Composition for preventing or alleviating hepatic dysfunction

Abstract

Disclosed is a composition for preventing or alleviating hepatic dysfunction, the composition containing a trisulfide compound represented by formula (1) or (6), a pharmaceutically acceptable salt thereof, or a cyclodextrin inclusion of the same.

Description

Composition for preventing or improving liver dysfunction

[0001] The present invention relates to a composition for preventing or ameliorating liver dysfunction.

[0002] The liver is a reddish-brown organ that is the largest in volume and weight of all organs, weighing 1,000 to 1,500 g in adult men and 900 to 1,300 g in adult women. The liver contributes to approximately 40% of the basal metabolic rate of all organs and plays a wide range of important roles, including respiration, maintenance of body temperature, metabolism of nutrients such as carbohydrates, lipids, proteins, and vitamins, and detoxification of harmful xenobiotics or nitrogen metabolites produced by vital activities.

[0003]

[0003] Decreased liver function can be caused by liver dysfunction and various associated liver dysfunction diseases. As these diseases progress, subjective symptoms such as fatigue, malaise, nausea, loss of appetite, and fever appear, and they are also known to cause complications associated with the diseases. Examples of liver dysfunction diseases include hepatitis B, hepatitis C, alcoholic liver disease, and non-alcoholic fatty liver disease. These liver dysfunction diseases, along with the increase in lifestyle-related diseases based on obesity, diabetes, dyslipidemia, and the like, have become one of the most common lifestyle diseases affecting modern people (Non-Patent Document 1).

[0004] If liver dysfunction is left untreated, there is a risk that it may develop into serious diseases such as cirrhosis and liver cancer. Therefore, it is important to protect the liver at an early stage and to appropriately and reliably prevent or improve liver dysfunction.

[0005] Reduced glutathione (GSH) is one of the substances known to have a protective effect on the liver (Non-Patent Documents 2 and 3), and α-lipoic acid (LA), which has been shown to produce glutathione in the body, has also been reported to have a protective effect on hepatocytes (Non-Patent Documents 4 and 5).

[0006] Furthermore, trisulfide compounds such as glutathione trisulfide are converted in vivo to reactive sulfur species such as glutathione persulfide. It has been reported that reactive sulfur species have strong antioxidant properties and may have physiological functions such as anti-aging (e.g., Non-Patent Documents 6 and 7). Furthermore, methods for producing trisulfide compounds are known, such as those described in Patent Document 1.

[0007] Alpha-lipoic acid trisulfide (LASSS), a type of trisulfide compound, is known to inhibit the nitration of tyrosine residues, which leads to the loss of physiological activity of hepatocyte growth factor (HGF), a growth factor involved in cell proliferation, etc. (Patent Document 2). On the other hand, the effects of administering alpha-lipoic acid trisulfide (LASSS), alpha-lipoic acid trisulfide choline ester (LASSS-CE), etc. on liver function have not been known at all until now.

[0008] International Publication No. 2018 / 117186 International Publication No. 2023 / 038088

[0009] Okanoue, et al., "Liver function tests and liver injury - focusing on AST / ALT and PLT count", General Health Examination, 42(2), 307-312, (2015)Sugimura, et al., "Effect of Orally Administered Reduced- and Oxidized-Glutathione against Acetaminophen-Induced Liver Injury in Rats", Journal of Nutritional Science and Vitaminology, 44, 613-624, (1998)Honda, et al., "Efficacy of glutathione for the treatment of nonalcoholic fatty liver disease: an open-label, single-arm, multicenter, pilot study", BMC Gastroenterology, 17:96, (2017)Kuo, et al., "Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells", European Journal of Pharmacology, 692:1-3, 10-18, (2012)Shi, et al., "α-Lipoic acid protects against the cytotoxicity and oxidative stress induced by cadmium in HepG2 cells through regeneration of glutathione by glutathione reductase via Nrf2 / ARE signaling pathway", Environmental Toxicology and Pharmacology, 45, 274-281, (2016)Ida, et al., "Reactive cysteine ​​persulfides and S-polythiolation regulate oxidative stress and redox signaling", Proceedings of the National Academy of Sciences of the United States of America, 111(21), 7606-7611, (2014) Kunikata et al., "Metabolomic profiling of reactive persulfides and polysulfides in the aqueous and vitreous humors", Scientific Reports, 7, 41984 (2017).

