WO2019024776A1 - Pharmaceutical composition and use thereof - Google Patents

Abstract

Disclosed is a pharmaceutical composition, comprising a first component and a second component, wherein the first component is selected from at least one of an ASK1 inhibitor and a precursor, an active metabolite, a stereisomer, a pharmaceutically acceptable salt, an ester and a solvate thereof; and the second component is selected from at least one of a PPAR modifier, and a precursor, an active metabolite, a stereisomer, a pharmaceutically acceptable salt, an ester and a solvate thereof. Also disclosed is a pharmaceutical preparation comprising the pharmaceutical composition. Also disclosed is a use of the pharmaceutical composition in the preparation of a medicament for treating and/or preventing nonalcoholic fatty liver or nonalcoholic steatohepatitis, reducing levels of total cholesterol, lowering liver coefficient, increasing high-density lipoprotein cholesterol, and/or improving NAS score and/or fibrosis score.

Description

Pharmaceutical composition and use thereof

This application claims the priority of the prior application filed on July 31, 2017, to the Chinese National Intellectual Property Office, the patent application number is 201710641321.3, entitled "Pharmaceutical Compositions and Uses". The entire contents of this prior application are incorporated herein by reference.

Technical field

The invention belongs to the field of biomedicine, and in particular to a pharmaceutical composition and use thereof, in particular to a pharmaceutical composition for effectively treating or preventing nonalcoholic fatty liver disease, a preparation method and application thereof.

Background technique

The liver is an important organ of fat metabolism. Under normal circumstances, fat accounts for 3% to 5% of the total weight of the liver. Normal people contain 4 to 5 g of lipid per 100 g of liver wet weight, of which phospholipids account for more than 50% and triacylglycerols account for 20%. The free fatty acid accounts for 20%, the cholesterol is about 7%, and the rest is cholesterol fat. Fatty liver is called when liver lipid accumulation exceeds 5% of liver wet weight, or histologically, per unit area sees more than 1/3 of hepatocyte steatosis. Nonalcoholic fatty liver disease (NAFLD) refers to diseases caused by excessive deposition of fat in hepatocytes caused by factors other than alcohol and other defined liver damage (such as drug-induced liver damage). Rapidly, becoming one of the most common liver diseases. The prevalence of NAFLD in adults in western countries such as Europe and the United States is 20% to 33%. The number of NAFLD patients in Asian countries is increasing rapidly and showing a trend of younger age. The prevalence of NAFLD in adults in Shanghai, Guangzhou and Hong Kong is about 15%. .

The disease spectrum of NAFLD ranges from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), resulting in the development of liver fibrosis and cirrhosis and even hepatocellular carcinoma. Its pathogenesis is related to insulin resistance, oxidative stress and lipid peroxidation, bile acid metabolism disorder, autophagy and so on. About one-third to one-half of NAFLD is NASH. The probability of developing cirrhosis with simple fatty liver for 10 to 20 years is 0.6% to 3%, and the incidence of cirrhosis with NASH for 10 to 15 years is as high as 15%. % to 25%, of which 30% to 40% will die of liver cancer (1% of cases per year after cirrhosis), liver failure, and recurrence after transplantation. Therefore, nonalcoholic fatty liver disease has become a new challenge in the field of contemporary liver disease.

Currently, drugs for the treatment of nonalcoholic fatty liver disease remain to be further studied.

Summary of the invention

The present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial choice. To this end, it is an object of the present invention to provide a pharmaceutical composition effective for treating or preventing nonalcoholic fatty liver disease and its associated disorders.

According to one aspect of the invention, the invention proposes a pharmaceutical composition. A pharmaceutical composition according to a particular embodiment of the invention comprises: a first component and a second component. Wherein the first component is at least one selected from the group consisting of an ASK1 inhibitor and a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, an ester, and a solvate; At least one of a PPAR modulator and a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, an ester, and a solvate.

Accordingly, the pharmaceutical composition of the present invention has a reduced level of total cholesterol, triglyceride, low density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase, and/or alkaline phosphatase, and/or Decreasing the liver coefficient, and/or reducing the expression level of the fibrosis marker Col1a1 and/or the expression level of α-SMA, and/or increasing HDL cholesterol, and/or improving the efficacy of the NAS score and/or fibrosis score, Further, it has a remarkable effect in preventing and/or treating NAFLD and its associated diseases. The NAFLD includes: nonalcoholic fatty liver, nonalcoholic steatohepatitis, nonalcoholic liver fibrosis, nonalcoholic cirrhosis. The NAFLD-associated disorders include, but are not limited to, secondary NAFLD, insulin resistance, hyperglycemia, metabolic syndrome, obesity with hyperlipidemia, type 2 diabetes, cardiovascular disease, liver failure, liver cancer, and the like.

The pharmaceutical composition of the present invention is particularly effective for preventing and/or treating nonalcoholic fatty liver disease, particularly for preventing, delaying and treating nonalcoholic fatty liver, nonalcoholic steatohepatitis and nonalcoholic liver fibrosis. The effect is excellent and the effect is significant in improving the NAS score.

Nonalcoholic fatty liver disease activity score (NAS) is defined as the unweighted sum of the values of steatosis (0 to 3), lobular inflammation (0 to 3), and bloating (0 to 2), thereby providing 0 to 8 NAS score (see Kleinen et al, Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321). Patients with different degrees of fibrosis have different NAS scores, and the higher the NAS score, the more severe the fibrosis.

Patients treated with NASH according to the present disclosure may exhibit a NAS score of > 4 prior to treatment, with a minimum score of 1 for both steatosis and lobular inflammation, plus a sinus fibrosis of at least 1 and possibly or The discovery of steatohepatitis. After administration/treatment (for example, one year), the patient can show a composite NAS score of ≤3, ≤2 or ≤1, and the fibrosis does not deteriorate. Alternatively, the patient may show that at least two of the NAS components in the NAS have improved by a value of ≥ 2 and that the fibrosis has not deteriorated. Alternatively, the patient may show an improvement in the NAS score of ≥3, 4, 5, 6, 7, or 8.

Steatosis can be broadly understood to describe a process involving the abnormal accumulation of lipids in the liver that hampers normal liver function. Liver biopsy can analyze and score patients with steatosis, with a score ranging from 0 to 3. The steatosis score of a patient treated with NASH according to the present disclosure may be 1, 2 or 3, such as from about 2 to about 3. After treatment, the patient is expected to show no deterioration in steatosis, or a steatosis score decreased by at least 1, or a steatosis score decreased by 2 or 3. Steatosis is usually graded as follows: a score of 1 indicates that less than 33% of the hepatocytes have fat droplets, a score of 2 indicates that 33% to 66% of fat cells are observed in the liver cells, and a score of 3 indicates that more than 66% of the liver cells are observed. To the fat drops.

Lobular inflammation can also be assessed by liver biopsy and scored on a scale of 0 to 3. The lobular inflammation score of a patient to be treated for NASH can be 1, 2 or 3, or a range of 1-2 or 2-3. After treatment, the patient's lobular inflammation score may be reduced by at least 1, or the lobular inflammation score is decreased by 2 or 3, and the lobular inflammation score is at least not aggravated.

The flatulence of hepatocytes is usually scored at a value of 0 to 2, and the patient's flatness score for treating NASH according to the present disclosure may be 0 to 2, including a specific value of 1 or 2, or a range of scores of 1 to 2. After treatment, the patient may show that the bloating score has not deteriorated at least, or that the bloating score has decreased by at least 1, or the bloating score has decreased by two.

Fibrosis can also be assessed by liver biopsy and scored on a scale of 0 to 4, defined as follows: 0 for no fibrosis; 1 for periorbital or portal vein fibrosis; 1a for sinus circumference with mild region 3 Fibrosis; 1b represents sinusoidal fibrosis in moderate area 3; 1c represents portal/portal perivascular fibrosis; 2 represents sinus and portal/portal perivascular fibrosis; 3 represents bridging fibrosis; and 4 represents cirrhosis. A patient treated according to the present disclosure may have a fibrosis stage score of 0 to 3, including 0, 1, 1a, 1b, 1c, 2, or 3, and the fibrosis stage score may be at least 1a. After treatment, the patient's fibrosis score may be at least no worse than the baseline score, or the fibrosis stage score may be reduced by at least one level, or at least two or three levels.

