Agent for preventing or ameliorating metabolic dysfunction-associated steatotic liver diseases

Abstract

The present invention addresses the problem of providing an agent for preventing or ameliorating MASLDs such as MASH, the agent containing, as an active ingredient, a low-molecular compound that can be produced relatively simply and inexpensively.　This agent for preventing or ameliorating MASLDs comprises one or more compounds selected from the group consisting of compounds represented by formula (Io) and physiologically acceptable salts thereof. [In the formula: R is a halogen atom, a C1-7 alkyl group, or a C1-7 alkoxyl group, and n is an integer of 0-3; Z is a halogen atom, a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an organic oxy group represented by OR8, m is an integer of 0-4, and R8 represents a C1-7 alkyl group, a C2-6 alkenyl group, or a C2-6 alkynyl group; in cases where n and m are pluralities, each R and each Z may be the same or different; R6 represents a hydrogen atom or a C1-6 alkyl group; and R3 is a group selected from any one of OH, OR4, NHR4, or NR4R5, and R4 and R5 are the same or different and are unsubstituted C1-4 alkyl groups.]

Description

Agent for preventing or improving fatty liver disease associated with metabolic dysfunction

[0001] The present invention relates to an agent for preventing or ameliorating metabolic dysfunction-associated steatotic liver disease (also referred to herein as "MASLD"), such as metabolic dysfunction-associated steatohepatitis (also referred to herein as "MASH"), which contains a specific indole compound as an active ingredient.

[0002] The liver is the largest organ in the body, possessing over 500 diverse functions. It reprocesses various nutrients absorbed by the intestine, supplies them to other organs, and stores any surplus. It also performs many other vital functions, including the production and secretion of bile, detoxification, and excretion. However, this organ is susceptible to damage caused by various factors, including an irregular lifestyle, stress, viruses, drugs, alcohol, malnutrition, and hepatic circulatory disorders, which can lead to diseases such as acute hepatitis, chronic hepatitis, fatty liver, and cirrhosis. In Japan, more than 15% of adults (an estimated 1.5 to 2 million people) are said to suffer from fatty liver, and its spread to younger generations has become a major social problem.

[0003] Fatty liver has been considered a pathological condition associated with alcoholic liver disease, which is primarily caused by excessive alcohol consumption. Alcoholic fatty liver is a condition in which fat accumulates in the liver of alcoholics, causing lipid droplets to appear in hepatocytes, and further leading to liver damage due to the deposition of fat (especially triglycerides) in hepatocytes. However, in recent years, it has been discovered that findings similar to alcoholic liver disease can also appear in non-alcoholics.

[0004] Metabolic dysfunction-associated fatty liver disease (MASLD) is known as one of the pathologies of fatty liver disease in non-alcoholic individuals. MASLD, formerly known as nonalcoholic fatty liver disease (NAFLD), is also known as metabolic dysfunction-associated fatty liver disease (MAFLD). MASLD manifests as fatty liver characterized by the deposition of triglycerides in hepatocytes, but approximately 90% of patients have simple fatty liver, which can be improved by appropriate dietary and exercise therapy.

[0005] However, it is known that approximately 10% of cases of MASLD are metabolically impaired steatohepatitis (MASH), a progressive condition. MASH is a disease previously known as nonalcoholic steatohepatitis (NASH). Although there is no consensus on the histopathological progression of MASH, there is a certain consensus that fat-accumulated hepatocytes undergo degeneration and necrosis, resulting in steatohepatitis accompanied by inflammation and fibrosis in the liver tissue. Furthermore, it is believed that subjects with multiple risk factors, such as metabolic syndrome, obesity, diabetes, hyperlipidemia, hypertension, and hyperuricemia, are more likely to progress from fatty liver to MASH, and even to more severe liver fibrosis, cirrhosis, and liver cancer.

[0006] Meanwhile, the present inventors have reported that 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic acid (i.e., MA5) has an effect of enhancing erythropoietin expression and a therapeutic effect for mitochondrial disease (Patent Document 1), an effect of suppressing organ fibrosis (Patent Document 2), and an effect of preventing or improving hearing loss (Patent Document 3). However, it has not been known until now that the compound of the present invention has a therapeutic effect against MASLD.

