Compositions for combating metabolic diseases and their uses

Abstract

A composition for combating metabolic diseases and its use (or method). The pharmaceutical composition comprises a therapeutic agent A or a pharmaceutically acceptable salt thereof; a therapeutic agent B or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. The pharmaceutical composition can effectively treat or prevent metabolic syndrome diseases caused by obesity, non-alcoholic fatty liver disease, polycystic ovary syndrome, type 2 diabetes and insulin resistance.

Description

Technical Field

[0001] The present invention relates to the field of small molecule drugs, and in particular, to a composition for reducing the risk of metabolic syndrome and its uses. Background Art

[0002] Metabolic syndrome refers to a pathological state in which substances such as proteins, fats, and carbohydrates in the human body undergo metabolic disorders. It is not a single disease, but a group of complex metabolic disorder syndromes: abdominal fat accumulation, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hypertension, hyperglycemia, etc. The central link of metabolic syndrome is obesity and insulin resistance, and its main component is obesity, especially central obesity. Subjects with metabolic syndrome have risk factors for diabetes, cardiovascular and cerebrovascular diseases, fatty liver, polycystic ovary syndrome, and the prevalence of cardiovascular events and the risk of death are about 2-3 times that of non-metabolic syndrome subjects.

[0003] Obesity and type 2 diabetes are prevalent worldwide (see National Diabetes Data Group, Diabetes in America, United States: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 1994; Mokdad et al., Diabetes Care 23(9):1278 - 12 S3(2000); Mokdad et al., JAMA 284(13):1650 - 1651(2000); Mokdad et al., JAMA286(10):1195 - 1200(2001); Mokdad et al., JAMA 289(1):76 - 79(2003)). In 2000, it was estimated that 2.9 million people died from diabetes-related causes (Roglic et al., Diabetes Care 28:2130 - 2135, 2005), and it is estimated that the global diabetes burden will double in the next 25 years (King et al., Diabetes Care 21:1414 - 1431, 1998; Amos et al., Diabet Med 14 Suppl 5:S1 - 85, 1997; Wild et al., Diabetes Care 27:1047 - 1053, 2004), in parallel with the increase in obesity. Type 2 diabetes (T2DM) is an increasingly common disease, and due to the high frequency of complications, it leads to a significant reduction in life expectancy. Due to diabetes-related microvascular complications, type 2 diabetes is currently the most common cause of adult-onset vision loss, renal failure, and amputation in the industrialized world. In addition, the presence of type 2 diabetes is associated with an increased risk of cardiovascular disease.

[0004] After a disease has persisted for a long time, most type 2 diabetes subjects will eventually become insulin-dependent due to oral treatment failure, requiring daily injections and multiple daily glucose measurements. The UKPDS (United Kingdom Prospective Diabetes Study) demonstrated that intensive treatment with metformin, sulfonylureas, or insulin resulted in only limited improvement in glycemic control (a difference in HbA1c of ~0.9%). Additionally, even among subjects in the intensive treatment group, glycemic control deteriorated significantly over time, which was attributed to the deterioration of β-cell function. Importantly, intensive treatment was not associated with a significant reduction in macrovascular complications (i.e., cardiovascular events).

[0005] Accordingly, there is an unmet medical need for methods, drugs, and pharmaceutical compositions that have good efficacy in terms of glycemic control, in terms of the nature of disease improvement, and in terms of a safety profile that shows improvement while reducing cardiovascular morbidity and mortality.

[0006] In current research, inflammation induced by obesity plays a crucial role, especially the link between elevated blood glucose levels and the activation of Cox-2 in pancreatic β-cells has been well established. High glucose-induced prostaglandin E2 (PGE2) leads to a decrease in β-cell mass by inhibiting β-cell proliferation and inducing β-cell apoptosis (Oshima, H. et al., 2006). Indomethacin, a non-selective cyclooxygenase inhibitor, can prevent high-fat diet (HFD)-induced obesity and insulin resistance in C57BL / 6J mice (Fjaere E. et al., 2014). It has been demonstrated that treatment with the non-steroidal anti-inflammatory drug (NSAID) celecoxib can partially restore insulin sensitivity in both preclinical models and obese type 2 diabetes mellitus (T2DM) subjects (Gonzalez-Ortiz et al., 2005). Hyperglycemia activates Cox-2 in pancreatic β-cells and causes β-cell dysfunction. Treatment with NS-398, a selective Cox-2 inhibitor, may reverse β-cell dysfunction by reducing PGE2-mediated β-cell apoptosis (Tian, V.F. et al., 2014). Inflammation induced by obesity leads to non-alcoholic hepatic steatosis, which is a pathological hallmark of insulin resistance. Non-alcoholic steatohepatitis (NASH) is a condition that coexists with T2DM. Celecoxib can reverse steatohepatitis and inflammation in a NASH model of HFD-induced Wistar rats (Chen, J. et al., 2011). Overexpression of NAG-1 / GDF-15 (NSAID-activated gene-1) has been shown to improve glycemic parameters and prevent the development of obesity by increasing thermogenesis, lipolysis, and oxidative metabolism in obese C57BL / 6J mice (Chrysovergis, K. et al., 2014). The activation of inducible form of Cox-2 plays a crucial role in the initiation of cellular dysfunction, including adipocyte dysfunction, pancreatic islet β-cell dysfunction, and macrophage dysfunction. Cellular dysfunction contributes to the development of insulin resistance and systemic glucose intolerance. Deletion of Cox-2 in C57BL / 6J obese mice reduces blood glucose levels (Fujta et al., 2007). More importantly, in the same preclinical models, the selective Cox-2 inhibitor celecoxib can slightly reduce HbA1c levels, improve glucose tolerance, and increase insulin levels (Fujita, H. et al., 2007). Based on preclinical and clinical data, targeting the potential inflammatory components of the complex pathophysiology of type 2 diabetes with anti-inflammatory therapies is one of the strategies for partial remission and management of type 2 diabetes.

[0007] In addition, many publications have supported the importance of adipocytes and inflammation in the development of insulin resistance. For example, JNK-1 deficiency in adipocytes inhibits high-fat diet-induced insulin resistance in the liver due to JNK-dependence (Sabio, C. et al., 2008), adipocyte-specific Glut4 deletion or MCP-1 overexpression leads to systemic insulin resistance (Qi, L. et al., 2009), and TNF-α deficiency improves insulin sensitivity in diet-induced obesity and Lep ob / ob models of obesity (Hotamisiligil, G.S. et al., 1995). Elevated IL-1β, IL-6, and CRP predict the development of T2DM (Visser, M. et al., 1999), and TLR4 knockout mice are protected from inflammation and insulin resistance (Shi, H. et al., 2006). Current hypoglycemic drugs do not involve anti-inflammatory agents at all, and thus lack sufficient efficacy and overall clinical benefit. This is because they cannot inhibit the pro-inflammatory components of the complex pathophysiology that initiates and maintains systemic insulin resistance.

[0008] Inflammation is not only a pathophysiology of type 2 diabetes, but also an important component of clinically relevant comorbidities. More importantly, pro-inflammatory signals contribute to the initiation and maintenance of complications associated with type 2 diabetes, such as diabetic retinopathy, skin ulcers, coronary heart disease (CHD), stroke, fatty liver, polycystic ovary syndrome, chronic kidney disease (CKD), diabetic peripheral neuropathy, and diabetic angiopathy. Pro-inflammatory signals determine the severity and duration of diabetes-related complications. There is a direct association between elevated pro-inflammatory biomarkers and impaired glucose metabolism. Therefore, for effective clinical management, it is important to jointly regulate inflammation and metabolism, and to treat metabolic diseases including type 2 diabetes, including all metabolic syndrome diseases with complex pathophysiology with strong pro-inflammatory components, rather than treating each metabolic disease as an individual disease. Currently, single use of NSAID drugs cannot comprehensively inhibit numerous inflammatory factors and inflammatory pathways, and single use of hypoglycemic drugs cannot comprehensively restore glucose and lipid metabolism. For example, in order to obtain sufficient therapeutic effects in subjects with type 2 diabetes, fatty liver, polycystic ovary syndrome, or obesity, it is necessary to treat with a combination of drugs that jointly regulate inflammation and metabolism, which can not only correct impaired blood glucose and lipid homeostasis, but also reduce clinically relevant metabolic comorbidities. More importantly, it can reduce the severity of diabetes-related complications. Therefore, it is not advisable to treat subjects with metabolic syndrome with only one drug, and it is necessary to use a combination of anti-inflammatory drugs and anti-metabolic disease drugs to treat the subjects. SUMMARY OF THE INVENTION

[0009] The object of the present invention is to overcome the defects existing in the prior art, that is, all drugs on the market cannot effectively treat or prevent metabolic syndrome diseases caused by obesity, type 2 diabetes and insulin resistance, so as to provide a pharmaceutical composition that can not only correct impaired glucose homeostasis, but also alleviate clinically relevant complications, and more importantly, can reduce the severity of diabetes-related complications.

[0010] To achieve the above object, in one aspect, the present invention provides a pharmaceutical composition comprising a therapeutic agent A or a pharmaceutically acceptable salt thereof; a therapeutic agent B or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor.

[0011] In one embodiment, in the pharmaceutical composition, the therapeutic agent A and the therapeutic agent B are contained in a single dosage form.

[0012] In another embodiment, in the pharmaceutical composition, the therapeutic agent A and the therapeutic agent B exist in separate dosage forms.

[0013] In one embodiment, the therapeutic agent A is selected from at least one of salicylates, ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenoprofen, ketoprofen, ketorolac, tiaprofenic acid, sulindac and tolmetin.

[0014] In another embodiment, the therapeutic agent B is selected from trimetazidine, etomoxir, aminocarnitine or a phosphonyloxy derivative of carnitine.

[0015] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutic agent A or a pharmaceutically acceptable salt thereof; a therapeutic agent B or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient, wherein the therapeutic agent A is selected from salicylates and the therapeutic agent B is selected from trimetazidine.

[0016] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutic agent A or a pharmaceutically acceptable salt thereof; a therapeutic agent B or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient, wherein the therapeutic agent A is selected from aspirin and the therapeutic agent B is selected from trimetazidine.

[0017] In some embodiments, the weight ratio of the therapeutic agent A (e.g., salicylate, such as aspirin) and the therapeutic agent B (e.g., trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (e.g., salicylate, such as aspirin) and the therapeutic agent B (e.g., trimetazidine) is 6:1.

[0018] In some embodiments, the pharmaceutical dosage form is an oral dosage form. In some embodiments, the pharmaceutical dosage form is an injectable dosage form.