According to an embodiment of the invention, the first component is selected from the group consisting of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-[6-(4-isopropyl- 4H-1,2,4-triazol-3-yl)pyridin-2-yl]-4-methylbenzamide (GS-4997), 4-(4-cyclopropyl-1H-imidazole-1- -N-[3-(4-cyclopropyl-4H-1,2,4-triazol-3-yl)phenyl]pyridine-2-carboxamide and 6-amino-9-[(3R) At least one of -1-(but-2-ynyl)pyrrolidin-3-yl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-indole.

According to an embodiment of the invention, the second component is a PPAR alpha / delta dual agonist and / or a PPAR alpha / delta / gamma agonist. According to a particular embodiment of the invention, the second component is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propene Phenoxy]-2-methylpropionic acid (GFT505), 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl Butyric acid (IVA337) and 2-[2-(4-fluorobenzoyl)phenyl)amino]-3-(4-(2-carbazolylethoxy)phenyl)-propionate (CS) At least one of -038).

Apoptosis signal-regulated kinase-1 (ASK1), also known as MAP3K5, is a kinase with a broad biological effect in vivo. Numerous studies have shown that ASK1 plays an important role in many stress-related diseases, including tumors, cardiovascular diseases, and neurodegenerative diseases.

Peroxisome proliferator-activated receptor (PPAR) δ as an energy regulator can reduce the deposition of TG in cells, increase the uncoupling of β-oxidation and oxidative phosphorylation, and reduce body weight. At the same time, PPARδ can be accurate. Regulates the body's inflammatory response, inhibits macrophage production of MCP1, IL1β, MMP9 and other inflammatory mediators, while also inhibiting endotoxin-induced COX2 expression, thereby reducing inflammation. PPARα and PPARδ play a cross-talk role in hepatocytes and macrophages. PPARα can increase liver fatty acid and lipoprotein metabolism, and PPARδ inhibits macrophage production of inflammatory factors, thereby improving metabolic syndrome. Commonly used PPAR alpha / delta dual agonists are: natural agonists, such as: linoleic acid, linolenic acid and eicosapentaenoic acid (EPA); synthetic agonists, such as: thiazolidinedione (TZD), carboxy Acid agonist.

Recent studies have shown that the efficacy and safety of ASK1 inhibitors and PPAR agonists have reached a good conclusion. It has been reported that ASK1 inhibitor (GS-4997) has a certain effect on patients with NASH: after 24 weeks of treatment with GS-4997, patients showed improvement in multiple indicators of liver disease severity, including regression of liver fibrosis (regression) ), cirrhosis progression, liver stiffness (assessed by magnetic resonance elastography, MRE), liver fat content (magnetic resonance imaging - proton density fat fraction evaluation, MRI-PDFF). However, the combination of GS-4997 and the monoclonal antibody simtuzumab (SIM) showed no better results than GS-4997 alone.

In addition, it has been reported that the PPARα/δ dual agonist GFT505 also plays a role in improving the NAS score. The PPAR alpha/delta/gamma pan agonist (IVA337) also showed an improvement in NAS scores on MCD and foz/foz animal models.

The experiment of the present invention specifically studied the combination of GS-4997 and GFT505, or GS-4997 and IVA337, or GS-4997 and CS038, and surprisingly found that the above-mentioned combination drug has achieved unexpected technical effects, and the two exerted Synergistically, the treatment of NAFLD, including nonalcoholic fatty liver and nonalcoholic steatohepatitis, is significantly more effective than the separate active ingredients.

According to the present invention, the weight ratio of the first component to the second component in the pharmaceutical composition is 1: (0.1 to 400), preferably 1: (0.5 to 200). Therefore, by adopting the above ratio, the synergistic effect of the two can be further improved, thereby improving the effect of treating NAFLD, especially nonalcoholic fatty liver and NASH.

In a preferred embodiment of the present invention, the weight ratio of GS-4997 to GFT505 in the pharmaceutical composition is 1: (0.5 to 40), preferably 1: (1.5 to 20); GS- in the pharmaceutical composition The weight ratio of 4997 to IVA337 is 1: (2 to 400), preferably 1: (10 to 400), more preferably 1: (20 to 200); weight ratio of GS-4997 and CS038 in the pharmaceutical composition It is 1: (2 to 40), preferably 1: (4 to 20).

According to the present invention, the therapeutically or prophylactically effective dose per day of the various active ingredients (i.e., ASK1 inhibitory or PPAR modulator) in the above pharmaceutical composition is not limited to 0.01 to 100 mg/kg body weight, preferably 0.02 to 80 mg/kg, respectively. The kg body weight is more preferably 0.05 to 50 mg/kg body weight. It is worth noting that the measured value may vary depending on the severity of the symptoms to be alleviated and on factors such as the disease state, age, sex and weight of the subject.

In a preferred embodiment of the present invention, the therapeutically or prophylactically effective dose of GS-4997 is not limited to 0.01 to 1 mg/kg body weight per day, preferably 0.02 to 0.6 mg/kg body weight, more preferably Preferably, the therapeutically or prophylactically effective dose of IVA337 is in a non-limiting range of 1 to 50 mg/kg body weight, preferably 3 to 35 mg/kg body weight, more preferably 5 to 25 mg/kg body weight; GFT505 per day. A non-limiting range of therapeutically or prophylactically effective doses is from 0.1 to 10 mg/kg body weight, preferably from 0.25 to 5 mg/kg body weight, more preferably from 0.5 to 3 mg/kg body weight; a non-limiting range of therapeutically or prophylactically effective doses per day for CS038 It is 0.05 to 5 mg/kg body weight, preferably 0.1 to 3.5 mg/kg body weight, more preferably 0.2 to 2.5 mg/kg body weight.

It will be further understood that for any particular subject, the particular dosage regimen should be adjusted over time to improve the optimal therapeutic response, depending on the individual needs and the professional judgment of the administration or supervising composition administration, and the dosage ranges described herein. The illustrations are not intended to limit the scope or practice of the claimed compositions.

In a second aspect of the invention, the invention provides a pharmaceutical preparation comprising the pharmaceutical composition described above as an active ingredient. Thus, the preparation exerts the pharmacological action of the active ingredient and further has the efficacy of the pharmaceutical composition. Specifically, the preparation has a lower total cholesterol, triglyceride, low density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase and/or alkaline phosphatase levels, and/or a reduced liver coefficient, and / or reduce the expression level of fibrosis markers Col1a1 and / or alpha-SMA expression levels, and / or increase high-density lipoprotein cholesterol, and / or improve the efficacy of NAS scores and / or fibrosis scores, and thus in prevention and / Or has a significant effect in the treatment of NAFLD and its associated disorders. The NAFLD includes: nonalcoholic fatty liver, nonalcoholic steatohepatitis, nonalcoholic liver fibrosis, nonalcoholic cirrhosis. The NAFLD-associated disorders include, but are not limited to, secondary NAFLD, insulin resistance, hyperglycemia, metabolic syndrome, obesity with hyperlipidemia, type 2 diabetes, cardiovascular disease, liver failure, liver cancer, and the like.

The pharmaceutical preparation of the invention is particularly effective for preventing and/or treating nonalcoholic fatty liver disease, in particular for preventing, delaying and treating nonalcoholic fatty liver, nonalcoholic steatohepatitis and nonalcoholic liver fibrosis. Excellent, it is effective in improving the NAS score.

According to an embodiment of the present invention, the above pharmaceutical preparation comprises, in addition to the pharmaceutical composition described above as an active ingredient, a pharmaceutically acceptable carrier. According to an embodiment of the present invention, the pharmaceutically acceptable carrier is selected from the group consisting of pharmaceutically acceptable solvents, propellants, solubilizers, solubilizers, emulsifiers, colorants, binders, disintegrators, fillers, Lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH Value modifier, buffer, plasticizer, surfactant, foaming agent, antifoaming agent, thickener, inclusion agent, moisturizer, absorbent, diluent, flocculant and deflocculant, filter aid, At least one of a retarder, a polymer skeleton material, and a film-forming material is released. Thereby, the pharmaceutical composition can be formulated into a form suitable for administration of a clinical pharmaceutical preparation.

According to the invention, the content of the first component in the pharmaceutical preparation is from 1 mg to 100 mg, preferably from 5 mg to 50 mg; and the second component is from 1 mg to 1500 mg, preferably from 5 mg to 1200 mg.

