Physiological activity enhancer of hepatocyte growth factor

Abstract

The invention discloses a physiological activity enhancer of a hepatocyte growth factor. The physiological activity enhancer contains a trisulfide compound. The trisulfide compound is selected from glutathione trisulfide and pharmaceutically acceptable salt thereof, compound represented by formula (1) and pharmaceutically acceptable salt thereof, cyclodextrin inclusion compound of compound and pharmaceutically acceptable salt thereof, and formula (6) The compound is at least one selected from the group consisting of a pharmaceutically acceptable salt of the compound and cyclodextrin inclusion compounds of the compound and the pharmaceutically acceptable salt.

Description

Technical Field

[0001] The present invention relates to a physiological activity enhancer of hepatocyte growth factor. Background Art

[0002] Hepatocyte Growth Factor (hereinafter referred to as "HGF"), a cytokine that promotes the proliferation of primary cultured hepatocytes, was isolated and purified from the plasma of patients with acute severe hepatitis in the 1980s, and its cDNA was cloned. It is secreted as a single-chain, inactive form (pro-HGF) and converted by protease cleavage into an active HGF heterodimer consisting of a light chain and a heavy chain with disulfide bonds. Subsequent research has clarified that HGF has diverse functions, including promoting cell proliferation, cell migration, inhibiting cell death, anti-fibrosis, and angiogenesis, which contribute to the regeneration and repair of tissues and organs. Research and development of its therapeutic applications for a variety of diseases, including cirrhosis, chronic renal failure, pulmonary fibrosis, myocardial infarction, obliterative arteriosclerosis, amyotrophic lateral sclerosis, and acute spinal cord injury, is progressing. Furthermore, HGF's diverse biological effects are exerted through its binding to c-Met, a product of the proto-oncogene c-Met, which possesses tyrosine kinase activity (Non-Patent Documents 1 and 2). HGF is reported to be an activating factor for muscle stem cells (satellite cells), and satellite cells are known to contribute to the regeneration of muscle fibers, etc. This effect can also be exerted through c-Met (Non-Patent Document 3).

[0003] Trisulfide compounds such as glutathione trisulfide can be converted into active sulfur species such as glutathione persulfide in vivo. Active sulfur species are reported to have strong antioxidant properties and may have diverse physiological functions, such as anti-aging (e.g., Non-Patent Documents 4-7). Also known methods for producing trisulfide compounds include those described in Patent Documents 1 and 2.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2018 / 117186

[0007] Patent Document 2: International Publication No. 2022 / 045212

[0008] Non-patent literature

[0009] Non-patent document 1: Eiichi Koda, “Function and production control of hepatocyte growth factor (HGF)”, Folia Pharmacol. Jpn. 119, 287-294 (2002)

[0010] Non-patent document 2: Mizuno Nobuya et al., "Clinical application of regenerative factor HGF", Drug Delivery System 16-6, 473-484 (2001)

[0011] Non-patent document 3: R. Tatsumi, "Mechano-biology of skeletal musclehypertrophy and regeneration: possible mechanism of stretch-induced activation of resident myogenic stem cells", Animal Science Journal 81(1),11-20(2010).

[0012] Non-patent document 4: T. Ida, et al., "Reactive cysteine ​​persulfides and S-polythiolation regulate oxidative stress and redox signaling", PNAS, May27, 2014, 111(21)7606-7611

[0013] Non-patent document 5: T. Akaike, et al., "Cysteinyl-tRNA synthetase governscysteine ​​polysulfidation and mitochondrial bioenergetics", NatureCommunications, 2017, Oct27; 8(1): 1177

[0014] Non-patent document 6: M. Ezaka, et al., "Oral Administration of GlutathioneTrisulfide Increases Reactive Sulfur Levels in Dorsal Root Ganglion andAmeliorates Paclitaxel-Induced Peripheral Neuropathy in Mice", Antioxidants11, 2122(2022)

[0015] Non-Patent Literature 7: F. Nagashima, et al., “Sulfide:quinone oxidoreductaseameliorates neurodegeneration in a murine model of Parkinson's disease”, Redox Biol. Feb; 59: 102562. (2023) Summary of the Invention

[0016] Problems to be solved by the invention

[0017] An object of the present invention is to provide an enhancer for the physiological activity of hepatocyte growth factor.

[0018] Methods used to solve problems

[0019] The present inventors have discovered that trisulfide compounds can enhance the binding of HGF to its receptor c-Met, and as a result, can enhance the cell proliferation effect of HGF in a concentration-dependent manner, thereby completing the present invention.

[0020] The present invention relates to, for example, the following [1] to

[23] .

[0021] [1] A hepatocyte growth factor physiological activity enhancer comprising a trisulfide compound, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and a pharmaceutically acceptable salt thereof, a compound represented by the following formula (1) (hereinafter also referred to as compound (1)) and a pharmaceutically acceptable salt thereof and a cyclodextrin inclusion compound thereof, and a compound represented by formula (6) (hereinafter also referred to as compound (6)) and a pharmaceutically acceptable salt thereof and a cyclodextrin inclusion compound thereof,

[0022]

[0023] [In formula (1), R 1 is a group represented by the following formula (2), a group represented by the following formula (3), a group represented by the following formula (4), or a group represented by the following formula (5),

[0024]

[0025] [In formula (2), R 2 and R 3 are each independently an alkylene group having 1 to 4 carbon atoms, Indicates a bond],

[0026]

[0027] [In formula (3), R 4and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a substituent, R 4 and R 5 can form rings together with the atoms to which they are bonded, Indicates a bond],

[0028]

[0029] [In formula (4), Indicates a bond],

[0030]

[0031] [In formula (5), Indicates a bond]],

[0032]

[0033] [In formula (6), X represents -OR 6 or -NR 7 R 8 , R 6 、R 7 and R 8 Each independently represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5].

[0034] [2] The physiological activity enhancer according to [1], further comprising a hepatocyte growth factor.

[0035] [3] A hepatocyte growth factor physiological activity enhancer, which is administered in combination with hepatocyte growth factor and contains a trisulfide compound, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and cyclodextrin inclusion compounds thereof, and compound (6) and its pharmaceutically acceptable salts and cyclodextrin inclusion compounds thereof.

