Application of combination of rosmeltirol and metformin or pharmaceutically acceptable salts thereof in preparation of medicines

Abstract

The invention belongs to the field of biological medicine research, and particularly relates to application of combination of rosmeltirol and metformin or pharmaceutically acceptable salt thereof in preparation of drugs. According to the invention, through the combination of the rosmeltirol (abbreviated as Res) and the metformin or the pharmaceutically acceptable salt thereof, the curative effect on inflammation caused by metabolic dysfunction related fatty liver diseases is remarkably improved on the basis of reducing the dosage of the rosmeltirol. And moreover, the inhibition effect on inflammation after addition of the two is obviously superior to that of independent medication of the two, and a synergistic interaction effect is generated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine research, and in particular to the use of resmetiro combined with metformin or a pharmaceutically acceptable salt thereof in the preparation of medicines. Background Art

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common chronic liver disease with a global prevalence of about 30% and an increasing trend year by year. The impact of MASLD on health is not limited to the liver, but is also closely related to cardiovascular disease, chronic kidney disease, obesity, and type 2 diabetes. MASLD is a progressive disease. MASLD includes simple fatty infiltration (a benign disease called fatty liver disease) and metabolic dysfunction-associated steatohepatitis (MASH). MASH is a more advanced stage of MASLD. The pathogenesis of MASH is complex, and its main characteristics include excessive accumulation of lipids in the liver, inflammatory infiltration, and fibrosis. If not intervened in time, it will further develop into cirrhosis or hepatocellular carcinoma (HCC). On December 2, 2022, the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, the First Affiliated Hospital of Anhui Medical University, and the First Affiliated Hospital of Wenzhou Medical University published a collaborative paper titled A multi-omic landscape of steatosis-to-NASH progression in the journal Life Metabolism, showing that early high-fat, high-cholesterol, high-fructose (HFHC) mice only showed obesity and liver lipid deposition, similar to the commonly used high-fat diet (HFD) 12-week mice, but no obvious liver inflammation and damage. HFHC 28-week mice showed typical liver inflammation and damage, accompanied by the occurrence of liver fibrosis.

[0003] At the same time, the global prevalence of MASLD has become a huge economic burden, exceeding $100 billion in the United States alone, and the direct cost of this disease is $103 billion per year. It is estimated that in the next decade, the burden of MASLD in the United States may reach 1.005 trillion US dollars and 334 billion euros in Europe. Due to the series of extrahepatic manifestations caused by MASLD (including cardiovascular disease, chronic kidney disease, and related cancers in and outside the liver), its economic burden will far exceed the scope of liver-related complications, emphasizing the need to develop culturally appropriate interventions and strengthen the need for MASH treatment.

[0004] Although MASH has received widespread attention due to its high prevalence and heavy economic burden, few effective treatments have been approved to date. Resmetirom (Res) is a thyroid hormone receptor β agonist that was approved for marketing by the US FDA in March 2024 and became the only marketed drug for the treatment of MASH. The clinically recommended dose of Res is 80 mg / kg or 100 mg / kg, but the drug is expensive and therefore limited in use. In addition, MASH is a chronic disease and patients need to take medication for a long time, which may lead to an increased risk of adverse reactions. Summary of the invention

[0005] Therefore, the object of the present invention is to provide the use of resmetiro combined with metformin or a pharmaceutically acceptable salt thereof in the preparation of a drug, specifically referring to the use of resmetiro combined with metformin or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, treating or alleviating fatty liver disease associated with metabolic dysfunction, so as to improve the therapeutic effect and reduce the dosage.

[0006] To solve the above technical problems, the present invention is achieved through the following technical solutions: The present invention provides the use of resmetiro combined with metformin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, treating or alleviating fatty liver disease associated with metabolic dysfunction.

[0007] Use of resmetiro combined with metformin or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for preventing, treating or alleviating fatty liver disease associated with metabolic dysfunction.