[0019] In another aspect, the present invention also provides the use (or method) of the therapeutic agent A and the therapeutic agent B in the preparation of a medicament for treating type 1 diabetes, type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, postprandial hyperglycemia, overweight, obesity and metabolic syndrome in a subject in need thereof; or improving blood glucose control and / or reducing fasting plasma glucose, postprandial plasma glucose and / or glycated hemoglobin HbA1c; or slowing, delaying or reversing the progression from impaired glucose tolerance, impaired fasting glucose, insulin resistance and / or metabolic syndrome to type 2 diabetes; or treating diabetic complications such as cataracts, and microvascular and macrovascular diseases such as nephropathy, retinopathy, neuropathy, tissue ischemia, arteriosclerosis, myocardial infarction, stroke and peripheral arterial occlusive disease; or reducing weight or promoting weight loss; or treating pancreatic β-cell degeneration and / or decline of pancreatic β-cell function, and / or restoring pancreatic β-cell function, and / or restoring pancreatic insulin secretion function; or treating diseases or disorders leading to abnormal accumulation of liver fat; or maintaining and / or improving insulin sensitivity and / or treating hyperinsulinemia and / or insulin resistance; or treating atherosclerosis and atherosclerotic complications; or treating glaucoma and glaucoma complications; treating dyslipidemia / hyperlipidemia and dyslipidemia / hyperlipidemia complications, or treating reproductive-related metabolic diseases, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor.

[0020] In one embodiment, the subject is an individual diagnosed with one or more conditions selected from overweight, obesity, visceral obesity and abdominal obesity.

[0021] In another embodiment, the subject is an individual diagnosed with one or more of the following conditions:

[0022] (a) Fasting blood glucose or serum glucose concentration greater than 110 mg / dL, particularly greater than 125 mg / dL;

[0023] (b) Postprandial plasma glucose concentration equal to or greater than 140 mg / dL; and

[0024] (c) The HbA1c value is equal to or greater than 6.5%, especially equal to or greater than 8.0%.

[0025] In another embodiment, the subject is an individual having one or more of the following conditions:

[0026] (a) Obesity, visceral obesity, and / or abdominal obesity;

[0027] (b) Plasma triglyceride concentration ≥ 150 mg / dL;

[0028] (c) For female subjects, the blood level of HDL-cholesterol < 40 mg / dL, and for male subjects, the blood level of HDL-cholesterol < 50 mg / dL;

[0029] (d) Systolic blood pressure ≥ 130 mmHg and diastolic blood pressure ≥ 85 mmHg;

[0030] (e) Fasting blood glucose level ≥ 110 mg / dL; and

[0031] (f) LDL-cholesterol blood level ≥ 130 mg / dL.

[0032] In another embodiment, the subject is an individual who is disabled from metformin monotherapy and / or intolerant to therapeutic doses of metformin.

[0033] In another embodiment, the subject is an individual with insufficient blood glucose control after treatment with one or more anti-diabetic drugs selected from the group consisting of:

[0034] (a) Biguanides;

[0035] (b) Sulfonylureas;

[0036] (c) Meglitinides;

[0037] (d) Thiazolidinediones;

[0038] (e) Alpha-glucosidase inhibitors;

[0039] (f) Insulin and insulin analogs;

[0040] (g) Dipeptidyl peptidase-IV inhibitors;

[0041] (h) SGLT2 inhibitors;

[0042] (i) PPARα modulators;

[0043] (j) Glucose-dependent insulinotropic polypeptide agonists;

[0044] (k) Beta-3 agonists;

[0045] (l) GLP1 and GLP1 analogs;

[0046] (m) PPARγ modulators; and

[0047] (n) HMG-CoA reductase inhibitors.

[0048] The pharmaceutical composition according to the present invention exhibits very good efficacy in blood glucose control, especially in terms of reducing fasting plasma glucose, postprandial plasma glucose, and / or glycated hemoglobin (HbA1c), and at the same time can effectively treat or prevent metabolic syndrome diseases caused by obesity, non-alcoholic fatty liver, polycystic ovary syndrome, type 2 diabetes, and insulin resistance.

[0049] In some embodiments, the therapeutic agent A is selected from salicylates, and the therapeutic agent B is selected from trimetazidine.

[0050] In some embodiments, the therapeutic agent A is selected from aspirin, and the therapeutic agent B is selected from trimetazidine.

[0051] In some embodiments, the weight ratio of the therapeutic agent A (e.g., salicylates, such as aspirin) to the therapeutic agent B (e.g., trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (e.g., salicylates, such as aspirin) to the therapeutic agent B (e.g., trimetazidine) is 6:1.

[0052] In some embodiments, the therapeutic agent A and the therapeutic agent B are administered simultaneously. In some embodiments, the therapeutic agent A and the therapeutic agent B are contained in a single dosage form. In some embodiments, the single pharmaceutical dosage form is an oral dosage form.

[0053] In some embodiments, the therapeutic agent A and the therapeutic agent B are administered separately. In some embodiments, the therapeutic agent A is administered before the therapeutic agent B. In some embodiments, the therapeutic agent A is administered after the therapeutic agent B. In some embodiments, the therapeutic agent A and the therapeutic agent B are administered orally separately. In some embodiments, the therapeutic agent A and the therapeutic agent B are administered by injection separately.

[0054] Other features and advantages of the present invention will be described in detail in the subsequent detailed description section. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification, and are used together with the following detailed description to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0056] Figure 1 It shows the results of the weight change of diet-induced obesity (DIO) mice after 10 days of drug therapeutic agents A + B in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the control group.

[0057] Figure 2 It shows the results of fat reduction induced by drug therapeutic agents A + B in DIO mice in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the control group.

[0058] Figure 3 It shows the results of the adipocyte morphology of the gonadal fat pad in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the DIO control group.

[0059] Figure 4 It shows the results of fatty liver and hepatocyte morphology in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the DIO control group.

[0060] Figure 5 It shows the results of the cardiac muscle cell morphology in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the DIO control group.

[0061] Figure 6 It shows the results of the skeletal muscle cell morphology in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the DIO control group.

[0062] Figure 7 It shows the results of the kidney toxicity indices serum creatinine (CREA) and blood urea nitrogen (BUN) in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the DIO control group.

[0063] Figure 8 It shows the results of the weight change of DIO mice in diabetic patients after 4 weeks of drug therapeutic agents A + B in the test group using the pharmaceutical composition according to an embodiment of the present invention, compared with the control group.

[0064] Figure 9 It shows the comparison of the total serum cholesterol, an index related to hyperlipidemia, in DIO mice of each group.

[0065] Figure 10 It shows the comparison of serum LDL-cholesterol, an index related to hyperlipidemia, in DIO mice of each group.

[0066] Figure 11 It shows the comparison of serum alanine aminotransferase (ALT), an index related to fatty liver, in DIO mice of each group.

[0067] Figure 12Shows the comparison of the serum albumin / globulin ratio, an index related to nitrogen metabolism function, among groups of DIO mice.

[0068] Figure 13 Shows the comparison of total serum protein, an index related to nitrogen metabolism function, among groups of DIO mice.

[0069] Figure 14 Shows the results of glucose tolerance in DIO mice of diabetic patients.

[0070] Figure 15 Shows the results of insulin sensitivity in DIO mice of diabetic patients.

[0071] Figure 16 Shows the comparison of the body weights of DIO mice after administration of various combinations of therapeutic agents.

[0072] Figure 17 Shows the comparison of the fasting blood glucose levels of DIO mice after administration of various combinations of therapeutic agents.

[0073] Figure 18 Shows the results of the weight change induced by the drug therapeutic agents A + B in normal wild-type mice in the test group using the pharmaceutical composition according to an embodiment of the present invention compared with the control group.

[0074] Figure 19 Shows the results of the fat reduction induced by the drug therapeutic agents A + B in normal wild-type mice in the test group using the pharmaceutical composition according to an embodiment of the present invention compared with the control group.

[0075] Figure 20 Shows the results of the weight change induced by the drug therapeutic agents A + B in rats with polycystic ovary syndrome in the test group using the pharmaceutical composition according to an embodiment of the present invention compared with the control group.

[0076] Figure 21 Shows the comparison of the fasting blood glucose levels among groups of rats with polycystic ovary syndrome.

[0077] Figure 22 Shows the comparison of serum aspartate aminotransferase (AST) and total cholesterol, indices related to fatty liver, among groups of rats with polycystic ovary syndrome.

[0078] Figure 23 Shows the comparison of total serum protein, an index related to liver nitrogen metabolism function, among groups of rats with polycystic ovary syndrome.

[0079] Figure 24 Shows the comparison of serum creatinine, an index related to kidney function, among groups of rats with polycystic ovary syndrome.

[0080] Figure 25 The comparison of the indicators related to heart disease, namely serum lactate dehydrogenase (LDH), creatine kinase (CK), myocardial creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH), in each group of polycystic ovary syndrome rats is shown.

[0081] Figure 26 The results of insulin sensitivity in polycystic ovary syndrome rats are shown.

[0082] Figure 27 The results of the estrous cycle in polycystic ovary syndrome rats are shown.

[0083] Figure 28 The results of serum hormone ELISA in polycystic ovary syndrome rats are shown.

[0084] Figure 29 The results of Western blot analysis of skeletal muscle protein in polycystic ovary syndrome rats are shown.

[0085] Figure 30 The results of left ventricular wall thickness analysis in polycystic ovary syndrome rats are shown.

[0086] Figure 31 The results of myocardial fibrosis analysis in polycystic ovary syndrome rats are shown.

[0087] Figure 32 The results of blood routine analysis in polycystic ovary syndrome rats are shown.

[0088] Figure 33 The results of the weight change in NASH mice treated with drug therapeutic agents A + B are shown.

[0089] Figure 34 The results of food intake test in NASH mice are shown.

[0090] Figure 35 The results of liver weight test in NASH mice are shown.

[0091] Figure 36 The results of liver section analysis in NASH mice are shown.

[0092] Figure 37 The results of heart section analysis in NASH mice are shown.

[0093] Figure 38 The comparison of two indicators related to liver injury in each group of mice is shown.

[0094] Figure 39 The comparison of the indicators related to hyperlipidemia in each group of mice is shown.

[0095] Figure 40Shows the comparison of indicators related to organ damage in each group of mice.

[0096] Figure 41 Shows the results of fasting blood glucose in each group of mice.

[0097] Figure 42 Shows the results of glucose tolerance in NASH mice.

[0098] Figure 43 Shows the results of insulin sensitivity in NASH mice.

[0099] Figure 44 Shows the results of Western blot analysis of skeletal muscle proteins in NASH mice.

[0100] Figure 45 Shows the results of transcriptomic sequencing analysis in NASH mice.

[0101] Figure 46 Shows the results of Western blot analysis of proteins 24 hours after drug administration in primary human skeletal muscle cells in vitro.

[0102] Figure 47 Shows the results of Western blot analysis of proteins 7 days after drug administration in primary human skeletal muscle cells induced to have insulin resistance in vitro.

[0103] Figure 48 Shows the results of transcriptomic sequencing analysis of skeletal muscle in PCOS polycystic ovary syndrome rats induced by a high-fat and high-sugar diet.

[0104] Figure 49 Shows the results of insulin enzyme-linked immunosorbent assay and insulin resistance index HOMA-IR results in serum samples of NASH mice.

[0105] Figure 50 Shows the results of adiponectin enzyme-linked immunosorbent assay in serum samples of NASH mice.