[0007] International Publication No. 2014 / 080640 Pamphlet Japanese Patent Application Laid-Open No. 2015-189670 Japanese Patent Application Laid-Open No. 2019-116453

[0008] An object of the present invention is to provide an agent for preventing or ameliorating MASLD such as MASH, which contains as an active ingredient a low molecular weight compound that can be produced relatively simply and inexpensively.

[0009] The present inventors have been conducting intensive research to solve the above-mentioned problems. In the course of their research, they have found that the compound described below 1) has the effect of improving weight gain and increases in blood aspartate aminotransferase (herein also referred to as "AST") activity and blood alanine aminotransferase (herein also referred to as "ALT") activity caused by MASH, 2) has the effect of improving inflammation and fibrosis in liver tissue caused by MASH, 3) has the effect of improving increases in blood AST activity and blood ALT activity caused by MASLD, 4) has the effect of improving steatosis and fibrosis in liver tissue caused by MASLD, and 5) has the effect of improving weight loss and shortened lifespan caused by MASLD (fatty liver induced by reduced nutrient intake), and have thus completed the present invention.

[0010] That is, the present invention is as follows: [1] An agent for preventing or ameliorating fatty liver disease associated with metabolic dysfunction, comprising one or more compounds selected from the group consisting of compounds represented by the following formula (Io) and physiologically acceptable salts thereof: [In the formula, R is a halogen atom, an alkyl group having 1 to 7 carbon atoms, or an alkoxyl group having 1 to 7 carbon atoms, n is an integer of 0 to 3, and Z is a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or OR ８ m is an integer of 0 to 4, and R ８ represents an alkyl group having 1 to 7 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; when n and m are plural, each R and each Z may be the same or different; R ６represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ３ OH, OR ４ , N.H.R. ４ and NR ４ R ５ and R ４ and R ５ and are the same or different and each represents an unsubstituted alkyl group having 1 to 4 carbon atoms.] [2] The prophylactic or ameliorating agent according to the above-mentioned [1], wherein the compound represented by formula (Io) is a compound represented by the following formula (I-1): [3] The prophylactic or ameliorating agent according to the above-mentioned [1] or [2], wherein the fatty liver disease associated with metabolic dysfunction is metabolic disorder-related steatohepatitis. [4] The prophylactic or ameliorating agent according to any of the above-mentioned [1] to [3], which is orally administered.

[0011] Further, other embodiments of the present invention include: a method for preventing or ameliorating (treating) fatty liver disease associated with metabolic dysfunction, comprising ingesting (e.g., administering) one or more compounds (herein also referred to as "the compound") selected from the group consisting of compounds represented by the following formula (Io) and physiologically acceptable salts thereof to a mammal (e.g., a human, particularly a patient) in need of prevention or amelioration (treatment) of fatty liver disease associated with metabolic dysfunction; the compound of the present invention for use in the prevention or amelioration (treatment) of fatty liver disease associated with metabolic dysfunction; and use of the compound of the present invention in the manufacture of an agent for preventing or ameliorating (treating) fatty liver disease associated with metabolic dysfunction.

[0012]

[0013] In the above formula (Io), R is a halogen atom, an alkyl group having 1 to 7 carbon atoms, or an alkoxyl group having 1 to 7 carbon atoms, n is an integer of 0 to 3; Z is a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or OR ８ m is an integer of 0 to 4, and R ８represents an alkyl group having 1 to 7 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; when n and m are plural, each R and each Z may be the same or different; R ６ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ３ OH, OR ４ , N.H.R. ４ and NR ４ R ５ and R ４ and R ５ are the same or different and each is an unsubstituted alkyl group having 1 to 4 carbon atoms.

[0014] According to the present invention, an agent for preventing or ameliorating fatty liver disease associated with metabolic dysfunction can be provided. Furthermore, the present invention uses a low-molecular-weight compound that can be produced relatively simply and with high yield as an active ingredient for preventing or ameliorating fatty liver disease associated with metabolic dysfunction, and therefore the agent can be produced relatively simply and inexpensively.

