WO2019024776A1 - Pharmaceutical composition and use thereof - Google Patents
Pharmaceutical composition and use thereof Download PDFInfo
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- WO2019024776A1 WO2019024776A1 PCT/CN2018/097380 CN2018097380W WO2019024776A1 WO 2019024776 A1 WO2019024776 A1 WO 2019024776A1 CN 2018097380 W CN2018097380 W CN 2018097380W WO 2019024776 A1 WO2019024776 A1 WO 2019024776A1
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- JPSOSJUCPVQRSP-NRFANRHFSA-N COC([C@H](Cc(cc1)ccc1O)Nc(cccc1)c1C(c(cc1)ccc1F)=O)=O Chemical compound COC([C@H](Cc(cc1)ccc1O)Nc(cccc1)c1C(c(cc1)ccc1F)=O)=O JPSOSJUCPVQRSP-NRFANRHFSA-N 0.000 description 1
- OPAKXXJCWSHZGJ-QHCPKHFHSA-N COC([C@H](Cc(cc1)ccc1OCCBr)Nc(cccc1)c1C(c(cc1)ccc1F)=O)=O Chemical compound COC([C@H](Cc(cc1)ccc1OCCBr)Nc(cccc1)c1C(c(cc1)ccc1F)=O)=O OPAKXXJCWSHZGJ-QHCPKHFHSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/428—Thiazoles condensed with carbocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- 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.
- 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.
- NASH Nonalcoholic fatty liver disease
- NAFLD nonalcoholic fatty liver
- NASH nonalcoholic steatohepatitis
- 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.
- 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.
- a pharmaceutical composition according to a particular embodiment of the invention comprises: a first component and a second component.
- 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 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 ⁇ -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 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.
- the patient can show a composite NAS score of ⁇ 3, ⁇ 2 or ⁇ 1, and the fibrosis does not deteriorate.
- 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.
- 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.
- 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.
- 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.
- 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.
- the second component is a PPAR alpha / delta dual agonist and / or a PPAR alpha / delta / gamma agonist.
- 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
- MAP3K5 Apoptosis signal-regulated kinase-1
- 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) ⁇ as an energy regulator can reduce the deposition of TG in cells, increase the uncoupling of ⁇ -oxidation and oxidative phosphorylation, and reduce body weight. At the same time, PPAR ⁇ can be accurate. Regulates the body's inflammatory response, inhibits macrophage production of MCP1, IL1 ⁇ , MMP9 and other inflammatory mediators, while also inhibiting endotoxin-induced COX2 expression, thereby reducing inflammation. PPAR ⁇ and PPAR ⁇ play a cross-talk role in hepatocytes and macrophages.
- PPAR ⁇ can increase liver fatty acid and lipoprotein metabolism, and PPAR ⁇ 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.
- ASK1 inhibitor 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).
- MRE magnetic resonance elastography
- MRI-PDFF magnetic resonance imaging - proton density fat fraction evaluation
- PPAR ⁇ / ⁇ 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.
- 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.
- 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).
- 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.
- 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
- 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.
- the invention provides a pharmaceutical preparation comprising the pharmaceutical composition described above as an active ingredient.
- the preparation exerts the pharmacological action of the active ingredient and further has the efficacy of the pharmaceutical composition.
- 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.
- the above pharmaceutical preparation comprises, in addition to the pharmaceutical composition described above as an active ingredient, a pharmaceutically acceptable carrier.
- 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.
- the pharmaceutical composition can be selected from the group consisting of pharmaceutically acceptable solvents, propellants
- 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.
- 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.
- 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 ⁇ -SMA expression level, and / Or raise high density lipoprotein cholesterol, and / or improve NAS score and / or fibrosis score.
- 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.
- 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.
- 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.
- 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.
- the pharmaceutical composition in the use of a pharmaceutical composition for the preparation of a medicament, is administered orally, or as a mixture, or administered in a sequential manner at different times.
- the pharmaceutical composition in the use of the pharmaceutical composition for the preparation of a medicament, is administered in an amount of from 1 to 1500 mg/day.
- 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.
- 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 ⁇ -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.
- the weight ratio of the ASK1 inhibitor to the PPAR modulator is 1: (0.1 to 400), preferably 1: (0.5 to 200).
- the weight ratio of GS-4997 to GFT505 is 1: (0.5 to 40), preferably 1: (1.5 to 20).
- the weight ratio of GS-4997 to IVA337 is 1: (2 to 400), preferably 1: (10 to 400), more preferably 1: (20 to 200).
- the weight ratio of GS-4997 and CS038 is 1: (2 to 40), preferably 1: (4 to 20).
- 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
- 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+2.5m
- Figure 2 shows serum ALT levels of mice in each group according to Examples 5-7 of the present invention
- 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
- CS MCD+10mg/kg CS-038
- GC MCD+2.5mg/kg GS-4997+5mg/kg CS-038
- MCS normal methionine-choline supplement feed control group
- MCD methionine-choline-deficient feed model group
- GS MCD +5mg/kg GS-4997
- 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 ⁇ -SMA levels in mice of each group according to Examples 5-7 of the present invention.
- 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
- MCD+2.5mg/kg GS-4997+5mg/kg CS-038 MCD+2.5mg/kg GS-4997+5mg/kg CS-0
- 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.
- MCS normal methionine-choline supplement feed control group
- MCD methionine-choline-deficient feed model group
- GS MCD+5mg/kg
- the compounds of the present invention were prepared by the prior art (CN104080771A, CN1257893C, CN101248044A, CN1688532A).
- 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 (ariessertib, GS-4997)
- Step 1 Preparation of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-hydroxyphenyl]prop-2-en-1-one (Intermediate 3)
- Step 2 Preparation of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropoxycarbonyldimethylmethoxyphenyl]propan-2- En-1-one (intermediate 4)
- Step 3 Preparation of 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy]-2 -methylpropionic acid (Elafibranor, GFT505)
- Step 1 Preparation of methyl 6-(5-chloro-2-nitrophenyl)-hex-5-ynoate (intermediate 3)
- Step 2 Preparation of methyl 6-(2-amino-5-chlorophenyl)-hex-5-ynoate (intermediate 4)
- Step 3 Preparation of methyl 6-[5-chloro-2-(1,3-benzothiazol-6-ylsulfonylamino)phenyl]-hex-5-ynoate (intermediate 5)
- Step 1 Preparation of 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl)-propionic acid methyl ester (CHIG-003)
- 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.
- Step 2 2-[(2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-bromoethoxy)phenyl)-propionic acid methyl ester (CHIG- Preparation of 005)
- 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.
- 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 ⁇ -SMA in this animal model to a certain extent. Regulates the role of fatty acid synthesis and lipid metabolism. GFT505, as a PPAR ⁇ / ⁇ dual agonist, can exert lipid synthesis, metabolic regulation, and improve liver fibrosis in this model at a dose concentration of 3 mg/kg.
- mice 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).
- MCS methionine-choline supplemented feed control group
- 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.
- 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.
- 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.
- 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).
- 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).
- 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-1 ⁇ , IL-6, MCP-1, etc., and reduce tissue fibrosis scores.
- mice 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).
- MCS methionine-choline supplemented feed control group
- 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.
- 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.
- 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).
- 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).
- 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).
- CS-038 is a PPAR ⁇ / ⁇ / ⁇ total agonist for type 2 diabetes, and there is no relevant research data disclosed in the field of NASH treatment.
- 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.
- 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.
- mice 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).
- MCS methionine-choline supplemented feed control group
- 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.
- 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.
- 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.
- 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).
- 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. .
- the schematic representation of the above terms does not necessarily mean the same embodiment or example.
- the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.
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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
本申请要求2017年7月31日向中国国家知识产权局提交的专利申请号为201710641321.3,发明名称为“药物组合物及其用途”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。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.
本发明属于生物医药领域,具体而言,本发明涉及一种药物组合物及其用途,尤其是涉及一种有效治疗或预防非酒精性脂肪肝病的药物组合物及其制备方法和应用。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.
肝脏是脂肪代谢的重要器官,正常情况下脂肪占肝脏总重量的3%~5%,正常人每100g肝湿重含4~5g脂类,其中磷脂占50%以上,三酰甘油占20%,游离脂肪酸占20%,胆固醇约7%,其余为胆固醇脂等。当肝细胞内脂质蓄积超过肝湿重的5%,或组织学上每单位面积见1/3以上肝细胞脂肪变时即称为脂肪肝。非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)指除酒精和其他明确的肝损伤(如药物性肝损伤)之外的因素所导致的肝细胞内脂肪过度沉积而引起的疾病,近年来发展迅速,成为最普遍的肝脏疾病之一。欧美等西方发达国家的成人NAFLD患病率为20%~33%,亚洲国家NAFLD患者数增长迅速且呈低龄化发病趋势,中国上海、广州和香港等发达地区成人NAFLD患病率约在15%。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%. .