[0106] Figure 51 Shows the comparison results of liquid chromatography-mass spectrometry analysis of serum samples in mice of control group 1 and control group 3. Detailed implementation manners

[0107] The following details the specific implementation manners of the present invention. It should be understood that the specific implementation manners described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0108] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0109] The present invention is largely based on the unexpected effects obtained by the inventors from the combined use of a non-steroidal anti-inflammatory drug (therapeutic agent A) and a fatty acid oxidation inhibitor (therapeutic agent B). The inventors surprisingly found that when therapeutic agent A and therapeutic agent B are simultaneously administered to a subject with diabetes, cardiovascular and cerebrovascular diseases, fatty liver, or polycystic ovary syndrome, multiple indicators of the subject are improved, and the drug combination can also achieve the effect of reversing multiple symptoms. The inventors also surprisingly found that the mechanism of action of the drug combination is not limited to regulating inflammatory responses and metabolism. Administering the drug combination daily can specifically cause the p38 and AMPK signaling pathways and related metabolic changes to cycle repeatedly, forming an excitatory effect cycle, just like mimicking exercise. After short-term drug administration (within 60 minutes), the drug therapeutic agent A+B provided by the present invention regulates the p38 and AMPK signaling pathways through fatty acid oxidation (FAO), fatty acid metabolites (acyl-metabolites), and adenosine triphosphate (ATP) in a combined manner, causing a simultaneous sharp increase in the p38 and AMPK signaling pathways, thereby promoting catabolism such as lipolysis and fatty acid oxidation. After long-term drug administration (3 - 24 hours), the drug therapeutic agent A+B provided by the present invention regulates the p38 and AMPK signaling pathways through inflammatory cytokines (Infcytokines), fatty acid oxidation (FAO), mitochondrial reactive oxygen species (mtROS), and glycolysis in a combined manner, causing a simultaneous sharp decrease in the p38 and AMPK signaling pathways, thereby promoting anabolism and muscle repair. Therefore, this patent application provides a novel and effective pharmaceutical composition on the one hand and a treatment method using this pharmaceutical composition on the other hand.

[0110] Pharmaceutical composition

[0111] In one aspect, the present invention provides a pharmaceutical composition comprising therapeutic agent A or a pharmaceutically acceptable salt thereof; therapeutic agent B or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient, wherein therapeutic agent A is a non-steroidal anti-inflammatory drug and therapeutic agent B is a fatty acid oxidation inhibitor.

[0112] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio. Therapeutic agent A and therapeutic agent B can form stable pharmaceutically acceptable acid or base salts, and in such cases, it may be appropriate to administer the compound as a salt. Examples of acid addition salts include acetate, adipate, ascorbate, benzoate, benzenesulfonate, bicarbonate, bisulfate, butyrate, camphorate, camphorsulfonate, choline, citrate, cyclohexylsulfamate, diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethanesulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate, malate, maleate, mesylate, meglumine, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfonate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate and undecanoate. Examples of base salts include ammonium salts; alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as aluminum, calcium and magnesium salts; salts with organic bases such as dicyclohexylamine salts and N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, ornithine and the like.

[0113] In one embodiment, therapeutic agent A and therapeutic agent B in the pharmaceutical composition may be present in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means an amount of a compound or composition sufficient to significantly and positively alter the symptoms and / or condition to be treated (e.g., provide a positive clinical response). The effective amount of the active ingredient in a pharmaceutical composition will vary depending on the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the specific active ingredient employed, the specific pharmaceutically acceptable excipient utilized, and like factors within the knowledge and expertise of the attending physician.

[0114] As used herein, the term "treatment" refers to a method for obtaining a beneficial or desired clinical outcome. The term "treatment" refers to inhibiting, preventing or arresting the development or progression of a pathology (disease, disorder or condition) and / or causing the alleviation, remission or regression of a pathology. Those skilled in the art will understand that various methods and assays can be used to assess the development of a pathology, and similarly, various methods and assays can be used to assess the alleviation, remission or regression of a pathology.

[0115] As used herein, the term "prevention" refers to preventing a disease, disorder or condition from occurring in a subject who is at risk of the disease but has not been diagnosed as having the disease. Prevention (and the dose for effective prevention) can be demonstrated in population studies. For example, with respect to an untreated control population, an effective amount for preventing a given disease or medical condition is an amount that reduces the incidence rate in the treated population.

[0116] As used herein, the term "subject" can include mammals, preferably humans of any age with a pathological characteristic. Preferably, the term can also include individuals at risk of developing a pathological characteristic.

[0117] Therapeutic agent A and therapeutic agent B

[0118] According to the present invention, therapeutic agent A is a non-steroidal anti-inflammatory drug. As used herein, the term "non-steroidal anti-inflammatory drug (NSAID)" is a class of anti-inflammatory drugs that do not contain a steroid structure and can be mainly divided into three categories: namely, acetylsalicylates, including acetyl salicyclic acid; non-acetylsalicylates, including magnesium salicylate, sodium salicylate, choline magnesium salicylate, diflunisal, salsalate; non-salicylates, including ibuprofen, indomethacin, flurbiprofen, fenoprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenoprofen, ketoprofen, ketorolac, meclofenamic acid, sulindac, tolmetin, etc. And therapeutic agent A in the present invention can be selected from any one of these non-steroidal anti-inflammatory drugs. Preferably, therapeutic agent A can be selected from at least one of salicylates, ibuprofen, indomethacin, flurbiprofen, fenoprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenoprofen, ketoprofen, ketorolac, meclofenamic acid, sulindac and tolmetin. More preferably, therapeutic agent A can be a salicylate, such as aspirin or magnesium salicylate, etc.

[0119] According to the present invention, therapeutic agent B is a fatty acid oxidation inhibitor, including, but not limited to, Etomoxir, Meldonium, Oxfenicine, Perhexiline, Ranolazine, substituted piperazines, Trimetazidine, and derivatives of carnitine. Preferably, therapeutic agent B can be Trimetazidine, aminocarnitines (such as those described in WO85 / 04396), phosphinyloxy derivatives of carnitine (such as those described in EP0574355B1), or complex products of carnitine with other compounds (such as those described in JP5127093B2 / US6369073B1). More preferably, therapeutic agent B can be Trimetazidine.

[0120] According to the present invention, in the pharmaceutical composition of the present invention, there is no particular limitation on the combination mode of therapeutic agent A and therapeutic agent B. That is to say, in the pharmaceutical composition of the present invention, therapeutic agent A and therapeutic agent B can be included in a single dosage form, or can also exist in separate dosage forms, so that when the pharmaceutical composition of the present invention is subsequently used, therapeutic agent A and therapeutic agent B can be administered to the subject simultaneously, separately, or sequentially. For example, therapeutic agent A and therapeutic agent B can be administered to the subject simultaneously (such as administered in the same dosage form), therapeutic agent A can be administered to the subject and then therapeutic agent B can be immediately administered to the subject, or therapeutic agent B can be administered to the subject within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after therapeutic agent A is administered to the subject. In some embodiments, therapeutic agent B can be administered to the subject and then therapeutic agent A can be immediately administered to the subject, or therapeutic agent A can be administered to the subject within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after therapeutic agent B is administered to the subject.

[0121] Route of administration

[0122] The pharmaceutical composition of the present invention may be in a form suitable for oral administration (e.g., as tablets, lozenges, hard or soft gelatin capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical administration (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (e.g., as finely divided powders or liquid aerosols), administration by insufflation (e.g., as finely divided powders), or parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular or intra - muscular administration, or as suppositories for rectal administration). In a preferred embodiment, the pharmaceutical composition of the present invention is administered orally.

[0123] The pharmaceutical composition of the present invention can be obtained by conventional procedures using conventional pharmaceutical excipients known in the art. Suitable pharmaceutically acceptable excipients for tablet formulations include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch; lubricants such as magnesium stearate, stearic acid or talc; preservatives such as ethyl or propyl p - hydroxybenzoate; and antioxidants such as ascorbic acid. Tablet formulations can be uncoated or coated, and the coating is either to modify their disintegration in the gastrointestinal tract and subsequent absorption of the active ingredient, or to improve their stability and / or appearance, and in both cases, conventional coating agents and methods known in the art can be used.

[0124] There are no strict limitations on the dosage and frequency of administration of the pharmaceutical composition of the present invention, and it can vary according to many factors such as age, weight, general health, diet, gender, the drug to be administered, the route (or method) of administration, and the severity of the condition being treated, as well as the judgment of the attending physician. Generally, the pharmaceutical composition of the present invention can be administered once or multiple times a day, e.g., once a day, twice a day, three times a day or more, and can also be administered once every two days, once every three days, once a week or other frequencies. In terms of the daily dose, the amount of therapeutic agent A can be from 0.1 mg to 5000 mg per day, or preferably from 10 mg to 3000 mg, more preferably from 80 mg to 2000 mg, or even more preferably from 500 mg to 1500 mg, and the amount of therapeutic agent B can be from 0.1 mg to 1000 mg per day, or preferably from 10 mg to 1000 mg, more preferably from 100 mg to 500 mg, or even more preferably from 35 mg to 300 mg. Therefore, the above - mentioned dosages can be reasonably achieved according to the different administration frequencies and methods, such as administering 5 mg to 500 mg of therapeutic agent A once a day, or administering 10 mg to 250 mg of therapeutic agent A twice a day, etc.

[0125] In some embodiments, the pharmaceutical composition comprises a salicylate (such as 100, 200, 300, 400, 500, or 600 mg of salicylate), and trimetazidine (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg of trimetazidine). In some embodiments, the pharmaceutical composition comprises aspirin (such as 100, 200, 300, 400, 500, or 600 mg of aspirin), and trimetazidine (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg of trimetazidine). In some embodiments, the weight ratio of the salicylate (such as aspirin) to trimetazidine is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0126] Use (or method)

[0127] In another aspect, the present invention also provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, postprandial hyperglycemia, type 1 diabetes, overweight, obesity and metabolic syndrome in a subject in need thereof; or improving glycemic control and / or reducing fasting plasma glucose, postprandial plasma glucose and / or glycated hemoglobin HbA1c; or slowing, delaying or reversing the progression from impaired glucose tolerance, impaired fasting glucose, insulin resistance and / or metabolic syndrome to type 2 diabetes; or treating diabetic complications such as cataracts, as well as microvascular and macrovascular diseases such as nephropathy, retinopathy, neuropathy, tissue ischemia, arteriosclerosis, myocardial infarction, stroke and peripheral arterial occlusive disease; or reducing or promoting weight loss; or treating pancreatic β-cell degeneration and / or decline in pancreatic β-cell function, and / or restoring pancreatic β-cell function, and / or restoring pancreatic insulin secretion function; or treating diseases or disorders leading to abnormal accumulation of fat in the liver; or maintaining and / or improving insulin sensitivity and / or treating hyperinsulinemia and / or insulin resistance; or treating atherosclerosis and atherosclerotic complications; or treating glaucoma and glaucoma complications; or treating dyslipidemia / hyperlipidemia and dyslipidemia / hyperlipidemia complications, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor.