[0015] These figures show the results of measuring mouse body weight (FIG. 1A), blood AST activity after 15 weeks of intervention (FIG. 1B), blood AST activity after 25 weeks of intervention (FIG. 1C), blood ALT activity after 15 weeks of intervention (FIG. 1D), and blood ALT activity after 25 weeks of intervention (FIG. 1E) for three types of intervention groups (control group ["NC" in the figure], FFC group ["FFC" in the figure], and FFC+MA5 group ["FFC+MA5" in the figure]). In FIGS. 1B to 1E, ● indicates individual mice, bars indicate mean values, and "*" and "**" indicate statistically significant differences (p<0.05 and p<0.01, respectively). FIG. 2A shows microscopic images of Masson's trichrome stained mouse liver tissues from three intervention groups (control group ["NC" in the figure], FFC group ["FFC" in the figure], and FFC+MA5 group ["FFC+MA5" in the figure]). FIG. 2B shows the results of measuring the inflammation level of mouse liver tissues from the three intervention groups after hematoxylin-eosin (HE) staining. FIG. 2C shows the results of measuring the fibrosis level of mouse liver tissues based on the microscopic images in FIG. 2A. In FIGS. 2B and 2C, ● indicates an individual mouse, the center bar indicates the mean value, the bars on both ends indicate the standard deviation (SD), and "*" and "**" indicate statistically significant differences (p<0.05 and p<0.01, respectively). These figures show the results of measuring blood AST activity after 13 weeks of intervention (FIG. 3A), after 22 weeks of intervention (FIG. 3B), after 13 weeks of intervention (FIG. 3C), and after 22 weeks of intervention (FIG. 3D) for three types of intervention groups (control group ["NC" in the figure], HF-MRTD group ["HF-MRTD" in the figure], and HF-MRTD+MA5 group ["HF-MRTD+MA5" in the figure]). In FIGS. 3A to 3D, ● indicates individual mice, bars indicate mean values, and "*" and "**" indicate statistically significant differences (p<0.05 and p<0.01), respectively. Figure 4A shows microscopic images of Masson's trichrome stained mouse liver tissues from three intervention groups (control group ["NC" in the figure], HF-MRTD group ["HF-MRTD" in the figure], and HF-MRTD+MA5 group ["HF-MRTD+MA5" in the figure]).The dotted line indicates the area where fibrosis was observed in the fatty region. Figure 4B shows the results of measuring the level of steatosis in mouse liver tissue after HE staining for the mouse liver tissue from the three intervention groups. Figure 4C shows the results of measuring the level of fibrosis in mouse liver tissue based on the microscopic image of Figure 4A. In Figures 4B and 4C, ● indicates an individual mouse, the center bar indicates the mean value, the bars on both ends indicate the standard deviation (SD), and "**" indicates a statistically significant difference (p<0.01). Figure 5A shows the results of measuring the mouse body weight (Figure 5A) and survival rate (Figure 5B) for the three intervention groups (control group ["NC" in the figure], MCD group ["MCD" in the figure], and MCD+MA5 group ["MCD+MA5" in the figure]). Figures 6A and 6B show the results of measuring blood AST activity (Figure 6A) and blood ALT activity (Figure 6B) for two intervention groups (the severe fibrosis group ["FFC + CCl4" in the figure] and the severe fibrosis + MA5 group ["FFC + CCl4 + MA5" in the figure]). Figure 6C shows microscopic images of Masson's trichrome-stained mouse liver tissue from the two intervention groups.

[0016] The preventive or ameliorating agent of the present invention is an agent containing the present compound (hereinafter, may be referred to as the "present preventive / ameliorating agent"), whose use is specified as "for preventing or ameliorating fatty liver disease associated with metabolic dysfunction."

[0017] The agent of the present invention may be used alone as a livestock feed, food or drink, cosmetic, quasi-drug, or pharmaceutical (preparation) containing the compound of the present invention as an active ingredient, or may be used in the form of a composition (a livestock feed composition, a food or drink composition, a cosmetic composition, a quasi-topical composition, or a pharmaceutical composition) by mixing with an additive. Examples of the food or drink include health foods (functional foods, nutritional supplements, health supplements, nutrient-enhanced foods, nutritionally regulated foods, supplements, etc.) and health-promoting foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.).

[0018] As used herein, "fatty liver disease associated with metabolic dysfunction" refers to fatty liver not caused by alcohol, and refers to a state (pathological condition) in which deposition of triglycerides in hepatocytes (usually deposition of triglycerides in at least 5% or more of hepatocytes [e.g., 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more]) is observed in the liver tissue of a mammal with no history of alcohol drinking or a mammal with little or no history of alcohol drinking (e.g., in the case of a human mammal, at least less than 20 g per day in terms of ethanol [e.g., less than 20 g per day, less than 25 g per day, less than 30 g per day, etc.]).