NAFLD的疾病谱从非酒精性单纯性肝脏脂肪变性(Non alcoholic fatty liver,NAFL)到非酒精性脂肪性肝炎(Non alcoholic steatohepatitis,NASH),以致发展为肝纤维化及肝硬化甚至肝细胞癌,其发病机制与胰岛素抵抗、氧化应激和脂质过氧化、胆汁酸代谢紊乱、自噬等有关。NAFLD中约有1/3~1/2为NASH,单纯性脂肪肝随访10~20年发展为肝硬化的概率为0.6%~3%,而NASH随访10~15年肝硬化的发生率高达15%~25%,其中30%~40%将会死于肝癌(发生肝硬化后以每年1%病例发生)、 肝功能衰竭和移植后复发。因此非酒精性脂肪肝病已成为当代肝病领域的新挑战。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.
目前,治疗非酒精性脂肪肝病的药物尚待进一步研究。Currently, drugs for the treatment of nonalcoholic fatty liver disease remain to be further studied.
发明内容Summary of the invention
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种有用的商业选择。为此,本发明的一个目的在于提出一种有效治疗或预防非酒精性脂肪肝病及其关联病症的药物组合物。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.
根据本发明的一个方面,本发明提出了一种药物组合物。根据本发明具体实施例的药物组合物包括:第一组分和第二组分。其中第一组分为选自ASK1抑制剂及其前体、活性代谢产物、立体异构体、药学上可接受的盐、酯类和溶剂合物中的至少一种;第二组分为选自PPAR调节剂及其前体、活性代谢产物、立体异构体、药学上可接受的盐、酯类和溶剂合物中的至少一种。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.
因此,本发明所述药物组合物具有降低总胆固醇、甘油三酯、低密度脂蛋白胆固醇、丙氨酸氨基转氨酶、天门冬氨酸氨基转移酶和/或碱性磷酸酶的水平,和/或降低肝系数,和/或降低纤维化标志物Col1a1表达水平和/或α-SMA表达水平,和/或升高高密度脂蛋白胆固醇,和/或改善NAS评分和/或纤维化评分的功效,进而在预防和/或治疗NAFLD及其关联的病症方面具有显著效果。所述NAFLD包括:非酒精性脂肪肝、非酒精性脂肪性肝炎、非酒精性肝纤维化、非酒精性肝硬化。所述NAFLD关联的病症,包括但不限于,二次NAFLD,胰岛素抗性,高血糖,代谢综合征,肥胖伴高脂血症,2型糖尿病,心血管疾病,肝衰竭,肝癌等。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.
本发明所述药物组合物尤其对预防和/或治疗非酒精性脂肪肝病效果优异,特别是对预防、延缓和治疗非酒精性脂肪肝、非酒精性脂肪性肝炎和非酒精性肝纤维化上效果优异,在改善NAS评分上效果显著。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.
非酒精性脂肪肝病活动性评分(NAS)被定义为脂肪变性(0~3)、小叶炎症(0~3)和气胀(0~2)的值的未加权和,由此提供0~8的NAS评分(参见Kleinen等,Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease,Hepatology,第41卷,第6期,2005,第1313-1321页)。不同程度纤维化患者NAS积分不同,其中NAS评分越高,纤维化程度越重。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.
根据本公开治疗NASH的患者在治疗前可显示出≥4的NAS评分,其中对于脂肪变性和小叶炎症各自的最小评分为1,加上气胀或至少为1的窦纤维变性,以及可能或确 切的脂肪性肝炎的发现。在给药/治疗后(例如一年),患者可显示出的复合NAS评分为≤3、≤2或≤1,而且纤维变性没有恶化。作为另一选择,患者可显示出NAS中至少两个NAS分量改善了≥2的值,而且纤维变性没有恶化。作为另一选择,患者可显示出NAS评分改善了≥3、4、5、6、7或8。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.
脂肪变性可广义理解为描述了涉及脂质在肝脏中的异常积聚的过程,其妨碍了正常的肝功能。肝活组织检查能够分析患者的脂肪变性并对其评分,评分范围为0~3。根据本公开治疗NASH的患者的脂肪变性评分可以为1、2或3,如约2~约3。治疗后,期望患者表现出脂肪变性没有恶化,或者脂肪变性评分下降了至少1,或者脂肪变性评分下降了2或3。脂肪变性通常分级如下:评分为1表示小于33%的肝细胞存在脂肪滴,评分为2表示33%~66%的肝细胞中观察到脂肪滴,评分为3表示大于66%的肝细胞中观察到脂肪滴。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.
小叶炎症也可经肝活组织检查评价,并以0~3的值来评分。要治疗NASH的患者的小叶炎症评分可以是1、2或3,或者1~2或2~3的范围。治疗后,患者的小叶炎症评分可下降至少1,或者小叶炎症评分下降2或3,以及小叶炎症评分至少没有恶化。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.
肝细胞的气胀通常以0~2的值来评分,并且根据本公开治疗NASH的患者的气胀评分可为0~2,包括1或2的具体值,或者1~2的分数范围。治疗后,患者可显示出气胀评分至少没有恶化,或者气胀评分值下降了至少1,或者气胀评分值下降了2。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.
纤维变性也可经肝活组织检查评价,并以0~4的值评分,该评分定义如下:0表示无纤维变性;1表示窦周或门静脉周纤维变性;1a表示轻度区域3的窦周纤维变性;1b表示中度区域3的窦周纤维变性;1c表示门静脉/门静脉周纤维变性;2表示窦周和门静脉/门静脉周纤维变性;3表示桥脉纤维变性;且4表示肝硬化。根据本公开治疗的患者的纤维变性阶段评分可以为0~3,包括0、1、1a、1b、1c、2或3,并且纤维变性阶段评分可至少为1a。治疗后,患者的纤维变性评分可以至少不比基线评分差,或者纤维变性阶段评分下降了至少一级,或者至少两或三级。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.
根据本发明的实施例,所述第一组分为选自5-(4-环丙基-1H-咪唑-1-基)-2-氟-N-[6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-基]-4-甲基苯甲酰胺(GS-4997)、4-(4-环丙基-1H-咪唑-1-基)-N-[3-(4-环丙基-4H-1,2,4-三唑-3-基)苯基]吡啶-2-甲酰胺和6-氨基-9-[(3R)-1-(丁-2-炔酰基)吡咯烷-3-基]-7-(4-苯氧基苯基)-7,9-二氢-8H-嘌呤中的至少一种。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.
根据本发明的实施例,所述第二组分为PPARα/δ双重激动剂和/或PPARα/δ/γ泛激 动剂。根据本发明的具体实施例,所述第二组分为2-[2,6-二甲基-4-[3-[4-(甲硫基)苯基]-3-氧-1-丙烯基]苯氧基]-2-甲基丙酸(GFT505)、4-[1-(1,3-苯并噻唑-6-基磺酰基)-5-氯-1H-吲哚-2-基]丁酸(IVA337)和2-[2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-卡巴唑基乙氧基)苯基)-丙酸钠(CS-038)中的至少一种。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).
凋亡信号调节激酶-1(ASK1)又被称为MAP3K5,是一种在体内具有广泛生物学效应的激酶。大量研究表明,ASK1在许多应激相关疾病中发挥了重要作用,包括肿瘤,心血管疾病,神经退行性疾病等。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.
过氧化物酶体增殖物激活受体(PPAR)δ作为能量调节剂,可减少细胞内TG的沉积,提高细胞β氧化和氧化磷酸化的解偶联作用,以及减轻体重;同时PPARδ又能精确调控机体的炎症反应,抑制巨噬细胞产生MCP1、IL1β、MMP9等炎症介质,同时还能抑制内毒素诱导的COX2表达,从而减轻炎症。PPARα与PPARδ在肝实质细胞和巨噬细胞发挥交叉对话(cross-talk)作用,PPARα能增加肝脂肪酸和脂蛋白代谢,PPARδ抑制巨噬细胞产生炎症因子,从而改善代谢综合症。常用的PPARα/δ双重激动剂有:天然激动剂,例如:亚油酸、亚麻酸和二十碳五烯酸(EPA);合成激动剂,例如:噻唑烷二酮(thiazolidinedione,TZD),羧酸类激动剂。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.
近来研究表明ASK1抑制剂及PPAR激动剂的效果及安全性取得较好结论。据报道,ASK1抑制剂(GS-4997)对NASH患者有一定疗效:患者接受GS-4997治疗24周后,在肝脏疾病严重程度多个评价指标方面均表现出改善,包括肝纤维化回归(regression)、肝硬化进程、肝脏硬度(采用磁共振弹性成像技术评价,MRE)、肝脂肪含量(采用磁共振成像-质子密度脂肪分数评价,MRI-PDFF)。但GS-4997和单抗类药物simtuzumab(SIM)联合用药,效果没有显示比单独使用GS-4997更好。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.
另外,据报道,PPARα/δ双重激动剂GFT505在改善NAS评分方面也具有一定作用。PPARα/δ/γ泛激动剂(IVA337)在MCD和foz/foz动物模型上也显示对NAS评分有改善。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.
本发明实验具体研究了GS-4997与GFT505、或者GS-4997与IVA337、或者GS-4997与CS038的联合用药,令人惊讶的发现,上述联合用药取得了意料不到的技术效果,二者发挥协同作用,对NAFLD,包括非酒精性脂肪肝和非酒精性脂肪性肝炎的治疗比各有效成分单独用药有明显促进作用。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.