[0008] Among them, Resmetirom, English name: Resmetirom, CAS number is 920509-32-6, molecular formula is C 17 H 12 Cl 2 N 6 O 4 .

[0009] Furthermore, the mass ratio of resmetiro to metformin or a pharmaceutically acceptable salt thereof is 6-10:180-250; preferably 8.2:205.

[0010] Furthermore, the dosage form of the drug includes powder, tablet, solution, capsule, granule or suspension.

[0011] Furthermore, the resmetiro and metformin or a pharmaceutically acceptable salt thereof are respectively used as active ingredients in the same preparation unit or in different preparation units.

[0012] Furthermore, the drug includes resmetiro and a pharmaceutically acceptable carrier to form a first preparation, and the metformin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier to form a second preparation; preferably, the pharmaceutically acceptable carrier is selected from one or more of pharmaceutically acceptable solvents, cosolvents, emulsifiers, flavoring agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, preservatives, suspending agents, coating materials, pH regulators, absorbents, flocculants, and deflocculating agents.

[0013] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of pharmaceutically acceptable solvents, cosolvents, emulsifiers, flavoring agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, preservatives, suspending agents, coating materials, pH regulators, absorbents, flocculants, and deflocculating agents.

[0014] Furthermore, the metabolic dysfunction-related fatty liver disease includes one or more of simple fatty infiltration, metabolic dysfunction-related fatty hepatitis or metabolic dysfunction-related liver fibrosis.

[0015] The present invention also provides a drug for preventing, treating or alleviating fatty liver disease associated with metabolic dysfunction, wherein the drug comprises resmetiro and metformin or a pharmaceutically acceptable salt thereof.

[0016] Furthermore, the mass ratio of resmetiro to metformin or a pharmaceutically acceptable salt thereof is 6-10:180-250; preferably 8.2:205.

[0017] Furthermore, the resmetiro and metformin or a pharmaceutically acceptable salt thereof are respectively used as active ingredients in the same preparation unit or in different preparation units.

[0018] Furthermore, the metabolic dysfunction-related fatty liver disease includes one or more of simple fatty infiltration, metabolic dysfunction-related fatty hepatitis or metabolic dysfunction-related liver fibrosis.

[0019] Furthermore, when the drug is used to treat metabolic dysfunction-related fatty hepatitis in mice, the solution of resmetiro is gavaged at a volume of 6-10 mg / kg (e.g., 8.2 mg / kg); the solution of metformin hydrochloride is gavaged at a volume of 180-250 mg / kg (e.g., 205 mg / kg).

[0020] The administration frequency is 1 to 2 times (e.g., 1 time) per day by gavage for 7 to 12 weeks (e.g., 9 weeks).

[0021] The first preparation is a solution containing resmetiro, which uses 0.1-1.0 wt% (e.g. 0.5 wt%) CMC-Na (sodium carboxymethyl cellulose) solution as solvent and has a concentration of 1-4 mg / mL (e.g. 1.64 mg / mL); the second preparation is a solution containing metformin hydrochloride, which uses pure water as solvent and has a concentration of 60-100 mg / mL (e.g. 41 mg / mL).

[0022] In the present invention, the pharmaceutically acceptable salt includes hydrochloride, that is, the pharmaceutically acceptable salt of metformin is metformin hydrochloride (abbreviated as Met).

[0023] The technical solution of the present invention has the following advantages: 1. The present invention uses resmetiro (abbreviated as Res) in combination with metformin or a pharmaceutically acceptable salt thereof, and significantly improves the efficacy of inflammation caused by metabolic dysfunction-related fatty liver disease on the basis of reducing the dosage of resmetiro. It is also proved that the inhibitory effect of the combination of the two on inflammation is significantly better than that of the two alone, producing a synergistic effect.