In a preferred embodiment of the present invention, the active ingredient in the pharmaceutical preparation has a content of GS-4997 of 1 mg to 50 mg, preferably 2 mg to 20 mg; and the content of IVA337 is 100 mg to 1500 mg, preferably 200 mg to 1200 mg; The content is 10 mg to 150 mg, preferably 40 mg to 120 mg; and the content of CS038 is 10 mg to 150 mg, preferably 15 mg to 100 mg.

In a third aspect of the invention, the invention provides the use of a pharmaceutical composition or a pharmaceutical preparation as described above for the preparation of a medicament for: reducing total cholesterol, triglycerides, low density lipoprotein cholesterol, C Aminotransferase, aspartate aminotransferase and/or alkaline phosphatase levels, and/or decreased liver coefficient, and/or decreased fibrosis marker Col1a1 expression level and/or α-SMA expression level, and / Or raise high density lipoprotein cholesterol, and / or improve NAS score and / or fibrosis score.

In a fourth aspect of the invention, the invention provides the use of a pharmaceutical composition or pharmaceutical formulation as hereinbefore described in the manufacture of a medicament for the prevention and/or treatment of a NAFLD or NAFLD associated disorder.

According to a specific embodiment of the present invention, the medicament is for preventing and/or treating nonalcoholic fatty liver, nonalcoholic steatohepatitis, nonalcoholic liver fibrosis, nonalcoholic cirrhosis, insulin resistance, high Blood sugar, metabolic syndrome, obesity with hyperlipidemia, type 2 diabetes, cardiovascular disease, liver failure, and/or liver cancer.

In a fifth aspect of the invention, the invention provides the use of a pharmaceutical composition or pharmaceutical formulation as described above for the preparation of a medicament for the prophylaxis and/or treatment of NASH.

In a sixth aspect of the invention, the invention provides the use of a pharmaceutical composition or pharmaceutical preparation as hereinbefore described for the preparation of a medicament for the prevention and/or treatment of non-alcoholic fatty liver.

According to a particular embodiment of the invention, in the use of a pharmaceutical composition for the preparation of a medicament, the pharmaceutical composition is administered orally, or as a mixture, or administered in a sequential manner at different times.

According to a specific embodiment of the present invention, in the use of the pharmaceutical composition for the preparation of a medicament, the pharmaceutical composition is administered in an amount of from 1 to 1500 mg/day.

In a seventh aspect of the invention, the invention provides at least one selected from the group consisting of an ASK1 inhibitor and a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, an ester, and a solvate. Use of a combination of at least one selected from the group consisting of a PPAR modulator and a precursor, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, an ester, and a solvate for the preparation of a medicament.

The medicament for: reducing total cholesterol, triglyceride, low density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase and/or alkaline phosphatase levels, and/or reducing liver coefficient, and / or reduce the fibrosis marker Col1a1 expression level and / or α-SMA expression level, and / or increase high density lipoprotein cholesterol, and / or improve NAS score and / or fibrosis score.

The medicament is for preventing or treating NAFLD or a related condition thereof, for example, preventing and/or treating nonalcoholic fatty liver, nonalcoholic steatohepatitis, nonalcoholic liver fibrosis, nonalcoholic cirrhosis, insulin Resistance, hyperglycemia, metabolic syndrome, obesity with hyperlipidemia, type 2 diabetes, cardiovascular disease, liver failure, and/or liver cancer.

The medicament is for the prevention and/or treatment of NASH.

The medicament is for the prevention and/or treatment of nonalcoholic fatty liver.

The ASK1 inhibitor is preferably 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-[6-(4-isopropyl-4H-1,2,4-tri Zyrid-3-yl)pyridin-2-yl]-4-methylbenzamide (GS-4997), 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-[3-( 4-cyclopropyl-4H-1,2,4-triazol-3-yl)phenyl]pyridine-2-carboxamide and 6-amino-9-[(3R)-1-(but-2-yne) At least one of acyl)pyrrolidin-3-yl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-indole.

The PPAR modulator is preferably a PPAR alpha / delta dual agonist and / or a PPAR alpha / delta / gamma pan agonist.

The PPAR modulator is further preferably 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy]- 2-methylpropionic acid (GFT505), 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl]butyric acid (IVA337) and At least one of 2-[2-(4-fluorobenzoyl)phenyl)amino]-3-(4-(2-carbazolylethoxy)phenyl)-propionate (CS-038) Kind.

In the medicament, the weight ratio of the ASK1 inhibitor to the PPAR modulator is 1: (0.1 to 400), preferably 1: (0.5 to 200).

Preferably, in the drug, the weight ratio of GS-4997 to GFT505 is 1: (0.5 to 40), preferably 1: (1.5 to 20).

Preferably, in the drug, the weight ratio of GS-4997 to IVA337 is 1: (2 to 400), preferably 1: (10 to 400), more preferably 1: (20 to 200).

Preferably, in the drug, the weight ratio of GS-4997 and CS038 is 1: (2 to 40), preferably 1: (4 to 20).

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

1 shows serum AST levels of mice in each group according to Examples 5-7 of the present invention; wherein: MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD +5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505; GG: MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA: MCD+10mg/kg IVA337; GI:MCD+2.5mg /kg GS-4997+5mg/kg IVA337;CS:MCD+10mg/kg CS-038;GC:MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 2 shows serum ALT levels of mice in each group according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD +5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505; GG: MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA: MCD+10mg/kg IVA337; GI:MCD+2.5mg /kg GS-4997+5mg/kg IVA337;CS:MCD+10mg/kg CS-038;GC:MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 3 shows liver TG levels of mice in each group according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD +5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505; GG: MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA: MCD+10mg/kg IVA337; GI:MCD+2.5mg /kg GS-4997+5mg/kg IVA337;CS:MCD+10mg/kg CS-038;GC:MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 4 shows liver TC levels in mice of each group according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplemented feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD +5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505; GG: MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA: MCD+10mg/kg IVA337; GI:MCD+2.5mg /kg GS-4997+5mg/kg IVA337;CS:MCD+10mg/kg CS-038;GC:MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 5 shows NAS scores for each group of mice according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline deficiency feed model group; GS: MCD+ 5 mg/kg GS-4997; GFT: MCD + 3 mg/kg GFT505; GG: MCD + 2.5 mg/kg GS-4997 + 1.5 mg/kg GFT505; IVA: MCD + 10 mg/kg IVA337; GI: MCD + 2.5 mg/ Kg GS-4997+5mg/kg IVA337; CS: MCD+10mg/kg CS-038; GC: MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 6 shows the ratio of Sirius red staining area of mice in each group according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD + 5 mg / kg GS-4997; GFT: MCD + 3 mg / kg GFT505; GG: MCD + 2.5 mg / kg GS-4997 + 1.5 mg / kg GFT505; IVA: MCD + 10 mg / kg IVA337; GI: MCD +2.5mg/kg GS-4997+5mg/kg IVA337;CS:MCD+10mg/kg CS-038;GC:MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 7 shows α-SMA levels in mice of each group according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD+5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505; GG: MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA: MCD+10mg/kg IVA337; GI:MCD+2.5 Mg/kg GS-4997+5mg/kg IVA337; CS: MCD+10mg/kg CS-038; GC: MCD+2.5mg/kg GS-4997+5mg/kg CS-038;

Figure 8 shows the expression level of Col1a1 in the liver of each group of mice according to Examples 5-7 of the present invention; wherein, MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD+5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505; GG: MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA: MCD+10mg/kg IVA337; GI:MCD+2.5 Mg/kg GS-4997+5 mg/kg IVA337; CS: MCD+10 mg/kg CS-038; GC: MCD+2.5 mg/kg GS-4997+5 mg/kg CS-038.

Detailed ways

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. In addition, it is to be understood that various changes and modifications may be made by those skilled in the art in the form of the invention.

The compounds of the present invention were prepared by the prior art (CN104080771A, CN1257893C, CN101248044A, CN1688532A).

Example 1 Preparation of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-[6-(4-isopropyl-4H-1,2,4-triazole-3 -yl)pyridin-2-yl]-4-methylbenzamide (Selonsertib, GS-4997)

(1) Step 1: Preparation of Intermediate C

Preparation of Compound A: A solution of 2-methoxycarbonyl-6-aminopyridine (480 g, 3.15 mol) in MeOH (9 L) was obtained, and hydrazine hydrate (315 g, 6.30 mol) was added. The reaction mixture was heated under reflux for 5 hours and then cooled to room temperature. The precipitate formed in the mixture was collected by filtration, washed with EA then dried in vacuo to afford Compound A (460 g, yield: 96%).