[0010] An object of the present invention is to provide a composition for preventing or ameliorating liver dysfunction.

[0011] The present inventors have found that α-lipoic acid trisulfide and its derivatives improve the cell viability of a human liver cancer cell line (HepG2 cells) that has been damaged by hydrogen peroxide, and have completed the present invention. The present invention relates to the following items [1] to

[14] . [1] A composition for preventing or ameliorating liver dysfunction, comprising a trisulfide compound, wherein the trisulfide compound is represented by the following formula (1): A compound represented by the formula (1) (hereinafter also referred to as compound (1)) [wherein R １ is expressed by the following formula (2): [wherein R ２ and R ３ are each independently an alkylene group having 1 to 4 carbon atoms, and * represents a bond], [wherein R ４ and R ５ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms which may have a substituent, and R ４ and R ５ may form a ring together with the atom to which they are attached, and * represents a bond], the following formula (4) [In formula (4), * represents a bond] or a group represented by the following formula (5): [wherein * represents a bond]], pharmaceutically acceptable salts thereof, and cyclodextrin inclusion compounds thereof; and (hereinafter, also referred to as compound (6)) [wherein X is —OR ６ or -NR ７ R ８ represents R ６ , R ７ and R ８ are each independently a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R １５ represents R ９ , R １０ , R １１ , R １２ , R １３ , R １４ and R １５each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5], a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof. [2] The composition according to [1], wherein the liver dysfunction is a state in which abnormal liver function is observed without liver dysfunction, or a state in which abnormal liver function is observed with liver dysfunction. [3] The composition according to [2], wherein the liver dysfunction is at least one selected from the group consisting of hepatitis B, hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease, and chronic hepatitis. [4] The composition according to [1] or [2], wherein the liver dysfunction is liver dysfunction caused by reactive oxygen species. [5] The composition according to any of [1] to [4], wherein the composition is a food composition. [6] The composition according to any of [1] to [4], wherein the composition is a pharmaceutical composition. [7] The composition according to any one of [1] to [6], wherein the trisulfide compound is α-lipoic acid trisulfide or a pharmaceutically acceptable salt thereof, or α-lipoic acid trisulfide choline ester or a pharmaceutically acceptable salt thereof. [8] A method for preventing or ameliorating liver dysfunction, comprising administering a trisulfide compound to a subject in need thereof, wherein the trisulfide compound is at least one selected from the group consisting of compound (1), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and compound (6), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof. [9] A trisulfide compound for use in preventing or ameliorating liver dysfunction, wherein the trisulfide compound is at least one selected from the group consisting of compound (1), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and compound (6), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof.[9a] Non-therapeutic use of a trisulfide compound in preventing or ameliorating liver dysfunction, wherein the trisulfide compound is at least one selected from the group consisting of compound (1), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and compound (6), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof.

[10] Use of a trisulfide compound for producing a composition for preventing or ameliorating liver dysfunction, wherein the trisulfide compound is at least one selected from the group consisting of compound (1), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and compound (6), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof.

[11] The method, compound for use, or use according to any of [8] to

[10] , wherein the liver dysfunction is a state in which abnormalities in liver function are observed without liver dysfunction, or a state in which abnormalities in liver function are observed with liver dysfunction.

[12] The method, compound for use, or use according to

[11] , wherein the liver function disease is at least one selected from the group consisting of hepatitis B, hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease, and chronic hepatitis.