[0019] As used herein, "metabolic disorder-associated steatohepatitis" refers to fatty liver disease associated with metabolic dysfunction that exhibits a progressive pathology, and refers to fatty liver disease associated with metabolic dysfunction in a state in which inflammation occurs in liver tissue. Metabolic disorder-associated steatohepatitis is, for example, a condition in which one or more steatohepatitis findings selected from macrovesicular fat deposition; inflammatory cell infiltration; and balloon-like swelling of hepatocytes are observed in liver tissue, and in some cases is accompanied by liver fibrosis or its progression. Note that "metabolic disorder-associated steatohepatitis" does not include hepatitis caused by factors other than non-alcoholic fatty liver disease (e.g., viral hepatitis, autoimmune hepatitis, etc.).

[0020] The fatty liver diseases associated with the above metabolic dysfunction include not only metabolic disorder-associated steatohepatitis, but also pathological conditions that do not meet the definition of metabolic disorder-associated steatohepatitis but are thought to be in the transitional process to MASH, in which localized fatty degeneration and necrosis of the liver parenchyma or interstitial dilation in the centrilobular or portal vein region is observed, as well as conditions in which the activity of liver enzymes (e.g., AST, ALT) in the blood is abnormal (e.g., increased by about two times or more compared to a healthy state), weight gain or loss, shortened lifespan, etc.

[0021] As used herein, "mammals" include humans and non-human mammals (e.g., cows, pigs, sheep, goats, mice, rats, hamsters, guinea pigs, etc.). Other examples of non-human mammals include livestock (e.g., cows, pigs, sheep, goats, etc.).

[0022] As used herein, "preventing or ameliorating fatty liver disease associated with metabolic dysfunction" means one or more selected from preventing the onset of fatty liver disease associated with metabolic dysfunction; delaying the onset of fatty liver disease associated with metabolic dysfunction; preventing or delaying the progression of severity of fatty liver disease associated with metabolic dysfunction; improving the level of severity of fatty liver disease associated with metabolic dysfunction; and remission of fatty liver disease associated with metabolic dysfunction.

[0023] The compounds encompassed by the present compound are not particularly limited as long as they are one or more compounds selected from the group consisting of compounds represented by the following formula (Io) and physiologically acceptable salts thereof.

[0024]

[0025] In the above formula (Io), R is a halogen atom, an alkyl group having 1 to 7 carbon atoms, or an alkoxyl group having 1 to 7 carbon atoms. n is an integer of 0 to 3. Z is a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or OR ８ m is an integer of 0 to 4. R ８ represents an alkyl group having 1 to 7 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms. When n and m are plural, each R and each Z may be the same or different. R ６ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ３ OH, OR ４ , N.H.R. ４ and NR ４ R ５ R is a group selected from any one of ４ and R ５ are the same or different and each is an unsubstituted alkyl group having 1 to 4 carbon atoms.

[0026] (R) in the compound represented by the above formula (Io) ｎWhen n is 0, it means that the compound is unsubstituted, and when n is an integer of 1 to 3, it means that 1 to 3 of the hydrogen atoms bonded to the carbon atoms constituting the benzene ring are substituted with a halogen atom, an alkyl group having 1 to 7 carbon atoms (C), or an alkoxyl group having 1 to 7 carbon atoms. When n is 2 or 3 (i.e., when the substituted benzene ring has 2 or 3 substituents), the substituents may be the same or different. (R) in the compound represented by the above formula (Io) ｎ Examples of the alkyl group include 1 to 3 halogen atoms, 1 to 3 alkyl groups having 1 to 7 carbon atoms, and 1 to 3 alkoxy groups having 1 to 7 carbon atoms.

[0027] (Z) in the compound represented by the above formula (Io) ｍ When m is 0, it means that the ring is unsubstituted, and when m is an integer of 1 to 4, 1 to 4 of the hydrogen atoms bonded to the carbon atoms constituting the benzene ring are each a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or OR ８ An organic oxy group (R ８ represents an alkyl group having 1 to 7 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms). When m is an integer of 2 to 4 (i.e., when the substituted benzene ring has 2 to 4 substituents), the substituents may be the same or different. (Z) in the compound represented by the above formula (Io) ｍ Examples of the alkyl group include 1 to 3 halogen atoms, 1 to 3 alkyl groups having 1 to 6 carbon atoms, 1 to 3 alkenyl groups having 2 to 6 carbon atoms, 1 to 3 alkynyl groups having 2 to 6 carbon atoms, and 1 to 3 OR ８ Examples of the organic oxy group include organic oxy groups represented by the following formula:

[0028] The "alkyl group having 1 to 7 carbon atoms" in the above formula (Io) may be a linear or branched alkyl group, and examples thereof include a methyl group, an ethyl group, a propyl group, a 2-ethylbutyl group, a 2-methylpentyl group, and a 3-methylpentyl group, and preferably a methyl group, a 2-ethylbutyl group, a 2-methylpentyl group, or a 3-methylpentyl group.