Preparation of Compound B: A mixture of dimethylformamide-dimethyl acetal (DMF-DMA) (5 L) of Compound A (350 g, 2.30 mol). The residue was taken up in EtOAc (EtOAc (EtOAc)EtOAc.

Preparation of compound C: Compound take B (524.64g, 2mol) in CH ₃ CN-AcOH mixture (5L, 4: 1), was added propan-2-amine (591.1g, 10mL), the resulting mixture was heated at reflux for 24 hours, cooled At room temperature, the solvent was removed under reduced pressure. The residue was dissolved in water (2.5 L), and a saturated aqueous NaOH solution was added until pH 8.0. The precipitate was collected by filtration, and the filtrate was extracted with EA (400 mL×4). With ₂ SO ₄ the combined organic phases dried over anhydrous Na, then concentrated to 120mL volume. Under ice bath, PE (400 mL) was slowly added to the mixture, and the resulting suspension was filtered. The combined solid phases were recrystallized from EA-PE to afford Intermediate C (252 g, yield 62%) as white solid.

^{1 H-NMR (400MHz, CDCl} 3): δ8.24 (s, 1H), 7.52 (m, 2H), 6.51 (dd, J = 1.6,7.2Hz, 1H), 5.55 (m, 1H), 4.46 ( Bs, 2H), 1.45 (d, J = 6.8 Hz, 6H). MS (ESI +) M / Z : 204 (M + 1) +.

(2) Step 2: Preparation of 5-amino-2-fluoro-4-methylbenzonitrile-------(Compound 2)

Starting from 5-bromo-4-fluoro-2-methylaniline (1), it (31 g, 150 mmol) was dissolved in anhydrous 1-methylpyrrolidone (150 mL) and copper (I) cyanide was added (26.9) g, 300 mmol). The reaction was heated at 185 ° C for 3.5 hours, cooled to room temperature and water (350 mL) and concentrated ammonia (350 mL). The mixture was stirred for 50 minutes and extracted with EA (300 mL×3). The combined extracts were dried over magnesium sulfate, and then the solvent was evaporated under reduced pressure. The oily residue was washed with hexane (100 mL x 3), dissolved in dichloromethane and loaded onto silica gel column. Elution with a gradient of 0 to 25% EA in EtOAc afforded 5-amino-2-fluoro-4-methylbenzonitrile (15.32 g, yield 68%). MS (ESI +) M / Z : 151 (M + 1) +.

(3) Step 3: Preparation of 5-(2-cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile------- (Compound 3)

5-Amino-2-fluoro-4-methylbenzonitrile (6 g, 40 mmol) was dissolved in anhydrous N,N-dimethylformamide (100 mL) under nitrogen, then solid potassium carbonate was added ( 6.64 g, 48 mmol) and potassium iodide (7.31 mL, 44 mmol) with stirring. The reaction was stirred at room temperature for 10 minutes then bromomethylcyclopropyl ketone (10.12 mL, 90 mmol) was then added. The reaction mixture was heated to 65 ° C for 4 hours, then the solvent was removed under reduced pressure. The residue was dissolved in EA (100 mL) and washed with water (200 <RTIgt; The organic layer was dried over magnesium sulfate and solvent was evaporated under reduced pressure. The residue was redissolved in a minimum amount of EA and ethane was added in a volume ratio of ethane:EA = 3:1. The product was isolated from the solution and collected by filtration to give 5-(2-cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile (7.80 g, yield 84%) . MS (ESI+) M/Z: 233 (M+1) ⁺ .

(4) Step 4: Preparation of 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile------- (Compound 4 )

5-(2-Cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile (5.69 g, 24.5 mmol) was dissolved in EtOAc (150 mL). Solid potassium thiocyanate (4.76 g, 48.96 mmol) was added while stirring. The reaction mixture was heated to 115 ° C for 4 hours and then the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (100 mL) andEtOAc The aqueous extract was extracted with dichloromethane (200 mL×2) and organic extracts were combined and dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was crystallised from EtOAc (EtOAc) After forming a dark layer, a stir bar was added to the flask. The product precipitated as a pink solid with vigorous stirring. The product was collected by filtration to give 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile (5.83 g, yield: 87%). MS (ESI +) M / Z : 274 (M + 1) +.

(5) Step 5: Preparation of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile--(Compound 5)

Acetic acid (100 mL), water (20 mL) and hydrogen peroxide (30%, 4.98 mL, 43.92 mmol) were added to a three-neck round bottom flask. The mixture was stirred and heated to 45 ° C under a nitrogen atmosphere while detecting the internal temperature. Then, the solid 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile (4 g, 14.64 mmol) was added in small portions over 30 min. While maintaining the internal temperature below 55 ° C, after completion of the addition of the thioimidazole, stirring was carried out at 45 ° C for 30 minutes, then cooled to room temperature, and a 20% wt / wt aqueous solution of sodium sulfite (6 mL) was slowly added. After the mixture was stirred for 30 minutes, the solvent was removed under reduced pressure. The residue was suspended in 300 mL of water and a saturated aqueous solution of ammonium hydroxide was added until pH was approximately 10. The mixture was extracted with dichloromethane (150 mL × 3), and the organic phase was combined and dried over magnesium sulfate. The residue was dissolved in 30 mL of EA and 100 mL of hexane was added with stirring. The solvent was washed away and the oily residue remained. This procedure was repeated to give the product 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile (1.48 g, yield 42%). MS (ESI +) M / Z : 242 (M + 1) +.

(6) Step 6: Preparation of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid hydrochloride--(Compound 6)

5-(4-Cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile (7.48 g, 31.00 mmol) was placed in a round bottom flask equipped with a reflux condenser, and It was suspended in 38% hydrochloric acid (150 mL). The mixture was heated to 100 ° C for 5 hours and then cooled to room temperature. The solvent was removed under reduced pressure to give a pink solid. The solid product was collected by filtration and washed with 3×100 mL EA. To the obtained solid product, 100 mL of a dichloromethane solution containing 10% methanol was added, the mixture was stirred, and filtrate was collected by filtration. This step was repeated twice with 100 mL of a 10% methanol in dichloromethane solution. The filtrate was combined and the solvent was evaporated under reduced pressure to give crude 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoate. No further purification (7.86 g, yield 85%). MS (ESI +) M / Z : 261 (M + 1) +.

(7) Step 7: Preparation of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-[6-(4-isopropyl-4H-1,2,4-tri Zyrid-3-yl)pyridin-2-yl]-4-methylbenzamide (Selonsertib, GS-4997)

5-(4-Cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoate (1.25 g, 4.23 mmol) was taken and suspended in anhydrous 1 at room temperature. In 2-dichloroethane (23 mL). Stirred under nitrogen and oxalyl chloride (0.479 mL, 5.49 mmol) was added followed by N,N-dimethylformamide (0.037 mL, 0.423 mmol). The mixture was stirred at room temperature for 5 hours, then the solvent was removed under reduced pressure. The residue was dissolved in 25 mL of dry dichloromethane. Stir under nitrogen and quickly add 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (0.94 g, 4.65 mmol) (intermediate C) and 4-Dimethylpyridine (0.52 g, 4.23 mmol). The reaction mixture was stirred for 2 hours at room temperature, and water was added saturated NaHCO ₃ (18mL). The mixture was stirred for 10 minutes, the layers were separated and washed with dichloromethane (30×2 mL). And the combined organic layers were dried (MgSO _4), filtered and concentrated. The residue was dissolved in a minimum amount of CH ₃ CN, and water was slowly added until solid particles were precipitated in the mixture. The solid was collected by filtration and dried to give 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-[6-(4-isopropyl-4H-1,2). 4-Triazol-3-yl)pyridin-2-yl]-4-methylbenzamide (1.15 g, yield 61%). MS (ESI +) M / Z : 446.2 (M + 1) +.

¹ H-NMR (DMSO): δ 11.12 (s, 1H), 9.41 (s, 1H), 9.32 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.07 (t, J = 8.4) Hz, 1H), 7.95 (d, J = 6.4 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.79 (s, 1H), 7.59 (d, J = 10.4 Hz, 1H), 5.72 ( Sept, J=6.8Hz, 1H), 2.29(s,3H), 2.00-2.05(m,1H), 1.44(d,J=6.8Hz,6H),1.01-1.06(m,2H),0.85-0.89 (m, 2H).