根据本发明,所述药物组合物中第一组分与第二组份的重量比为1:(0.1~400),优选为1:(0.5~200)。由此通过采用上述配比,可以进一步提高二者协同效应,进而 提高治疗NAFLD,特别是非酒精性脂肪肝和NASH的效果。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.
在本发明的优选实施方式中,所述药物组合物中GS-4997和GFT505的重量比为1:(0.5~40),优选为1:(1.5~20);所述药物组合物中GS-4997和IVA337的重量比为1:(2~400),优选为1:(10~400),更优选为1:(20~200);所述药物组合物中GS-4997和CS038的重量比为1:(2~40),优选为1:(4~20)。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).
根据本发明,上述药物组合物中各种活性成分(即,ASK1抑制或PPAR调节剂)每天的治疗或预防有效剂量的非限制性范围分别为0.01~100mg/kg体重,优选为0.02~80mg/kg体重,更优选0.05~50mg/kg体重。值得注意的是,计量值可以随需要缓解的症状的严重程度和根据因素如受试者的疾病状态、年龄、性别和体重发生变化。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.
在本发明的优选实施方案中,所述药物组合物中,GS-4997每天的治疗或预防有效剂量的非限制性范围为0.01~1mg/kg体重,优选为0.02~0.6mg/kg体重,更优选0.04~0.4mg/kg体重;IVA337每天的治疗或预防有效剂量的非限制性范围为1~50mg/kg体重,优选为3~35mg/kg体重,更优选5~25mg/kg体重;GFT505每天的治疗或预防有效剂量的非限制性范围为0.1~10mg/kg体重,优选为0.25~5mg/kg体重,更优选0.5~3mg/kg体重;CS038每天的治疗或预防有效剂量的非限制性范围为0.05~5mg/kg体重,优选为0.1~3.5mg/kg体重,更优选0.2~2.5mg/kg体重。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.
应当进一步理解,对于任何特定的受试者,应当根据个体需要和给药或监督组合物施用人的专业判断,随着时间调整具体剂量方案以提高最佳的治疗反应,且本文所述剂量范围仅是示例性的并不旨在限制请求保护的组合物的范围或实践。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.
在本发明的第二方面,本发明提出了一种药物制剂,该药物制剂包括前面所述的药物组合物作为活性成分。由此该制剂发挥活性成分的药理作用,进而具有该药物组合物的所用功效。具体地,该制剂具有降低总胆固醇、甘油三酯、低密度脂蛋白胆固醇、丙氨酸氨基转氨酶、天门冬氨酸氨基转移酶和/或碱性磷酸酶水平,和/或降低肝系数,和/或降低纤维化标志物Col1a1表达水平和/或α-SMA表达水平,和/或升高高密度脂蛋白胆固醇,和/或改善NAS评分和/或纤维化评分的功效,进而在预防和/或治疗NAFLD及其关联的病症方面具有显著效果。所述NAFLD包括:非酒精性脂肪肝、非酒精性脂肪性肝炎、非酒精性肝纤维化、非酒精性肝硬化。所述NAFLD关联的病症,包括但不限于,二次NAFLD,胰岛素抗性,高血糖,代谢综合征,肥胖伴高脂血症,2型糖尿病,心血管疾病,肝衰竭,肝癌等。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.
本发明所述药物制剂尤其对预防和/或治疗非酒精性脂肪肝病效果优异,特别是 对预防、延缓和治疗非酒精性脂肪肝、非酒精性脂肪性肝炎和非酒精性肝纤维化上效果优异,在改善NAS评分上效果显著。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.
根据本发明的实施例,上述药物制剂除以前面所述的药物组合物作为活性成分外,还包括药学上可接受的载体。根据本发明的实施例,所述药学可接受的载体为选自药学上可接受的溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、粘合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗粘合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂、高分子骨架材料和成膜材料中的至少一种。由此可以使得所述药物组合物能够制剂成型,呈现临床用药物制剂适于给药的形式。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.
根据本发明,所述药物制剂中的活性成分中的第一组分的含量为1mg~100mg,优选为5mg~50mg;第二组分的含量为1mg~1500mg,优选为5mg~1200mg。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.
在本发明的优选实施方式中,所述药物制剂中的活性成分中GS-4997的含量为1mg~50mg,优选为2mg~20mg;IVA337的含量为100mg~1500mg,优选为200mg~1200mg;GFT505的含量为10mg~150mg,优选为40mg~120mg;CS038的含量为10mg~150mg,优选为15mg~100mg。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.
在本发明的第三方面,本发明提出了前面所述的药物组合物或药物制剂在制备药物中的用途,所述药物用于:降低总胆固醇、甘油三酯、低密度脂蛋白胆固醇、丙氨酸氨基转氨酶、天门冬氨酸氨基转移酶和/或碱性磷酸酶水平,和/或降低肝系数,和/或降低纤维化标志物Col1a1表达水平和/或α-SMA表达水平,和/或升高高密度脂蛋白胆固醇,和/或改善NAS评分和/或纤维化评分。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.
在本发明的第四方面,本发明提出了前面所述的药物组合物或药物制剂在制备药物中的用途,所述药物用于:预防和/或治疗NAFLD或NAFLD关联病症。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.
根据本发明的具体实施例,所述药物用于:预防和/或治疗非酒精性脂肪肝、非酒精性脂肪性肝炎、非酒精性肝纤维化、非酒精性肝硬化、胰岛素抗性、高血糖、代谢综合征、肥胖伴高脂血症、2型糖尿病、心血管疾病、肝衰竭、和/或肝癌。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.
在本发明的第五方面,本发明提出了前面所述的药物组合物或药物制剂在制备预防和/或治疗NASH药物中的用途。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.
在本发明的第六方面,本发明提出了前面所述的药物组合物或药物制剂在制备预 防和/或治疗非酒精性脂肪肝药物中的用途。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.
根据本发明的具体实施例,药物组合物在制备药物中的用途中,药物组合物的给药方式采用口服的方式,或以混合物形式给药,或给予不同时间的先后次序形式给药。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.
根据本发明的具体实施例,药物组合物在制备药物中的用途中,药物组合物的用药剂量为1~1500mg/天。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.
在本发明的第七方面,本发明提出了选自ASK1抑制剂及其前体、活性代谢产物、立体异构体、药学上可接受的盐、酯类和溶剂合物中的至少一种与选自PPAR调节剂及其前体、活性代谢产物、立体异构体、药学上可接受的盐、酯类和溶剂合物中的至少一种的联合在制备药物中的用途。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.
所述药物用于:降低总胆固醇、甘油三酯、低密度脂蛋白胆固醇、丙氨酸氨基转氨酶、天门冬氨酸氨基转移酶和/或碱性磷酸酶水平,和/或降低肝系数,和/或降低纤维化标志物Col1a1表达水平和/或α-SMA表达水平,和/或升高高密度脂蛋白胆固醇,和/或改善NAS评分和/或纤维化评分。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.
所述药物用于:预防或治疗NAFLD或其关联的病症,例如:预防和/或治疗非酒精性脂肪肝、非酒精性脂肪性肝炎、非酒精性肝纤维化、非酒精性肝硬化、胰岛素抗性、高血糖、代谢综合征、肥胖伴高脂血症、2型糖尿病、心血管疾病、肝衰竭、和/或肝癌。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.
所述药物用于预防和/或治疗NASH。The medicament is for the prevention and/or treatment of NASH.
所述药物用于预防和/或治疗非酒精性脂肪肝。The medicament is for the prevention and/or treatment of nonalcoholic fatty liver.
所述ASK1抑制剂优选为5-(4-环丙基-1H-咪唑-1-基)-2-氟-N-[6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-基]-4-甲基苯甲酰胺(GS-4997)、4-(4-环丙基-1H-咪唑-1-基)-N-[3-(4-环丙基-4H-1,2,4-三唑-3-基)苯基]吡啶-2-甲酰胺和6-氨基-9-[(3R)-1-(丁-2-炔酰基)吡咯烷-3-基]-7-(4-苯氧基苯基)-7,9-二氢-8H-嘌呤中的至少一种。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.
所述PPAR调节剂优选为PPARα/δ双重激动剂和/或PPARα/δ/γ泛激动剂。The PPAR modulator is preferably a PPAR alpha / delta dual agonist and / or a PPAR alpha / delta / gamma pan agonist.
所述PPAR调节剂进一步优选为2-[2,6-二甲基-4-[3-[4-(甲硫基)苯基]-3-氧-1-丙烯基]苯氧基]-2-甲基丙酸(GFT505)、4-[1-(1,3-苯并噻唑-6-基磺酰基)-5-氯-1H-吲哚-2-基]丁酸(IVA337)和2-[2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-卡巴唑基乙氧基)苯基)-丙酸钠(CS-038)中的至少一种。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.
在所述药物中,ASK1抑制剂与PPAR调节剂的重量比为1:(0.1~400),优选为1:(0.5~200)。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).