[0029] The "alkoxyl group having 1 to 7 carbon atoms" in the above formula (Io) may be a linear or branched alkoxyl group, and examples thereof include a methoxy group, an ethoxy group, and a propoxy group.

[0030] The "alkyl group having 1 to 6 carbon atoms" in the above formula (Io) means a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and as the alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 3 carbon atoms such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or a benzyl group is preferred.

[0031] The "alkenyl group having 2 to 6 carbon atoms" in the above formula (Io) means a linear or branched alkenyl group having 2 to 6 carbon atoms which may have a substituent, and examples thereof include an ethenyl group (vinyl group), a 1-propenyl group, and a 2-propenyl group (allyl group).

[0032] The "alkynyl group having 2 to 6 carbon atoms" in the above formula (Io) means a linear or branched alkynyl group having 2 to 6 carbon atoms which may have a substituent, and examples thereof include an ethynyl group, a 1-propynyl group, a 1-butynyl group, a 1-pentynyl group, and a 1-hexynyl group.

[0033] Examples of the "halogen atom" in the above formula (Io) include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0034] Examples of the "unsubstituted alkyl group having 1 to 4 carbon atoms" in the above formula (Io) include a methyl group, a monochloromethyl group, an ethyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 2-methoxyethyl group, a 2,2,2-trichloroethyl group, a propyl group, an isopropyl group, a hexafluoroisopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, R ４ and R ５ Examples include pyrrolidine, in which the nitrogen atom is bonded to the aryl group.

[0035] When the present compound has an asymmetric carbon atom and an asymmetric center related to axial chirality, such a compound includes all possible optical isomers, and these optical isomers can be used in any ratio. For example, a certain optically active compound can be used as an enantiomer, a racemic compound, or a mixture of enantiomers in any ratio, and when there are multiple asymmetric centers, it can be used as a mixture of diastereomers in any ratio.

[0036] Physiologically acceptable salts of the compound represented by formula (Io) above include metal salts formed from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, and organic salts formed from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine, etc.

[0037] The compound of the present invention can be synthesized using the methods disclosed in Patent Document 1, International Publication No. 2019 / 235455, etc., but is not limited to these methods, and any commonly known synthesis method can be used.

[0038] As the compound represented by the above formula (Io), the effect of which has been demonstrated in the examples of the present specification, a compound represented by the following formula (I-1) (i.e., in the above formula (Io), R is a fluorine atom, n is 2, m is 0, and R ６ is a hydrogen atom, and R ３ is OH) (also referred to herein as "MA5").

[0039]

[0040] Examples of additives for the present preventive / ameliorative agent include physiologically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonicity agents, coating agents, solubilizers, lubricants, glidants, solubilizers, flavorings, sweeteners, solvents, gelling agents, nutrients, various oils, surfactants, preservatives, antioxidants, dispersants, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, colorants, fragrances, etc. Specific examples of such additives include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0041] The subject to receive the present preventive / ameliorating agent (e.g., the subject to which it is administered) may be any subject (mammal) in need of prevention or amelioration of metabolic dysfunction-associated fatty liver disease, preferably a subject at risk of or already suffering from metabolic dysfunction-associated fatty liver disease (MASLD patients), and more preferably a subject at risk of or already suffering from metabolic disorder-associated steatohepatitis (MASH patients). These subjects may be elderly (e.g., over 60, over 65, over 70, over 75, or over 80 years old), or may even be a subject suffering from cancer (e.g., liver cancer) (cancer patients).

[0042] The method of ingesting (e.g., administering) the present preventive / ameliorating agent may be any method that allows the present compound contained in the present preventive / ameliorating agent to be taken into the body of a mammal, and may be an oral administration method (e.g., oral administration in the form of powder, granules, tablets, capsules, syrup, etc.) or a parenteral administration method (e.g., parenteral administration in the form of topical skin preparation, transdermal preparation, transmucosal preparation, nasal preparation, enteral preparation, injection, suppository, inhalant, patch, etc.). However, the oral administration method can be preferably exemplified because its effect has been demonstrated in the Examples described below.