[0024] 2. The present invention uses metabolic dysfunction-related fatty hepatitis model mice (db / db mice) as an example for research. The combined administration of half the clinical dose of resmetiro and metformin to db / db mice can effectively alleviate the progression of the disease: the liver lipid metabolism disorder of db / db mice is significantly improved, such as the reduction of liver fat droplets, down-regulation of lipid synthesis gene expression, and up-regulation of lipid decomposition gene expression; the inflammation of db / db mice is relieved, the level of inflammatory factor IL-6 decreases, and the level of IL10 increases; liver fibrosis and liver function are improved, and the degree of liver lesions is alleviated. In addition, the combined treatment of half-dose resmetiro and metformin shows the superiority of combined administration in terms of liver inflammation, and the effect is better than the use of resmetiro alone. Therefore, the combined administration of half-dose resmetiro and metformin can be used as a new treatment for metabolic dysfunction-related fatty hepatitis, which greatly reduces the economic burden of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The effect of combined administration of Res and Met on metabolic dysfunction-related fatty liver hepatitis in Experimental Example 1: Part A is a schematic diagram of the combined administration of Res and Met to treat MASH; Part B is a representative picture of mouse liver tissue; Figure 2 The results of HE (hematoxylin-eosin staining), Oil Red O staining and transmission electron microscopy (TEM) observation of mouse liver tissue in Experimental Example 1 and the ELISA (enzyme-linked immunosorbent assay) results of mouse serum inflammatory factors; Part A is a representative picture of HE staining; Part B is a representative picture of Oil Red O staining; Part C is a representative picture of TEM; Part D is the result of the determination of TNFα content in serum; Part E is the result of the determination of IL10 content in serum; Figure 3 The results of Sirius red staining and Masson staining of mouse liver tissue in Experimental Example 1: Part A is a representative picture of Sirius red staining; Part B is a representative picture of Masson staining; Figure 4 The test results of TG and TC in mouse serum in Experimental Example 2: Part A is the test result of TG content in serum; Part B is the test result of TC content in serum; Figure 5 The RT-qPCR results of lipid metabolism-related genes and inflammation-related genes in mouse liver tissue in Experimental Example 3: Part A is the mRNA expression level of gene CPT1A; Part B is the mRNA expression level of gene ACADL; Part C is the mRNA expression level of gene PPARG; Part D is the mRNA expression level of gene TNFα; Part E is the mRNA expression level of gene IL10; Figure 6 The RT-qPCR results of genes related to the expression of fibrosis markers in mouse liver tissue in Experimental Example 3: Part A is the mRNA expression level of gene COL1A1; Part B is the mRNA expression level of gene COL1A2 in liver tissue; Part C is the mRNA expression level of gene TIMP1 in liver tissue; In the figures, # indicates p≤0.05 compared with the normal group; ## indicates p≤0.01 compared with the normal group; ### indicates p≤0.001 compared with the normal group; * indicates p≤0.05 compared with the model group; ** indicates p≤0.01 compared with the model group; *** indicates p≤0.001 compared with the model group; $ indicates p≤0.05 compared with the combined medication group; $$ indicates p≤0.01 compared with the combined medication group; $$$ indicates p≤0.001 compared with the combined medication group. DETAILED DESCRIPTION

[0027] The following examples are provided to further understand the present invention better, are not limited to the best mode, and are not intended to limit the content and scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the enlightenment of the present invention or by combining the features of the present invention with other prior arts, all falls within the protection scope of the present invention. If no specific experimental steps or conditions are indicated in the examples, the operation or conditions of the conventional experimental steps described in the literature in this area can be carried out. If the manufacturer is not indicated for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0028] Experimental Materials: Metformin hydrochloride tablets were purchased from Shanghai Bristol-Myers Squibb Pharmaceuticals Co., Ltd. (Batch No.: ACM2249), and resmetirox standard was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Cat. No.: 920509-32-6).