[13] The method, compound for use, or use according to any of [8] to

[11] , wherein the liver function disorder is liver function disorder caused by reactive oxygen species.

[14] The method, compound for use, or use according to any of [8] to

[13] , wherein the trisulfide compound is α-lipoic acid trisulfide or a pharmaceutically acceptable salt thereof, or α-lipoic acid trisulfide choline ester or a pharmaceutically acceptable salt thereof.

[0012] According to the present invention, a composition for preventing or ameliorating liver dysfunction can be provided. The composition for preventing or ameliorating liver dysfunction of the present invention contains at least one trisulfide compound selected from the group consisting of compound (1), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and compound (6), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and thereby protects the liver through a high anti-inflammatory or antioxidant effect in the body, thereby preventing or ameliorating liver dysfunction. Furthermore, the composition of the present invention can be used routinely when it is a health food or a food composition ingested with a focus on the specific function of the present invention.

[0013] Figure 1 shows the effect of adding LASSS or LASSS-CE to HepG2 cells in which liver dysfunction was induced by the addition of aqueous hydrogen peroxide. The graph on the left shows the cell viability when LASSS was added, and the graph on the right shows the cell viability when LASSS-CE was added. Figure 2 shows the effect of adding LASSS or LASSS-CE to HepG2 cells in which aqueous hydrogen peroxide was not added. The graph on the left shows the cell viability when LASSS was added, and the graph on the right shows the cell viability when LASSS-CE was added.

[0014] The present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0015] The trisulfide compound contained in the composition for preventing or ameliorating liver dysfunction of the present invention is at least one selected from the group consisting of compound (1), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof, and compound (6), a pharmaceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof.

[0016] Compound (1): [In formula (1), R １ is expressed by the following formula (2): [wherein R ２ and R ３ are each independently an alkylene group having 1 to 4 carbon atoms, and * represents a bond], [wherein R ４ and R ５ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms which may have a substituent, and R ４ and R ５ may form a ring together with the atom to which they are attached, and * represents a bond], the following formula (4) [In formula (4), * represents a bond] or a group represented by the following formula (5): [wherein * represents a bond]

[0017] Compound (6): [In formula (6), X is —OR ６ or -NR ７ R ８ represents R ６ , R ７ and R ８ are each independently a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R １５ represents 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.

[0018] Compound (1) can be said to be a compound in which two molecules of α-lipoic acid trisulfide are bonded via a linker.

[0019] In compound (1), R ２ and R ３are each independently an alkylene group having 1 to 4 carbon atoms. Examples of the alkylene group having 1 to 4 carbon atoms include a methylene group, an ethylene group, an n-propylene group, an isopropylene group, a cyclopropylene group, an n-butylene group, an isobutylene group, a sec-butylene group, a tert-butylene group, and a cyclobutylene group.

[0020] R ４ and R ５ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms which may have a substituent, and R ４ and R ５ may form a ring together with the atom to which they are bonded. Examples of the substituent include a hydroxy group; an alkoxy group having 1 to 4 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, or a butoxy group. Examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, and a cyclobutyl group. R ４ and R ５ When they form a ring together with the atom to which they are attached, the ring formed may be, for example, an oxetane ring.

[0021] Compound (1) can be produced by: (A) a step (Step 1) of condensing a compound represented by formula (1a) (α-lipoic acid trisulfide) 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, or the like); or (B) a step (Step 1′) of condensing a compound represented by formula (1b) (lipoic acid) with the above-described molecule for forming a linker, a step (Step 2) of oxidizing the obtained disulfide compound with an oxidizing agent to obtain a sulfoxide compound, and a step (Step 3) of reacting the obtained sulfoxide compound with a sulfur source to obtain a trisulfide compound.

