 hours, the mixture was filtered, and the flask and the crystals were washed with 12 mL of water. The obtained filtrate was frozen in a freezer at -20°C for 15 hours. The mixture was freeze-dried at an external temperature of 20°C for about 2 days, and 1040.0 mg of the inclusion compound (white solid) was obtained.

[0080] Reference Example 6: Me-β-CD inclusion complex of lipoic acid trisulfide (racemic body) In a 50 mL recovery flask, 1616.0 mg of Me-β-CD (several methylated mixture) and 12 mL of water were charged. After confirming that the contents of the flask were dissolved, 101.0 mg (0.424 mmol) of lipoic acid trisulfide was added, and the flask was washed with 4 mL of water. After stirring for 21 hours, the mixture was filtered, and the flask and crystals were washed with 12 mL of water. The obtained filtrate was frozen in a freezer at -20°C for 20 hours. The mixture was freeze-dried at an external temperature of 20°C for about 4 days, and 1665.2 mg of the inclusion compound (white solid) was obtained.

[0081] Reference Example 7: (R)-Lipoic acid trisulfide Me-β-CD inclusion complex A 50 mL recovery flask was charged with 1616.0 mg of Me-β-CD (several methylated mixture) and 16 mL of water. After confirming that the contents of the flask were dissolved, 99.9 mg (0.420 mmol) of (R)-lipoic acid trisulfide was added, and the flask was washed with 4 mL of water. After stirring at room temperature for 6 hours, the mixture was filtered, and the flask and crystals were washed with 13 mL of water. The obtained filtrate was frozen in a freezer at -20°C for 28 hours. The mixture was freeze-dried at an external temperature of 20°C for about 3 days, and 1610.9 mg of the inclusion compound (white solid) was obtained.

[0082] Reference Example 8: Mal-β-CD inclusion complex of lipoic acid trisulfide (racemic form) A 50 mL eggplant flask was charged with 24.2 mg (0.839 mmol) of Mal-β-CD12 and 14 mL of water. After confirming that the contents of the flask had dissolved, 100.4 mg (0.421 mmol) of lipoic acid trisulfide was added, and the flask was washed with 2 mL of water. After stirring at room temperature for 31 hours, the mixture was filtered, and the flask and the crystals were washed with 10 mL of water. The obtained filtrate was frozen in a freezer at -20°C for 22 hours. The mixture was freeze-dried at an external temperature of 20°C for approximately 46 hours, and 1180.0 mg of the inclusion compound (white solid) was obtained.

[0083] Reference Example 9: Mal-β-CD inclusion complex of (R)-lipoic acid trisulfide A 50 mL eggplant flask was charged with 24.2 mg (0.839 mmol) of Mal-β-CD12 and 10 mL of water. After confirming that the contents of the flask had dissolved, 100.1 mg (0.420 mmol) of (R)-lipoic acid trisulfide was added, and the flask was washed with 5 mL of water. After stirring at room temperature for 4.5 hours, the mixture was filtered, and the flask and the crystals were washed with 11 mL of water. The obtained filtrate was frozen in a freezer at -20°C for 24 hours. The mixture was freeze-dried at an external temperature of 20°C for about 41 hours, and 1319.6 mg of the inclusion compound (white solid) was obtained.

[0084] The yields and solubilities of the inclusion compounds obtained in Reference Examples 3 to 9 are shown in Table 2.

[0085] [Table 2]

Claims

1. An agent for suppressing nitration of a tyrosine residue in hepatocyte growth factor, comprising a trisulfide compound, The trisulfide compound is glutathione trisulfide or a pharma- ceutically acceptable salt thereof, or a compound represented by the formula (1): 【Chemistry 1】 [wherein X is -OR １ Or -NR ２ R ３ indicates R １ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ２ and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and the alkyl group may have one or more substituents selected from the group consisting of an amino group and a carboxy group.], a pharma- ceutical acceptable salt thereof, or a cyclodextrin clathrate thereof.

2. The nitration inhibitor according to claim 1, wherein the trisulfide compound is glutathione trisulfide or a pharma- ceutically acceptable salt thereof.

3. The nitration inhibitor according to claim 1, wherein the trisulfide compound is lipoic acid trisulfide or a pharma- ceutically acceptable salt thereof.

4. A preventive or therapeutic agent for muscle atrophy and / or muscle regeneration failure, comprising the nitration inhibitor according to any one of claims 1 to 3.

5. A method for preventing or treating muscle atrophy and / or muscle regeneration failure in a companion animal, comprising administering the nitration inhibitor according to any one of claims 1 to 3 to the companion animal.

6. 4. An agent for suppressing or ameliorating muscle growth inhibition (reduction in meat productivity) caused by heat stress in livestock or poultry, comprising the nitration inhibitor according to any one of claims 1 to 3.

7. A method for suppressing or improving muscle growth inhibition (reduced meat productivity) caused by heat stress in livestock or poultry, comprising administering the nitration inhibitor according to any one of claims 1 to 3 to livestock or poultry.

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