[0128] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating one or more of type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, type 1 diabetes, obesity, non-alcoholic steatohepatitis, atherosclerosis, glaucoma, dyslipidemia / hyperlipidemia, and hyperglycemia in a subject in need thereof, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) of treating one or more of type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, type 1 diabetes, obesity, non-alcoholic steatohepatitis, atherosclerosis, glaucoma, dyslipidemia / hyperlipidemia, and hyperglycemia in a subject in need thereof, comprising administering therapeutic agent A and therapeutic agent B to an affected individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0129] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating type 1 diabetes or type 2 diabetes in a subject in need thereof, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) of treating type 1 diabetes or type 2 diabetes in a subject in need thereof, comprising administering therapeutic agent A and therapeutic agent B to an affected individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0130] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating a disease or disorder causing abnormal accumulation of fat in the liver in a subject in need thereof, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) of treating a disease or disorder causing abnormal accumulation of fat in the liver in a subject in need thereof, comprising administering therapeutic agent A and therapeutic agent B to an affected individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, like aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, like aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0131] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating reproductive-related metabolic diseases in a subject in need thereof, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) of treating reproductive-related metabolic diseases in a subject in need thereof, comprising administering therapeutic agent A and therapeutic agent B to an affected individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. Reproductive-related metabolic diseases include, but are not limited to, polycystic ovary syndrome (PCOS), gestational diabetes, preeclampsia, recurrent spontaneous abortion, fetal growth restriction, ovarian insufficiency, premature ovarian failure, and male infertility. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, like aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, like aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0132] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating polycystic ovary syndrome (PCOS) in a diseased individual in need thereof, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a method for treating polycystic ovary syndrome (PCOS), comprising administering therapeutic agent A and therapeutic agent B to a diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0133] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for restoring skeletal muscle insulin sensitivity and protein anabolism (or alleviating muscle inflammation, insulin resistance, sarcopenia and / or metabolic syndrome) in a diseased individual in need thereof (such as a patient with polycystic ovary syndrome), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for restoring skeletal muscle insulin sensitivity and protein anabolism (or alleviating muscle inflammation, insulin resistance, sarcopenia and / or metabolic syndrome) in a diseased individual in need thereof (such as a patient with polycystic ovary syndrome), comprising administering therapeutic agent A and therapeutic agent B to the diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0134] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for improving (including reversing) cardiovascular diseases such as myocardial fibrosis, ventricular hypertrophy, and heart failure in a diseased individual in need thereof (such as a patient suffering from polycystic ovary syndrome or NASH), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for improving (including reversing) cardiovascular diseases such as myocardial fibrosis, ventricular hypertrophy, and heart failure in a diseased individual in need thereof (such as a patient suffering from polycystic ovary syndrome or NASH), comprising administering therapeutic agent A and therapeutic agent B to the diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0135] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for reversing insulin resistance, sarcopenia, and metabolic syndrome in a diseased individual in need thereof (such as a patient suffering from polycystic ovary syndrome or NASH), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for reversing insulin resistance, sarcopenia, and metabolic syndrome in a diseased individual in need thereof (such as a patient suffering from polycystic ovary syndrome or NASH), comprising administering therapeutic agent A and therapeutic agent B to the diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0136] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a diseased individual in need thereof, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for treating non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), comprising administering therapeutic agent A and therapeutic agent B to a diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0137] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for curbing weight gain (or reversing hyperlipidemia) in a diseased individual in need thereof (such as a patient with non-alcoholic steatohepatitis (NASH)), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for treating curbing weight gain (or reversing hyperlipidemia) in a diseased individual in need thereof (such as a patient with non-alcoholic steatohepatitis (NASH)), comprising administering therapeutic agent A and therapeutic agent B to a diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0138] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for reversing hepatomegaly (or liver injury) in a diseased individual in need thereof (such as a patient with non-alcoholic steatohepatitis (NASH)), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for treating and reversing hepatomegaly (or liver injury) in a diseased individual in need thereof (such as a patient with non-alcoholic steatohepatitis (NASH)), comprising administering therapeutic agent A and therapeutic agent B to the diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0139] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for increasing glucose tolerance (or reversing insulin resistance, restoring skeletal muscle and liver insulin sensitivity, alleviating muscle inflammation) in a diseased individual in need thereof (such as a patient with non-alcoholic steatohepatitis (NASH)), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for treating and increasing glucose tolerance (or reversing insulin resistance, restoring skeletal muscle and liver insulin sensitivity, alleviating muscle inflammation) in a diseased individual in need thereof (such as a patient with non-alcoholic steatohepatitis (NASH)), comprising administering therapeutic agent A and therapeutic agent B to the diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0140] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for promoting angiogenesis (or promoting neuromuscular tissue sensitivity) in a diseased individual in need thereof (such as a patient suffering from non-alcoholic steatohepatitis (NASH)), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for promoting angiogenesis (or promoting neuromuscular tissue sensitivity) in a diseased individual in need thereof (such as a patient suffering from non-alcoholic steatohepatitis (NASH)), comprising administering therapeutic agent A and therapeutic agent B to the diseased individual, wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0141] In some embodiments, the present invention provides the use (or method) of therapeutic agent A and therapeutic agent B in the preparation of a medicament for regulating the p38 and AMPK signaling pathways in an individual in need thereof (such as a patient suffering from non-alcoholic steatohepatitis (NASH)), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for regulating the p38 and AMPK signaling pathways, comprising administering therapeutic agent A and therapeutic agent B to the desired individual (such as a patient suffering from non-alcoholic steatohepatitis (NASH)), wherein the therapeutic agent A is a non-steroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the therapeutic agent A is selected from salicylates (such as aspirin), and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylate, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1.

[0142] Since the definitions of therapeutic agent A and therapeutic agent B, as well as the administration frequency, mode, and dosage, etc. can be the same as those described above.

[0143] In some embodiments, the therapeutic agent A is selected from salicylates, and the therapeutic agent B is selected from trimetazidine. In some embodiments, the therapeutic agent A is selected from aspirin, and the therapeutic agent B is selected from trimetazidine. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is from 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratio of the therapeutic agent A (such as salicylates, such as aspirin) to the therapeutic agent B (such as trimetazidine) is 6:1. In some embodiments, the therapeutic agent A and the therapeutic agent B are administered simultaneously (such as orally). In some embodiments, the therapeutic agent A and the therapeutic agent B are included in a single dosage form. In some embodiments, the single pharmaceutical dosage form is an oral dosage form. In some embodiments, the therapeutic agent A and the therapeutic agent B are administered separately (such as orally).

[0144] The treatment course may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer. In some embodiments, the therapeutic agent A may be administered once a day, twice a day, three times a day or more, and may also be administered once every two days, once every three days, once a week or other frequencies. In some embodiments, the therapeutic agent B may be administered once a day, twice a day, three times a day or more, and may also be administered once every two days, once every three days, once a week or other frequencies. When the therapeutic agent A and the therapeutic agent B are administered separately, the administration frequencies of the two may be the same or different.

[0145] According to the present invention, the subject in need can be of many types. For example, a subject who has been diagnosed with or is at risk of having various metabolic syndromes, or a subject whose various indices indicating various metabolic syndromes exceed the normal range, or a subject who is intolerant to, unavailable for, or has poor effects after using certain antidiabetic drugs, etc. Thus, in one embodiment, the subject can be an individual diagnosed with one or more conditions selected from overweight, obesity, visceral obesity, and abdominal obesity. In another embodiment, the subject can be an individual diagnosed with one or more of the following conditions: (a) fasting blood glucose or serum glucose concentration greater than 110 mg / dL, especially greater than 125 mg / dL; (b) postprandial plasma glucose concentration equal to or greater than 140 mg / dL; and (c) HbA1c value equal to or greater than 6.5%, especially equal to or greater than 8.0%. In another embodiment, the subject can be an individual having one or more of the following conditions: (a) obesity, visceral obesity, and / or abdominal obesity; (b) triglyceride blood drug concentration ≥ 150 mg / dL; (c) HDL-cholesterol blood level < 40 mg / dL in female subjects; HDL-cholesterol blood level < 50 mg / dL in male subjects; (d) systolic blood pressure ≥ 130 mmHg, diastolic blood pressure ≥ 85 mmHg; (e) fasting blood glucose level ≥ 110 mg / dL; and (f) LDL-cholesterol blood level ≥ 130 mg / dL. In another embodiment, the subject can be an individual who is prohibited from metformin monotherapy and / or intolerant to therapeutic doses of metformin. In another embodiment, the subject can be an individual with insufficient blood glucose control after treatment with one or more antidiabetic drugs selected from the group consisting of: (a) biguanides; (b) sulfonylureas; (c) meglitinides; (d) thiazolidinediones; (e) α-glucosidase inhibitors; (f) insulin and insulin analogs; (g) dipeptidyl peptidase-IV inhibitors; (h) SGLT2 inhibitors; (i) PPARα modulators; (j) glucose-dependent insulinotropic polypeptide agonists; (k) β-3 adrenergic receptor agonists; (l) GLP1 and GLP1 analogs; (m) PPARγ modulators; and (n) HMG-CoA reductase inhibitors.

[0146] In some embodiments, the subject is a human, such as 20, 30, 40, 50, 60, 70, or over 80 years old.

[0147] The present invention will be described in detail below by way of examples.

[0148] Examples

[0149] In the following examples, all animal (e.g., mouse) procedures were based on the animal care guidelines approved by the Institutional Animal Care and Use Committee. For histology, tissue samples were fixed in 10% neutral formalin or Bouin's solution and embedded in paraffin.

[0150] In addition, in the examples of the present invention, both the control group and the test group used intraperitoneal injection. The injection volume started at 4 μl / g body weight and was adjusted according to the dose. Among them, the therapeutic agent A in the test group (Aspirin, trade name) was diluted in PBS to a dose between 0.3 mg / kg and 120 mg / kg body weight, and the therapeutic agent B (Trimetazidine, trade name) was diluted in PBS to a dose between 0.05 mg / kg and 500 mg / kg body weight. In the control group, usually only an equal amount of therapeutic agent A or an equal amount of therapeutic agent B, or only an equal amount of PBS solvent was used as a blank control.

[0151] For the results in each chart, the data are presented as the mean ± standard error of the mean (SEM), and the Student's t-test (two-tailed distribution, two-sample unequal variance) was used to calculate the p-value. Statistical significance is shown as p > 0.05 (not significant, n.s.), p < 0.05 (one asterisk, i.e., *), or p < 0.01 (two asterisks, i.e., **), or p < 0.001 (three asterisks, i.e., ***). The tests were performed using Microsoft Excel, where the test type was always set to two-sample equal variance.

[0152] Example 1

[0153] Twenty 6-week-old diet-induced obese (Jackson Lab’s Diet-Induced Obese, DIO) male mice from the Jackson Laboratory with similar physical conditions in all aspects were selected and divided into four groups, with 5 mice in each group. For the test group, each mouse was effectively administered 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B per day; for control group 1, each mouse was effectively administered 30 mg / kg of therapeutic agent A per day; for control group 2, each mouse was effectively administered 5 mg / kg of therapeutic agent B per day; and for control group 3, each mouse was only administered an equal amount of PBS solvent as a blank control for 10 days. The body weight of each mouse was recorded daily, and a graph of body weight over time was plotted based on the body weight of each mouse, as Figure 1 shown.

[0154] Figure 1The results of the weight change of the drug therapeutic agents A + B in the test group using the pharmaceutical composition according to an embodiment of the present invention compared with the control group in diet-induced obesity (DIO) mice are shown. It can be seen that the pharmaceutical composition of the present invention can effectively reduce the weight of DIO mice by more than about 10% within 10 days, while the control groups 1 - 3 have almost no significant effect on the weight of the mice. Therefore, it can be considered that the therapeutic agent A and therapeutic agent B provided by the present invention have a synergistic effect on the therapeutic effect of weight reduction when used in combination.