[0036] [4] A hepatocyte growth factor physiological activity enhancer, which is administered in combination with a trisulfide compound and contains hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and cyclodextrin inclusion compounds thereof, and compound (6) and its pharmaceutically acceptable salts and cyclodextrin inclusion compounds thereof.

[0037] [5] The physiological activity enhancer according to [3] or [4], wherein the trisulfide compound and the hepatocyte growth factor are administered simultaneously or separately.

[0038] [6] The physiological activity enhancer according to any one of [1] to [5], which is an enhancer for the binding between the hepatocyte growth factor and c-Met.

[0039] [7] A kit for enhancing the physiological activity of hepatocyte growth factor, comprising a preparation containing a trisulfide compound and a preparation containing hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salt, compound (1) and its pharmaceutically acceptable salt and their cyclodextrin inclusion complex, and compound (6) and its pharmaceutically acceptable salt and their cyclodextrin inclusion complex.

[0040] [8] The kit according to [7], wherein the preparation containing the trisulfide compound and the preparation containing the hepatocyte growth factor are administered simultaneously or separately.

[0041] [9] The kit according to [7] or [8], which is used to enhance the binding between the hepatocyte growth factor and c-Met.

[0042]

[10] The physiological activity enhancer or kit according to any one of [1] to [9], wherein the trisulfide compound is lipoic acid trisulfide or a pharmaceutically acceptable salt thereof.

[0043]

[11] The physiological activity enhancer or kit according to any one of [1] to [9], wherein the trisulfide compound is glutathione trisulfide or a pharmaceutically acceptable salt thereof.

[0044]

[12] A method for enhancing the physiological activity of hepatocyte growth factor, wherein a trisulfide compound is administered to a subject in need thereof, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0045]

[13] A trisulfide compound for enhancing the physiological activity of hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0046]

[14] Use of a trisulfide compound in the manufacture of a physiological activity enhancer of hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0047]

[15] The method for enhancing physiological activity, the trisulfide compound for enhancing the physiological activity of hepatocyte growth factor, or the use according to any one of

[12] to

[14] , wherein the enhancement of the physiological activity of the hepatocyte growth factor is the enhancement of the binding between the hepatocyte growth factor and cMet.

[0048]

[16] A method for enhancing the physiological activity of hepatocyte growth factor, wherein a trisulfide compound and hepatocyte growth factor are administered to a patient in need thereof, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0049]

[17] A method for enhancing the physiological activity of a hepatocyte growth factor administered in combination with a trisulfide compound, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and a pharmaceutically acceptable salt thereof, compound (1) and a pharmaceutically acceptable salt thereof and a cyclodextrin inclusion compound thereof, and compound (6) and a pharmaceutically acceptable salt thereof and a cyclodextrin inclusion compound thereof.

[0050]

[18] A trisulfide compound for enhancing the physiological activity of hepatocyte growth factor, administered in combination with hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and cyclodextrin inclusion compounds thereof, and compound (6) and its pharmaceutically acceptable salts and cyclodextrin inclusion compounds thereof.

[0051]

[19] A combination of a trisulfide compound and a hepatocyte growth factor for enhancing the physiological activity of hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0052]

[20] Use of hepatocyte growth factor in the manufacture of a hepatocyte growth factor physiological activity enhancer for administration in combination with a trisulfide compound, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0053]

[21] Use of a trisulfide compound in the manufacture of a hepatocyte growth factor physiological activity enhancer for administration in combination with hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0054]

[22] The method for enhancing physiological activity, the hepatocyte growth factor used in the method for enhancing the physiological activity of the hepatocyte growth factor, the trisulfide compound for enhancing the physiological activity of the hepatocyte growth factor, the combination or use according to any one of

[16] to

[21] , wherein the trisulfide compound and the hepatocyte growth factor are administered simultaneously or separately.

[0055]

[23] Use of a combination of hepatocyte growth factor and a trisulfide compound in the manufacture of a physiological activity enhancer of hepatocyte growth factor, wherein the trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salts, compound (1) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes, and compound (6) and its pharmaceutically acceptable salts and their cyclodextrin inclusion complexes.

[0056] Effects of the Invention

[0057] According to the present invention, a physiological activity enhancer of hepatocyte growth factor can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a graph showing the effect of the addition of GSSSG or LA-SSS on the binding (affinity) between HGF and c-Met.

[0059] Figure 2 This is a graph showing the effect of the addition of GSSSG or LA-SSS on the muscle stem cell activation effect of HGF (3 ng / ml).

[0060] Figure 3 Representative bright-field microscopy images of the cultured cells of Example 2.

[0061] Figure 4 This is a graph showing the effect of the addition of GSSSG or LA-SSS on the muscle stem cell activation effect of HGF (1 ng / ml). DETAILED DESCRIPTION

[0062] The present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0063] The hepatocyte growth factor physiological activity enhancer and the hepatocyte growth factor physiological activity enhancer of the present invention can be administered or applied to humans, pets, livestock or poultry.

[0064] The trisulfide compound contained in the enhancer of the physiological activity of hepatocyte growth factor of the present invention is at least one selected from the group consisting of glutathione trisulfide and a pharmaceutically acceptable salt thereof, compound (1) and a pharmaceutically acceptable salt thereof and a cyclodextrin inclusion complex thereof, and compound (6) and a pharmaceutically acceptable salt thereof and a cyclodextrin inclusion complex thereof.

[0065] Compound (1) can be said to be a compound in which two molecules of lipoic acid trisulfide are bonded via a linking group.

[0066] In compound (1), R 2 and R 3 Each independently represents an alkylene group having 1 to 4 carbon atoms. Examples of the alkylene group having 1 to 4 carbon atoms include methylene, ethylene, n-propylene, isopropylene, cyclopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene, and cyclobutylene.

[0067] R 4 and R 5are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a substituent, R 4 and R 5 They can form a ring together with the atoms to which they are bonded. Examples of substituents include hydroxyl groups; alkoxy groups having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, and butoxy. Examples of alkyl groups having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and cyclobutyl. 4 and R 5 When forming a ring together with the atoms to which they are bonded, the formed ring may be, for example, an oxetane ring.