优选,在所述药物中,GS-4997和GFT505的重量比为1:(0.5~40),优选为1:(1.5~20)。Preferably, in the drug, the weight ratio of GS-4997 to GFT505 is 1: (0.5 to 40), preferably 1: (1.5 to 20).
优选,在所述药物中,GS-4997和IVA337的重量比为1:(2~400),优选为1:(10~400),更优选为1:(20~200)。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).
优选,在所述药物中,GS-4997和CS038的重量比为1:(2~40),优选为1:(4~20)。Preferably, in the drug, the weight ratio of GS-4997 and CS038 is 1: (2 to 40), preferably 1: (4 to 20).
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。The additional aspects and advantages of the invention will be set forth in part in the description which follows.
图1显示了根据本发明的实施例5-7,各组小鼠血清AST水平;其中:MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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;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;
图2显示了根据本发明的实施例5-7,各组小鼠血清ALT水平;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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;
图3显示了根据本发明的实施例5-7,各组小鼠肝脏TG水平;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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;
图4显示了根据本发明的实施例5-7,各组小鼠肝脏TC水平;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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;
图5显示了根据本发明的实施例5-7,各组小鼠NAS评分;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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;
图6显示了根据本发明的实施例5-7,各组小鼠肝脏天狼星红染色面积比;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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 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;
图7显示了根据本发明的实施例5-7,各组小鼠α-SMA水平;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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 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;
图8显示了根据本发明的实施例5-7,各组小鼠肝脏Col1a1表达水平;其中,MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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 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.
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外,应理解,在阅读了本发明所记载的内容之后, 本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本发明所限定的范围。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.
本发明化合物采用现有技术进行制备(CN104080771A、CN1257893C、CN101248044A、CN1688532A)。The compounds of the present invention were prepared by the prior art (CN104080771A, CN1257893C, CN101248044A, CN1688532A).
实施例1 制备5-(4-环丙基-1H-咪唑-1-基)-2-氟-N-[6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-基]-4-甲基苯甲酰胺(Selonsertib,GS-4997)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)步骤一:中间体C的制备(1) Step 1: Preparation of Intermediate C
制备化合物A:取含有2-甲氧基羰基-6-氨基吡啶(480g,3.15mol)的MeOH溶液(9L),加入水合肼(315g,6.30mol)。将此反应混合物回流加热5小时,然后冷却至室温。过滤收集该混合物中形成的沉淀物,以EA洗涤,然后真空干燥得到白色固体状的化合物A(460g,收率96%)。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%).
制备化合物B:取化合物A(350g,2.30mol)的二甲基甲酰胺-二甲基乙缩醛(DMF-DMA)混合物(5L),回流加热24小时,冷却至室温,然后减压浓缩。用EA(500mL×2)吸收残留物并以55℃加热30分钟,冷却到室温后,过滤收集固体并真空干燥得到白色固体状的化合物B(531g,收率88%)。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.
制备化合物C:取化合物B(524.64g,2mol)的CH
3CN-AcOH混合物(5L,4:1),加入丙-2-胺(591.1g,10mL),将所得混合物回流加热24小时,冷却至室温,然后减压去除溶剂。用水(2.5L)溶解残留物,并加入饱和NaOH水溶液直到pH为8.0。过滤收集沉淀,并用EA(400mL×4)提取滤液。用无水Na
2SO
4干燥合并的有机相,然后浓缩至120mL体积。冰浴下,缓慢地向该混合物加入PE(400mL),并过滤所得悬液。然后合并固相从EA-PE重结晶,得到白色固体状的中间体C(252g,收率62%)。
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.
1H-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=6.8Hz,6H)。MS(ESI+)M/Z: 204(M+1)
+。
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)步骤二:制备5-氨基-2-氟-4-甲基苯甲腈-------(化合物2)(2) Step 2: Preparation of 5-amino-2-fluoro-4-methylbenzonitrile-------(Compound 2)
从5-溴-4-氟-2-甲基苯胺(1)开始,将其(31g,150mmol)溶解在无水1-甲基吡咯烷酮(150mL)中,并加入铜(I)氰化物(26.9g,300mmol)。在185℃下加热反应3.5小时,冷却至室温,加入水(350mL)和浓氢氧化氨(350mL)。将混合物搅拌50分钟,并用EA(300mL×3)萃取。用硫酸镁干燥所得的合并萃取物,然后减压去除溶剂。用乙烷(100mL×3)洗涤油状残留物,用二氯甲烷溶解该固体并装载到硅胶柱上。用0至25%EA的乙烷梯度溶液洗脱,得到5-氨基-2-氟-4-甲基苯甲腈(15.32g,收率68%)。MS(ESI+)M/Z:151(M+1)
+。
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)步骤三:制备5-(2-环丙基-2-氧代乙基氨基)-2-氟-4-甲基苯甲腈-------(化合物3)(3) Step 3: Preparation of 5-(2-cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile------- (Compound 3)
取5-氨基-2-氟-4-甲基苯甲腈(6g,40mmol),在氮气环境下溶解在无水N,N-二甲基甲酰胺(100mL)中,然后加入固体碳酸钾(6.64g,48mmol)和碘化钾(7.31mL,44mmol),同时搅拌。在室温下搅拌反应10分钟,然后加入溴甲基环丙基酮(10.12mL,90mmol)。将反应混合物加热至65℃反应4小时,然后减压除去溶剂。将残余物溶解于EA(100mL)中,并用水(200mL×2)洗涤。将有机层用硫酸镁干燥,并在 减压下除去溶剂。将残余物重新溶解于最小量的EA中,并以乙烷:EA=3:1的体积比加入乙烷。产物从溶液中析出,并通过过滤收集,得到5-(2-环丙基-2-氧代乙基氨基)-2-氟-4-甲基苯甲腈(7.80g,收率84%)。MS(ESI+)M/Z:233(M+1)
+。
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)步骤四:制备5-(4-环丙基-2-巯基-1H-咪唑-1-基)-2-氟-4-甲基苯甲腈-------(化合物4)(4) Step 4: Preparation of 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile------- (Compound 4 )
取5-(2-环丙基-2-氧代乙基氨基)-2-氟-4-甲基苯甲腈(5.69g,24.5mmol)溶解于冰醋酸(150mL)。加入固体硫氰酸钾(4.76g,48.96mmol),同时搅拌。将反应混合物加热至115℃并保持4小时,然后减压除去溶剂。将残余物溶于二氯甲烷(100mL)中,用150mL水洗涤。用二氯甲烷(200mL×2)萃取水性萃取液,合并有机萃取液并用硫酸镁干燥。减压除去溶剂,将油状残余物再溶解于EA(40mL)和120mL乙烷中。形成深色层后,向烧瓶中加入搅拌棒。剧烈搅拌下,产品沉淀为桃红色固体。过滤收集产物,得到5-(4-环丙基-2-巯基-1H-咪唑-1-基)-2-氟-4-甲基苯甲腈(5.83g,收率87%)。MS(ESI+)M/Z:274(M+1)
+。
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)步骤五:制备5-(4-环丙基-1H-咪唑-1-基)-2-氟-4-甲基苯甲腈-------(化合物5)(5) Step 5: Preparation of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile--(Compound 5)
在三颈圆底烧瓶中加入乙酸(100mL)、水(20mL)及过氧化氢(30%,4.98mL,43.92mmol)。在氮气环境下将混合物搅拌加热至45℃,同时检测内部温度。然后,在30分钟内分小份加入固体5-(4-环丙基-2-巯基-1H-咪唑-1-基)-2-氟-4-甲基苯甲腈(4g,14.64mmol),同时保持内部温度低于55℃,当完成硫代咪唑的加入后,在45℃下搅拌30分钟,然后冷却至室温,并缓慢加入20%wt/wt的亚硫酸钠(6mL)水溶液。将混合物搅拌30分钟后,减压除去溶剂。将残余物悬浮在300mL水中,并加入饱和氢氧化铵水溶液直至pH约为10。用二氯甲烷(150mL×3)萃取混合物,合并有机相,并用硫酸镁干燥,然后在减压下除去溶剂。将残余物溶解在30mL的EA中,边搅拌边加入100mL乙烷。将溶剂洗掉,余下油状残余物。重复该过程,获得粘稠油状的产物5-(4-环丙基-1H-咪唑-1-基)-2-氟-4-甲基苯甲腈(1.48g,收率42%)。MS(ESI+)M/Z:242(M+1)
+。
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)步骤六:制备5-(4-环丙基-1H-咪唑-1-基)-2-氟-4-甲基苯甲酸盐酸盐-------(化合物6)(6) Step 6: Preparation of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid hydrochloride--(Compound 6)
取5-(4-环丙基-1H-咪唑-1-基)-2-氟-4甲基苯甲腈(7.48g,31.00mmol)置于装配有回流冷凝器的圆底烧瓶中,并将其悬浮在38%的盐酸(150mL)中。将混合物加热至100℃并保持5小时,然后冷却至室温。减压除去溶剂,得到粉红色固体,向其中加 入100mL EA。过滤收集固体产物,并用3×100mL的EA洗涤。向所得固体产物中加入100mL含10%甲醇的二氯甲烷溶液,搅拌混合物,并过滤收集滤液。用100mL含10%甲醇的二氯甲烷溶液重复该步骤2次。合并滤液,并在减压下除去溶剂,得到粗5-(4-环丙基-1H-咪唑-1-基)-2-氟-4-甲基苯甲酸盐酸盐。未进行进一步纯化(7.86g,收率85%)。MS(ESI+)M/Z:261(M+1)
+。
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)步骤七:制备5-(4-环丙基-1H-咪唑-1-基)-2-氟-N-[6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-基]-4-甲基苯甲酰胺(Selonsertib,GS-4997)(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)
取5-(4-环丙基-1H-咪唑-1-基)-2-氟-4-甲基苯甲酸盐酸盐(1.25g,4.23mmol),在室温下悬浮在无水1,2-二氯乙烷(23mL)中。在氮气搅拌并加入草酰氯(0.479mL,5.49mmol),然后加入N,N-二甲基甲酰胺(0.037mL,0.423mmol)。在室温下搅拌混合物5小时,然后减压除去溶剂。将残余物溶解在25mL无水二氯甲烷中。在氮气环境下搅拌并迅速加入6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-胺(0.94g,4.65mmol)(中间体C)和4-二甲基吡啶(0.52g,4.23mmol)。在室温下搅拌反应混合物2小时,并加入水饱和的NaHCO
3(18mL)。搅拌混合物10分钟,分离各层,并用二氯甲烷(30×2mL)洗涤水性层。并将合并的有机层干燥(MgSO
4)、过滤并浓缩。将残余物溶解在最小量的CH
3CN中,并缓缓加入水,直至混合物中析出固体颗粒。过滤收集固体并干燥,得到96%纯度的5-(4-环丙基-1H-咪唑-1-基)-2-氟-N-[6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-基]-4-甲基苯甲酰胺(1.15g,收率61%)。MS(ESI+)M/Z:446.2(M+1)
+。
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) +.