[0022] Examples of the solvent used in Step 1 and Step 1' include methylene chloride, chloroform, and tetrahydrofuran, and preferably methylene chloride. The amount of the solvent can be 1 mL to 200 mL, and preferably 3 mL to 35 mL, per 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, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and salts thereof, N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP), 1,1'-carbonyldiimidazoledi(1H-imidazol-1-yl)methanone (CDI), and the like. When these compounds are used as condensing agents, N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), or the like may be used as an additive. The amount of the condensing agent used in Step 1 and Step 1' can be 0.8 to 2.0 equivalents, and 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, and preferably 15°C to 25°C. The reaction time in Step 1 and Step 1' can be 1 hour to 3 days, and preferably 1 hour to 24 hours.

[0023] 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 α-lipoic acid trisulfide. However, other equivalent amounts may be used as long as the target compound is obtained.

[0024] The compound represented by the following formula (1a) (α-lipoic acid trisulfide) can be produced by the method described in WO 2022 / 045212. The procedures and conditions for steps 2 and 3 in production method (B) can be similar to, for example, the procedures and conditions for steps 1 and 2 in WO 2022 / 045212.

[0025] R in formula (2a) ２ and R ３ is R in formula (2) ２ and R ３ and are the same as those shown in the formula (2a). Examples 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 and linear or branched dipropylene glycol.

[0026] R in formula (3a) ４ and R ５ is R in formula (3). ４ and R ５ Examples of the compound represented by formula (3a) include 2,2-dimethyl-1,3-propanediamine, 2-oxetane-1,3-propanediamine, and 2-methoxy-1,3-propanediamine.

[0027] Examples of compound (1) include compounds represented by the following formulas (11) to (16) (compounds (11) to (16)).

[0028] The molecular weights and ClogP of compounds (11) to (16) are shown in Table 1.

[0029] The molecular weight of compound (1) and its pharmaceutically acceptable salts may be 800 or less, 750 or less, or 700 or less, and preferably 600 or less. The molecular weight of compound (1) and its pharmaceutically acceptable salts may be 400 or more, 450 or more, or 500 or more. When the molecular weight of compound (1) and its pharmaceutically acceptable salts is within these ranges, it is believed that the absorption rate in the body will be further improved.

[0030] The ClogP of compound (1) and its pharmaceutically acceptable salts may be 1 or more, 2 or more, 3 or more, or 4 or more. The ClogP of compound (1) and its pharmaceutically acceptable salts may be 8 or less, 7 or less, or 6 or less. When the ClogP of compound (1) and its pharmaceutically acceptable salts is within these ranges, it can be said that compound (1) and its pharmaceutically acceptable salts have a better balance of water solubility and lipid solubility. ClogP is a computer-calculated partition coefficient and can be determined in accordance with the principles described in "CLOGP Reference Manual Daylight Version 4.9 (Release Date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )."

[0031] The compounds herein may exist as optical isomers (R-, S-, R,R-, R,S-, S,R-, and S,S-isomers). The compounds herein may be replaced with their optical isomers or mixtures containing their optical isomers in any ratio (e.g., racemates). 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 with a mixture containing these optical isomers in any ratio (e.g., racemates).

[0032] In one embodiment, compound (6) is represented by formula (7): [wherein R ６ represents a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R１５ represents 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. ６ 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. ６ is a hydrogen atom, and in this case, the compound represented by formula (7) is α-lipoic acid trisulfide represented by formula (8). For example, R ６ Ha-(CH ２ ) ２ N ＋ (CH ３ ) ３ In this case, the compound represented by formula (7) is α-lipoic acid trisulfide choline ester represented by formula (8'). Compound (6) is preferably a compound selected from the group consisting of α-lipoic acid trisulfide (LASSS) and α-lipoic acid trisulfide choline ester (LASSS-CE).

[0033] In another embodiment, compound (6) is represented by formula (9): A compound represented by the formula ７ and R ８ are each independently a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R １５ represents R ９ , R １０ , R １１ , R １２ , R １３ , R １４ and R １５each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 2 to 5. ７ and R ８ 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 a substituent. ７ and R ８ may be, for example, a group represented by formula (10) (where * represents a bond). Specific examples of the compound represented by formula (9) include, for example, R ７ and R ８ are both hydrogen atoms, R ７ is a hydrogen atom and R ８ is a group represented by formula (10).