[0068] Compound (1) can be produced by the following (A) or (B):

[0069] (A): A step of subjecting the compound represented by formula (1a) (lipoic acid trisulfide) to a condensation reaction with a molecule for forming a linker (a compound represented by the following formula (2a), a compound represented by the following formula (3a), o-xylylenediamine, m-xylylenediamine, p-xylylenediamine, piperazine, etc.) (step 1);

[0070] (B): a step of condensing the compound represented by formula (1b) (lipoic acid) with the above-mentioned molecule for forming a linking group (step 1'); a step of oxidizing the obtained disulfide compound with an oxidizing agent to obtain a sulfoxide compound (step 2); and a step of reacting the obtained sulfoxide compound with a sulfur source to obtain a trisulfide compound (step 3).

[0071] Examples of the solvent used in Step 1 and Step 1' include dichloromethane, chloroform, and tetrahydrofuran, with dichloromethane being preferred. The amount of the solvent can be 1 mL to 200 mL, preferably 3 mL to 35 mL, relative to 1 g of Compound (1a) or Compound (1b). Examples of the condensing agent used in Step 1 and Step 1' include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and its salts, N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP), and 1,1'-carbonyldiimidazolebis(1H-imidazol-1-yl)methanone (CDI). When using these compounds as the condensing agent, N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), and the like can be used as additives. The amount of the condensing agent used in Step 1 and Step 1' can be 0.8 to 2.0 equivalents, preferably 1.0 to 1.5 equivalents, relative to 1 equivalent of Compound (1a) or Compound (1b). The reaction temperature in Step 1 and Step 1' can be -10°C to 40°C, preferably 15°C to 25°C. The reaction time in Step 1 and Step 1' can be 1 hour to 3 days, preferably 1 hour to 24 hours.

[0072]

[0073]

[0074]

[0075]

[0076] In order to bond two molecules of Compound (1a) or Compound (1b) via a linker, the amount of the molecule for forming the linker in Step 1 and Step 1' may be 0.45 to 1.00 equivalents relative to the amount of lipoic acid trisulfide. However, other equivalents may be used as long as the target compound can be obtained.

[0077] The compound represented by the following formula (1a) (lipoic acid trisulfide) can be produced by the method described in Patent Document 2. The procedures and conditions of Steps 2 and 3 in Production Method (B) can be based on the procedures and conditions of Steps 1 and 2 of Patent Document 2, for example.

[0078] R in formula (2a) 2 and R 3 Respectively with R in formula (2) 2 and R 3Examples of the compound represented by formula (2a) include dialkylene glycols having 4 to 6 carbon atoms. Examples of the compound represented by formula (2a) include diethylene glycol, linear or branched dipropylene glycol, and the like.

[0079] R in formula (3a) 4 and R 5 and R in formula (3) 4 and R 5 Examples of the compound represented by formula (3a) include 2,2-dimethyl-1,3-propylenediamine, 2-oxetane-1,3-propylenediamine, and 2-methoxy-1,3-propylenediamine.

[0080] Examples of the compound (1) include compounds represented by the following formulae (11) to (16) (compounds (11) to (16)”).

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Table 1 shows the molecular weights and ClogPs of compounds (11) to (16).

[0088] [Table 1]

[0089]

[0090] The molecular weight of compound (1) and its salt may be 800 or less, 750 or less, or 700 or less, preferably 600 or less. The molecular weight of compound (1) and its salt may be 400 or more, 450 or more, or 500 or more. It is believed that when the molecular weight of compound (1) and its salt is within these ranges, the absorption rate in the body is further improved.

[0091] The ClogP of compound (1) and its salts may be 1 or greater, 2 or greater, 3 or greater, or 4 or greater. The ClogP of compound (1) and its salts may be 8 or less, 7 or less, or 6 or less. It can be said that when the ClogP of compound (1) and its salts is within these ranges, the balance between water solubility and fat solubility of compound (1) and its salts is more excellent. ClogP is a computer-calculated partition coefficient and can be determined according to the rules described in "CLOGP Reference Manual Daylight Version 4.9 (Release Date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )".

[0092] The salt of compound (1) may be any pharmacologically acceptable salt, and examples thereof include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; salts with inorganic acids such as hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; and 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. When compound (1) is obtained in the form of a free form, it can be converted into a salt by conventional methods. Alternatively, when compound (1) is obtained in the form of a salt, it can also be converted into a free form by conventional methods.

[0093] The compounds in this specification may exist in optical isomers (R isomer, S isomer, R,R isomer, R,S isomer, S,R isomer and S,S isomer). The compounds in this specification may be replaced by their optical isomers or a mixture containing the optical isomers in any ratio (e.g., a racemic form). For example, compound (1) may be an optical isomer represented by the following formula (1-1), an optical isomer represented by the following formula (1-2), an optical isomer represented by the following formula (1-3), or an optical isomer represented by the following formula (1-4), and may be replaced by a mixture containing these optical isomers in any ratio (e.g., a racemic form).

[0094]

[0095]

[0096]

[0097]

[0098] In one embodiment, compound (6) may be a compound represented by formula (7),

[0099]

[0100] [In formula (7), R6 represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5],

[0101] R 6 For example, it can be a hydrogen atom, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a hexyl group. 6 is a hydrogen atom, and in this case, the compound represented by formula (7) is lipoic acid trisulfide represented by formula (8).

[0102]

[0103] In another embodiment, compound (6) may be a compound represented by formula (9),

[0104]

[0105] [Where R 7 and R 8 Each independently represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5],

[0106] R7 and R 8 It can be a hydrogen atom, methyl, ethyl, propyl, butyl, pentyl, hexyl or other alkyl group. These alkyl groups may have one or both of amino and carboxyl groups as substituents. 7 and R 8 For example, it may be a group represented by formula (10) (wherein, represents a bond). Specific examples of the compound represented by formula (9) include, for example, R 7 and R 8 Compounds containing only hydrogen atoms, R 7 is a hydrogen atom and R 8 A compound having a group represented by formula (10).