[0155] Example 2

[0156] The mice in the test group and the control group 3 after completing Example 1 were selected, dissected, and the fat accumulation in various parts of the mice in the test group and the control group 3 was observed, and then compared. The photographed images of the fat accumulation in various parts after dissection are shown in Figure 2 .

[0157] Figure 2 The results of the reduction of fat in DIO mice induced by the drug therapeutic agents A + B in the test group using the pharmaceutical composition according to an embodiment of the present invention compared with the control group are shown. It can be seen that there are obvious fat accumulations in many parts of the mice in the control group 3, such as fatty liver, visceral fat, and subcutaneous fat, etc., while the fat accumulation in the mice in the test group is significantly reduced. Therefore, it can be considered that the pharmaceutical composition provided by the present invention has the effect of significantly reducing the fat content in the body.

[0158] Example 3

[0159] Similarly, the mice in the test group and the control groups 1, 2, and 3 after completing Example 1 were selected, the dissected mice were observed under a microscope, and the morphology and size of the fat globules in the gonadal fat pads were compared. The diagrams of the morphology of the fat globules under the microscope are shown in Figure 3 .

[0160] Figure 3 The results of the gonadal fat pads in the test group using the pharmaceutical composition according to an embodiment of the present invention compared with the control group are shown. It can be seen that compared with the fat globules in the control groups 1, 2, and 3, the size of the fat globules in the test group is significantly reduced, that is, the therapeutic agent A + B has the therapeutic effect of inducing fat reduction.

[0161] Example 4

[0162] Similarly, the mice in the test group and the control groups 1, 2, and 3 after completing Example 1 were selected, the dissected mice were observed under a microscope, and the morphology and size of the fat globules in the liver cells were compared. The diagrams of the morphology of the liver cells under the microscope are shown in Figure 4 .

[0163] Figure 4The results of the liver in the test group using the pharmaceutical composition according to an embodiment of the present invention are shown as compared with the control group. It can be seen that, compared with the liver cells in control groups 1, 2, and 3, the number of fat granules in the liver cells in the test group is reduced, and the size is significantly decreased, and the inflammatory cells are reduced. That is to say, the therapeutic agents A + B have a therapeutic effect of inducing reduction of fat and inflammation in fatty liver.

[0164] Example 5

[0165] Similarly, the mice in the test group and control groups 1, 2, and 3 after completing Example 1 were selected, the dissected mice were observed under a microscope, and the morphology of the heart muscle was compared. The illustration of the myocardial morphology under the microscope is shown in Figure 5 in.

[0166] Figure 5 The results of the cardiomyocyte morphology in the test group using the pharmaceutical composition according to an embodiment of the present invention are shown as compared with the control group. It can be seen that the colors and morphologies in the muscle images in the test group and control groups 1, 2, and 3 are basically the same. That is to say, the therapeutic agents A + B do not cause myocardial toxicity.

[0167] Example 6

[0168] Similarly, the mice in the test group and control groups 1, 2, and 3 after completing Example 1 were selected, the dissected mice were observed under a microscope, and the morphology of the skeletal muscle was compared. The illustration of the skeletal muscle morphology under the microscope is shown in Figure 6 in.

[0169] Figure 6 The results of the skeletal muscle cell morphology in the test group using the pharmaceutical composition according to an embodiment of the present invention are shown as compared with the control group. It can be seen that the colors and morphologies in the muscle images in the test group and control groups 1, 2, and 3 are basically the same. That is to say, the therapeutic agents A + B do not cause muscle loss and muscle toxicity.

[0170] Example 7

[0171] Similarly, the mice in the test group and control groups 1, 2, and 3 after completing Example 1 were selected, the serum of the dissected mice was subjected to blood biochemical tests, and the kidney toxicity indicators serum creatinine (CREA) and blood urea nitrogen (BUN) were compared. The comparison of the two indicators is shown in Figure 7 in.

[0172] Figure 7The results of serum creatinine (CREA) and blood urea nitrogen (BUN) in the test group using the pharmaceutical composition according to an embodiment of the present invention are shown as compared with the control group. It can be seen that the amounts of serum creatinine (CREA) and blood urea nitrogen (BUN) in the test group and control groups 1, 2, and 3 are basically the same, without significant differences. That is to say, the therapeutic agent A + B did not cause kidney toxicity.

[0173] Example 8

[0174] The weight change test of diabetic DIO mice was carried out according to the same method in Example 1, except that 12 mice were used in each group and the treatment time was increased to 4 weeks. The weight of each mouse was recorded every day, and a graph of weight change over time was plotted according to the weight of each mouse, as Figure 8 shown.

[0175] Figure 8 The results of the weight change induced by the drug therapeutic agent A + B in diabetic DIO mice in the test group using the pharmaceutical composition according to an embodiment of the present invention are shown as compared with the control group. It can be seen that the pharmaceutical composition of the present invention can effectively and stably reduce the weight of diabetic DIO mice by more than about 20% within 3 weeks, while control groups 1-3 had almost no obvious effect on the weight of the mice. Therefore, it can be considered that the therapeutic agent A and therapeutic agent B provided by the present invention have a synergistic effect on the treatment effect of weight reduction in diabetic patients when used in combination.

[0176] Figure 9 The comparison of the total serum cholesterol, an index related to hyperlipidemia, in each group of DIO mice is shown.

[0177] Figure 10 The comparison of serum LDL-cholesterol, an index related to hyperlipidemia, in each group of DIO mice is shown.

[0178] Figure 11 The comparison of serum alanine aminotransferase (ALT), an index related to fatty liver, in each group of DIO mice is shown.

[0179] Figure 12 The comparison of the serum albumin / globulin ratio, an index related to nitrogen metabolism function, in each group of DIO mice is shown.

[0180] Figure 13 The comparison of total serum protein, an index related to nitrogen metabolism function, in each group of DIO mice is shown.

[0181] Examples 9-13

[0182] Subsequent tests were performed on each group of mice that had completed the treatment in Example 8. In Examples 9 - 10, two indicators related to obesity and hyperlipidemia were detected: total cholesterol and LDL - cholesterol. In Example 11, the indicator alanine aminotransferase (ALT) related to fatty liver disease was detected. And in Examples 12 - 13, two indicators related to nitrogen metabolism function were detected: albumin / globulin ratio and total protein. The comparison of each indicator for each group is shown respectively in Figures 9 - 13 as follows.

[0183] Figures 9 - 13 The comparison of each indicator related to metabolic syndromes such as obesity and hyperlipidemia, fatty liver disease, and nitrogen metabolism function in each group of mice is shown respectively. It can be seen that after 4 weeks of administration of Therapeutic Agent A + B, compared with the blank control group 3, each indicator in the test group of mice showed a significant decrease, while no obvious therapeutic or recovery effect was achieved in the single use of Therapeutic Agent A in control group 1 or Therapeutic Agent B in control group 2. Thus, it can be seen that the pharmaceutical composition provided by the present invention has obvious medical uses in the treatment or improvement of diseases related to obesity and hyperlipidemia, fatty liver disease, and nitrogen metabolism function.

[0184] Example 14

[0185] Similarly, a glucose tolerance test was also performed on each group of mice that had completed the treatment in Example 8. In the test group and control groups 1 - 3, intraperitoneal glucose injection (2 mg / g body weight) was performed on mice that had fasted overnight for the test. At every 15 - 30 minutes thereafter, the glucose content (mM) in the mice was measured at each time point using a Lifescan One Touch blood glucose meter, and a line graph was plotted based on the glucose content and time. The results are shown in Figure 14 as follows.

[0186] Figure 14 The results of glucose tolerance in diabetic DIO mice are shown. It can be seen that compared with control groups 1 - 3, the glucose content in the test group of mice did not increase significantly to a very high glucose content, and it also quickly decreased to a lower glucose content subsequently. Therefore, it can be seen that the mice after 4 - week treatment with the pharmaceutical composition of the present invention showed excellent performance in terms of glucose tolerance.

[0187] Example 15

[0188] Similarly, groups of mice that had completed the treatment of Example 8 were also tested for insulin sensitivity. In the experimental group and Control Groups 1-3, insulin (Humulin) at a dose of 0.75 U insulin / kg body weight was intraperitoneally administered to 5-hour fasted rats using a 27G syringe needle. Every 15-30 minutes thereafter, the glucose content (mM) in the mice was measured at each time point using a Lifescan One Touch blood glucose meter, and a line graph was plotted based on the glucose content and time. The results are shown in Figure 15 as follows.

[0189] Figure 15 The results of insulin sensitivity in DIO mice with diabetes are shown. It can be seen that compared with Control Groups 1-3, the glucose content in the experimental group of mice always remained at a lower level and had a greater variation. Therefore, it can be seen that the mice treated with the pharmaceutical composition of the present invention for 4 weeks showed excellent performance in terms of insulin sensitivity.

[0190] Example 16

[0191] DIO mice were treated for 10 days using different combinations of therapeutic agents: (1) blank control; (2) 30 mg / kg A; (3) 5 mg / kg B; (4) 30 mg / kg A + 5 mg / kg B; (5) 3 mg / kg A + 5 mg / kg B; (6) 0.3 mg / kg A + 5 mg / kg B; (7) 30 mg / kg A + 0.5 mg / kg B; (8) 30 mg / kg A + 0.05 mg / kg B. After the treatment ended, the body weight (%) in each group treated with each combination of therapeutic agents was measured. The results are as Figure 16 shown below.

[0192] Figure 16 The comparison of the body weights of DIO mice after the administration of various combinations of therapeutic agents is shown. It can be seen that the expected therapeutic effect cannot be achieved when using commonly used doses. In contrast, in the case of the combination of 30 mg / kg A + 5 mg / kg B in Group (4), excellent performance can be shown in terms of body weight reduction.

[0193] Example 17

[0194] Use different combinations of therapeutic agents: (1) blank control; (2) 30 mg / kg A; (3) 5 mg / kg B; (4) 30 mg / kg A + 5 mg / kg B; (5) 3 mg / kg A + 5 mg / kg B; (6) 0.3 mg / kg A + 5 mg / kg B; (7) 30 mg / kg A + 0.5 mg / kg B; (8) 30 mg / kg A + 0.05 mg / kg B to treat DIO mice for 10 days. After the treatment, measure the fasting blood glucose content (mM) in each group of each combination of therapeutic agents. The results are as Figure 17 shown.

[0195] Figure 17 shows the comparison of the fasting blood glucose content in DIO mice after the administration of various combinations of therapeutic agents. It can be seen that the expected therapeutic effect cannot be achieved under the condition of using general common doses. In contrast, in the case of the combination of 30 mg / kg A + 5 mg / kg B in group (4), excellent performance can be shown in reducing the fasting blood glucose content.

[0196] Example 18

[0197] Select ordinary wild-type C57BL / 6 mice with similar physical conditions in all aspects. For the experimental group, effectively administer 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B to each mouse every day; for the blank control group, administer only an equal amount of PBS solvent to each mouse every day for a total of 13 days. Record the weight of each mouse every day, and draw a graph of the weight change over time based on the weight of each mouse, as Figure 18 shown.