[0029] Male db / db mice and male C57 / BL6J mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All animals were kept in the Animal Center of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. All mice were kept in a constant temperature (22±2℃), constant humidity (56±5%), and 12-hour alternating dark and light environment, with free access to water and food. The use of experimental animals followed the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health and the regulations of the Animal Care and Use Committee of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. This experiment was approved by the Animal Welfare and Laboratory Animal Management and Animal Welfare Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College (approval number: SLXD-20240430010).

[0030] Experimental Example 1 1. Experimental Methods 1. Preparation of test drug solution Resmetiro solution (Res solution): The resmetiro standard was dissolved in 0.5% CMC-Na solution to prepare Res solutions with concentrations of 1.64 mg / mL and 3.28 mg / mL, respectively.

[0031] Metformin hydrochloride solution (Met solution): Crush metformin hydrochloride tablets and dissolve them in water to prepare Met solutions with concentrations of 41 mg / mL and 82 mg / mL, respectively.

[0032] 2. Grouped dosing regimen The db / db mice were randomly divided into the model group (Model group), the low-dose Met group (205 mg / kg), the high-dose Met group (410 mg / kg), the single-dose Res group (also known as the Res clinical equivalent dose group, 16.4 mg / kg) and the combined administration group (half-dose Res: 8.2 mg / kg + Met: 205 mg / kg), and the C57 / BL6J mice were used as the control group (Control), with 12 mice in each group. After one week of adaptive feeding of all mice, the drug treatment was performed as follows: Met low-dose group: intragastrically administered Met solution with a concentration of 41 mg / mL; the intragastrically administered dose was 205 mg / kg animal body weight based on the mass of Met; Met high-dose group: intragastrically administered Met solution with a concentration of 82 mg / mL; the intragastrically administered dose was 410 mg / kg animal body weight based on the mass of Met; Res clinical equivalent dose group: intragastrically administered Res solution with a concentration of 3.28 mg / mL; the intragastrically administered dose was 16.4 mg / kg animal body weight based on the mass of Res; combined administration group: intragastrically administered Res solution with a concentration of 1.64 mg / mL; the intragastrically administered dose was 8.2 mg / kg animal body weight based on the mass of Res, and then intragastrically administered Met solution with a concentration of 41 mg / mL; the intragastrically administered dose was 205 mg / kg animal body weight based on the mass of Met; mice in the control group (C57 / BL6J mice) and model group were given an equal volume of normal saline (5 mL / kg animal body weight), respectively. db / db mice and C57 / BL6J mice were dosed once a day starting at 7 weeks of age for 9 weeks.

[0033] Sample collection method: After 9 weeks of administration, blood was collected from the eyeballs of mice to collect serum. Liver tissues of 6 mice in each group were fixed, and the rest were frozen in liquid nitrogen.

[0034] 3. Test methods (1) HE staining, Oil Red O staining and TEM analysis Mouse liver tissue was fixed and sliced, and HE staining, Oil Red O staining and TEM analysis were performed using conventional methods in the field. The specific steps of HE staining are as follows: 1) Dewaxing of paraffin sections to water: Put the sections into environmentally friendly dewaxing solution I for 20 min-environmentally friendly dewaxing solution II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min, and wash with tap water. Rewarming and fixing of frozen sections: Take the frozen sections out of the -20℃ refrigerator and restore them to room temperature, fix them with tissue fixative for 15 min, and then rinse with running water. 2) Pretreatment: The sections were treated with high-definition constant staining pretreatment solution for 1 min. 3) Hematoxylin staining: The sections were stained with hematoxylin staining solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blueing solution, and rinsed with running water. 4) Eosin staining: The sections were dehydrated in 95% alcohol for 1 min, and stained in eosin staining solution for 15 s. 5) Dehydration and sealing: the slices were placed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, xylene II for 2 min, and transparent, neutral gum for sealing. 6) Microscope examination, image acquisition and analysis.