Example 2 2-[2,6-Dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy]-2-methylpropane Acid (Elafibranor, GFT505)

(1) Step 1: Preparation of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-hydroxyphenyl]prop-2-en-1-one (Intermediate 3)

Compound 1 (4.2 g, 25 mmol) and Compound 2 (3.8 g, 25 mmol) were dissolved in a saturated aqueous solution of hydrochloric acid. The reaction was stirred at room temperature for about 8 hours and then the solvent was evaporated in vacuo. Purification by silica gel chromatography gave Intermediate 3 (6.5 g, yield 88%).

(2) Step 2: Preparation of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropoxycarbonyldimethylmethoxyphenyl]propan-2- En-1-one (intermediate 4)

Intermediate 3 (8.67 g, 29.1 mmol) was dissolved in acetonitrile. Then a halogenated derivative (30.229 g, 145.3 mmol) and potassium carbonate (20.086 g, 145.3 mmol) were added. The reaction medium was vigorously stirred under reflux for about 12 hours. The salt was removed by filtration, and the solvent was evaporated, evaporated,jjjjjjj

(3) Step 3: Preparation of 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy]-2 -methylpropionic acid (Elafibranor, GFT505)

Intermediate 4 (13.3 g, 31.2 mmol) was dissolved in dichloromethane, trifluoroacetic acid (45.668 g, 400.6 mmol). The obtained product was purified by silica gel chromatography or recrystallization. GFT-505 (11.0 g, yield 92%) was obtained.

¹ H NMR DMSO δ ppm: 1.39 (s, 6H), 2.22 (s, 6H), 2.57 (s, 3H), 7.40 (d, J = 8.55 Hz, 2H), 7.57 (s, 2H), 7.62 (d, J) =15.5 Hz, 1H), 7.83 (d, J = 15.5 Hz, 1H) 8.10 (d, J = 8.55 Hz, 2H), 12.97 (s, 1H). MS (ESI-) M / Z: 383.3 (M-1) -.

Example 3 4-[1-(1,3-Benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl]butyric acid (IVA337)

(1) Step 1: Preparation of methyl 6-(5-chloro-2-nitrophenyl)-hex-5-ynoate (intermediate 3)

Mix 25.4 g (89 mmol) of 4-chloro-2-iodo-nitrobenzene, 370 mL of triethylamine, 2.06 g (1.8 mmol) of tetrakis(triphenylphosphine)palladium, 0.51 g of cuprous iodide and 37 mL of dimethyl Formamide (DMF). Then, 10 g (89 mmol) of methyl 5-hexynoate (Compound 2) was added with stirring at room temperature, and the reaction mixture was stirred at room temperature for 24 hours. 75 mL of toluene was added and the solvent was removed under reduced pressure. The evaporation residue was treated with 120 mL ethyl acetate and 60 mL 1N hydrochloric acid. The organic phase was separated, washed with water then dried over magnesium sulfate Purification by silica gel chromatography using a cyclohexane/ethyl acetate mixture (9/1, V/V) as eluent to afford 17.0 g of Intermediate 3 as a yellow solid (yield 68%)

(2) Step 2: Preparation of methyl 6-(2-amino-5-chlorophenyl)-hex-5-ynoate (intermediate 4)

45.3 g (200 mmol) of stannous chloride, 35 mL of ethyl acetate and 11 mL of ethanol were added to a round bottom flask. The mixture was stirred at room temperature for 15 min then intermediate 3 (10.8 g) was slowly added. The reaction mixture was stirred at room temperature for 24 hours and then poured into a mixture of 200 g of ice and 200 mL of 1N sodium hydroxide solution. The resulting mixture was extracted with EtOAc EtOAc. Purification by silica gel chromatography using EtOAc/EtOAc (EtOAc/EtOAc)

(3) Step 3: Preparation of methyl 6-[5-chloro-2-(1,3-benzothiazol-6-ylsulfonylamino)phenyl]-hex-5-ynoate (intermediate 5)

A solution of Intermediate 4 (2.4 g, 10 mmol) in 30 mL of pyridine was added 2.8 g (12 mmol) of 1,3-benzothiazol-6-ylsulfonyl chloride. The mixture was stirred at room temperature for 1.5 hours and then concentrated under reduced pressure. The residual oil was purified by silica gel chromatography using EtOAc (EtOAc) (EtOAc)

(4) Step 4: Preparation of methyl 4-[5-chloro-1-(1,3-benzothiazol-6-ylsulfonyl)-1H-indol-2-yl]butanoate (Intermediate 6)

Intermediate 5 (200 mg, 0.45 mmol) in 35 mL of 1 ,2-dichloroethane was added, and 10 mg (0.05 mmol) of copper acetate was added, and the mixture was refluxed for 24 hours while stirring. The solvent was then removed under reduced pressure. EtOAc EtOAc m. ).

(5) Step 5: Preparation of 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl]butyric acid (IVA337)

90 mg (0.2 mmol) of intermediate 6 was mixed with 8 mL of THF and 2 mL of water, and 10 mg (0.24 mmol) of lithium hydroxide was added. The mixture was stirred at room temperature for 4 hours and then concentrated under pressure. The evaporation residue was treated in 12 mL of water and the solution was acidified with 1N hydrochloric acid. The white precipitate was extracted with EtOAc. EtOAc (EtOAc)

Example 4 Preparation of sodium 2-[2-(4-fluorobenzoyl)phenyl)amino]-3-(4-(2-carbazolylethoxy)phenyl)-propionate (CS-038 )

(1) Step 1: Preparation of 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl)-propionic acid methyl ester (CHIG-003)

39.0 g (0.2 mol) of L-tyrosine methyl ester, 49.5 g (0.23 mol) of 2-(4-fluorobenzoyl)cyclohexanone, 13.0 g of 5% palladium/carbon catalyst and 800 mL of benzene were added to the reaction flask. Methyl ether was refluxed for 2 hours while water formed during the reaction was removed with a water separator. The reaction mixture was cooled to 80 ° C and the palladium on carbon catalyst was removed by filtration. The reaction mixture was then cooled to 80 ° C and the palladium on carbon catalyst was removed by filtration. Then, the reaction mixture was further cooled to 40 ° C, 800 mL of n-hexane was added, and the mixture was allowed to stand at -20 ° C for 48 hours, filtered, and washed with n-hexane (200 mL × 5) to give a crude product. The crude product was added to 200 mL of methanol, refluxed for 30 minutes, filtered, washed with methanol (50 mL×2), and dried to give 40.1 g (yield: 51%) of title compound.

(2) Step 2: 2-[(2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-bromoethoxy)phenyl)-propionic acid methyl ester (CHIG- Preparation of 005)

0.1 g (1.74 mmol) of potassium hydroxide and 0.68 g (1.74 mmol) of 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl) were added to the reaction flask. Methyl propionate, 3.26 g (17.4 mmol) of 1,2-dibromoethane and 15 mL of ethanol, refluxing for 8 hours, removing solid insolubles by filtration, and concentrating the filtrate in vacuo to give a crude product. N-hexane: ethyl acetate = 4:1) gave 0.37 g (yield: 42%) of title compound.

(3) Step 3: 2-[2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-(9H-indazol-9-yl)ethoxy)phenyl) - Preparation of propionic acid (Chiglitazar)

0.084 g of carbazole (0.50 mmol) and 0.25 g (0.50 mmol) of 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-(2-bromoethyl) were added to the reaction flask. Ethyloxy)phenyl)-propionic acid methyl ester, 0.085 g (2.13 mmol) 50% NaOH, 0.08 g tetrabutylammonium bromide and 10 mL benzene, refluxing for 12 hours, adding 30 mL of benzene, washing with water (30 mL×3) The mixture was dried and concentrated in vacuo to give a crude product, which was purified (yield: methanol: chloroform = 4:1). _{HRMS (C 36 H 29 FN 2} O 4) Calcd. (%): 572.6357; Found (%): 572.6354. Elemental analysis (C ₃₆ H ₂₉ FN ₂ O ₄ ) calc. (%): C, 75.51%; H, 5.11%; N, 4.89%; found (%): C, 75.83%; H, 5.10%; , 4.90%.