[0107]

[0108] The compound represented by formula (9) can be produced by oxidizing the compound represented by formula (9a) with an oxidizing agent to obtain a sulfoxide compound (step 61), and reacting the obtained sulfoxide compound with a sulfur source (step 62).

[0109]

[0110] [Where R 7 and R 8 Each independently represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5.]

[0111] The above-mentioned production method can also allow step 61 and step 62 to be carried out in a one-pot reaction without isolating the sulfoxide compound.

[0112] The solvent used in step 61 is not particularly limited as long as it dissolves the compound represented by formula (9a) and the oxidant and does not inhibit the oxidation reaction. Examples of such solvents include water, aqueous sulfuric acid solution, aqueous ethanol solution, and aqueous acetonitrile solution, preferably water. The amount of the solvent used in step 61 can be 1 mL to 500 mL, preferably 10 mL to 20 mL, relative to 1 g of the compound represented by formula (9a).

[0113] As the oxidizing agent used in step 61, potassium peroxymonosulfate (sold under the trade name of Oxone (registered trademark)), peracetic acid, hydrogen peroxide and sodium periodate can be listed. Hydrogen peroxide can also 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 can be set to 0.8 equivalents to 2.0 equivalents, preferably 1.0 equivalents to 1.3 equivalents, relative to 1 equivalent of the compound represented by formula (9a).

[0114] The reaction temperature of step 61 can be set to -20°C to 30°C, preferably -5°C to 5°C.

[0115] The reaction time of step 61 can be set to 5 minutes to 24 hours, preferably 0.5 hours to 2 hours.

[0116] The solvent used in step 62 is not particularly limited as long as it dissolves the sulfoxide compound and the sulfur source and does not inhibit subsequent reactions. Examples of such solvents include water, aqueous sulfuric acid solution, aqueous ethanol solution, and aqueous acetonitrile solution, with water being preferred. The amount of solvent used in step 62 can be 1 mL to 500 mL, preferably 10 mL to 20 mL, per 1 g of the sulfoxide compound.

[0117] Examples of the sulfur source used in step 62 include sodium sulfide, potassium sulfide, sodium hydrosulfide, potassium hydrosulfide, and hydrogen sulfide. The amount of the sulfur source used is 0.5 to 4.0 equivalents, preferably 0.9 to 1.2 equivalents, per 1 equivalent of the sulfoxide compound.

[0118] The reaction temperature of step 62 can be set to -20°C to 30°C, preferably -5°C to 25°C.

[0119] The reaction time of step 62 can be set to 10 minutes to 2 days, preferably 0.5 hours to 2 hours.

[0120] When Step 61 and Step 62 are carried out in a one-pot process, examples of the reaction solvent include water, aqueous sulfuric acid solution, aqueous ethanol solution, and aqueous acetonitrile solution, preferably water. The amount of the solvent can be 1 mL to 500 mL, preferably 10 mL to 20 mL, relative to 1 g of the compound represented by Formula (9a). Examples of the oxidizing agent include potassium peroxymonosulfate, peracetic acid, hydrogen peroxide (which can be used together with a catalytic amount of methyltrioxorhenium), and sodium periodate, preferably potassium peroxymonosulfate. The amount of the oxidizing agent can be 0.8 to 2.0 equivalents, preferably 1.0 to 1.3 equivalents, relative to 1 equivalent of the compound represented by Formula (9a). Examples of the sulfur source include sodium sulfide, potassium sulfide, sodium hydrosulfide, potassium hydrosulfide, and hydrogen sulfide. The amount of the sulfur source can be 0.5 to 4.0 equivalents, preferably 0.9 to 1.2 equivalents, relative to 1 equivalent of the compound represented by Formula (9a). The reaction temperature can be set to -20° C. to 30° C., preferably -5° C. to 25° C. The reaction time can be set to 15 minutes to 2 days, preferably 1 hour to 4 hours.

[0121] In addition to Step 61 and Step 62, steps for protecting functional groups such as hydroxyl, carbonyl, amino, and carboxyl groups, and steps for deprotecting the protected functional groups may be included as needed. Protective groups and protection / deprotection reactions for these functional groups are well known to those skilled in the art. Suitable protecting groups and protection / deprotection reactions can be selected by referring to, for example, "Greene's Protective Groups in Organic Synthesis."

[0122] The compound represented by formula (9a) can be prepared by reacting lipoic acid with NHR 7 R 8 Condensation is used to produce the product. As solvents for the condensation reaction, for example, dichloromethane, chloroform, and tetrahydrofuran can be mentioned, preferably tetrahydrofuran. Regarding the amount of the solvent, it can be set to 1 mL to 200 mL relative to 1 g of the compound represented by formula (9a), preferably 3 mL to 35 mL. As condensing agents used, for example, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and its salts, N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) can be mentioned (N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBt) can be used as additives). Regarding the amount of the condensing agent used, it can be set to 0.8 equivalents to 2.0 equivalents relative to 1 equivalent of the compound represented by formula (9a), preferably 1.0 equivalents to 1.5 equivalents. The reaction temperature can be set to -10°C to 40°C, preferably 15°C to 25°C. The reaction time can be set to 1 hour to 3 days, and is preferably 1 hour to 24 hours.

[0123] The compound represented by formula (9) can also be prepared by reacting lipoic acid trisulfide with NHR 7 R 8 The condensation conditions are the same as above.

[0124] The compound represented by formula (7) can be produced by oxidizing the compound represented by formula (7a) with an oxidizing agent to obtain a sulfoxide compound (step 61), and reacting the obtained sulfoxide compound with a sulfur source (step 62). The reaction conditions are the same as above.

[0125]

[0126] [Where R 6 represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5.]

[0127] The compound represented by formula (7a) can be prepared by reacting lipoic acid with R 6 OH condensation to produce the same as above.

[0128] The compound represented by formula (7) can also be prepared by reacting lipoic acid trisulfide with R 6 The condensation conditions are the same as those described above.

[0129] In the present invention, examples of pharmaceutically 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.

[0130] 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) and the pharmaceutically acceptable salt of compound (6) are preferably salts with alkali metals, more preferably sodium salts.