[0035] The specific steps of Oil Red O staining are as follows: 1) Fixation of frozen sections: Take the frozen sections out of the -20℃ refrigerator and restore them to room temperature, fix them with tissue fixative for 15 minutes, wash with tap water, and dry them. 2) Oil Red O staining: Mix 6 volumes of saturated Oil Red O staining solution with 4 volumes of distilled water, let it stand overnight at 4℃, filter it once with qualitative filter paper the next day, let it stand at 4℃ for 24 hours and filter it again to obtain Oil Red O working solution. Slices are immersed in Oil Red staining solution for 8-10 minutes (cover and avoid light). 3) Background differentiation: Take out the slices, stay for 3 seconds, and then immerse them in two cylinders of 60% isopropanol for differentiation, 3 seconds and 5 seconds respectively. Slices are immersed in 2 cylinders of pure water for 10 seconds each. 4) Hematoxylin staining: Take out the slices, stay for 3 seconds, and then immerse them in hematoxylin for 3-5 minutes, and then immerse them in 3 cylinders of pure water for 5 seconds, 10 seconds, and 30 seconds respectively. Differentiation solution for 2-8 s, 2 cylinders of distilled water for 10 s each, blueing solution for 1 s, gently immerse the slices in 2 cylinders of tap water for 5 s and 10 s each, and examine the staining effect under a microscope. 5) Seal the slices: Seal the slices with glycerol gelatin sealant. 6) Microscope examination, image acquisition and analysis.

[0036] The specific steps of TEM analysis are as follows: pre-fixation of samples with 2.5% glutaraldehyde fixative - rinsing with 0.2 M phosphate buffer - post-fixation with 1% osmium tetroxide - rinsing with 0.2 M phosphate buffer - gradient alcohol dehydration (the volume percentage of ethanol is 30%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%) - alcohol acetone replacement - gradient embedding agent infiltration (1:2, 1:1) - pure resin embedding (812#) - polymerization - ultrathin sectioning (microtome model: Leica EM UC7) - staining (uranyl acetate, lead citrate) - TEM observation (electron microscope model: Hitachi HT7800).

[0037] (2) Determination of inflammatory factors Mouse serum was collected and the levels of inflammatory factors TNFα (tumor necrosis factor α) and IL10 (interleukin 10) in mouse serum were detected by ELISA according to the instructions of the kit. TNFα: Detected using the mouse TNFα detection kit (catalog number: HY-H0019) purchased from Beijing Huaying Biotechnology Institute. IL10: Detected using the mouse IL10 detection kit (catalog number: HY-H0009) purchased from Beijing Huaying Biotechnology Institute.

[0038] (3) Sirius red staining and Masson staining Mouse liver tissue was fixed and sliced, and then Sirius red staining and Masson staining were performed using conventional methods in the field. The specific steps of Sirius red staining are as follows: 1) Dewaxing of paraffin sections to water: Put the sections into environmentally friendly dewaxing solution I for 20 min-environmentally friendly dewaxing solution II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min, and wash with tap water. Rewarming and fixing of frozen sections: Take the frozen sections out of the -20℃ refrigerator and restore them to room temperature, fix them with tissue fixative for 15 min, and then rinse with running water. 2) Sirius red staining: Slices were stained in Sirius red staining solution for 8 min, and dehydrated with two or three cylinders of anhydrous ethanol. 3) Dehydration and sealing: The sections were then placed in clean xylene for 5 min, and sealed with neutral gum. 4) Microscope examination, image acquisition and analysis.