[0034] The compound represented by formula (9) can be produced by a step (step 61) of oxidizing the compound represented by formula (9a) with an oxidizing agent to obtain a sulfoxide compound, and a step (step 62) of reacting the obtained sulfoxide compound with a sulfur source. [In the formula, R ７ and R ８ are each independently a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R １５ represents 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.

[0035] In the above production method, the reactions in steps 61 and 62 may be carried out in one pot without isolating the sulfoxide compound.

[0036] The solvent used in Step 61 is not particularly limited as long as it dissolves the compound represented by Formula (9a) and the oxidizing agent and does not inhibit the oxidation reaction. Examples of such solvents include water, aqueous sulfuric acid, aqueous ethanol, and aqueous acetonitrile, with water being preferred. The amount of solvent used in Step 61 can be 1 mL to 500 mL, preferably 10 mL to 20 mL, per 1 g of the compound represented by Formula (9a).

[0037] Examples of oxidizing agents used in step 61 include potassium peroxymonosulfate (sold under trade names such as Oxone (registered trademark)), peracetic acid, hydrogen peroxide, and sodium periodate. Hydrogen peroxide may be used together with a catalytic amount of methyltrioxorhenium. From the viewpoints of safety and cost, potassium peroxymonosulfate is a preferred oxidizing agent. The amount of oxidizing agent used 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).

[0038] The reaction temperature in step 61 can be -20°C to 30°C, preferably -5°C to 5°C.

[0039] The reaction time in step 61 can be from 5 minutes to 24 hours, preferably from 0.5 hours to 2 hours.

[0040] 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 the subsequent reaction. Examples of such solvents include water, an aqueous sulfuric acid solution, an aqueous ethanol solution, and an aqueous acetonitrile solution, and water is 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.

[0041] The sulfur source used in step 62 includes 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, preferably 0.9 to 1.2 equivalents, relative to 1 equivalent of the sulfoxide compound.

[0042] The reaction temperature in step 62 can be between -20°C and 30°C, and is preferably between -5°C and 25°C.

[0043] The reaction time in step 62 can be from 10 minutes to 2 days, preferably from 0.5 hours to 2 hours.

[0044] When Step 61 and Step 62 are carried out in one pot, reaction solvents include, for example, water, aqueous sulfuric acid, aqueous ethanol, and aqueous acetonitrile, preferably water. The amount of the solvent can be 1 mL to 500 mL, preferably 10 mL to 20 mL, per 1 g of the compound represented by Formula (9a). Oxidizing agents used include potassium peroxymonosulfate, peracetic acid, hydrogen peroxide (which may be used together with a catalytic amount of methyltrioxorhenium), and sodium periodate, preferably potassium peroxymonosulfate. The amount of the oxidizing agent used can be 0.8 to 2.0 equivalents, preferably 1.0 to 1.3 equivalents, per equivalent of the compound represented by Formula (9a). Examples of the sulfur source that can be used include sodium sulfide, potassium sulfide, sodium hydrogen sulfide, potassium hydrogen sulfide, and hydrogen sulfide. The amount of the sulfur source that can be used can be 0.5 to 4.0 equivalents, and preferably 0.9 to 1.2 equivalents, relative to 1 equivalent of the compound represented by formula (9a). The reaction temperature can be −20° C. to 30° C., and preferably −5° C. to 25° C. The reaction time can be 15 minutes to 2 days, and preferably 1 to 4 hours.

[0045] In addition to Step 61 and Step 62, steps of protecting functional groups such as a hydroxy group, a carbonyl group, an amino group, a carboxy group, and the like, and a step of deprotecting the protected functional groups may be included, as necessary. Protecting groups for these functional groups and protection / deprotection reactions 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.