(4) Step 4: 2-[2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-(9H-indazol-9-yl)ethoxy)phenyl) -Preparation of sodium propionate (CS-038)

243.7 g (6.09 mol) of NaOH and 6 L of anhydrous methanol were placed in a 30 L plastic bucket, and dissolved by stirring at room temperature. Then, 150 L of tetrahydrofuran and 3.45 kg (6.03 mol) of 2-(2-(4-fluorobenzoyl)phenylamino)-3-(4-(2-) were added to a stainless steel reactor (lined with polytetrafluoroethylene). (9H-carbazol-9-yl)ethoxy)phenyl)propionic acid was dissolved by stirring at room temperature, and the above methanol solution in which sodium hydroxide was dissolved was added to the reaction vessel, and stirring was continued for 30 minutes. Concentration in vacuo, removal of tetrahydrofuran and methanol gave a concentrate. The concentrate was dissolved in 10 L of dichloromethane, and added dropwise to 65 L of isopropyl ether at room temperature with stirring, and the mixture was separated by centrifugation, and the solid was collected and dried under vacuum at 105 ° C for 3 h to give 2-(2-(4-fluorobenzoyl) Sodium phenylamino)-3-(4-(2-(9H-indazol-9-yl)ethoxy)phenyl)propionate, weight 3.50 kg, yield 97%.

¹ H NMR (DMSO-d ₆ ): δ 2.88 (dd, 1H, CH ₂ ), 3.03 (dd, 1H, CH ₂ ), 3.86 (m, 1H, CH), 4.25 (t, 2H, CH ₂ ) , 4.73 (t, 2H, CH ₂ ), 6.36 (t, 1H, Ar-H), 6.59 (d, 2H, Ar-H), 6.65 (d, 1H, Ar-H), 7.00 (d, 2H, Ar-H), 7.18 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 7.31 (m, 2H, Ar-H), 7.43 (m, 2H, Ar-H), 7.55 (m, 2H, Ar-H), 7.64 (d, 2H, Ar-H), 8.13 (d, 2H, Ar-H), 8.73 (d, 1H, NH).

Elemental analysis (C ₃₆ H ₂₈ FN ₂ NaO _4· 0.5 H ₂ O): C 71.63, H 4.83, N 4.64; Found: C 71.62, H 4.89, N 4.56.

Pharmacodynamic experiment:

The experimental design was carried out using the protocol shown in the table below.

Table 1: Pharmacological Examples

Among them, each group code is defined as: MCS: normal methionine-choline supplement feed control group; MCD: methionine-choline-deficient feed model group; GS: MCD+5mg/kg GS-4997; GFT: MCD+3mg/kg GFT505 ;GG:MCD+2.5mg/kg GS-4997+1.5mg/kg GFT505; IVA:MCD+10mg/kg IVA337;GI: MCD+2.5mg/kg GS-4997+5mg/kg IVA337;CS:MCD+10mg /kg CS-038; GC: MCD + 2.5 mg / kg GS-4997 + 5 mg / kg CS-038.

Example 5 Effect of GS-4997 in combination with GFT505 and drug alone on NASH

In this example, the methionine-choline-deficient (MCD) diet was used to construct the NASH model. This model is an internationally recognized animal model with simple operation, high rate of mold formation, and caused steatohepatitis. The lesion is very similar to human NASH, so it has become one of the most important animal models for studying the pathogenesis of NASH and its prevention and treatment. The dose of GS-4997 used in this example is the result of previous background experiments. It was found that 5 mg/kg of GS-4997 can reduce the level of fibrosis markers Col1a1 and α-SMA in this animal model to a certain extent. Regulates the role of fatty acid synthesis and lipid metabolism. GFT505, as a PPARα/δ dual agonist, can exert lipid synthesis, metabolic regulation, and improve liver fibrosis in this model at a dose concentration of 3 mg/kg.

Experimental procedure: In this example, C57BL/6J mice (Nanjing University-Nanjing Institute of Biomedical Research) were fed with methionine-choline-deficient feed, and a non-alcoholic fatty liver model was established by diet for 8 weeks. Mice were acclimated to normal diet at least 7 days prior to the start of the study, and then the mice were randomly divided into 5 groups by weight, each group of 10 mice, respectively, a methionine-choline supplemented feed control group (MCS, Researchdiets, A02082003B). , methionine-choline deficiency feed model group (MCD, Researchdiets, A02082002B), methionine-choline deficiency feed + GS-4997 group (GS, 5 mg/kg, poqd), methionine-choline deficiency feed + GFT505 group (GFT , 3 mg/kg, poqd), and methionine-choline-deficient feed + GS-4997 (2.5 mg/kg) + GFT505 (1.5 mg/kg) group (GG, poqd) (Table 1). Compound intervention and MCD feed induction were performed simultaneously, and the mice were orally administered daily according to the above grouping. After 8 weeks of continuous intervention, mice were anesthetized with ether, serum was collected, and then the animals were euthanized and their livers were harvested for morphological, histological, and biochemical analysis. All tissues used for biochemical analysis were rapidly frozen in liquid nitrogen and stored at -80 ° C; tissues for histological analysis were fixed with 4% neutral buffered paraformaldehyde.

RESULTS: The serum levels of AST and ALT in the MCD model group were significantly higher, while the serum TC and TG levels were not significantly different from those in the MCS group, but the liver TG and TC levels in the MCD model group were significantly higher than those in the MCS group. group. GS-4997 and GFT505 alone were able to reduce serum AST levels by 21% (ns) and 27% (P < 0.05), respectively (Figure 1), 24% (ns) and 33% (P < 0.05) of serum ALT levels ( Figure 2), 24% (ns) and 41% (P < 0.05) liver TG levels (Figure 3), 28% (ns) and 33% (P < 0.05) liver TC levels (Figure 4), while GS The inhibitory rate of the above indicators after the combination of -4997 and GFT505 was 63% (p < 0.001) (Fig. 1), 57% (p < 0.001) (Fig. 2), and 61% (p < 0.01) (Fig. 3). And 58% (p < 0.01) (Figure 4).

Liver tissue sections were observed by HE staining. It can be found that the MCD group has blurred hepatic lobules, narrowed hepatic sinus, and hepatocytes are swollen. Oil droplets of different sizes can be seen in the cells, showing balloon-like changes, and a large number of inflammatory cells. Infiltrate to form a inflammatory foci. Using the histological pathology score of NAS, the score of the MCD model group was 7.3±0.7, while the average score of the normal control MCS group was only 1.3±0.5. The mice with GS-4997 and GFT505 alone scored 5.9 ± 0.7 and 6.2 ± 1.0, respectively, after intervention with compound administration, and decreased by 19.2% (p < 0.001) and 15.7%, respectively, compared with the MCD group. 0.05). The NAS score of the GS-4997 and GFT505 co-administered groups was 4.6 ± 1.0, which was 36.3% lower than that of the MCD group (p < 0.001). Further, the NAS scores of the GS-4997 and GFT505 co-administered groups were reduced by 22% (P<0.01) and 26% (P<0.01), respectively, compared with the GS-4997 and GFT505 alone administration groups (Table 2 and Figure 5).

Using Sirius red staining to evaluate liver fibrosis in mice, it was found that the staining area of MCD model mice was significantly increased, accompanied by a significant increase in fibrosis markers α-SMA levels and Col1a1 expression. GS-4997 and GFT505 alone were able to reduce the staining area by 23% (ns) and 18% (ns), respectively (Figure 6), reducing α- induced by 43% (p < 0.05) and 16% (ns) models, respectively. Increased SMA levels (Figure 7), 55% (p < 0.05) and 22% (ns) models, respectively, induced an increase in Col1a1 expression (Figure 8). The combination of GS-4997 and GFT505 inhibited the above three indicators by 53% (p<0.01) (Fig. 6), 65% (p<0.01) (Fig. 7) and 83% (p<0.001). (Figure 8). Further, the fibrosis staining area of the GS-4997 and GFT505 co-administered groups was reduced by 38.9% (P < 0.001) and 42.8% (P<0.001), respectively, compared with the GS-4997 and GFT505 alone administration groups. <0.001) (Fig. 6).

Conclusion: From the above experimental results, it can be seen that the combined effects of GS-4997 and GFT505 on NASH liver lipid accumulation, liver function index, NAS score and liver fibrosis are significantly better than GS-4997 alone. Or GFT505. The specific performance was as follows: the combination of the most representative NAS scores and fibrosis in the NASH pathological group was better than the single administration group, and there was a statistically significant difference (NAS score: GS: GG = 5.9: 4.6, P < 0.01; GFT: GG = 6.2: 4.6, P < 0.01; Fibrosis staining area: GS: GG = 23%: 53%, P < 0.001; GFT: GG = 18%: 53%, P < 0.001). For other indicators such as AST (GS:GFT:GG=21%:27%:63%), ALT (GS:GFT:GG=24%:33%:57%), TG(GS:GFT:GG =24%: 41%: 61%), TC (GS: GFT: GG = 28%: 33%: 58%), α-SMA (GS: GFT: GG = 43%: 16%: 65%), Col1a1 The treatment intensity (GS: GFT: GG = 55%: 22%: 83%) was also greater than that of the single administration group. In summary, the combination of GS-4997 and GFT505 showed superior anti-NASH efficacy compared with monotherapy.