[0039] The specific steps of Masson staining are as follows: 1) Dewax the paraffin sections to water: put the sections into environmentally friendly dewaxing solution I for 20 min-environmentally friendly dewaxing solution II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min, and wash with tap water. Rewarming and fixing of frozen sections: take the frozen sections out of the -20℃ refrigerator and restore them to room temperature, fix them with tissue fixative for 15 min, and then rinse with running water. 2) Soak the sections in Masson A solution overnight and rinse with running water. 3) Put the sections into a dye solution mixed with Masson B solution and Masson C solution in equal proportions, soak for 1 min, wash with tap water, differentiate with differentiation solution for a few seconds, and wash with tap water. 4) Put the sections into Masson D solution for 6 min and rinse with tap water. 5) Soak for 1 min in Masson E solution. 6) Do not wash with water, drain slightly and directly put into Masson F solution for staining for 2-30 s. 7) The slices were rinsed and differentiated with 1% acetic acid, and dehydrated in two cylinders of anhydrous ethanol. 8) Transparent sealing: The slices were placed in the third cylinder of anhydrous ethanol for 5 minutes, xylene for 5 minutes to make them transparent, and neutral gum to seal the slices. 9) Microscope examination, image acquisition and analysis.

[0040] 4. Statistical data analysis GraphPad Prism 8.0 software was used for statistical processing and analysis of the data. The data were expressed as mean ± SD. Multiple comparisons and one-way analysis of variance were used to analyze and compare the data of each group.

[0041] 3. Experimental Results (1) Liver tissue status like Figure 1 As shown in the figure, compared with the control group, the liver of the mice in the model group was enlarged, the edges were blunted, the color was yellow, and there was a greasy feeling. After combined administration, the above-mentioned lesions in the liver tissue were significantly alleviated, and the oil content was significantly reduced. The efficacy was equivalent to that of the clinical equivalent dose group of Res and was superior to that of the low-dose Met group and the high-dose Met group ( Figure 1 These data indicate that combined administration of 100 mg / kg-1 ameliorates the pathological changes in the appearance of liver tissue in MASH mice and the effect is comparable to that of the clinical equivalent dose group of Res.

[0042] (2) HE staining, Oil Red O staining and TEM analysis results Compared with the control group, the model group mice showed balloon-like lesions and hepatocyte enlargement in the liver tissue, and the combined administration of drugs could improve the damage of liver tissue ( Figure 2 A in the figure). Excessive lipid accumulation is a prominent feature of MASH. Compared with the normal group, the oil red O staining area of ​​the liver tissue of the model group mice was larger and darker, and lipid accumulation was significantly increased ( Figure 2B). TEM observation of the liver tissue ultrastructure showed that compared with the control mice, the lipid droplets in the liver cells of the model mice were larger and more numerous, and the mitochondria were swollen ( Figure 2 However, after combined drug treatment, ballooning lesions in the liver tissue of db / db mice were improved and lipid accumulation was significantly reduced ( Figure 2 ). The results showed that the combination of Res and Met could inhibit the excessive accumulation of lipids in the liver tissue of MASH mice.

[0043] (3) Determination of inflammatory factors See Figure 2 D and Figure 2 As shown in Figure E, compared with the control group, the content of proinflammatory factor TNFα in the serum of mice in the model group was significantly increased, and the content of anti-inflammatory factor IL10 was significantly decreased. Compared with the model group, combined administration can inhibit the expression of proinflammatory factor TNFα in the serum of db / db mice, and increase the expression of anti-inflammatory factor IL10, thereby improving the inflammatory response of liver tissue, and the improvement effect on inflammatory factors is significantly better than the clinical equivalent dose of Res alone, low dose of Met and high dose of Met, indicating that the combined use of Res and Met plays a synergistic role.

[0044] (4) Sirius red staining and Masson staining results Compared with the normal group, the degree of liver fibrosis in the model group mice was significantly increased ( Figure 3 ), compared with the model group, the degree of fibrosis in the combined drug administration group was significantly improved, indicating that combined drug administration can significantly alleviate liver fibrosis in MASH mice.