[0198] Figure 18 shows the results of the change in body weight induced by the drug therapeutic agents A + B in ordinary wild-type C57BL / 6 mice in the experimental group using the pharmaceutical composition according to the embodiment of the present invention compared with the control group. It can be seen that the pharmaceutical composition of the present invention can effectively reduce the body weight of ordinary wild-type C57BL / 6 mice by more than about 10% within 13 days, while the control group has almost no obvious effect on the body weight of the mice. Therefore, it can be considered that the therapeutic agent A and therapeutic agent B provided by the present invention also have a synergistic effect on the therapeutic effect of reducing the body weight of ordinary wild-type C57BL / 6 mice when used in combination.

[0199] Example 19

[0200] Select the mice in the experimental group and the control group after completing Example 18, dissect them, and observe the accumulation of fat in various parts of the mice in the experimental group and the control group, and then make a comparison. The photographed images of the fat accumulation in various parts after dissection are shown in Figure 19 it.

[0201] Figure 19 It shows the results of the reduction of fat in normal wild-type C57BL / 6 mice induced by the drug therapeutics A + B in the test group using the pharmaceutical composition according to the embodiments of the present invention compared with the control group. It can be seen that there are obvious fat accumulations in many places in the mice of the control group, such as visceral fat and gonadal fat, etc., while the fat accumulation in the mice of the test group is significantly reduced. Thus, it can be considered that the pharmaceutical composition provided by the present invention has the effect of significantly reducing the fat content in the body.

[0202] Example 20

[0203] Twenty-four 8-week-old Sprague Dawley female rats with similar physical conditions in all aspects were selected and divided into two groups, with twelve rats in each group. Each rat was fed a high-fat and high-sugar diet (HFHSD, D11092103; Research Diet Inc) every day and subcutaneously injected with 60 mg / kg of dehydroepiandrosterone (DHEA; Sigma Aldrich) every day to simulate the occurrence of polycystic ovary syndrome (PCOS) (Zhang et al., Reproduction 2016). For the test group, each rat was effectively administered 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B every day; for the control group, each rat was effectively administered an equal amount of PBS solvent as a blank control every day for a total of three weeks. The body weight of each rat was recorded every day, and a graph of body weight change over time was plotted based on the body weight of each rat, as Figure 20 shown.

[0204] Figure 20 It shows the results of the body weight change in polycystic ovary syndrome rats induced by the drug therapeutics A + B in the test group using the pharmaceutical composition according to the embodiments of the present invention compared with the control group. It can be seen that the pharmaceutical composition of the present invention can effectively and stably avoid a weight gain of more than about 10% in polycystic ovary syndrome rats within 3 weeks. Thus, it can be considered that the therapeutic agent A and therapeutic agent B provided by the present invention have a synergistic effect on the preventive effect of obesity in polycystic ovary syndrome patients when used in combination.

[0205] Figure 21 It shows the comparison of the fasting blood glucose levels in PCOS polycystic ovary syndrome rats in each group.

[0206] Figure 22 It shows the comparison of serum aspartate aminotransferase (AST) and total cholesterol, which are indicators related to fatty liver, in polycystic ovary syndrome rats in each group.

[0207] Figure 23The comparison of serum total protein, an index related to liver nitrogen metabolism function, in each group of polycystic ovary syndrome rats is shown.

[0208] Figure 24 The comparison of serum creatinine, an index related to kidney function, in each group of polycystic ovary syndrome rats is shown.

[0209] Figure 25 The comparison of serum lactate dehydrogenase (LDH), creatine kinase (CK), myocardial creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH), indexes related to heart disease, in each group of polycystic ovary syndrome rats is shown.

[0210] Examples 21 - 25

[0211] Subsequent tests were performed on each group of rats that had completed the treatment in Example 20. In Example 21, fasting blood glucose, an index related to diabetes, was detected. In Example 22, serum aspartate aminotransferase (AST) and total cholesterol, indexes related to fatty liver disease, were detected. In Example 23, serum total protein, an index related to liver nitrogen metabolism function, was detected. In Example 24, serum creatinine, an index related to kidney function, was detected. And in Example 25, serum lactate dehydrogenase (LDH), creatine kinase (CK), myocardial creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH), indexes related to heart disease, were detected. The comparison of each index of each group is respectively shown in Figures 21 - 25 as follows.

[0212] Figures 21 - 25 The comparison of various indexes related to polycystic ovary syndrome, such as diabetes, obesity, hyperlipidemia, fatty liver disease, and heart disease, in each group of rats is shown. It can be seen that after 3 weeks of administration of therapeutic agent A + B, compared with the blank control group, each index in the experimental group of rats showed a significant decrease. Thus, it can be seen that the pharmaceutical composition provided by the present invention has obvious medical uses in preventing or improving polycystic ovary syndrome and related diseases such as diabetes, obesity, hyperlipidemia, fatty liver disease, and heart disease.

[0213] Example 26

[0214] Similarly, the insulin sensitivity of each group of rats that had completed the treatment in Example 20 was also tested. In the experimental group and the control group, insulin (Humulin) at a dose of 0.75 U insulin / kg body weight was intraperitoneally administered to 5-hour fasted rats using a 27G syringe needle. At every 15 - 30 minutes thereafter, the glucose content (mM) in the mice was measured at each time point using a Lifescan One Touch blood glucose meter, and a line graph was plotted based on the glucose content and time. The results are shown inFigure 26 in

[0215] Figure 26 The results of insulin sensitivity in rats with polycystic ovary syndrome are shown. It can be seen that compared with the control group, the glucose content in the test group of rats always remained at a relatively low level and showed significant variations. Therefore, it can be concluded that the rats treated with the pharmaceutical composition of the present invention for 3 weeks exhibited excellent performance in terms of insulin sensitivity.

[0216] Example 27

[0217] Similarly, vaginal cytology analysis was also performed on the rats in each group of Example 20. The estrous cycle was evaluated for 11 consecutive days, including D diestrus, P proestrus, E estrus, and M metestrus, and a line graph was plotted based on the estrous cycle stage and time. The results are shown in Figure 27 in

[0218] Figure 27 The results of the estrous cycle in rats with polycystic ovary syndrome are shown. It can be seen that compared with the normal control group (Control), the estrous cycle of rats with polycystic ovary syndrome (DHEA+HFHSD) was abnormal, and all 12 / 12 rats showed non-cyclic phenomena. The estrous cycle of the rats in the test group (DHEA+HFHSD+A+B) was relatively normal, and the estrous cycles of 4 / 12 rats completely returned to normal. Therefore, it can be concluded that some of the rats with polycystic ovary syndrome treated with the pharmaceutical composition of the present invention for 3 weeks could completely restore a normal estrous cycle.

[0219] Example 28

[0220] Similarly, serum hormone ELISA analysis was also performed on the rats in each group of Example 20, including testosterone (T), estradiol (E2), and follicle-stimulating hormone (FSH). The results are shown in Figure 28 in

[0221] Figure 28 The results of serum hormone ELISA in rats with polycystic ovary syndrome are shown. It can be seen that compared with the control group of rats with polycystic ovary syndrome (Control), there were no significant changes in testosterone (T) and estradiol (E2) in the test group of rats (A+B), but there was a significant increase in follicle-stimulating hormone (FSH) in 4 / 12 rats. Therefore, it can be concluded that some of the rats with polycystic ovary syndrome treated with the pharmaceutical composition of the present invention for 3 weeks could restore a normal estrous cycle because of the increase in follicle-stimulating hormone (FSH), rather than through the regulation of testosterone (T) and estradiol (E2).

[0222] Example 29

[0223] Similarly, Western blot analysis of skeletal muscle proteins, including phospho-Akt (S473), Akt, phospho-p38, muscle protein heavy chain (Myosin Heavy Chain, MHC), and GAPDH, was also performed on each group of rats in Example 20. The results are shown in Figure 29 as follows.

[0224] Figure 29 The results of Western blot analysis of skeletal muscle proteins in rats with polycystic ovary syndrome are shown. It can be seen that compared with the control group of rats with polycystic ovary syndrome (PCOS), the levels of phospho-Akt (S473), Akt, and muscle protein heavy chain (Myosin Heavy Chain, MHC) in the experimental group of rats (PCOS+A+B) were significantly increased, while the level of phospho-p38 was significantly decreased. Therefore, it can be seen that after 3 weeks of treatment with the pharmaceutical composition of the present invention, rats with polycystic ovary syndrome can restore normal skeletal muscle insulin sensitivity and protein synthesis metabolism, and relieve muscle inflammation, insulin resistance, sarcopenia, and metabolic syndrome.

[0225] Example 30

[0226] Similarly, heart section analysis, including ventricular wall thickness and fibrosis analysis by Masson trichrome staining, was also performed on each group of rats in Example 20. The results are shown in Figure 30 and Figure 31 as follows.

[0227] Figure 30 The results of left ventricular wall thickness analysis in rats with polycystic ovary syndrome are shown. It can be seen that compared with the control group of rats with polycystic ovary syndrome (Control), the left ventricular wall thickness of the experimental group of rats (A+B) was significantly decreased (P<0.001). Therefore, it can be seen that after 3 weeks of treatment with the pharmaceutical composition of the present invention, rats with polycystic ovary syndrome can significantly improve cardiovascular diseases such as ventricular hypertrophy and heart failure.

[0228] Figure 31 The results of myocardial fibrosis analysis in rats with polycystic ovary syndrome are shown. It can be seen that compared with the control group of rats with polycystic ovary syndrome (Control), the area of myocardial fibrosis in the experimental group of rats (A+B) was significantly decreased (P<0.05). Therefore, it can be seen that after 3 weeks of treatment with the pharmaceutical composition of the present invention, rats with polycystic ovary syndrome can significantly improve cardiovascular diseases such as myocardial fibrosis, ventricular hypertrophy, and heart failure caused by myocardial infarction.

[0229] Example 32

[0230] Similarly, blood routine analysis was also performed on each group of rats in Example 20, and the results are shown in Figure 32 .

[0231] Figure 32 The results of blood routine analysis in rats with polycystic ovary syndrome are shown. It can be seen that compared with the control group (Control) of rats with polycystic ovary syndrome, there were no significant changes in each blood routine index of the experimental group of rats (A + B) (P > 0.05). Therefore, it can be seen that there were no significant toxic reactions in the rats with polycystic ovary syndrome after 3 weeks of treatment with the pharmaceutical composition of the present invention.

[0232] Example 33

[0233] Twenty-six 6-week-old ob / ob obese male mice (Jackson Lab B6.Cg-Lepob / J, Stock No: 000632) with similar physical conditions in all aspects from the Jackson Laboratory were selected, and each was fed a high-fat and high-sugar diet (HFSD, D11092103; Research Diet Inc) every day for 45 days in order to simulate the occurrence of non-alcoholic steatohepatitis (NASH) (Kristiansen et al., 2016; doi: 10.4254 / wjh.v8.i16.673). After 45 days, they continued to be fed a high-fat and high-sugar diet and were divided into two groups, with 13 mice in each group. For the experimental group, each mouse was effectively administered 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B every day; for the control group, each mouse was only administered an equal amount of PBS solvent as a blank control every day for 37 days. The body weight of each mouse was recorded every day, and a graph of body weight change over time was plotted based on the body weight of each mouse, as shown in Figure 33 .