[0043] The intake amount of the present preventive / ameliorating agent or the present compound contained in the present preventive / ameliorating agent is appropriately determined depending on the species, age, body weight, sex, symptoms, drug sensitivity, etc. of the mammal to be ingested (e.g., administered) and is, for example, in the range of 1 μg to 200 mg / kg (body weight) / day (e.g., 500 μg / kg / day to 100 mg / kg / day, 1 mg / kg / day to 50 mg / kg / day, 3 mg / kg / day to 25 mg / kg / day, etc.). The present preventive / ameliorating agent is taken once or in divided doses (e.g., 2 to 4 times) per day, and the intake amount may be adjusted depending on the degree of effect.

[0044] The present preventive / ameliorating agent may contain, in addition to the present compound, a substance (e.g., a compound, a protein [e.g., an antibody], DNA, or RNA) that has an effect of preventing or ameliorating fatty liver diseases associated with metabolic dysfunction. However, since the present compound alone exhibits an excellent effect of preventing or ameliorating fatty liver diseases associated with metabolic dysfunction, it is preferable that the agent does not contain, in addition to the present compound, a substance that has an effect of preventing or ameliorating fatty liver diseases associated with metabolic dysfunction.

[0045] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0046] 1. Therapeutic Effect on FFC Diet Model To confirm that the present compounds are effective in treating MASLD, analyses were performed using a high-fructose, high-saturated fatty acid, and high-cholesterol diet (FFC diet) model, i.e., diet-induced MASH model mice.

[0047] 1-1 Methods 49-week-old C57BL / 6J mice (obtained from Nippon SLC) (male, n=16) and 50-week-old C57BL / 6J mice (obtained from Nippon CLEA) (male, n=10) were acclimated for one week and then randomly divided into three intervention groups (control group [n=6], FFC group [n=10], and FFC+MA5 group [n=10]) and underwent the intervention for 25 weeks. The control group was maintained on a standard diet (CE-2, manufactured by Japan CLEA) and water. The FFC group was maintained on a high-fat, high-cholesterol diet (Western Diet Powder [Research Diet, D12079BM]) and high-glucose, high-fructose water (18.9 g D-glucose / L, 23.1 g D-fructose / L). The FFC+MA5 group was maintained on the same high-fat, high-cholesterol diet supplemented with 0.012% MA5 by mass (equivalent to a human dose of approximately 5 mg / kg / day based on a conversion factor per body surface area) and the same high-glucose, high-fructose water. Mice were weighed and supplemented with various diets and water once a week. After 15 weeks of treatment (i.e., 64-65 weeks of age), mice from each group were anesthetized, and approximately 200 μL of blood samples were collected from the retro-orbital venous plexus. After 25 weeks of intervention (i.e., 74-75 weeks of age), mice were euthanized after 4 hours of fasting, and blood samples and liver tissues were promptly collected. Plasma was separated from the blood samples, and blood AST and ALT activities were measured using a SpotChem II Kenshin 2 (Arkray). Formalin-fixed, paraffin-embedded specimens were prepared from the collected liver tissues, stained with HE staining, and the level of liver inflammation was measured using an optical microscope based on the NAS (NAFLD activity score). Masson's trichrome staining was also performed, and the level of liver fibrosis was measured using an optical microscope according to the method described in "Hepatology. 2005 Jun;41(6):1313-21."

[0048] 1-2 Results Measurements of mouse body weight, blood AST activity, and blood ALT activity were performed for the three intervention groups (control, FFC, and FFC+MA5). Compared with the control group, the FFC group showed increased body weight (Fig. 1A) and significant increases in blood AST and ALT activity (Fig. 1C and Fig. 1E). In contrast, the FFC+MA5 group showed a decrease in body weight (Fig. 1A) and a significant decrease in elevated blood AST and ALT activity (Fig. 1C and Fig. 1E) compared with the FFC group, and these effects were maintained for at least 15 to 25 weeks (Fig. 1B-E). These results indicate that MA5 has the effect of ameliorating MASH-induced weight gain and increases in blood AST and ALT activity.