[0131] Glutathione trisulfide or its pharmaceutically acceptable salt, compound (1) or its pharmaceutically acceptable salt, or compound (6) or its pharmaceutically acceptable salt may sometimes exist in polymorphic forms, but are not limited to any single crystal form and may be a single substance of any crystal form or a mixture. In addition, glutathione trisulfide or its pharmaceutically acceptable salt, compound (1) or its pharmaceutically acceptable salt, or compound (6) or its pharmaceutically acceptable salt also includes amorphous forms. Glutathione trisulfide or its pharmaceutically acceptable salt, compound (1) or its pharmaceutically acceptable salt, or compound (6) or its pharmaceutically acceptable salt includes anhydrates and solvates (particularly hydrates).

[0132] The trisulfide compound may be a cyclodextrin inclusion complex (CD inclusion complex) in which at least one compound selected from the group consisting of compound (1) and a pharmaceutically acceptable salt thereof, and compound (6) and a pharmaceutically acceptable salt thereof is included in cyclodextrin.

[0133] The cyclodextrin may be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or a derivative thereof. Here, the term "derivative" means that the hydrogen atom of at least one hydroxyl group of each cyclodextrin is substituted with an alkyl group or a sugar which may have a substituent. As cyclodextrin derivatives, for example, 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, sulfobutyl ether-β-cyclodextrin, etc. can be used.

[0134] The cyclodextrin inclusion compound can be produced by the following steps: a step of dissolving cyclodextrin in a solvent (step a); a step of adding at least one selected from the group consisting of compound (1) and its pharmaceutically acceptable salt and compound (6) and its pharmaceutically acceptable salt to the obtained solution and stirring (step b); and a step of filtering the stirred liquid, washing it with the same solvent as used in step a, freezing the filtrate, and freeze-drying it (step c). It should be noted that the filtering and washing operations in step c can be omitted.

[0135] The solvent used in step a is preferably water.

[0136] The amount of the solvent used in step a can be 1 to 350 ml, preferably 1 to 80 ml, relative to 1 g of cyclodextrin.

[0137] In step b, the mass ratio of cyclodextrin to at least one selected from the group consisting of compound (1) and its pharmaceutically acceptable salt and compound (6) and its pharmaceutically acceptable salt can be set to 2 to 20, preferably 5 to 16.5.

[0138] The stirring temperature in step b can be set to 20-50°C, or room temperature.

[0139] The stirring time in step b can be set to 0.25 to 40 hours, preferably 2 to 35 hours.

[0140] In step b, after adding at least one selected from the group consisting of compound (1) and a pharmaceutically acceptable salt thereof and compound (6) and a pharmaceutically acceptable salt thereof and before stirring, the same solvent as used in step a may be added. In this case, the amount of the solvent may be 0 to 30 ml, preferably 0 to 20 ml, per 1 g of cyclodextrin.

[0141] The amount of the solvent used in step c can be 0 to 150 ml, preferably 0 to 20 ml, relative to 1 g of cyclodextrin.

[0142] The freezing temperature in step c can be set to -30 to -20°C, preferably -20°C.

[0143] The freezing time in step c can be set to 10 to 50 hours.

[0144] The freeze drying in step c can be carried out at an absolute pressure of 20 to 100 Pa and an external temperature of 10 to 40° C., preferably at an external temperature of 20° C.

[0145] The freeze-drying time in step c can be set to 1 to 5 days.

[0146] It is believed that enhancers of the physiological activity of hepatocyte growth factor can enhance the proliferation-promoting effect of hepatocyte growth factor on hepatocytes, renal tubular epithelial cells, mammary epithelial cells, bronchial epithelial cells, bile duct epithelial cells, keratinocytes, melanocytes, vascular endothelial cells, skin fibroblasts, chondrocytes, osteoclasts, muscle satellite cells, mesenchymal stem cells, and the like. It is also believed that enhancers of the physiological activity of hepatocyte growth factor also exhibit a neuroprotective effect. Enhancement of the physiological activity of hepatocyte growth factor may refer to increasing the affinity of hepatocyte growth factor for c-Met. c-Met is a receptor for hepatocyte growth factor, and it is believed that the aforementioned cell proliferation-promoting effect is exerted through the binding of HGF to c-Met.

[0147] In the present invention, "combination administration" or "combination use" means the combined use of active ingredients, including (1) a method of administering a single formulation containing HGF and a trisulfide compound and (2) a method of administering HGF and a trisulfide compound separately as separate formulations simultaneously or with a time difference. In case (2), either HGF or the trisulfide compound can be administered first. In case (2), any of the following methods can be used: (i) a method of administering HGF and a trisulfide compound separately as separate formulations simultaneously via the same administration route; (ii) a method of administering HGF and a trisulfide compound separately as separate formulations via the same administration route with a time difference; (iii) a method of administering HGF and a trisulfide compound separately as separate formulations simultaneously via different administration routes (administered from different sites on the same patient); (iv) a method of administering HGF and a trisulfide compound separately as separate formulations via different administration routes with a time difference. It should be noted that in case (i), the two agents may be mixed immediately before administration.

[0148] In other words, "combination administration" or "combination use" in the present invention can also be said to refer to a method of administering a drug while the action / effect of one drug is being manifested in the patient's body. Specifically, in the present invention, a method of administering the drugs so that HGF and the trisulfide compound are present simultaneously in the patient's body, for example, in the blood, is preferred, and a method of administering the drug within 24 hours of administration of one drug to the patient is preferred.

[0149] When administered in combination with a trisulfide compound, the daily dose of HGF is preferably 0.005 to 100 mg, more preferably 0.05 to 50 mg, and even more preferably 0.1 to 10 mg for adults.

[0150] When administered in combination with HGF, the daily dose of the trisulfide compound is preferably 0.1 to 5000 mg, more preferably 0.5 to 1000 mg, and even more preferably 1 to 500 mg for adults.

[0151] For example, for adults, the dosage of HGF is preferably 0.005 to 100 mg / day and the dosage of the trisulfide compound is preferably 0.1 to 5000 mg / day, more preferably 0.05 to 50 mg / day and the dosage of the trisulfide compound is preferably 0.5 to 1000 mg / day, and even more preferably 0.1 to 10 mg / day and the dosage of the trisulfide compound is preferably 1 to 500 mg / day. It is believed that when the dosages of HGF and the trisulfide compound are within this range, the physiological activity-enhancing effect of hepatocyte growth factor is further enhanced.