1H-NMR(DMSO):δ11.12(s,1H),9.41(s,1H),9.32(s,1H),8.20(d,J=8.4Hz,1H),8.07(t,J=8.4Hz,1H),7.95(d,J=6.4Hz,1H),7.92(d,J=7.6Hz,1H),7.79(s,1H),7.59(d,J=10.4Hz,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)。
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).
实施例2 2-[2,6-二甲基-4-[3-[4-(甲硫基)苯基]-3-氧-1-丙烯基]苯氧基]-2-甲基丙酸(Elafibranor,GFT505)Example 2 2-[2,6-Dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy]-2-methylpropane Acid (Elafibranor, GFT505)
(1)步骤一:制备1-[4-甲硫基苯基]-3-[3,5-二甲基-4-羟基苯基]丙-2-烯-1-酮(中间体3)(1) Step 1: Preparation of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-hydroxyphenyl]prop-2-en-1-one (Intermediate 3)
化合物1(4.2g,25mmol)和化合物2(3.8g,25mmol)溶于饱和有盐酸气的乙醇溶液中。在室温下搅拌反应约8小时,然后真空蒸发除去溶剂。通过硅胶色谱纯化得到中间体3(6.5g,收率88%)。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)步骤二:制备1-[4-甲硫基苯基]-3-[3,5-二甲基-4-异丙氧基羰基二甲基甲氧基苯基]丙-2-烯-1-酮(中间体4)(2) Step 2: Preparation of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropoxycarbonyldimethylmethoxyphenyl]propan-2- En-1-one (intermediate 4)
将中间体3(8.67g,29.1mmol)溶于乙腈中。然后加入卤化衍生物(30.229g,145.3mmol)和碳酸钾(20.086g,145.3mmol)。将反应介质在回流下有力搅拌约12小时。过滤除去盐,真空蒸发脱除溶剂和多余的试剂,目的产物通过硅胶色谱法纯化,得到中间体4(11.7g,收率94%)。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)步骤三:制备2-[2,6-二甲基-4-[3-[4-(甲硫基)苯基]-3-氧-1-丙烯基]苯氧基]-2-甲基丙酸(Elafibranor,GFT505)(3) Step 3: Preparation of 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1-propenyl]phenoxy]-2 -methylpropionic acid (Elafibranor, GFT505)
将中间体4(13.3g,31.2mmol)溶于二氯甲烷中,加入三氟乙酸(45.668g,400.6mmol),混合物在室温下搅拌12小时。所得产物通过硅胶色谱或者再结晶法纯化。得到GFT-505(11.0g,收率92%)。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.
1H NMR DMSOδppm:1.39(s,6H),2.22(s,6H),2.57(s,3H),7.40(d,J=8.55Hz,2H),7.57(s,2H),7.62(d,J=15.5Hz,1H),7.83(d,J=15.5Hz,1H)8.10(d,J=8.55Hz,2H),12.97(s,1H)。MS(ESI-)M/Z:383.3(M-1)
-。
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) -.
实施例3 4-[1-(1,3-苯并噻唑-6-基磺酰基)-5-氯-1H-吲哚-2-基]丁酸(IVA337)Example 3 4-[1-(1,3-Benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl]butyric acid (IVA337)
(1)步骤一:制备6-(5-氯-2-硝基苯基)-己-5-炔酸甲酯(中间体3)(1) Step 1: Preparation of methyl 6-(5-chloro-2-nitrophenyl)-hex-5-ynoate (intermediate 3)
混合25.4g(89mmol)4-氯-2-碘-硝基苯、370mL三乙胺、2.06g(1.8mmol)四(三苯基膦)合钯、0.51g碘化亚铜和37mL二甲基甲酰胺(DMF)。然后在室温下,搅拌加入10g(89mmol)5-己炔酸甲酯(化合物2),反应混合物在室温下搅拌24小时。加入75mL甲苯,减压除去溶剂。用120mL乙酸乙酯和60mL 1N盐酸处理蒸发残余物。分离有机相,用水洗涤,然后用硫酸镁干燥,减压浓缩。使用环己烷/乙酸乙酯混合物(9/1,V/V)作为洗脱剂,通过硅胶色谱纯化得到17.0g中间体3,黄色固体(收率68%)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)步骤二:制备6-(2-氨基-5-氯苯基)-己-5-炔酸甲酯(中间体4)(2) Step 2: Preparation of methyl 6-(2-amino-5-chlorophenyl)-hex-5-ynoate (intermediate 4)
将45.3g(200mmol)氯化亚锡、35mL乙酸乙酯和11mL乙醇加到圆底烧瓶中。混合物在室温下搅拌15min,然后缓慢加入中间体3(10.8g)。反应混合物在室温下搅拌24小时,然后倒入200g冰和200mL 1N氢氧化钠溶液的混合物中。得到的混合物用150mL乙酸乙酯萃取两次;合并有机相,用水洗涤,用硫酸镁干燥,减压浓缩。使用环己烷/乙酸乙酯混合物(80/20,v/v)作为洗脱剂,通过硅胶色谱纯化得到3.7g中间体4,橙黄色固体(收率为39%)。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)步骤三:制备6-[5-氯-2-(1,3-苯并噻唑-6-基磺酰基氨基)苯基]-己-5-炔酸甲酯(中间体5)(3) Step 3: Preparation of methyl 6-[5-chloro-2-(1,3-benzothiazol-6-ylsulfonylamino)phenyl]-hex-5-ynoate (intermediate 5)
中间体4(2.4g,10mmol)的30mL吡啶溶液,加入2.8g(12mmol)1,3-苯并噻唑-6-基磺酰氯。混合物在室温下搅拌1.5小时,然后减压浓缩。使用环己烷/乙酸乙酯混合物(8/2,v/v)作为洗脱剂,通过硅胶色谱纯化残余油,得到4.3g中间体5,浅褐色固体(收率96%)。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)步骤四:制备4-[5-氯-1-(1,3-苯并噻唑-6-基磺酰基)-1H-吲哚-2-基]丁酸甲酯 (中间体6)(4) Step 4: Preparation of methyl 4-[5-chloro-1-(1,3-benzothiazol-6-ylsulfonyl)-1H-indol-2-yl]butanoate (Intermediate 6)
中间体5(200mg,0.45mmol)的35mL1,2-二氯乙烷溶液,加入10mg(0.05mmol)醋酸铜,混合物搅拌下回流24小时。然后减压除去溶剂,使用甲苯/乙酸乙酯混合物(9/1,v/v)作为洗脱剂,通过硅胶色谱纯化残余粘稠固体,得到160mg中间体6,为黄色固体(收率79%)。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)步骤五:制备4-[1-(1,3-苯并噻唑-6-基磺酰基)-5-氯-1H-吲哚-2-基]丁酸(IVA337)(5) Step 5: Preparation of 4-[1-(1,3-benzothiazol-6-ylsulfonyl)-5-chloro-1H-indol-2-yl]butyric acid (IVA337)
将90mg(0.2mmol)中间体6与8mL THF和2mL水混合,加入10mg(0.24mmol)氢氧化锂。混合物在室温下搅拌4小时,然后加压浓缩。在12mL水中处理蒸发残余物,用1N盐酸溶液酸化溶液。用乙酸乙酯萃取白色沉淀,分离有机相,用硫酸镁干燥,减压浓缩,得到83mg目标产物,黄色固体(收率95%)。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)
实施例4 2-[2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-卡巴唑基乙氧基)苯基)-丙酸钠的制备(CS-038)Example 4 Preparation of sodium 2-[2-(4-fluorobenzoyl)phenyl)amino]-3-(4-(2-carbazolylethoxy)phenyl)-propionate (CS-038 )
(1)步骤一:2-[(2-(4-氟苯甲酰基)苯基)氨基]-3-(4-羟基苯基)-丙酸甲酯(CHIG-003)的制备(1) Step 1: Preparation of 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl)-propionic acid methyl ester (CHIG-003)