Example 6 Effect of GS-4997 in combination with IVA337 and drug alone on NASH

The dose of IVA337 in this example was determined by previous background experiments. The results showed that 10 mg/kg of IVA337 can effectively inhibit the expression levels of lipid metabolism genes such as SCD1, CPT1 and CPT2 in this animal model, and reduce liver lipid degeneration in mice. The scores inhibit the expression levels of inflammation-related genes such as IL-1β, IL-6, MCP-1, etc., and reduce tissue fibrosis scores.

Experimental procedure: In this example, C57BL/6J mice (Nanjing University-Nanjing Institute of Biomedical Research) were fed with methionine-choline-deficient feed, and a non-alcoholic fatty liver model was established by diet for 8 weeks. Mice were acclimated to normal diet at least 7 days prior to the start of the study, and then the mice were randomly divided into 5 groups by weight, each group of 10 mice, respectively, a methionine-choline supplemented feed control group (MCS, Researchdiets, A02082003B). , methionine-choline deficiency feed model group (MCD, Researchdiets, A02082002B), methionine-choline deficiency feed + GS-4997 group (GS, 5 mg/kg, poqd), methionine-choline deficiency feed + IVA337 group (IVA , 10 mg/kg, poqd) and methionine-choline-deficient feed + GS-4997 (2.5 mg/kg) + IVA337 (5 mg/kg) group (GI, poqd) (Table 1). Compound intervention and MCD feed induction were performed simultaneously, and the mice were orally administered daily according to the above grouping. After 8 weeks of continuous intervention, mice were anesthetized with ether, serum was collected, and then the animals were euthanized and their livers were harvested for morphological, histological, and biochemical analysis. All tissues used for biochemical analysis were rapidly frozen in liquid nitrogen and stored at -80 ° C; tissues for histological analysis were fixed with 4% neutral buffered paraformaldehyde.

RESULTS: The levels of AST and ALT in the serum of the MCD model group were significantly increased, while the levels of serum TC and TG were not significantly different from those in the MCS group, but the liver TG and TC levels in the MCD model group were significantly higher than those in the MCS group. group. GS-4997 and IVA337 alone were able to reduce serum AST levels by 21% (ns) and 36% (P < 0.05), respectively (Figure 1), 24% (ns) and 43% (P < 0.05) of serum ALT levels ( Figure 2), 24% (ns) and 48% (P < 0.05) liver TG levels (Figure 3), 28% (ns) and 34% (P < 0.05) liver TC levels (Figure 4), while GS The inhibition rates of the above indicators after the combination of -4997 and IVA337 were 65% (p < 0.001) (Fig. 1), 61% (p < 0.001) (Fig. 2), and 73% (p < 0.01) (Fig. 3). And 59% (p < 0.001) (Figure 4).

Liver tissue sections were observed by HE staining. It can be found that the MCD group has blurred hepatic lobules, narrowed hepatic sinus, and hepatocytes are swollen. Oil droplets of different sizes can be seen in the cells, showing balloon-like changes, and a large number of inflammatory cells. Infiltration, into the inflammatory foci, using NAS histopathological scoring results showed that the MCD model group score was 7.3 ± 0.7, while the normal control MCS group average score was only 1.3 ± 0.5. After administration of the compound, the mice with GS-4997 and IVA337 alone scored 5.9 ± 0.7 and 5.1 ± 0.9, respectively, which was 19.2% (p < 0.001) and 30.0% lower than the MCD group, respectively (p < 0.001). The NAS score of the GS-4997 and IVA337 co-administered groups was 3.9 ± 1.2, which was 46.7% lower than that of the MCD group (p < 0.001). Further, the NAS scores of the GS-4997 and IVA337 co-administered groups were reduced by 33.9% (P < 0.001) and 23.5% (P < 0.05), respectively, compared with the GS-4997 and IVA337 alone administration groups (Table 2 and Figure 5).

Using Sirius red staining to evaluate liver fibrosis in mice, it was found that the staining area of MCD model mice was significantly increased, accompanied by α-SMA levels and increased Col1a1 expression. GS-4997 and IVA337 alone were able to reduce the stained area by 23% (ns) and 17% (ns), respectively (Figure 6), reducing 43% (p < 0.05) and 37% (p < 0.05) of α-SMA, respectively. Elevated levels (Figure 7) reduced the expression of Col1a1 by 55% (p < 0.05) and 44% (p < 0.05), respectively (Figure 8). The inhibitory rates of GS-4997 and IVA337 for the above three indicators were 52% (p<0.01) (Fig. 6), 76% (p<0.001) (Fig. 7) and 94% (p<0.001). Figure 8). Further, the fibrotic staining area of the GS-4997 and IVA337 co-administered groups was reduced by 37.3% (P<0.01) and 42.7% (P<0.01) compared with the GS-4997 and IVA337 alone groups. 0.001) (Figure 6).

Conclusion: From the above experimental results, it can be seen that the combined effects of GS-4997 and IVA337 on NASH liver lipid accumulation, liver function index, NAS score and liver fibrosis are significantly better than GS-4997 alone. Or IVA337. The specific performance was as follows: the combination of the most representative NAS scores and fibrosis in the NASH pathological group was better than the single administration group, and there was a statistically significant difference (NAS score: GS: GI = 5.9: 3.9, P < 0.001; IVA: GI = 5.1: 3.9, P < 0.05; fibrotic staining area: GS: GI = 23%: 52%, P < 0.01; IVA: GI = 17%: 52%, P < 0.001). For other indicators such as AST (GS: IVA: GI = 21%: 36%: 65%), ALT (GS: IVA: GI = 24%: 43%: 61%), TG (GS: IVA: GI) =24%: 48%: 73%), TC (GS: IVA: GI = 28%: 34%: 59%), α-SMA (GS: IVA: GI = 43%: 37%: 76%), Col1a1 The treatment intensity (GS: IVA: GI = 55%: 44%: 94%) was also greater than that of the single administration group. In summary, the combination of GS-4997 and IVA337 showed superior anti-NASH efficacy compared with monotherapy.

Example 7 Effect of GS-4997 and CS-038 in combination with drug alone on NASH

In this example, CS-038 is a PPARα/δ/γ total agonist for type 2 diabetes, and there is no relevant research data disclosed in the field of NASH treatment. However, in view of the potential value of PPAR in the treatment of NASH, we found that 10 mg/kg of CS-038 can effectively reduce the levels of TG, TC and LDL-C in liver and serum and regulate lipid metabolism in mice. At the same time, it also has an inhibitory effect on the expression of inflammation-related genes, and can also effectively reduce the number of inflammation foci in histopathological analysis.

Experimental procedure: In this example, C57BL/6J mice (Nanjing University-Nanjing Institute of Biomedical Research) were fed with methionine-choline-deficient diet, and a non-alcoholic fatty liver model was established by diet for 8 weeks. Mice were acclimated to normal diet at least 7 days prior to the start of the study, and then the mice were randomly divided into 5 groups by weight, each group of 10 mice, respectively, a methionine-choline supplemented feed control group (MCS, Researchdiets, A02082003B). , methionine-choline-deficient feed model group (MCD, Researchdiets, A02082002B), methionine-choline-deficient feed + GS-4997 group (GS, 5 mg/kg, poqd), methionine-choline-deficient feed + CS-038 group (CS, 10 mg/kg, poqd) and methionine-choline-deficient diet + GS-4997 (2.5 mg/kg) + CS-038 (5 mg/kg) group (GC, poqd) (Table 1). Compound intervention and MCD feed induction were performed simultaneously, and the mice were orally administered daily according to the above grouping. After 8 weeks of continuous intervention, mice were anesthetized with ether, serum was collected, and then the animals were euthanized and their livers were harvested for morphological, histological, and biochemical analysis. All tissues used for biochemical analysis were rapidly frozen in liquid nitrogen and stored at -80 ° C; tissues for histological analysis were fixed with 4% neutral buffered paraformaldehyde.