[0152] The frequency of administration of HGF and the trisulfide compound can be 1 to 6 times per day or 1 to 4 times per day. It is believed that such a frequency of administration reduces the medication burden on the subject and improves medication compliance. As a result, the physiological activity-enhancing effect of the hepatocyte growth factor of the present invention is expected to be further enhanced. When discontinuing drug administration, the dosage can be gradually reduced. For example, one method can be used to administer another drug while the other is discontinued.

[0153] The hepatocyte growth factor physiological activity enhancer of the present invention can be used for the prevention or treatment of muscle atrophy and / or poor muscle regeneration.

[0154] As the amyotrophy in the present invention, amyotrophic lateral sclerosis, spinal muscular atrophy, spinal bulbar muscular atrophy and muscular dystrophy can be exemplified as a result of age, bed rest, trauma, postoperative immobilization, weightlessness, certain diseases, etc., which are caused by a state of not using muscles for a long time or a state of decreasing frequency and intensity of use. Amyotrophic lateral sclerosis, spinal muscular atrophy, spinal bulbar muscular atrophy and muscular dystrophy are representative examples of amyotrophic lateral sclerosis, spinal muscular atrophy and spinal bulbar muscular atrophy in the present invention.

[0155] Examples of pets include dogs, cats, hamsters, rabbits, ferrets, parrots, munias, and parrots.

[0156] The dosage of the hepatocyte growth factor physiological activity enhancer and the preventive or therapeutic agent for muscular atrophy and muscular dystrophy of the present invention is a therapeutically effective amount and varies depending on the symptoms, age, administration method, dosage form, etc. Generally, for adults, 0.1 to 5000 mg of glutathione trisulfide or a pharmaceutically acceptable salt thereof, compound (1) or a pharmaceutically acceptable salt thereof, or compound (6) or a pharmaceutically acceptable salt thereof can be administered daily, preferably 0.5 to 2000 mg once a day or divided into several doses for consecutive days. It is believed that when the dosage of the above-mentioned compound is within this range, the effect of enhancing the physiological activity of human hepatocyte growth factor and the preventive or therapeutic effect on muscular atrophy and muscular dystrophy are further improved.

[0157] The dosage of the hepatocyte growth factor physiological activity enhancer and the preventive or therapeutic agent for muscular atrophy and muscular dystrophy of the present invention is a therapeutically effective amount and varies depending on the symptoms, age, administration method, dosage form, etc. Generally, for pets, 0.01 to 500 mg / kg of glutathione trisulfide or a pharmaceutically acceptable salt thereof, compound (1) or a pharmaceutically acceptable salt thereof, or compound (6) or a pharmaceutically acceptable salt thereof can be administered daily, preferably 0.05 to 250 mg / kg once a day or divided into several times for consecutive days. It is believed that when the dosage of the above-mentioned compound is within this range, the physiological activity enhancing effect of hepatocyte growth factor on pets and the preventive or therapeutic effect on muscular atrophy and muscular dystrophy are further improved.

[0158] The hepatocyte growth factor physiological activity enhancer of the present invention can be used to suppress or improve muscle growth disorders (decreased meat productivity) caused by heat stress in livestock or poultry.

[0159] Causes of muscle growth impairment (reduced meat productivity) in livestock or poultry due to heat stress in the present invention include increases in the external temperature of the livestock or poultry breeding environment caused by summer heat, and abnormal or deviated temperature management in the livestock or poultry breeding and management areas. These factors are believed to lead to respiratory alkalosis, decreased thyroid hormone, increased corticosterone, altered immune responses, and excessive production of mitochondrial reactive oxygen species (ROS) in the livestock or poultry. As a result, muscle stem cell (satellite cell) activation is inhibited and proliferation is reduced, leading to disorders in muscle growth, enlargement, regeneration, or maintenance, and reduced meat productivity.

[0160] Examples of livestock or poultry include cattle, horses, pigs, donkeys, sheep, goats, chickens, ducks, geese, ostriches, turkeys, wild ducks, and quails.

[0161] The dosage of the hepatocyte growth factor physiological activity enhancer and the agent for suppressing or improving muscle growth disorder (decreased meat productivity) in livestock or poultry of the present invention is a therapeutically effective amount, which varies depending on the symptoms, administration method, etc. Generally, 0.01 to 500 mg / kg of glutathione trisulfide or a pharmaceutically acceptable salt thereof, compound (1) or a pharmaceutically acceptable salt thereof, or compound (6) or a pharmaceutically acceptable salt thereof can be administered to livestock or poultry daily, preferably 0.05 to 250 mg / kg once a day or divided into several doses for consecutive days. It is believed that when the dosage of the above-mentioned compound is within this range, the effect of enhancing the physiological activity of hepatocyte growth factor in livestock or poultry and the effect of suppressing or improving muscle growth disorder (decreased meat productivity) in livestock or poultry are further improved.

[0162] The physiological activity enhancer of the hepatocyte growth factor of the present invention, the preventive or therapeutic agent for amyotrophy and / or poor muscle regeneration, and the inhibitory or ameliorative agent for muscle growth disorder (decreased productivity of edible meat) can be administered orally in the form of tablets, capsules, powders, granules, liquids or syrups, or administered parenterally in the form of injections, infusions or suppositories. The physiological activity enhancer of the hepatocyte growth factor of the present invention, the preventive or therapeutic agent for amyotrophy and / or poor muscle regeneration, and the inhibitory or ameliorative agent for muscle growth disorder (decreased productivity of edible meat) can be added with acceptable additives in pharmaceutics as needed and formulated into these dosage forms by known formulation techniques. In the case of solid dosage forms, when formulated, acceptable excipients in pharmaceutics can be coordinated, such as starch, lactose, refined white sugar, glucose, microcrystalline cellulose, carboxycellulose, carboxymethyl cellulose, carboxyethyl cellulose, calcium phosphate, magnesium stearate, gum arabic, etc., and lubricants, binders, disintegrants, coating agents, colorants, etc. can be coordinated as needed. In the case of liquid preparations, stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives, and the like may be added.