[0045] Experimental Example 2 1. Experimental methods The serum of 12 mice in each group after 9 weeks of administration in the model group, control group and combined administration group in Experimental Example 1 was taken, and the contents of TG (triglyceride) and TC (total cholesterol) in the serum of mice were detected using Beckman Coulter biochemical analyzer AU480 according to the instructions of the kit. TG: The triglyceride assay kit (Cat. No.: 100020090) purchased from Biosino Biotech Co., Ltd. was used for detection. TC: The cholesterol assay kit (Cat. No.: 100020080) purchased from Biosino Biotech Co., Ltd. was used for detection.

[0046] 2. Statistical data analysis GraphPad Prism 8.0 software was used for statistical processing and analysis of the data. The data were expressed as mean ± SD. Multiple comparisons and one-way analysis of variance were used to analyze and compare the data of each group.

[0047] 3. Experimental results The experimental results are shown in Figure 4 As shown in Figure 2, TG and TC levels are closely related to blood lipid levels and are closely related to the progression of MASH. Therefore, we detected the levels of TG and TC in mouse serum to evaluate the blood lipid levels of mice. Figure 4 As shown in the figure, compared with the control group, the serum TG and TC levels of db / db mice were significantly increased. Combined administration can significantly reduce the levels of TG and TC in the serum of db / db mice. Therefore, combined treatment with Res and Met can improve the blood lipid levels of MASH model mice and delay the progression of MASH disease.

[0048] Experimental Example 3 1. Experimental methods The liver tissues of the model group, control group and combined drug administration group of 6 mice in each group were taken after 9 weeks of drug administration in Experimental Example 1 and the following tests were performed.

[0049] (1) Determination of transcriptional levels of lipid metabolism-related genes The acyl-CoA dehydrogenase long chain (ACADL) gene and carnitine palmitoyltransferase 1A (CPT1A) gene are genes related to lipid degradation, while the peroxisome proliferator-activated receptor (PPARG) gene is related to lipid synthesis. When the expression of genes related to lipid metabolism is abnormal, it will lead to liver lipid metabolism disorders and promote the progression of MASH.

[0050] Total RNA was extracted from mouse liver tissue using Trizol reagent. Then, the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (PrimeScript RT Reagent kit; RR036A; Takara Bio). Finally, we used a fluorescence quantitative kit (RR820A; Takara Bio) for real-time fluorescence quantitative analysis (RT-qPCR). The primer sequences used for RT-qPCR are shown in Table 1.

[0051] Table 1 Primer sequences of lipid metabolism related genes

[0052] (2) Determination of transcriptional levels of inflammation-related genes in liver tissue The protein encoded by the tumor necrosis factor-α (TNFα) gene is a key proinflammatory cytokine. The protein encoded by the interleukin-10 (IL10) gene is a key anti-inflammatory cytokine produced by many different types of immune cells and tissue epithelial cells. Total RNA was extracted from mouse liver tissue using Trizol reagent. Then, the extracted RNA was reverse transcribed into cDNA using PrimeScript RTReagent kit (RR036A; Takara Bio). Finally, we used a fluorescence quantitative kit (RR820A; Takara Bio) for real-time fluorescence quantitative analysis. The primer sequences used for RT-qPCR are shown in Table 2.

[0053] Table 2 Primer sequences of genes related to liver tissue inflammation

[0054] (3) Determination of transcriptional levels of genes related to liver fibrosis The proteins encoded by type I collagen alpha 1 chain (COL1A1) and type I collagen alpha 2 chain (COL1A2) are the main components of connective tissue and are essential for maintaining tissue integrity. The protein encoded by TIMP metallopeptidase inhibitor 1 (TIMP1) can also promote cell proliferation in a variety of cell types and may also have anti-apoptotic functions. Total RNA was extracted from mouse liver tissue using Trizol reagent. Then, the extracted RNA was reverse transcribed into cDNA using PrimeScript RT Reagentkit (RR036A; Takara Bio). Finally, we used the fluorescence quantitative kit (RR820A; Takara Bio) for real-time fluorescence quantitative analysis. The primer sequences used for RT-qPCR are shown in Table 3.