[0046] The compound represented by formula (9a) is a compound obtained by combining lipoic acid and NHR ７ R ８ The compound represented by formula (9a) can be produced by condensing the above compounds. Examples of solvents for the condensation reaction include dichloromethane, chloroform, and tetrahydrofuran, and tetrahydrofuran is preferred. The amount of solvent can be 1 mL to 200 mL, preferably 3 mL to 35 mL, per 1 g of the compound represented by formula (9a). Examples of condensing agents used include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and salts thereof, N,N'-dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) (N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBt) may also be used as additives). The amount of condensing agent used can be 0.8 to 2.0 equivalents, preferably 1.0 to 1.5 equivalents, per equivalent of the compound represented by formula (9a). The reaction temperature can be −10° C. to 40° C., preferably 15° C. to 25° C. The reaction time can be from 1 hour to 3 days, preferably from 1 hour to 24 hours.

[0047] The compound represented by formula (9) is a mixture of α-lipoic acid trisulfide and NHR ７ R ８ The condensation conditions are the same as above.

[0048] The compound of formula (7) can be produced by oxidizing the compound of 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 those described above. [In the formula, R ６ represents a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R １５ represents 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.

[0049] The compound represented by formula (7a) is a compound obtained by combining lipoic acid and R ６ It can be produced by condensing OH as described above.

[0050] The compound represented by formula (7) is a mixture of α-lipoic acid trisulfide and R ６ It can also be produced by condensing OH under the same condensation conditions as above.

[0051] 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. When compound (1) or compound (6) is obtained as a free form, it can be converted to a salt by a conventional method. Furthermore, when compound (1) or compound (6) is obtained as a salt, it can also be converted to a free form by a conventional method.

[0052] In the present invention, the trisulfide compound may have crystalline polymorphism, but is not limited to any one of these, and may be a single crystalline form or a mixture of crystalline forms. The trisulfide compound in the present invention also includes amorphous forms. The trisulfide compound in the present invention also includes anhydrides and solvates (particularly hydrates).

[0053] The trisulfide compound may be a cyclodextrin inclusion complex (CD inclusion complex) in which 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 is included in cyclodextrin.

[0054] 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 the cyclodextrin has been substituted with 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.

[0055] The cyclodextrin inclusion complex can be produced by the following steps: dissolving cyclodextrin in a solvent (step a), 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 to the resulting solution and stirring (step b), filtering the stirred solution, washing with the same solvent as used in step a, freezing the filtrate, and freeze-drying it (step c). Note that the filtration and washing steps in step c may be omitted.

[0056] The solvent used in step a is preferably water.

[0057] The amount of the solvent used in step a can be 1 mL to 350 mL, preferably 1 mL to 80 mL, per 1 g of cyclodextrin.

[0058] In step b, the mass ratio of cyclodextrin to 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 can be 2 to 20, and preferably 5 to 16.5.

[0059] The stirring temperature in step b can be 20°C to 50°C, and may be room temperature.

[0060] The stirring time in step b can be from 0.25 hours to 40 hours, preferably from 2 hours to 35 hours.

[0061] 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, the same solvent as used in step a may be added before stirring. In this case, the amount of solvent can be 0 mL to 30 mL, preferably 0 mL to 20 mL, per 1 g of cyclodextrin.

[0062] The amount of the solvent used in step c can be 0 mL to 150 mL, preferably 0 mL to 20 mL, per 1 g of cyclodextrin.

[0063] The freezing temperature in step c can be -30°C to -20°C, and is preferably -20°C.

[0064] The freezing time in step c can be from 10 hours to 50 hours.

[0065] The freeze-drying in step c can be carried out at an absolute pressure of 20 Pa to 100 Pa and an external temperature of 10°C to 40°C, preferably 20°C.

[0066] The freeze-drying period in step c can be 1 to 5 days.