于反应瓶中加入39.0g(0.2mol)L-酪氨酸甲脂、49.5g(0.23mol)2-(4-氟苯甲酰基)环己酮、13.0g 5%钯/碳催化剂及800mL苯甲醚,回流反应2小时,同时用分水器除去反应过程中生成的水。将反应混合物冷却至80℃,过滤除去钯/碳催化剂。然后再将反应混合物冷却至80℃,过滤除去钯/碳催化剂。然后再将反应混合物冷却至40℃,加入800mL正己烷,于-20℃静置48小时,过滤,正己烷(200mL×5)洗涤得粗产品。将粗产品加入200mL甲醇中,回流30分钟,过滤,甲醇洗涤(50mL×2),干燥得40.1g(收率51%)目标化合物。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)步骤二:2-[(2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-溴乙氧基)苯基)-丙酸甲酯(CHIG-005)的制备(2) Step 2: 2-[(2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-bromoethoxy)phenyl)-propionic acid methyl ester (CHIG- Preparation of 005)
于反应瓶中加入0.1g(1.74mmol)氢氧化钾、0.68g(1.74mmol)2-[(2-(4-氟苯甲酰基)苯基)氨基]-3-(4-羟基苯基)-丙酸甲酯、3.26g(17.4mmol)1,2-二溴乙烷及15mL乙醇,回流反应8小时,过滤去除固体不溶物,将滤液真空浓缩得粗产品,层析分离(展开剂:正己烷:乙酸乙酯=4:1)得0.37g(收率42%)目标化合物。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)步骤三:2-[2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-(9H-咔唑-9-基)乙氧基)苯基)-丙酸(Chiglitazar)的制备(3) Step 3: 2-[2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-(9H-indazol-9-yl)ethoxy)phenyl) - Preparation of propionic acid (Chiglitazar)
于反应瓶中加入0.084g咔唑(0.50mmol)、0.25g(0.50mmol)2-[(2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-溴乙氧基)苯基)-丙酸甲酯、0.085g(2.13mmol)50%NaOH、0.08g四丁基溴化铵及10mL苯,回流反应12小时,在加入30mL苯,水洗(30mL×3),干燥,真空浓缩得粗产品,层析分离(展开剂:甲醇:氯仿=4:1)得0.11g(收率37%)目标产物。HRMS(C
36H
29FN
2O
4)计算值(%):572.6357;实测值(%):572.6354。元素分析(C
36H
29FN
2O
4)计算值(%):C,75.51%;H,5.11%;N,4.89%;实测值(%):C,75.83%;H,5.10%;N,4.90%。
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)步骤四:2-[2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-(9H-咔唑-9-基)乙氧基)苯基)-丙酸钠(CS-038)的制备(4) Step 4: 2-[2-(4-Fluorobenzoyl)phenyl)amino]-3-(4-(2-(9H-indazol-9-yl)ethoxy)phenyl) -Preparation of sodium propionate (CS-038)
在30L塑料桶中加入243.7g(6.09mol)NaOH和6L无水甲醇,室温搅拌溶解。再在不锈钢反应釜(内衬聚四氟乙烯)中加入150L四氢呋喃和3.45kg(6.03mol)2-(2-(4-氟苯甲酰基)苯基氨基)-3-(4-(2-(9H-咔唑-9-基)乙氧基)苯基)丙酸,室温搅拌溶解,向反应釜中加入上述溶有氢氧化钠的甲醇溶液,加完后继续搅拌30min。真空浓缩,除去四氢呋喃和甲醇,得浓缩物。将浓缩物溶于10L二氯甲烷,在室温和搅拌下滴加到65L异丙醚中,离心分离,收集固体,于105℃真空干燥3h,得2-(2-(4-氟苯甲酰基)苯基氨基)-3-(4-(2-(9H-咔唑-9-基)乙氧基)苯基)丙酸钠,重量3.50kg,收率97%。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%.
1H NMR(DMSO-d
6):δ2.88(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)。
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).
元素分析(C
36H
28FN
2NaO
4·0.5H
2O)计算值:C 71.63,H 4.83,N 4.64;实测值:C71.62,H 4.89,N4.56。
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.
药效实验:Pharmacodynamic experiment:
采用下表所示方案进行实验设计。The experimental design was carried out using the protocol shown in the table below.
表1:药效实施例方案Table 1: Pharmacological Examples
其中,各组代号释义为:MCS:正常蛋氨酸-胆碱补充饲料对照组;MCD:蛋氨酸-胆碱缺乏饲料模型组;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。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.
实施例5 GS-4997与GFT505联合用药及单独用药对NASH的影响Example 5 Effect of GS-4997 in combination with GFT505 and drug alone on NASH
本实施例采用蛋氨酸-胆碱缺乏(methionine-choline-deficient,MCD)饮食喂养构建NASH模型,此模型是国际上被广泛认可的动物模型,操作简便,成模率高,且引起的脂肪性肝炎病变与人类NASH极为类似,因此成为研究NASH发病机制及防治药物的最主要动物模型之一。本实施例中所采用的GS-4997的剂量为前期背景实验成果,发现5mg/kg的GS-4997可以在一定程度上减少此动物模型中纤维化标志物Col1a1、α-SMA的水平,并发挥调节脂肪酸合成、脂质代谢的作用。而GFT505作为PPARα/δ双重激动剂,以3mg/kg的剂量浓度,在此模型上可发挥脂质合成、代谢调节作用,并改善肝纤维化程度。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.
实验过程:本实施例用蛋氨酸-胆碱缺乏饲料喂养C57BL/6J小鼠(南京大学—南京生物医药研究院),饮食诱导8周建立非酒精性脂肪肝模型。研究开始前至少7天给予正常饲料使小鼠适应,随后将小鼠按重量随机分为5组,每组10只小鼠,分别为蛋氨酸-胆碱补充饲料对照组(MCS,Researchdiets,A02082003B),蛋氨酸-胆碱缺乏饲料模型组(MCD,Researchdiets,A02082002B),蛋氨酸-胆碱缺乏饲料+GS-4997组(GS,5mg/kg,p.o.qd),蛋氨酸-胆碱缺乏饲料+GFT505组(GFT,3mg/kg,p.o.qd),和蛋氨酸-胆碱缺乏饲料+GS-4997(2.5mg/kg)+GFT505(1.5mg/kg)组(GG,p.o.qd)(表1)。化合物干预和MCD饲料诱导同时开展,根据上述分组每日对小鼠口服给药。在连续干预8周后,用乙醚麻醉小鼠,收集其血清,随后将动物安乐死,收获其肝脏,以供形态学、组织学和生化分析研究。所有用于生化分析的组织均以液氮快速冷冻,并-80℃保存;用于组织学分析的组织以4%中性缓冲多聚甲醛固定。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.
实验结果:MCD模型组小鼠血清中AST、ALT水平均显著升高,而血清TC和TG水平与MCS组无显著性差异,但MCD模型组小鼠的肝脏TG和TC水平则显著高于MCS组。单独给予GS-4997和GFT505分别能够减少21%(n.s.)和27%(P<0.05)的血清AST水平(图1),24%(n.s.)和33%(P<0.05)的血清ALT水平(图2),24%(n.s.)和41%(P<0.05)的肝脏TG水平(图3),28%(n.s.)和33%(P<0.05)的肝脏TC水平(图4),而GS-4997和GFT505联合用药后对以上指标的抑制率分别是63%(p<0.001)(图1),57%(p<0.001)(图2),61%(p<0.01)(图3)和58%(p<0.01)(图4)。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).
利用HE染色对肝组织切片进行病理观察,可以发现MCD组小鼠肝小叶边缘模糊,肝窦变窄,肝细胞肿胀,细胞内可见大小不一的油滴蓄积,呈气球样变,大量炎症细胞浸润,形成炎症灶。利用NAS组织学病理评分结果显示:MCD模型组的评分为7.3±0.7,而正常对照MCS组小鼠的平均分仅为1.3±0.5。在给予化合物干预后,单独给予GS-4997和GFT505的小鼠的NAS得分分别为5.9±0.7和6.2±1.0,与MCD组相比分别降低了19.2%(p<0.001)和15.7%(p<0.05)。而GS-4997和GFT505联合给药组的NAS得分为4.6±1.0,与MCD组相比降低了36.3%(p<0.001)。进一步,将GS-4997和GFT505联合给药组的NAS得分与GS-4997和GFT505单独给药组相比,分别降低了22%(P<0.01)和26%(P<0.01)(表2和图5)。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).