[0049] Furthermore, analysis of the levels of inflammation and fibrosis in the liver tissue of mice in the three intervention groups showed that the FFC group exhibited inflammation and fibrosis that were not observed in the control group, whereas the elevated levels of inflammation and fibrosis in the FFC+MA5 group were significantly reduced compared to the FFC group (Figure 2). These results indicate that MA5 has the effect of ameliorating the inflammation and fibrosis in liver tissue caused by MASH.

[0050] 2. Therapeutic Effect on HF-MRTD Diet Model To confirm that the present compound is effective in treating MASLD, analysis was performed using a high-fat, methionine-restricted, tyrosine-deficient diet (HF-MRTD diet) model, i.e., a diet-induced MASLD model mouse.

[0051] 2-1 Method Six-week-old C57BL / 6J mice (obtained from Japan SLC) (male, n = 15) were acclimated for one week and then randomly assigned to one of three intervention groups (control group [n = 5], HF-MRTD group [n = 5], and HF-MRTD + MA5 group [n = 5]) for 22 weeks. The control group was fed a standard diet (CE-2, manufactured by CLEA Japan) and water. The HF-MRTD group was fed a powdered MRTD diet (60% fat and 0.2% methionine by mass, prepared according to the method described in the literature, "Hepatology. 2021 Sep;74(3):1271-1286.") and high-glucose, high-fructose water (18.9 g D-glucose / L, 23.1 g D-fructose / L). The HF-MRTD+MA5 group was fed the MRTD diet powder with MA5 mixed at a mass ratio of 0.012% and the high-glucose, high-fructose water. The diets and water were replenished weekly. After 13 weeks of intervention (i.e., at 19 weeks of age), mice from each group were anesthetized, and approximately 200 μL of blood samples were collected from the retro-orbital venous plexus. After 22 weeks of intervention (i.e., at 28 weeks of age), mice were euthanized after 4 hours of fasting, and blood samples and liver tissue samples were promptly collected. Plasma was separated from the blood samples, and blood AST and ALT activities were measured using a SpotChem II Kenshin 2 (Arkray). Formalin-fixed, paraffin-embedded specimens were prepared from the collected liver tissue, stained with HE staining, and the level of liver steatosis was measured using an optical microscope based on NAS. Masson's trichrome staining was also performed, and the level of liver fibrosis was measured using an optical microscope according to the method described in "Hepatology. 2005 Jun;41(6):1313-21."

[0052] 2-2 Results Measurements of blood AST and ALT activities in the three intervention groups (control, HF-MRTD, and HF-MRTD+MA5) revealed significant increases in blood AST and ALT activities in the FFC group compared with the control group (Figures 3B and 3D). In contrast, the FFC+MA5 group showed a decrease in elevated blood AST activity (Figure 3B) and a significant decrease in elevated blood ALT activity (Figure 3D) compared with the FFC group, and these effects were maintained for at least 13 to 22 weeks (Figures 3A-D). These results indicate that MA5 has the effect of ameliorating the elevation of blood AST and ALT activities caused by MASLD.

[0053] Furthermore, analysis of the levels of fatty acid and fibrosis in the liver tissue of mice in the three intervention groups revealed that fatty acid and fibrosis in the liver tissue were observed in the HF-MRTD group, but not in the control group. In contrast, the HF-MRTD+MA5 group showed a significant reduction in elevated fatty acid levels (Figure 4B) and a significant reduction in elevated fibrosis levels (Figures 4A and 4C) compared with the HF-MRTD group. These results indicate that MA5 has the effect of ameliorating fatty acid and fibrosis in the liver tissue caused by MASLD.

[0054] 3. Therapeutic Effect on MCD Diet Model To confirm that the present compound is effective in treating MASLD, analysis was carried out using a methionine and choline deficient diet (MCD diet) model, i.e., a diet-induced MASLD model mouse.

[0055] 3-1 Methods: 54-week-old C57BL / 6J mice (obtained from Japan SLC) (male, n = 6) were acclimated for one week and then randomly assigned to one of three intervention groups (control group [n = 2], MCD group [n = 2], and MCD + MA5 group [n = 2]). The intervention was continued until the mice died. The control group was maintained on a standard diet (CE-2, Japan CLEA) and water. The MCD group was maintained on a methionine-choline-deficient diet (Research Diet, A02082002BRM) and water. The MCD + MA5 group was maintained on the methionine-choline-deficient diet supplemented with 0.012% MA5 by mass and water. Mice were weighed and supplemented with various diets and water once a week until the mice died.