[0067] The preventive or ameliorative composition and preventive or ameliorative method of the present invention may be administered or applied to humans.

[0068] In the present invention, liver dysfunction refers to a state in which abnormalities in liver function are observed, including, for example, a state in which hepatocytes are destroyed and extraneous enzymes are leaked into the blood, a state in which a large amount of neutral fat is accumulated in liver tissue compared with a normal liver, and a state in which hepatic parenchymal cells are degenerated or necrotic.

[0069] The liver dysfunction in the present invention may be a state in which abnormal liver function is observed without liver function disease, or a state in which abnormal liver function is observed with liver function disease. The liver dysfunction in the present invention may be liver function disorder caused by reactive oxygen species. Reactive oxygen disorder refers to disorder caused by reactive oxygen species. Examples of liver dysfunction include hepatitis B, hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, chronic hepatitis, etc.

[0070] The composition of the present invention can also prevent or ameliorate symptoms associated with liver dysfunction. That is, the present invention can also be said to be a composition for preventing or ameliorating symptoms associated with liver dysfunction. Examples of symptoms associated with liver dysfunction include subjective symptoms caused by decreased liver function, such as fatigue, tiredness, malaise, nausea, loss of appetite, gastrointestinal symptoms, jaundice, rough skin, swelling, and fever, as well as cirrhosis, liver cancer, and their complications.

[0071] The preventive or ameliorating composition of the present invention can be provided as a food composition for preventing or ameliorating liver dysfunction and associated symptoms, or as a pharmaceutical composition for preventing or ameliorating liver dysfunction and associated symptoms.

[0072] Food compositions include not only general foods, but also functional foods, foods for specified health uses, etc. The food composition may be in the form suitable for consumption, such as solid, liquid, granular, powder, capsule, cream, or paste.

[0073] When the composition for preventing or ameliorating liver dysfunction of the present invention is provided as a pharmaceutical composition, it can be mixed with pharmaceutically acceptable additives and formulated into a dosage form suitable for application to the affected area. The composition for preventing or ameliorating liver dysfunction of the present invention, which is a pharmaceutical composition, can be administered orally, for example, as tablets, capsules, powders, granules, liquids, or syrups, or parenterally as injections, infusions, or suppositories. Oral administration is preferred due to its ease of administration. These dosage forms can be formulated using known formulation techniques. In the case of solid formulations, pharmaceutically acceptable excipients such as starch, lactose, refined sucrose, glucose, crystalline cellulose, carboxycellulose, carboxymethylcellulose, carboxyethylcellulose, calcium phosphate, magnesium stearate, gum arabic, etc. can be added during formulation, and lubricants, binders, disintegrants, coating agents, colorants, etc. can also be added if necessary. In the case of liquid formulations, stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives, etc. can also be added.

[0074] The dosage of the composition for preventing or ameliorating liver dysfunction of the present invention varies depending on symptoms, age, administration method, dosage form, etc., but typically, the compound can be administered to an adult at 0.5 to 2000 mg, preferably 10 to 1000 mg, and most preferably 50 to 800 mg per day, with 1 to 800 mg being preferably administered once or in divided doses daily. Dosages of LASSS and LASSS-CE within these ranges are believed to further enhance the preventive or ameliorative effects on liver dysfunction and related symptoms.

[0075] Human hepatocellular carcinoma cell line (HepG2:HB-8065 ＴＭ , Lot No. 70029112) was obtained from the American Type Culture Collection, and Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) containing 10% FBS was used as the basal medium. Cells were cultured at 37°C and 5% CO ２ The experiment was carried out in an incubator, and cells that had been passaged for 17 to 22 generations were used for the experiment.

[0076] The initial cell concentration was 2 x 10 ４ The cell suspension adjusted to 0.15 cells / mL was seeded in a 96-well plate (Thermo Fisher Scientific Co., Ltd.).