利用天狼星红染色对小鼠肝脏纤维化进行评价,发现MCD造模小鼠染色面积明显增加,并伴随有纤维化标志物α-SMA水平和Col1a1表达的显著增加。单独给予GS-4997和GFT505分别能够减少23%(n.s.)和18%(n.s.)的染色面积(图6),分别减少43%(p<0.05)和16%(n.s.)模型诱导引起的α-SMA水平升高(图7),分别减少55%(p<0.05)和22%(n.s.)模型诱导引起Col1a1表达增加(图8)。而GS-4997、GFT505联合用药对以上3个指标的抑制率分别可达到53%(p<0.01)(图6),65%(p<0.01)(图7)和83%(p<0.001)(图8)。进一步,将GS-4997和GFT505联合给药组的纤维化染色面积与GS-4997和GFT505单独给药组的相比,纤维化染色面积分别降低了38.9%(P<0.001)和42.8%(P<0.001)(图6)。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).
结论:从以上实验结果可以看出,GS-4997和GFT505联合给药对NASH的肝脂质蓄积、肝功能指标、NAS打分以及肝纤维化等方面的疗效均显著的优于单独给予GS-4997或GFT505。具体表现为:联合给药组在NASH病理指标中最具代表性的NAS打分和纤维化程度这两方面的治疗效果均优于单独给药组,且有统计学意义的显著差异(NAS打分:GS:GG=5.9:4.6,P<0.01;GFT:GG=6.2:4.6,P<0.01;纤维化染色面积:GS:GG=23%:53%,P<0.001;GFT:GG=18%:53%,P<0.001)。而对其他各项指标如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(GS:GFT:GG=55%:22%:83%)的治疗强度也均大于单独给药组。综合可知,与单药治疗相比,GS-4997与GFT505联合用药显示了更优越的抗NASH疗效。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.
实施例6 GS-4997与IVA337联合用药及单独用药对NASH的影响Example 6 Effect of GS-4997 in combination with IVA337 and drug alone on NASH
本实施例中IVA337的剂量由前期背景实验决定,实验结果显示10mg/kg的IVA337可以有效地抑制此动物模型中脂质代谢基因如SCD1、CPT1、CPT2等表达水平,降低小鼠肝脏脂质变性打分,抑制炎症相关基因如IL-1β,IL-6,MCP-1等的表达水平,降低组织纤维化打分。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.
实验过程:本实施例用蛋氨酸-胆碱缺乏饲料喂养C57BL/6J小鼠(南京大学—南京生物医药研究院),饮食诱导8周建立非酒精性脂肪肝模型。研究开始前至少7天给予正常饲料使小鼠适应,随后将小鼠按重量随机分为5组,每组10只小鼠,分别为蛋氨酸-胆碱补充饲料对照组(MCS,Researchdiets,A02082003B),蛋氨酸-胆碱缺乏饲料模型组(MCD,Researchdiets,A02082002B),蛋氨酸-胆碱缺乏饲料+GS-4997组(GS,5mg/kg,p.o.qd),蛋氨酸-胆碱缺乏饲料+IVA337组(IVA,10mg/kg,p.o.qd)和蛋氨酸-胆碱缺乏饲料+GS-4997(2.5mg/kg)+IVA337(5mg/kg)组(GI,p.o.qd)(表1)。化合物干预和MCD饲料诱导同时开展,根据上述分组每日对小鼠口服给药。在连续干预8周后,用乙醚麻醉小鼠,收集其血清,随后将动物安乐死,收获其肝脏,以供形态学、组织学和生化分析研究。所有用于生化分析的组织均以液氮快速冷冻,并-80℃保存;用于组织学分析的组织以4%中性缓冲多聚甲醛固定。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.
实验结果:MCD模型组小鼠血清中AST、ALT水平均显著升高,而血清TC和TG水平与MCS组无显著性差异,但MCD模型组小鼠的肝脏TG和TC水平则明显高于MCS组。单独给予GS-4997和IVA337分别能够减少21%(n.s.)和36%(P<0.05)的血清AST水平(图1),24%(n.s.)和43%(P<0.05)的血清ALT水平(图2),24%(n.s.)和48%(P<0.05)的肝脏TG水平(图3),28%(n.s.)和34%(P<0.05)的肝脏TC水平(图4),而GS-4997和IVA337联合用药后对以上指标的抑制率分别是65%(p<0.001)(图1),61%(p<0.001)(图2),73%(p<0.01)(图3)和59%(p<0.001)(图4)。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).
利用HE染色对肝组织切片进行病理观察,可以发现MCD组小鼠肝小叶边缘模糊,肝窦变窄,肝细胞肿胀,细胞内可见大小不一的油滴蓄积,呈气球样变,大量炎症细胞浸润,成炎症灶,利用NAS组织学病理评分结果显示:MCD模型组的评分为7.3±0.7,而正常对照MCS组小鼠的平均分仅为1.3±0.5。在给予化合物干预后,单独给予GS-4997和IVA337的小鼠的NAS得分分别为5.9±0.7和5.1±0.9,与MCD组相比分别降低了19.2%(p<0.001)和30.0%(p<0.001)。而GS-4997和IVA337联合给药组的NAS得分 为3.9±1.2,与MCD组相比降低了46.7%(p<0.001)。进一步,将GS-4997和IVA337联合给药组的NAS得分与GS-4997和IVA337单独给药组相比,分别降低了33.9%(P<0.001)和23.5%(P<0.05)(表2和图5)。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).
利用天狼星红染色对小鼠肝脏纤维化进行评价,发现MCD造模小鼠染色面积明显增加,并伴随有α-SMA水平和Col1a1表达增加。单独给予GS-4997和IVA337分别能够减少23%(n.s.)和17%(n.s.)的染色面积(图6),分别减少43%(p<0.05)和37%(p<0.05)的α-SMA水平升高(图7),分别减少55%(p<0.05)和44%(p<0.05)的Col1a1表达增加(图8)。而GS-4997、IVA337联合用药对以上3个指标的抑制率分别是52%(p<0.01)(图6),76%(p<0.001)(图7)和94%(p<0.001)(图8)。进一步,将GS-4997和IVA337联合给药组的纤维化染色面积与GS-4997和IVA337单独给药组相比,纤维化染色面积分别降低了37.3%(P<0.01)和42.7%(P<0.001)(图6)。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).
结论:从以上实验结果可以看出,GS-4997和IVA337联合给药对NASH的肝脂质蓄积、肝功能指标、NAS打分以及肝纤维化等方面的疗效均显著的优于单独给予GS-4997或IVA337。具体表现为:联合给药组在NASH病理指标中最具代表性的NAS打分和纤维化程度这两方面的治疗效果均优于单独给药组,且有统计学意义的显著差异(NAS打分:GS:GI=5.9:3.9,P<0.001;IVA:GI=5.1:3.9,P<0.05;纤维化染色面积:GS:GI=23%:52%,P<0.01;IVA:GI=17%:52%,P<0.001)。而对其他各项指标如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(GS:IVA:GI=55%:44%:94%)的治疗强度也均大于单独给药组。综合可知,与单药治疗相比,GS-4997与IVA337联合用药显示了更优越的抗NASH疗效。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.
实施例7 GS-4997与CS-038联合用药及单独用药对NASH的影响Example 7 Effect of GS-4997 and CS-038 in combination with drug alone on NASH
本实施例中CS-038为针对2型糖尿病的PPARα/δ/γ全激动剂,目前尚无其在NASH治疗领域的相关研究数据披露。但鉴于PPAR在NASH治疗中的潜在价值,我们在前期剂量摸索实验中发现10mg/kg的CS-038能够有效的降低肝脏和血清中的TG、TC、LDL-C水平,调节小鼠脂质代谢,同时对炎症相关基因的表达也具有抑制作用,在组织病理分析中也可有效的减少炎症灶的数量。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.
实验过程:本实施例用蛋氨酸-胆碱缺乏饲料喂养C57BL/6J小鼠(南京大学—南 京生物医药研究院),饮食诱导8周建立非酒精性脂肪肝模型。研究开始前至少7天给予正常饲料使小鼠适应,随后将小鼠按重量随机分为5组,每组10只小鼠,分别为蛋氨酸-胆碱补充饲料对照组(MCS,Researchdiets,A02082003B),蛋氨酸-胆碱缺乏饲料模型组(MCD,Researchdiets,A02082002B),蛋氨酸-胆碱缺乏饲料+GS-4997组(GS,5mg/kg,p.o.qd),蛋氨酸-胆碱缺乏饲料+CS-038组(CS,10mg/kg,p.o.qd)和蛋氨酸-胆碱缺乏饲料+GS-4997(2.5mg/kg)+CS-038(5mg/kg)组(GC,p.o.qd)(表1)。化合物干预和MCD饲料诱导同时开展,根据上述分组每日对小鼠口服给药。在连续干预8周后,用乙醚麻醉小鼠,收集其血清,随后将动物安乐死,收获其肝脏,以供形态学、组织学和生化分析研究。所有用于生化分析的组织均以液氮快速冷冻,并-80℃保存;用于组织学分析的组织以4%中性缓冲多聚甲醛固定。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.