[0163] Example

[0164] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0165] <Example 1>

[0166] The effect of the addition of glutathione trisulfide (GSSSG) or lipoic acid trisulfide (LA-SSS) on the binding (affinity) of HGF to c-Met was investigated by the following method. GSSSG or LA-SSS was added to recombinant HGF (R&D Systems) to a molar ratio of 1:4000 (HGF:GSSSG or HGF:LA-SSS) ( Figure 1(A)) or 1:8000 (HGF:GSSSG or HGF:LA-SSS) ( Figure 1 (B)) An ELISA-like HGF-c-Met binding assay was performed using a 96-well strip plate immobilized with recombinant c-Met (R&D Systems) (Tatsumi et al. 2002; the principle of this method is schematically shown in Figure 1 (C)). Table 2 and Figure 1 In (A) and (B), the negative control (NC) 450-535nm The value obtained by subtracting the absorbance at a wavelength of 535 nm from the absorbance at a wavelength of 450 nm was set to 1, and the values ​​of the other experimental areas were shown as relative values.

[0167] [Table 2]

[0168]

[0169] Table 2 and Figure 1 In the figure, NC: negative control (no addition area), PC: positive control (only HGF addition area), PC': solvent for dissolving GSSSG and the addition area of ​​HGF, A': solvent for dissolving GSSSG, the addition area of ​​GSSSG and HGF (1 ng / ml), A: solvent for dissolving GSSSG and the addition area of ​​GSSSG, PC'': solvent for dissolving LA-SSS and the addition area of ​​HGF, B': solvent for dissolving LA-SSS, the addition area of ​​LA-SSS and HGF (1 ng / ml), B: solvent for dissolving LA-SSS and the addition area of ​​LA-SSS.

[0170] When GSSSG or LA-SSS was added in an amount of 8000 mol relative to 1 mol of HGF, A, which indicates the binding of HGF to c-Met, 450-535nm value( Figure 1 (B)) significantly increased (p < 0.01 vs. PC' or PC" =. This effect was more significant in LA-SSS (refer to Figure 1 (A', B' in (B)). When GSSSG or LA-SSS was added in an amount of 4000 mol relative to 1 mol of HGF, A indicating the binding of HGF to c-Met 450-535nm value( Figure 1 (A) increased (p < 0.01 vs. PC' or PC"). This effect was more significant in LA-SSS (refer to Figure 1 (Bar graphs A' and B' in (A)) Thus, it was found that when GSSSG or LA-SSS was added at a high molar ratio relative to HGF, the binding affinity between HGF and c-Met was significantly increased.

[0171] <Example 2>

[0172] The effect of GSSSG or LA-SSS on the muscle stem cell activation effect of HGF (3 ng / ml) was investigated by the following method. GSSSG or LA-SSS was added to recombinant HGF (R&D Systems) to a molar ratio of 1:4000 (HGF:GSSSG or HGF:LA-SSS) ( Figure 2 (A)) or 1:8000 (HGF:GSSSG or HGF:LA-SSS) ( Figure 2 (B)). The activation effect of HGF was measured using a muscle stem cell (satellite cell) culture system. Quiescent muscle stem cells were isolated from the skeletal muscle of 8-9 month old mature rats according to a modified method of Allen et al. (1997) (Tatsumi et al. 2006) and cultured for 24 hours in DMEM (DMEM-10% HS, pH 7.2) containing 10% normal horse serum. HGF mixed with GSSSG or LA-SSS was added to a final concentration of 3 ng / ml and cultured for a further 24 hours. The activation rate of muscle stem cells was then measured using a bromodeoxyuridine (BrdU) pulse labeling method. Cells that had taken up BrdU were visualized using an anti-BrdU monoclonal antibody, and the proportion of positive cells was used as an indicator of muscle stem cell activation (Tatsumi et al. 1998).

[0173] Figure 2 In the figure, NC: negative control (area without addition), PC: positive control (area with only HGF addition), A': area with GSSSG and HGF (3 ng / ml), A: area with GSSSG addition, B': area with LA-SSS and HGF (3 ng / ml), B: area with LA-SSS addition.

[0174] When GSSSG or LA-SSS was added at a concentration of 4000 or 8000 mol relative to 1 mol of HGF, the ratio of BrdU-positive cells, an index indicating the muscle stem cell activation effect of HGF, increased significantly (p < 0.05 vs. NC), and this value was comparable to that of the positive control (PC) (refer to Figure 2 (A' and B' in (A) and (B)). Therefore, it was confirmed that GSSSG and LA-SSS had no adverse effects on the physiological activity of HGF and the responsiveness of muscle stem cells (such as c-Met receptor signaling activity). This was also supported by the following cell morphological observations.

[0175] When GSSSG or LA-SSS was added at a concentration of 8000 mol per 1 mol of HGF, the enhancing effect observed in Example 1 was not observed. It is believed that the expression level (number of molecules) of c-Met on the cell surface in the cell culture system is controlled within an appropriate range, and therefore the results are different from those in the c-Met binding assay system in which a large amount of c-Met is immobilized on an ELISA plate.

[0176] <Example 3>

[0177] The morphology of muscle stem cells cultured after addition of GSSSG or LA-SSS and HGF (3 ng / ml) was observed by the following method: A representative field of view of the culture system used in Example 2 was photographed. Figure 3 a is Figure 2 (A) and (B) are representative fields of view of NC. Figure 3 b is Figure 2 (A) and (B) are representative visual fields of PC. Figure 3 c is Figure 2 (B) Representative fields of A' (A+HGF (3 ng / ml)), Figure 3 c is Figure 2 (B) Representative fields of B' (B + HGF (3 ng / ml)).

[0178] like Figure 3 As shown in c and d, the morphology of cells cultured after adding GSSSG or LA-SSS and HGF (3 ng / ml) was similar to that in the control area (NC and PC: Figure 3 There was no difference in the observed morphology between a and b. Therefore, it was confirmed that GSSSG and LA-SSS had no adverse effects on the physiological activity of HGF and the responsiveness of muscle stem cells (such as the signal transduction activity of the c-Met receptor).