[0056] 3-2 Results Compared with the control group, the MCD group showed a decrease in mouse body weight and a shortened lifespan, whereas the MCD+MA5 group showed an increase in mouse body weight and an extension of the shortened lifespan compared with the MCD group (Figure 5). These results indicate that MA5 has the effect of ameliorating the weight loss and shortened lifespan caused by MASLD (nutrient-depleted fatty liver disease).

[0057] 4. Therapeutic Effects on Diet- and Drug-Induced MASH Model Mice with Severe Fibrosis To confirm that the present compounds are effective in treating MASLD, analyses were performed using diet- and drug-induced MASH model mice with severe fibrosis.

[0058] 4-1 Methods Twelve-week-old C57BL / 6J mice (obtained from Japan SLC) (male, n = 2) were acclimated for 1 week and then randomly assigned to two intervention groups (severe fibrosis group [n = 1] and severe fibrosis + MA5 group [n = 1]) for 6 weeks. To generate MASLD model mice with severe fibrosis, the two intervention groups were maintained on a high-fat, high-cholesterol diet (Western Diet Powder [Research Diet, D12079BM]) and high-glucose, high-fructose water (18.9 g D-glucose / L, 23.1 g D-fructose / L). Carbon tetrachloride, a drug that induces liver damage and liver fibrosis, was administered intraperitoneally. In addition, the severe fibrosis group received oral DMSO three times a week via a probe, and the severe fibrosis + MA5 group received oral MA5 (25 mg / kg) three times a week via a probe. Food and water were replenished once a week. After 6 weeks of intervention, mice were euthanized after a 4-hour fast, and blood samples and liver tissue were promptly collected. Plasma was separated from the blood samples, and blood AST and ALT activities were measured using a SpotChem II Kenshin 2 (Arkray). Formalin-fixed, paraffin-embedded specimens were prepared from the collected liver tissue, stained with Masson's Trichrome, and the level of liver fibrosis was observed under an optical microscope.

[0059] 4-2 Results Measurement of blood AST and ALT activities in the two intervention groups (severe fibrosis group and severe fibrosis + MA5 group) revealed that the severe fibrosis group had elevated blood AST and ALT activities compared with the control group shown in Figures 1C and 1E, whereas the severe fibrosis + MA5 group had reduced elevated blood AST and ALT activities compared with the severe fibrosis group (Figures 6A and 6B). These results indicate that MA5 has the effect of improving the elevated blood AST and ALT activities caused by MASH accompanied by severe fibrosis.

[0060] Furthermore, analysis of the fibrosis level in the mouse liver tissues for the two intervention groups revealed that the severe fibrosis group exhibited fibrosis at a level close to cirrhosis (equivalent to F3-F4), which was not observed in the control group shown in Figure 4A. In contrast, in the severe fibrosis + MA5 group, although fibrosis itself was observed, its progression was suppressed, resulting in a level equivalent to F1 (Figure 6C). These results indicate that MA5 has the effect of ameliorating the fibrosis in liver tissues caused by MASH, which is accompanied by severe fibrosis.

[0061] The present invention contributes to the prevention or amelioration of MASLD.

Claims

1. A prophylactic or ameliorating agent for fatty liver disease associated with metabolic dysfunction, comprising one or more compounds selected from the group consisting of a compound represented by the following formula (Io) and a physiologically acceptable salt thereof. [In the formula, R is a halogen atom, an alkyl group having 1 to 7 carbon atoms, or an alkoxyl group having 1 to 7 carbon atoms, and n is any integer from 0 to 3; Z is a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or an organic oxy group represented by OR ８ , m is any integer from 0 to 4, and R ８ represents an alkyl group having 1 to 7 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; when n and m are plural, each R and each Z may be the same or different; R ６ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ３ is a group selected from any one of OH, OR ４ , NHR ４ and NR ４ R ５ , and R ４ and R ５ are the same or different and are unsubstituted alkyl groups having 1 to 4 carbon atoms. ] 2. The prophylactic or ameliorating agent according to claim 1, wherein the compound represented by formula (Io) is a compound represented by the following formula (I-1).

3. The prophylactic or ameliorating agent according to claim 1 or 2, wherein the fatty liver disease associated with metabolic insufficiency is metabolic disorder-related steatohepatitis.

4. The prophylactic or ameliorating agent according to claim 1 or 2, which is orally administered.

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