实验结果:MCD模型组小鼠血清中AST、ALT水平均显著升高,而血清TC和TG水平与MCS组无显著性差异,但MCD模型组小鼠的肝脏TG和TC水平则明显高于MCS组。单独给予GS-4997和CS-038分别能够减少21%(n.s.)和18%(n.s.)的血清AST水平(图1),24%(n.s.)和28%(n.s.)的血清ALT水平(图2),24%(n.s.)和33%(n.s.)的肝脏TG水平(图3),28%(n.s.)和38%(n.s.)的肝脏TC水平(图4),而GS-4997和CS-038联合用药后对以上指标的抑制率分别是55%(p<0.001)(图1),48%(p<0.001)(图2),57%(p<0.01)(图3)和53%(p<0.01)(图4)。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).
利用HE染色对肝组织切片进行病理观察,可以发现MCD组小鼠肝小叶边缘模糊,肝窦变窄,肝细胞肿胀,细胞内可见大小不一的油滴蓄积,呈气球样变,大量炎症细胞浸润,成炎症灶,利用NAS组织学病理评分结果显示:MCD模型组的评分为7.3±0.7,而正常对照MCS组小鼠的平均分仅为1.3±0.5。在给予化合物干预后,单独给予GS-4997和CS-038的小鼠的NAS得分分别为5.9±0.7和6.0±1.2,与MCD组相比分别降低了19.2%(p<0.001)和17.7%(p<0.01)。而GS-4997和CS-038联合给药组的NAS得分为4.4±0.8,与MCD组相比降低了39.0%(p<0.001)。进一步,将GS-4997和CS-038联合给药组的NAS得分与GS-4997和CS-038单独给药组相比,分别降低了25.4%(P<0.001)和26.7%(P<0.01)(表2和图5)。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).
利用天狼星红染色对小鼠肝脏纤维化进行评价,发现MCD造模小鼠染色面积明显增加,并伴随有α-SMA水平和Col1a1表达增加。单独给予GS-4997和CS-038分别能够减少23%(n.s.)和15%(n.s.)的染色面积(图6),分别减少43%(p<0.05)和21%(n.s.)的α-SMA水平升高(图7),分别减少55%(p<0.05)和24%(n.s.)的Col1a1表达增加(图8)。 而GS-4997、CS-038联合用药对以上3个指标的抑制率分别是49%(p<0.01)(图6),67%(p<0.01)(图7)和79%(p<0.01)(图8)。进一步,将GS-4997和CS-038联合给药组的纤维化染色面积与GS-4997和CS-038单独给药组相比,纤维化染色面积分别降低了33.5%(P<0.01)和38.0%(P<0.001)(图6)。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).
结论:从以上实验结果可以看出,GS-4997和CS-038联合给药对NASH的肝脂质蓄积、肝功能指标、NAS打分以及肝纤维化等方面的疗效均显著的优于单独给予GS-4997或CS-038。具体表现为:联合给药组在NASH病理指标中最具代表性的NAS打分和纤维化程度这两方面的治疗效果均优于单独给药组,且有统计学意义的显著差异(NAS打分:GS:GC=5.9:4.4,P<0.001;CS:GC=6.0:4.4,P<0.01;纤维化染色面积:GS:GC=23%:49%,P<0.01;CS:GC=15%:49%,P<0.001)。而对其他各项指标如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(GS:CS:GC=55%:24%:79%)的治疗强度也均大于单独给药组。综合可得,与单药治疗相比,GS-4997与CS-038联合用药显示了更优越的抗NASH疗效。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.
表2:各组小鼠NAS打分Table 2: NAS scores for each group of mice
在本说明书的描述中,参考术语“实施例”、“实例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。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.
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。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)
- 一种药物组合物,其特征在于,包括第一组分、第二组分,其中,A pharmaceutical composition comprising a first component and a second component, wherein第一组分为选自ASK1抑制剂及其前体、活性代谢产物、立体异构体、药学上可接受的盐、酯类和溶剂合物中的至少一种;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;第二组分为选自PPAR调节剂及其前体、活性代谢产物、立体异构体、药学上可接受的盐、酯类和溶剂合物中的至少一种。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.
- 根据权利要求1所述的药物组合物,其特征在于,所述第一组分为选自The pharmaceutical composition according to claim 1, wherein said first component is selected from the group consisting of5-(4-环丙基-1H-咪唑-1-基)-2-氟-N-[6-(4-异丙基-4H-1,2,4-三唑-3-基)吡啶-2-基]-4-甲基苯甲酰胺、4-(4-环丙基-1H-咪唑-1-基)-N-[3-(4-环丙基-4H-1,2,4-三唑-3-基)苯基]吡啶-2-甲酰胺和6-氨基-9-[(3R)-1-(丁-2-炔酰基)吡咯烷-3-基]-7-(4-苯氧基苯基)-7,9-二氢-8H-嘌呤中的至少一种;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;任选地,所述第二组分为PPARα/δ双重激动剂和/或PPARα/δ/γ泛激动剂;Optionally, the second component is a PPAR alpha / delta dual agonist and / or a PPAR alpha / delta / gamma pan agonist;优选的,所述第二组分为2-[2,6-二甲基-4-[3-[4-(甲硫基)苯基]-3-氧-1-丙烯基]苯氧基]-2-甲基丙酸、4-[1-(1,3-苯并噻唑-6-基磺酰基)-5-氯-1H-吲哚-2-基]丁酸和2-[2-(4-氟苯甲酰基)苯基)氨基]-3-(4-(2-卡巴唑基乙氧基)苯基)-丙酸钠中的至少一种。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.
- 根据权利要求1~2中任一项所述的药物组合物,其特征在于,所述第一组分与所述第二组分的重量比为1:(0.1~400),优选为1:(0.5~200);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);优选所述药物组合物中GS-4997和GFT505的重量比为1:(0.5~40),优选为1:(1.5~20);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);优选所述药物组合物中GS-4997和IVA337的重量比为1:(2~400),优选为1:(10~400),更优选为1:(20~200);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);优选所述药物组合物中GS-4997和CS038的重量比为1:(2~40),优选为1:(4~20)。Preferably, the weight ratio of GS-4997 to CS038 in the pharmaceutical composition is 1: (2 to 40), preferably 1: (4 to 20).
- 一种药物制剂,其包括权利要求1~3任一项所述的药物组合物作为活性成分,任选地,所述药物制剂进一步包含药学可接受的载体。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.
- 根据权利要求4所述的药物制剂,所述第一组分的含量为1mg~100mg,优选为5mg~50mg;第二组分的含量为1mg~1500mg,优选为5mg~1200mg;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;优选所述药物制剂中GS-4997的含量为1mg~50mg,优选为2mg~20mg;Preferably, the pharmaceutical preparation has a content of GS-4997 of 1 mg to 50 mg, preferably 2 mg to 20 mg;优选所述药物制剂中IVA337的含量为100mg~1500mg,优选为200mg~1200mg;Preferably, the content of IVA337 in the pharmaceutical preparation is from 100 mg to 1500 mg, preferably from 200 mg to 1200 mg;优选所述药物制剂中GFT505的含量为10mg~150mg,优选为40mg~120mg;Preferably, the pharmaceutical preparation has a GFT505 content of 10 mg to 150 mg, preferably 40 mg to 120 mg;优选所述药物制剂中CS038的含量为10mg~150mg,优选为15mg~100mg。Preferably, the pharmaceutical preparation has a CS038 content of 10 mg to 150 mg, preferably 15 mg to 100 mg.
- 权利要求1~3任一项所述的药物组合物在制备药物中的用途,所述药物用于:降低总胆固醇、甘油三酯、低密度脂蛋白胆固醇、丙氨酸氨基转氨酶、天门冬氨酸氨基转移酶、和/或碱性磷酸酶水平,和/或降低肝系数,和/或降低纤维化标志物Col1a1表达水平和/或α-SMA表达水平,和/或升高高密度脂蛋白胆固醇,和/或改善NAS评分和/或纤维化评分。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.
- 权利要求1~3任一项所述的药物组合物在制备药物中的用途,所述药物用于:预防和/或治疗NAFLD或NAFLD关联病症,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,任选地,所述药物用于预防和/或治疗:非酒精性脂肪肝、非酒精性脂肪性肝炎、非酒精性肝纤维化、非酒精性肝硬化、胰岛素抗性、高血糖、代谢综合征、肥胖伴高脂血症、2型糖尿病、心血管疾病、肝衰竭、和/或肝癌。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.
- 权利要求1~3任一项所述的药物组合物在制备预防和/或治疗NASH药物中的用途。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.
- 权利要求1~3任一项所述的药物组合物在制备预防和/或治疗非酒精性脂肪肝药物中的用途。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.
- 根据权利要求6~9任一项所述的用途,其特征在于,所述药物组合物的给药方式采用口服的方式,或以混合物形式给药,或给予不同时间的先后次序形式给药,任选地,所述药物组合物的用药剂量为1~1500mg/天。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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