RESULTS: The levels of AST and ALT in the serum of the MCD model group were significantly increased, while the levels of serum TC and TG were not significantly different from those in the MCS group, but the liver TG and TC levels in the MCD model group were significantly higher than those in the MCS group. group. Administration of GS-4997 and CS-038 alone reduced serum AST levels (Figure 1), 24% (ns) and 28% (ns) serum ALT levels by 21% (ns) and 18% (ns), respectively (Figure 2 ), 24% (ns) and 33% (ns) of liver TG levels (Figure 3), 28% (ns) and 38% (ns) of liver TC levels (Figure 4), while GS-4997 and CS-038 The inhibition rates of the above indicators after combination therapy were 55% (p < 0.001) (Fig. 1), 48% (p < 0.001) (Fig. 2), 57% (p < 0.01) (Fig. 3) and 53% (Fig. 3). p < 0.01) (Fig. 4).

Liver tissue sections were observed by HE staining. It can be found that the MCD group has blurred hepatic lobules, narrowed hepatic sinus, and hepatocytes are swollen. Oil droplets of different sizes can be seen in the cells, showing balloon-like changes, and a large number of inflammatory cells. Infiltration, into the inflammatory foci, using NAS histopathological scoring results showed that the MCD model group score was 7.3 ± 0.7, while the normal control MCS group average score was only 1.3 ± 0.5. After administration of compound intervention, the NAS scores of mice given GS-4997 and CS-038 alone were 5.9 ± 0.7 and 6.0 ± 1.2, respectively, which was 19.2% (p < 0.001) and 17.7% lower than the MCD group, respectively. p<0.01). The NAS score of the GS-4997 and CS-038 co-administered groups was 4.4 ± 0.8, which was 39.0% lower than that of the MCD group (p < 0.001). Further, the NAS scores of the GS-4997 and CS-038 co-administered groups were reduced by 25.4% (P < 0.001) and 26.7% (P < 0.01), respectively, compared with the GS-4997 and CS-038 alone administration groups. (Table 2 and Figure 5).

Using Sirius red staining to evaluate liver fibrosis in mice, it was found that the staining area of MCD model mice was significantly increased, accompanied by α-SMA levels and increased Col1a1 expression. GS-4997 and CS-038 alone were able to reduce the stained area by 23% (ns) and 15% (ns), respectively (Figure 6), reducing 43% (p < 0.05) and 21% (ns) of α-SMA, respectively. Elevated levels (Figure 7) reduced the expression of Col1a1 by 55% (p < 0.05) and 24% (ns), respectively (Figure 8). The inhibition rates of GS-4997 and CS-038 for the above three indicators were 49% (p<0.01) (Fig. 6), 67% (p<0.01) (Fig. 7) and 79% (p<0.01). ) (Figure 8). Further, the fibrotic staining area of the GS-4997 and CS-038 co-administered groups was reduced by 33.5% (P<0.01) and 38.0, respectively, compared with the GS-4997 and CS-038 alone administration groups. % (P < 0.001) (Fig. 6).

Conclusion: From the above experimental results, it can be seen that the combined effects of GS-4997 and CS-038 on NASH liver lipid accumulation, liver function index, NAS score and liver fibrosis are significantly better than GS alone. -4997 or CS-038. The specific performance was as follows: the combination of the most representative NAS scores and fibrosis in the NASH pathological group was better than the single administration group, and there was a statistically significant difference (NAS score: GS: GC = 5.9: 4.4, P < 0.001; CS: GC = 6.0: 4.4, P < 0.01; Fibrosis staining area: GS: GC = 23%: 49%, P < 0.01; CS: GC = 15%: 49%, P < 0.001). For other indicators such as AST (GS:CS:GC=21%:18%:55%), ALT (GS:CS=GC=24%:28%:48%), TG(GS:CS:GC =24%: 33%: 57%), TC (GS: CS: GC = 28%: 38%: 53%), α-SMA (GS: CS: GC = 43%: 21%: 67%), Col1a1 The treatment intensity (GS:CS:GC=55%: 24%: 79%) was also greater than that of the single administration group. Comprehensively, the combination of GS-4997 and CS-038 showed superior anti-NASH efficacy compared with monotherapy.

Table 2: NAS scores for each group of mice

In the description of the present specification, the description of the terms "embodiment", "example" and the like means that a specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. . In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, it is understood that the foregoing embodiments are illustrative and not restrictive Variations, modifications, alterations and variations of the above-described embodiments are possible within the scope of the invention.

Claims (10)

1. A pharmaceutical composition comprising a first component and a second component, wherein

   The first component is at least one selected from the group consisting of an ASK1 inhibitor and a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, an ester, and a solvate;

   The second component is at least one selected from the group consisting of a PPAR modulator and a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, an ester, and a solvate.
2. The pharmaceutical composition according to claim 1, wherein said first component is selected from the group consisting of

   5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-[6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine -2-yl]-4-methylbenzamide, 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-[3-(4-cyclopropyl-4H-1,2, 4-triazol-3-yl)phenyl]pyridine-2-carboxamide and 6-amino-9-[(3R)-1-(but-2-ynyl)pyrrolidin-3-yl]-7- At least one of (4-phenoxyphenyl)-7,9-dihydro-8H-indole;

   Optionally, the second component is a PPAR alpha / delta dual agonist and / or a PPAR alpha / delta / gamma pan agonist;

   Preferably, the second component is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy ]-2-methylpropionic acid, 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl]butyric acid and 2-[2 At least one of -(4-fluorobenzoyl)phenyl)amino]-3-(4-(2-carbazolylethoxy)phenyl)-propionate.
3. The pharmaceutical composition according to any one of claims 1 to 2, wherein the weight ratio of the first component to the second component is 1: (0.1 to 400), preferably 1: (0.5 to 200);

   Preferably, the weight ratio of GS-4997 and GFT505 in the pharmaceutical composition is 1: (0.5 to 40), preferably 1: (1.5 to 20);

   Preferably, the weight ratio of GS-4997 to IVA337 in the pharmaceutical composition is 1: (2 to 400), preferably 1: (10 to 400), more preferably 1: (20 to 200);

   Preferably, the weight ratio of GS-4997 to CS038 in the pharmaceutical composition is 1: (2 to 40), preferably 1: (4 to 20).
4. A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 1 to 3 as an active ingredient, optionally, the pharmaceutical preparation further comprising a pharmaceutically acceptable carrier.
5. The pharmaceutical preparation according to claim 4, wherein the content of the first component is from 1 mg to 100 mg, preferably from 5 mg to 50 mg; and the content of the second component is from 1 mg to 1500 mg, preferably from 5 mg to 1200 mg;

   Preferably, the pharmaceutical preparation has a content of GS-4997 of 1 mg to 50 mg, preferably 2 mg to 20 mg;

   Preferably, the content of IVA337 in the pharmaceutical preparation is from 100 mg to 1500 mg, preferably from 200 mg to 1200 mg;

   Preferably, the pharmaceutical preparation has a GFT505 content of 10 mg to 150 mg, preferably 40 mg to 120 mg;

   Preferably, the pharmaceutical preparation has a CS038 content of 10 mg to 150 mg, preferably 15 mg to 100 mg.
6. The use of the pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for: reducing total cholesterol, triglyceride, low density lipoprotein cholesterol, alanine aminotransferase, aspartame Acid aminotransferase, and/or alkaline phosphatase levels, and/or decreased liver coefficient, and/or decreased fibrosis marker Col1a1 expression level and/or α-SMA expression level, and/or elevated high density lipoprotein Cholesterol, and / or improve NAS score and / or fibrosis score.
7. Use of the pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for: preventing and/or treating a disease associated with NAFLD or NAFLD,

   Optionally, the medicament is for prevention and/or treatment: nonalcoholic fatty liver, nonalcoholic steatohepatitis, nonalcoholic liver fibrosis, nonalcoholic cirrhosis, insulin resistance, hyperglycemia, metabolic synthesis Signs, obesity with hyperlipidemia, type 2 diabetes, cardiovascular disease, liver failure, and / or liver cancer.
8. Use of the pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for the prevention and/or treatment of NASH.
9. Use of the pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for preventing and/or treating nonalcoholic fatty liver.
10. The use according to any one of claims 6 to 9, wherein the pharmaceutical composition is administered orally, or as a mixture, or administered in a sequential manner at different times. Optionally, the pharmaceutical composition is administered in an amount of from 1 to 1500 mg/day.

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