[0179] <Example 4>

[0180] The effect of adding GSSSG or LA-SSS on the muscle stem cell activation effect of HGF (1 ng / ml) was investigated in the same manner as in Example 2, except that the final concentration of HGF was set to 1 ng / ml instead of 3 ng / ml. The results are shown in Tables 3 and Figure 4 .

[0181] [Table 3]

[0182]

[0183] Table 3 and Figure 4In the figure, NC: negative control (area without addition), PC': positive control (area with only HGF (1 ng / ml) added), A': area with GSSSG and HGF (1 ng / ml), A: area with GSSSG added, B': area with LA-SSS and HGF (1 ng / ml), B: area with LA-SSS added, PC: positive control (area with only HGF (3 ng / ml) added).

[0184] In the 1 ng / ml HGF-added area (PC'), the ratio of BrdU-positive cells, which is an index indicating the muscle stem cell activation effect of HGF, increased significantly (p < 0.05 vs. NC), and this value was significantly lower than that in the 3 ng / ml HGF-added area (PC). This result is consistent with the results of Ryuichi Tatsumi, et al., “Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide.”, Molecular Biology of the Cell, 13(8): 2909-2918 (2002). Figure 3 The results are consistent with those shown, confirming again that HGF at a concentration of 1 ng / ml cannot sufficiently activate satellite cells.

[0185] In the area (A') where GSSSG was added at a 1:8000 molar ratio, the proportion of BrdU-positive cells, an index indicating the HGF muscle stem cell activation effect, increased. In the area (B') where LA-SSS was added at a 1:8000 molar ratio, the proportion of BrdU-positive cells, an index indicating the HGF muscle stem cell activation effect, increased significantly (p < 0.05 vs. NC), with this value comparable to that in the area (PC) where HGF was added at 3 ng / ml. This confirms that LA-SSS further enhances the HGF muscle stem cell activation effect.

[0186] These results in cell culture systems are consistent with Figure 1 The results show that GSSSG and LA-SSS have similar effects on enhancing HGF-c-Met affinity. This suggests that the addition of trisulfide compounds such as GSSSG and LA-SSS facilitates myocardial activation even at low concentrations of HGF. LA-SSS is particularly effective against HGF.

Claims

1. A physiological activity enhancer of hepatocyte growth factor, comprising a trisulfide compound, The trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salt, the compound represented by the following formula (1) and its pharmaceutically acceptable salt and its cyclodextrin inclusion compound, and the compound represented by formula (6) and its pharmaceutically acceptable salt and its cyclodextrin inclusion compound, In formula (1), R 1 is a group represented by the following formula (2), a group represented by the following formula (3), a group represented by the following formula (4), or a group represented by the following formula (5), In formula (2), R 2 and R 3 are each independently an alkylene group having 1 to 4 carbon atoms, Indicates a bond, In formula (3), R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a substituent, R 4 and R 5 can form rings together with the atoms to which they are bonded, Indicates a bond, In formula (4), Indicates a bond, In formula (5), Indicates a bond, In formula (6), X represents -OR 6 or -NR 7 R 8 , R 6 、R 7 and R 8 Each independently represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5.

2. The physiological activity enhancer according to claim 1, further comprising hepatocyte growth factor.

3. A hepatocyte growth factor physiological activity enhancer, which is administered in combination with hepatocyte growth factor and contains a trisulfide compound, The trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salt, the compound represented by the following formula (1) and its pharmaceutically acceptable salt and its cyclodextrin inclusion compound, and the compound represented by formula (6) and its pharmaceutically acceptable salt and its cyclodextrin inclusion compound, In formula (1), R 1 is a group represented by the following formula (2), a group represented by the following formula (3), a group represented by the following formula (4), or a group represented by the following formula (5), In formula (2), R 2 and R 3 are each independently an alkylene group having 1 to 4 carbon atoms, Indicates a bond, In formula (3), R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a substituent, R 4 and R 5 can form rings together with the atoms to which they are bonded, Indicates a bond, In formula (4), Indicates a bond, In formula (5), Indicates a bond, In formula (6), X represents -OR 6 or -NR 7 R 8 , R 6 、R 7 and R 8 Each independently represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5.

4. A physiological activity enhancer of hepatocyte growth factor, which is administered in combination with a trisulfide compound and contains hepatocyte growth factor, The trisulfide compound is at least one selected from the group consisting of glutathione trisulfide and its pharmaceutically acceptable salt, the compound represented by the following formula (1) and its pharmaceutically acceptable salt and its cyclodextrin inclusion compound, and the compound represented by formula (6) and its pharmaceutically acceptable salt and its cyclodextrin inclusion compound, In formula (1), R 1 is a group represented by the following formula (2), a group represented by the following formula (3), a group represented by the following formula (4), or a group represented by the following formula (5), In formula (2), R 2 and R 3 are each independently an alkylene group having 1 to 4 carbon atoms, Indicates a bond, In formula (3), R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a substituent, R 4 and R 5 can form rings together with the atoms to which they are bonded, Indicates a bond, In formula (4), Indicates a bond, In formula (5), Indicates a bond, In formula (6), X represents -OR 6 or -NR 7 R 8 , R 6 、R 7 and R 8 Each independently represents a hydrogen atom, may have a group selected from carboxyl, -OR 9 、-NR 10 R 11 and -N + R 12 R 13 R 14 At least one substituent in the group consisting of an alkyl group having 1 to 6 carbon atoms, or -(CH2CH2O) n R 15 , R 9 、R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5.

5. The physiological activity enhancer according to claim 3, wherein The trisulfide compound and the hepatocyte growth factor are administered simultaneously or separately. The physiological activity enhancer according to claim 1 , which is an enhancer for the binding between the hepatocyte growth factor and c-Met.

7. The physiological activity enhancer according to any one of claims 1 to 6, wherein The trisulfide compound is lipoic acid trisulfide or a pharmaceutically acceptable salt thereof.

8. The physiological activity enhancer according to any one of claims 1 to 6, wherein The trisulfide compound is glutathione trisulfide or a pharmaceutically acceptable salt thereof.

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