[0077] After 20 hours of culture from seeding, α-lipoic acid trisulfide (LASSS) or α-lipoic acid trisulfide choline ester (LASSS-CE) (final concentration 0.025 mM addition group, final concentration 0.05 mM addition group) was added to the medium. Separately, a group receiving only the LASSS or LASSS-CE solvent (phosphate-buffered saline (PBS) containing 4% DMSO (Fujifilm Wako Pure Chemical Corporation)) was designated the solvent-added group. After 4 hours of culture from sample addition, the sample-added and solvent-added groups were replaced with medium (not containing LASSS or LASSS-CE) containing hydrogen peroxide (Fujifilm Wako Pure Chemical Corporation) at a final concentration of 0.1 mM to induce hepatocellular damage. The control group received the same amount of PBS instead of hydrogen peroxide as the solvent-added group. After 2 hours of incubation following the addition of hydrogen peroxide or PBS, cell viability was measured by the MTT assay to evaluate the degree of hepatocellular damage. The results are shown in Figure 1 and Table 2. In the table, SD represents standard deviation.

[0078]

[0079] Furthermore, to evaluate the effects of LASSS and LASSS-CE on cell proliferation itself, a similar experiment was performed except that the hydrogen peroxide solution was replaced with an equal volume of PBS, and cell viability was measured by the MTT assay. The results are shown in Figure 2 and Table 3.

[0080]

[0081] As shown in Figure 1 and Table 2, the addition of hydrogen peroxide solution induced reactive oxygen damage, resulting in a decrease in cell viability of HepG2 cells. The LASSS and LASSS-CE groups showed improved cell viability compared to the hydrogen peroxide group. This suggests that LASSS and LASSS-CE have excellent effects in suppressing or ameliorating liver damage caused by oxidative stress.

[0082] As shown in FIG. 2 and Table 3, under experimental conditions where there was no influence of hydrogen peroxide solution that causes oxidative stress damage, LASSS and LASSS-CE did not affect the proliferation of HepG2 cells.

Claims

1. A composition for preventing or improving liver dysfunction, comprising a trisulfide compound, the trisulfide compound being represented by the following formula (1): [wherein R １ is expressed by the following formula (2): [wherein R ２ and R ３ each independently represents an alkylene group having 1 to 4 carbon atoms, and * represents a bond, [wherein R ４ and R ５ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms which may have a substituent, R ４ and R ５ may form a ring together with the atom to which they are attached, and * represents a bond], [wherein * represents a bond] or a group represented by the following formula (5): [wherein * represents a bond]], pharma- ceutically acceptable salts thereof, and cyclodextrin inclusion complexes thereof; and [wherein X is -OR ６ Or -NR ７ R ８ represents R ６ , R ７ and R ８ each independently represents a hydrogen atom; a carboxy group, -OR ９ , -NR １０ R １１ and -N ＋ R １２ R １３ R １４ an alkyl group having 1 to 6 carbon atoms, which may have at least one substituent selected from the group consisting of: ２ CH ２ O) ｎ R １５ represents 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], a pharma- ceutically acceptable salt thereof, and a cyclodextrin inclusion complex thereof.

2. The composition according to claim 1, wherein the liver dysfunction is a state in which abnormalities in liver function are observed without liver function disease, or a state in which abnormalities in liver function are observed with liver function disease.

3. The composition according to claim 2, wherein the liver functional disease is at least one selected from the group consisting of hepatitis B, hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease and chronic hepatitis.

4. The composition according to claim 1, wherein the liver dysfunction is caused by reactive oxygen species.

5. The composition according to any one of claims 1 to 4, which is a food composition.

6. The composition according to any one of claims 1 to 4, which is a pharmaceutical composition.

7. The composition according to any one of claims 1 to 4, wherein the trisulfide compound is α-lipoic acid trisulfide or a pharma-ceutically acceptable salt thereof, or α-lipoic acid trisulfide choline ester or a pharma-ceutically acceptable salt thereof.

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