[0234] Figure 33 The results of the change in body weight of NASH mice treated with the drug therapeutic agent A + B are shown. It can be seen that compared with the NASH control group, the pharmaceutical composition of the present invention can effectively reduce the body weight of NASH mice by more than about 3% within 5 days (P < 0.001), and can effectively reduce the body weight of NASH mice by about 20% within 37 days (P = 3.8e-10). While 1-3 had almost no obvious effect on the body weight of the mice. Thus, it can be considered that the drug therapeutic agent A + B provided by the present invention can effectively curb weight gain.

[0235] Example 34

[0236] Similarly, the food intake of each group of mice that had completed the treatment in Example 33 was also tested for three days, and the results are shown in Figure 34 .

[0237] Figure 34The results of the food intake test of NASH mice are shown. It can be seen that, compared with the NASH control group, the food intake of the mice in the experimental group did not decrease, but was slightly higher (P = 0.109). Therefore, it can be seen that the appetite of NASH mice after 40 days of treatment with the pharmaceutical composition of the present invention did not change significantly.

[0238] Example 35

[0239] Similarly, each group of mice that had completed the treatment of Example 33 was dissected, and the liver weight was measured. The results are shown in Figure 35 .

[0240] Figure 35 The results of the liver weight test of NASH mice are shown. It can be seen that, compared with the NASH control group, the liver weight of the mice in the experimental group was significantly reduced (P < 0.001), and the liver volume was also significantly reduced. Therefore, it can be seen that NASH mice after 40 days of treatment with the pharmaceutical composition of the present invention can reverse hepatomegaly.

[0241] Example 36

[0242] Similarly, each group of mice that had completed the treatment of Example 33 was dissected, tissue-fixed and sectioned, stained with hematoxylin-eosin and Picro-Sirius red (Sigma-Aldrich), and microscopically observed and scored by experts. The results of the liver analysis sections are shown in Figure 36 and the results of the heart analysis sections are shown in Figure 37 .

[0243] Figure 36 The results of the liver section analysis of NASH mice are shown. It can be seen that, compared with the NASH control group, the fatty liver grade and fatty liver activity score of the mice in the experimental group were both significantly reduced (P < 0.01), that is, the therapeutic agent A + B had a therapeutic effect of inducing the reduction of fat and inflammation in fatty liver. At the same time, it can be seen that, compared with the NASH control group, the liver fibrosis area (%) and fibrosis stage of the mice in the experimental group were also significantly reduced (P < 0.01), that is, the therapeutic agent A + B had a therapeutic effect of inducing the reduction of fibrosis in fatty liver. Mice after 40 days of treatment with the pharmaceutical composition of the present invention can reverse NASH fatty liver and liver fibrosis even if they continue to consume a large amount of high-fat and high-sugar feed.

[0244] Figure 37The results of the analysis of heart sections of NASH mice are shown. It can be seen that compared with the NASH control group, the area of cardiac fibrous tissue in the test group of mice was significantly reduced (P<0.01), that is to say, the therapeutic agent A+B has a therapeutic effect of inducing a reduction in cardiac fibrosis. After 40 days of treatment with the pharmaceutical composition of the present invention, even if the mice continue to consume a large amount of high-fat and high-sugar feed, they can reverse cardiovascular diseases such as cardiac fibrosis and heart failure.

[0245] Example 38

[0246] Similarly, blood biochemical tests were performed on each group of mice that had completed the treatment of Example 33. In Example 38, indicators related to liver injury were detected: alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In Example 39, indicators related to hyperlipidemia were detected: triglyceride (TG), total cholesterol (CHOL), HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C). In Example 40, indicators related to organ damage were detected: albumin / globulin ratio, urea, creatinine, lactate dehydrogenase (LDH), creatine kinase (CK), myocardial creatine kinase isoenzyme MB (CKMB) and α-hydroxybutyrate dehydrogenase (HBDH). The comparison of each indicator of each group is shown respectively in Figures 38 - 40 below.

[0247] Figure 38 The comparison of two indicators related to liver injury in each group of mice is shown. Compared with the NASH control group, both alanine aminotransferase (ALT, P<0.001) and aspartate aminotransferase (AST, P=0.02) in the test group of mice were significantly decreased. After 40 days of treatment with the pharmaceutical composition of the present invention, even if the mice continue to consume a large amount of high-fat and high-sugar feed, they can reverse liver injury.

[0248] Figure 39 The comparison of indicators related to hyperlipidemia in each group of mice is shown. Compared with the NASH control group, triglyceride (TG), total cholesterol (CHOL, P<0.01), HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C, P=0.016) in the test group of mice were all decreased. After 40 days of treatment with the pharmaceutical composition of the present invention, even if the mice continue to consume a large amount of high-fat and high-sugar feed, they can reverse hyperlipidemia.

[0249] Figure 40The comparison of indicators related to organ damage in each group of mice is shown. Compared with the NASH control group, there were no significant changes in the albumin / globulin ratio, urea, creatinine, lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyric acid dehydrogenase (HBDH) in the experimental group of mice (P>0.05). After 40 days of treatment with the pharmaceutical composition of the present invention, no toxic reactions occurred in any of the organs of the mice.

[0250] Example 41

[0251] Similarly, for each group of mice in Example 33, a fasting blood glucose test was performed. Every 3 - 7 days, the glucose content (mM) in the mice that had fasted overnight was measured using a Lifescan OneTouch blood glucose meter, and a line graph was plotted based on the glucose content and time. The results are shown in Figure 41 .

[0252] Figure 41 The results of fasting blood glucose in each group of mice are shown. It can be seen that compared with the NASH control group, the glucose content in the body of the mice in the A + B experimental group not only did not increase significantly, but instead decreased significantly to a lower glucose content within 14 days (P<0.001). Therefore, it can be seen that after 40 days of treatment with the pharmaceutical composition of the present invention, the mice showed significant improvement in blood glucose control and prevented the occurrence of type 2 diabetes.

[0253] Example 42

[0254] Similarly, a glucose tolerance test was also performed on each group of mice in Example 33. In the experimental group and the control group, an equal amount of glucose (2 mg / g body weight) was injected intraperitoneally into the mice that had fasted overnight for the experiment. Every 15 - 30 minutes thereafter, the glucose content (mM) in the mice was measured at each time point using a Lifescan OneTouch blood glucose meter, and a line graph was plotted based on the glucose content and time. The results are shown in Figure 42 .

[0255] Figure 42 The results of glucose tolerance in NASH mice are shown. It can be seen that compared with the NASH control group, the glucose content in the body of the mice in the A + B experimental group did not increase significantly to a very high glucose content, and subsequently decreased rapidly to a lower glucose content (P<0.001). Therefore, it can be seen that after 40 days of treatment with the pharmaceutical composition of the present invention, the NASH mice recovered normal glucose tolerance.

[0256] Example 43

[0257] Similarly, groups of mice in Example 33 were tested for insulin sensitivity. In the experimental group and the control group, an equal amount of insulin (Humulin, 0.75 U / kg body weight) was intraperitoneally administered to 6-hour fasted rats using a 27G syringe needle. Every 15 - 30 minutes thereafter, the glucose content (mM) in the mice was measured at each time point using a Lifescan One Touch blood glucose meter, and a line graph was plotted based on the glucose content and time. The results are shown in Figure 43 .

[0258] Figure 43 The results of insulin sensitivity in NASH mice are shown. It can be seen that compared with the control group, the glucose content in the experimental group of mice remained at a relatively low level and changed significantly after 60 minutes (P < 0.01). Therefore, it can be seen that NASH mice after 40 days of treatment with the pharmaceutical composition of the present invention recovered normal insulin sensitivity and reversed insulin resistance.

[0259] Example 44

[0260] Similarly, after 6-hour fasting, insulin (Humulin, 0.75 U / kg body weight) was injected into groups of mice in Example 33. After 15 minutes, skeletal muscle and liver samples were taken for protein Western blot analysis, including phospho-Irs1 (S307), phospho-Akt (S473), Akt, phospho-p38, and GAPDH. The results are shown in Figure 44 .

[0261] Figure 44 The results of protein Western blot analysis of skeletal muscle in NASH mice are shown. It can be seen that compared with the control group of NASH mice (Control), the levels of phospho-Irs1 (S307), phospho-Akt (S473), and Akt in the skeletal muscle of the experimental group of mice (A + B) increased significantly, while the level of phospho-p38 decreased significantly. It can also be seen that compared with the control group of NASH mice (Control), the levels of phospho-Akt (S473) and phospho-Irs1 (S307) in the liver of the experimental group of mice (A + B) increased significantly. Therefore, it can be seen that NASH mice after 40 days of treatment with the pharmaceutical composition of the present invention can recover normal insulin sensitivity in skeletal muscle and liver, relieve muscle inflammation, systemic insulin resistance, and metabolic syndrome.

[0262] Example 45

[0263] Six 6-week-old ob / ob obese male mice (Jackson Lab B6.Cg-Lepob / J, Stock No: 000632) with similar physical conditions in all aspects were selected from Jackson Laboratory. Each mouse was fed a high-fat and high-sugar diet (HFSD, D11092103; Research Diet Inc) every day for 45 days to simulate the occurrence of non-alcoholic steatohepatitis (NASH) (Kristiansen et al., 2016; doi: 10.4254 / wjh.v8.i16.673). After 45 days, they continued to be fed the high-fat and high-sugar diet and were divided into two groups, with 3 mice in each group. For the experimental group, each mouse was effectively administered 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B every day; for the control group, each mouse was only administered an equal amount of PBS solvent as a blank control every day for 7 days. After the experiment, skeletal muscle and liver samples were taken, RNAseq transcriptomics sequencing analysis and GSEA (Gene Set Enrichment Analysis) were performed, and the most significant gene tags and markers were taken out, such as Figure 45 as shown

[0264] Figure 45 The results of transcriptomics sequencing analysis in NASH mice are shown. It can be seen that compared with the NASH control group (C), the pharmaceutical composition (F) of the present invention can effectively up-regulate the VEGF signaling pathway in the skeletal muscle and liver of NASH mice within 7 days (P<0.001, FDR<0.001), the Axon Guidance (nerve axon guidance, P<0.001, FDR<0.001) and Ephrin signaling pathway in skeletal muscle (P<0.001, FDR = 0.0068), and the IGF (insulin-like) signaling pathway in the liver (P = 0.0039, FDR = 0.07). It can be considered that the pharmaceutical therapeutic agents A+B provided by the present invention can effectively promote angiogenesis, neuromuscular tissue, and insulin sensitivity, just like mimicking exercise (Hoier & Hellsten 2014 doi:10.1111 / micc.12117; Stark et al. 2015 doi:10.1083 / jcb.201502036; Lavin et al. 2020 doi:10.3389 / fphys.2020.00653; Sarvas et al. 2015 doi:10.14814 / phy2.12277).