[0055] Table 3 Primer sequences of genes related to liver fibrosis

[0056] 2. Statistical data analysis GraphPad Prism 8.0 software was used for statistical processing and analysis of the data. The data were expressed as mean ± SD. Multiple comparisons and one-way analysis of variance were used to analyze and compare the data of each group.

[0057] 3. Experimental results Results Figure 5 and Figure 6 As shown, from Figure 5 A- Figure 5As can be seen from the C in the figure, the combined administration of Res and Met can increase the expression levels of ACADL and CPT1A in the liver tissue of the model group mice, reduce the expression level of PPARG, and further improve liver lipid metabolism, thereby reducing the excessive accumulation of lipids in the liver of db / db mice. The results of the study showed that combined drug treatment can improve the lipid metabolism disorder in the liver of MASH mice. Compared with the normal group, the level of the pro-inflammatory gene TNFα in the liver tissue of the model group mice was significantly increased, and the level of the anti-inflammatory gene IL10 was significantly decreased. Compared with the model group, the combined drug administration can reduce the expression level of the pro-inflammatory gene TNFα in the liver tissue and promote the expression of the anti-inflammatory gene IL10 ( Figure 5 D- Figure 5 E in the liver), thereby improving the inflammatory response of liver tissue.

[0058] from Figure 6 It can be seen that compared with the normal group, the expression levels of liver fibrosis markers COL1A1, COL1A2, and TIMP1 in the model group mice were significantly increased. Compared with the model group, combined administration can reduce the expression level of fibrosis genes in liver tissue. The results show that combined administration can significantly alleviate liver fibrosis in MASH mice.

[0059] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of resmetiro combined with metformin or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, treating or alleviating fatty liver disease associated with metabolic dysfunction.

2. The use according to claim 1, characterized in that: The mass ratio of resmetiro to metformin or a pharmaceutically acceptable salt thereof is 6-10:180-250; and / or, the pharmaceutically acceptable salt comprises hydrochloride.

3. The use according to claim 1 or 2, characterized in that: The dosage form of the drug includes powder, tablet, solution, capsule, granule or suspension.

4. The use according to claim 1 or 2, characterized in that: The resmetiro and metformin or a pharmaceutically acceptable salt thereof are respectively used as active ingredients in the same preparation unit or in different preparation units.

5. The use according to claim 4, characterized in that: The drug comprises resmetiro and a pharmaceutically acceptable carrier to form a first preparation, and the metformin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier to form a second preparation.

6. The use according to claim 1 or 2, characterized in that: The metabolic dysfunction-related fatty liver disease includes one or more of simple fatty infiltration, metabolic dysfunction-related fatty hepatitis or metabolic dysfunction-related liver fibrosis.

7. A drug for preventing, treating or alleviating fatty liver disease associated with metabolic dysfunction, characterized in that: The drugs include resmetiro and metformin or a pharmaceutically acceptable salt thereof.

8. The drug according to claim 7, characterized in that The mass ratio of resmetiro to metformin or a pharmaceutically acceptable salt thereof is 6-10:180-250; and / or, the pharmaceutically acceptable salt comprises hydrochloride.

9. The drug according to claim 7, characterized in that The resmetiro and metformin or a pharmaceutically acceptable salt thereof are respectively used as active ingredients in the same preparation unit or in different preparation units; and / or, the drug further includes a pharmaceutically acceptable carrier.

10. The drug according to claim 9, characterized in that The pharmaceutically acceptable carrier is selected from one or more of pharmaceutically acceptable solvents, cosolvents, emulsifiers, flavoring agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, preservatives, suspending agents, coating materials, pH regulators, absorbents, flocculants, and deflocculating agents.

11. The drug according to any one of claims 7 to 9, characterized in that The metabolic dysfunction-related fatty liver disease includes one or more of simple fatty infiltration, metabolic dysfunction-related fatty hepatitis or metabolic dysfunction-related liver fibrosis.

Images