[0265] Example 46

[0266] Using in vitro human primary skeletal muscle cells, they were divided into four groups, with six time points in each group. For the experimental group, 100 mg / L of therapeutic agent A and 2 mg / L of therapeutic agent B were administered; for control group 1, 100 mg / L of therapeutic agent A was administered; for control group 2, 2 mg / L of therapeutic agent B was administered; and for control group 3, an equal amount of DMSO solvent was used as a blank control, lasting for 24 hours in total. Then, protein Western blot analysis was performed on the samples at each time point, as Figure 46 shown.

[0267] Figure 46 The results of protein Western blot analysis of in vitro human primary skeletal muscle cells are shown.

[0268] Figure 46 A shows that after short-term drug administration (within 60 minutes), compared with control groups 1 - 3, in the experimental group using the pharmaceutical composition according to the embodiments of the present invention, the drug therapeutic agents A + B can rapidly and significantly up-regulate phospho-p38, phospho-AMPK, phospho-ACC, and PGC1a. Compared with control group 3, although control groups 1 and 2 using therapeutic agent A alone or therapeutic agent B alone can also up-regulate phospho-p38, phospho-AMPK, phospho-ACC, and PGC1a, the effects are relatively weak or slow, and the time is not uniform. Thus, it can be seen that after short-term drug administration, the drug therapeutic agents A + B provided by the present invention can more effectively promote the p38 and AMPK signaling pathways and downstream targets compared with using therapeutic agent A alone or therapeutic agent B alone, thereby promoting catabolism such as lipolysis and fatty acid oxidation. After long-term drug administration (3 - 24 hours), compared with control groups 1 - 3, in the experimental group using the pharmaceutical composition according to the embodiments of the present invention, the drug therapeutic agents A + B can significantly down-regulate phospho-p38, phospho-AMPK, phospho-ACC, and PGC1a. Compared with control group 3, although control groups 1 and 2 using therapeutic agent A alone or therapeutic agent B alone can also down-regulate phospho-p38, phospho-ACC, and PGC1a within 24 hours, the effects are relatively weak or slow, and the time is not uniform. Thus, it can be seen that after long-term drug administration, the drug therapeutic agents A + B provided by the present invention can more effectively inhibit the p38 and AMPK signaling pathways and downstream targets compared with using therapeutic agent A alone or therapeutic agent B alone, thereby promoting anabolism.

[0269] Figure 46Panel B shows the mechanism model of the combined regulation of p38 and AMPK signaling pathways by the pharmaceutical therapeutic agents A+B provided by the present invention through fatty acid oxidation (FAO), fatty acid metabolites (acyl-metabolites), and adenosine triphosphate (ATP) after short-term administration (within 60 minutes). It can be seen that the pharmaceutical therapeutic agents A+B can cause a sudden increase in both p38 and AMPK signaling pathways after short-term administration, thereby promoting catabolism such as lipolysis and fatty acid oxidation.

[0270] Figure 46 Panel C shows the mechanism model of the combined regulation of p38 and AMPK signaling pathways by the pharmaceutical therapeutic agents A+B provided by the present invention through inflammatory factors (Inf cytokines), fatty acid oxidation (FAO), mitochondrial reactive oxygen species (mtROS), and glycolysis after long-term administration (3 - 24 hours). It can be seen that the pharmaceutical therapeutic agents A+B can cause a sudden decrease in both p38 and AMPK signaling pathways after long-term administration, thereby promoting anabolism and muscle repair.

[0271] Therefore, daily administration of the pharmaceutical therapeutic agents A+B can specifically make the p38 and AMPK signaling pathways and related metabolic changes cycle repeatedly, forming an excitatory effect cycle, just like mimicking exercise.

[0272] Example 47

[0273] Using in vitro human primary skeletal muscle cells, they were divided into six groups. For control group 1, an equal amount of DMSO solvent was administered for 7 days as a blank control. For control group 2, an equal amount of bovine serum albumin (BSA) was administered for 7 days as a fat solvent control. For control group 3, palmitic acid (Pal) and TNFα were administered for 7 days as a high-fat and inflammation-induced insulin resistance control. For control group 4, palmitic acid (Pal) and TNFα were administered for 7 days and the therapeutic agent A at 100 mg / L was administered on the 4th day. For control group 5, palmitic acid (Pal) and TNFα were administered for 7 days and the therapeutic agent B at 2 mg / L was administered on the 4th day. For the experimental group, palmitic acid (Pal) and TNFα were administered for 7 days and the therapeutic agent A at 100 mg / L and the therapeutic agent B at 2 mg / L were administered on the 4th day; then protein Western blot analysis was performed on each group of samples, as Figure 47 shown.

[0274] Figure 47The results of protein Western blot analysis of in vitro human primary skeletal muscle cells induced to insulin resistance and 7 days after drug administration are shown. After the 7-day experiment, compared with control group 3, in the experimental groups using the pharmaceutical composition according to the embodiments of the present invention, the drug therapeutic agents A+B could more significantly up-regulate the insulin sensitivity indexes phospho-Akt, phospho-S6 and myosin heavy chain (MHC) and down-regulate the cell senescence index H3K9me3, restoring to the levels of control groups 1-2. Compared with control group 3, although the single use of therapeutic agent A in control group 4 or the single use of therapeutic agent B in control group 5 could also up-regulate the insulin sensitivity indexes phospho-Akt, phospho-S6 and myosin heavy chain, the effects were relatively weak. Compared with control group 3, the single use of therapeutic agent A in control group 4 could down-regulate the cell senescence index H3K9me3, restoring to the level of control group 1, but the single use of therapeutic agent B in control group 5 could not. Thus, it can be seen that the drug therapeutic agents A+B provided by the present invention can more effectively promote insulin sensitivity and reverse cell senescence in the case of insulin resistance than the single use of therapeutic agent A or therapeutic agent B.

[0275] Example 48

[0276] Similarly, RNAseq transcriptomics sequencing analysis and GSEA (Gene Set Enrichment Analysis) analysis were also performed on each group of rats in Example 20, and the most significant gene tags and markers were taken out, such as Figure 48 shown.

[0277] Figure 48The results of RNAseq transcriptome sequencing analysis of skeletal muscle in PCOS polycystic ovary syndrome rats induced by a high-fat and high-sugar diet are shown. It can be seen that compared with the PCOS rat control group (M2), the IRS1 and IRS2 insulin receptor pathway targets (P<0.0001, FDR = 4.9e-5), Rapamycin-sensitive PI3K-Akt-mTOR pathway targets (P<0.0001, FDR = 2.6e-5), and Mitochondria mitochondrial genes in the A+B experimental group rats (M1) were significantly increased. It can also be seen that compared with the polycystic ovary syndrome rat control group (M2), the inflammation-related Interferon interferon targets (P<0.0001, FDR<0.0001), fibrosis-related Collagen collagen pathway targets (P<0.0001, FDR = 3.5e-5), and mesenchymal cell division targets (P<0.0001, FDR<0.0001) in the A+B experimental group rats (M1) were significantly decreased. Therefore, it can be seen that the polycystic ovary syndrome rats treated with the pharmaceutical composition of the present invention can restore normal insulin (insulin / IGF-IRS-PI3K-mTOR) sensitivity and mitochondrial metabolism, relieve inflammaging and fibrosis, thereby reversing insulin resistance, sarcopenia, and metabolic syndrome.

[0278] Example 49

[0279] Similarly, enzyme-linked immunosorbent assays (ELISA, Abcam) for fasting insulin in the serum samples of each group of mice in Example 33 were performed, and the insulin resistance index HOMA-IR was calculated, as Figure 49 shown.

[0280] Figure 49 The results of the insulin enzyme-linked immunosorbent assay and the insulin resistance index HOMA-IR in the serum samples of NASH mice are shown. It can be seen that compared with the NASH control group, the pharmaceutical composition A+B of the present invention can effectively reduce fasting serum insulin (P<0.05) and the insulin resistance index (P<0.05). It can thus be considered that the pharmaceutical therapeutic agent A+B provided by the present invention can effectively promote insulin sensitivity in NASH patients and relieve insulin resistance and hyperinsulinemia, just like mimicking exercise (van der Windt et al. 2018, doi:10.3727 / 105221617X15124844266408).

[0281] Example 50

[0282] Similarly, enzyme-linked immunosorbent assay (ELISA, Abcam) for adiponectin was performed on the serum samples of each group of mice in Example 33, as Figure 50 shown.

[0283] Figure 50 The results of the adiponectin enzyme-linked immunosorbent assay for serum samples in NASH mice are shown. It can be seen that, compared with the NASH control group, the pharmaceutical composition A+B of the present invention can effectively up-regulate serum adiponectin (P<0.001). It can thus be considered that the pharmaceutical therapeutic agent A+B provided by the present invention can effectively promote glycolipid metabolism and inhibit inflammation through adiponectin, just like mimicking exercise (Simpson&Singh 2008, doi:10.1038 / oby.2007.53).

[0284] Example 51

[0285] Mice were grouped and administered drugs according to the same method in Example 8, except that 1 hour later, liquid chromatography-mass spectrometry (LC-MS, Waters XBridge C18 column and Xevo G2-XS) analysis was performed on the serum samples of each group of mice, and the most significant small molecule markers were taken out, as Figure 51 shown.

[0286] Figure 51 The results of the comparison of liquid chromatography-mass spectrometry analysis of serum samples in mice in Control Group 1 and Control Group 3 are shown. It can be seen that, compared with Control Group 3 (blank control), drug A (Control Group 1) can effectively form a variety of salicylate derivatives in the serum, and the retention times of most of them are between 5 and 5.5 minutes, similar to acetylsalicylic acid (https: / / mona.fiehnlab.ucdavis.edu / spectra / display / EQ357853). It can thus be considered that any salicylate derivative can mimic the drug effect of drug A in the obese body.

[0287] Based on the above examples, it can be easily seen that, compared with the use of individual components in the pharmaceutical composition of the present invention, the pharmaceutical composition according to the present invention shows very good synergistic effects in blood glucose control, especially in reducing fasting plasma glucose and postprandial plasma glucose, and at the same time can effectively treat or prevent metabolic syndrome diseases caused by obesity, fatty liver, type 2 diabetes and insulin resistance.

[0288] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0289] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

[0290] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. Use of therapeutic agent A and therapeutic agent B in the preparation of a medicament for treating overweight, obesity, non-alcoholic fatty liver, non-alcoholic steatohepatitis, polycystic ovary syndrome in a subject in need thereof, wherein, the therapeutic agent A is aspirin or a pharmaceutically acceptable salt thereof, and the therapeutic agent B is trimetazidine or a pharmaceutically acceptable salt thereof; wherein, the therapeutic agent A and the therapeutic agent B are administered simultaneously or separately and daily.

2. The use according to claim 1, wherein, the obesity is visceral obesity.

3. The use according to claim 1, wherein, the subject is a human.

4. The use according to claim 1, wherein, the therapeutic agent A is administered before the therapeutic agent B.

5. The use according to claim 1, wherein, the therapeutic agent A is administered after the therapeutic agent B.

6. The use according to any one of claims 1-5, wherein, the therapeutic agent A and therapeutic agent B are administered orally or by injection.

7. The use according to any one of claims 1-5, wherein, the therapeutic agent A and therapeutic agent B can achieve the following effects: restoring the estrous cycle and / or increasing follicle-stimulating hormone in patients with polycystic ovary syndrome.

Images