Compositions used to combat metabolic diseases and uses of the compositions

Abstract

A composition used to combat metabolic diseases and a method for using the composition. The pharmaceutical composition includes 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 nonsteroidal anti-inflammatory drug and the therapeutic agent B is a fatty acid oxidation inhibitor. The pharmaceutical composition effectively treats or prevents metabolic syndrome diseases caused by obesity, nonalcoholic 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, specifically to compositions and uses thereof for reducing the risk of metabolic syndrome. [Background technology]

[0002] Metabolic syndrome refers to a medical condition in which the metabolism of proteins, fats, carbohydrates, and other substances in the human body is disturbed. It is not a single disease, but a complex group of metabolic disorders, including abdominal fat accumulation, high blood lipids, high triglycerides, high cholesterol, high blood pressure, high blood glucose levels, etc. The central part of metabolic syndrome is obesity and insulin resistance, which mainly includes the components of obesity, especially central obesity. Subjects with metabolic syndrome have risk factors for diabetes, cardiovascular and cerebrovascular diseases, fatty liver, and polycystic ovarian syndrome, and the prevalence of cardiovascular events and the risk of death are about 2 to 3 times higher than those with non-metabolic syndrome.

[0003] The prevalence of obesity and type 2 diabetes is worldwide (see National Diabetes Data Set, American Diabetes, USA: National Institute of Diabetes and Digestive 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, JAMA 286(10):1195-1200(2001); Mokdad et al, JAMA 289(1):76-79(2003)). In 2000, an estimated 2.9 million people died from diabetes-related causes (Roglic et al., Diabetes Care 28:2130-2135, 2005). The global burden of diabetes is estimated to double over 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). This occurs in conjunction with an increase in obesity. Type 2 diabetes mellitus (T2DM) is a disease that is becoming common. Due to the high frequency of complications, the life expectancy of patients is significantly reduced. Type 2 diabetes is currently the most common cause of vision loss, renal failure, and amputation in adults in industrialized countries due to microvascular complications associated with diabetes. In addition, the presence of type 2 diabetes is associated with an increased risk of cardiovascular disease.

[0004] After a long period of disease, most subjects with type 2 diabetes eventually become insulin dependent after failing oral therapy, requiring daily injections and multiple daily glucose measurements. The UK Prospective Diabetes Study (UKPDS) shows that intensive treatment with metformin, sulfonylureas, or insulin may only result in limited improvements in glycemic control (HbA1c difference of approximately 0.9%). Moreover, even in subjects within the intensive treatment group, glycemic control significantly worsened over time. This is attributed to a decline in beta cell function. Importantly, intensive treatment is not associated with a significant reduction in macrovascular complications (i.e., cardiovascular events).

[0005] Thus, there is an unmet medical need for methods, agents, and pharmaceutical compositions that have superior efficacy in glycemic control, disease modifying properties, and reduced cardiovascular morbidity and mortality, while exhibiting an improved safety profile.

[0006] Current research indicates that obesity-induced inflammation plays a key role, as the link between elevated blood glucose levels and Cox-2 activation, especially in pancreatic beta cells, is well established. High glucose-induced prostaglandin E2 (PGE2) can result in reduced beta cell mass by inhibiting beta cell proliferation and inducing beta cell apoptosis (Oshima, H. et al., 2006). Indomethacin, a nonselective cyclooxygenase inhibitor, can prevent HFD (high fat diet)-induced obesity and insulin resistance in C57BL / 6J mice (Fjaere E. et al., 2014). It has been shown that treatment with certain nonsteroidal anti-inflammatory drugs (NSAIDs), such as celecoxib, partially restores insulin sensitivity in both translational preclinical models as well as obese T2DM subjects (Gonzalez-Ortiz et al., 2005). Hyperglycemia activates Cox-2 in pancreatic beta cells, leading to beta cell dysfunction. Treatment with NS-398, a selective Cox-2 inhibitor, can reverse β-cell dysfunction by reducing PGE2-mediated β-cell apoptosis (Tian, ​​VF et al., 2014). Obesity-induced inflammation can lead to nonalcoholic fatty liver disease, a pathological hallmark of insulin resistance. Nonalcoholic steatohepatitis (NASH) is a co-morbid condition with T2DM. Celecoxib can reverse steatohepatitis and inflammation in HFD-induced Wistar rat NASH model (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). Inducible activation of Cox-2 may play an important role in initiating cellular dysfunction, including adipocyte dysfunction, pancreatic beta cell dysfunction, and macrophage dysfunction. Cellular dysfunction contributes to the development of insulin resistance and systemic glucose tolerance. Cox-2 deletion in C57BL / 6J obese mice reduced blood glucose levels (Fujita et al., 2007).More importantly, celecoxib, a selective Cox-2 inhibitor, can slightly reduce HbA1c levels, improve glucose tolerance, and increase insulin levels in the same translational preclinical model (Fujita, H. et al., 2007). Based on preclinical and clinical data, treating the inflammatory components underlying the complex pathophysiology of type 2 diabetes with anti-inflammatory therapy may be one of the strategies for partial remission and management of type 2 diabetes.

[0007] In addition, many publications support the importance of adipocytes and inflammation in the development of insulin resistance. For example, JNK-1 deficiency in adipocytes suppresses high-fat diet-induced insulin resistance in the liver through JNK dependence (Sabio, C. et al., 2008), adipocyte-specific deletion of Glut4 or overexpression of MCP-1 causes whole-body insulin resistance (Qi, L. et al., 2009), and TNF-α deficiency suppresses diet-induced obesity and the Leptocarcinoma of obesity (Kei et al., 2010). ob / ob It improves insulin sensitivity in models (Hotamisiligil, GS et al., 1995), elevations of IL-1β, IL-6, and CRP predict the development of T2DM (Visser, M. et al., 1999), and TLR4 knockout mice can be protected from inflammation and insulin resistance (Shi, H. et al., 2006). Current hypoglycemic drugs, which do not contain any inflammation inhibitors, lack sufficient efficacy and sufficient overall clinical benefit because they fail to inhibit the proinflammatory components of the complex pathophysiology that initiates and maintains whole-body insulin resistance.

[0008] Inflammation is not only an important part of the pathophysiology of type 2 diabetes, but also a key part of clinically relevant comorbidities. Moreover, proinflammatory signals help initiate and maintain complications associated with type 2 diabetes, such as diabetic retinopathy, skin ulcers, coronary heart disease (CHD), stroke, fatty liver, polycystic ovarian syndrome, chronic kidney disease (CKD), diabetic peripheral neuropathy, and diabetic vascular disease. Proinflammatory signals can determine the severity and duration of diabetes-related complications. There is a direct association between elevated proinflammatory biomarkers and impaired glucose metabolism. Therefore, to achieve effective clinical management, it is important to regulate inflammation and metabolism together and uniformly treat metabolic diseases, including type 2 diabetes, which may include all metabolic syndrome disorders of complex pathophysiology with a strong proinflammatory component, rather than treating each metabolic disease as a separate individual disease. Currently, NSAID drugs alone cannot comprehensively inhibit many inflammatory factors and inflammatory pathways, and hypoglycemic drugs alone cannot fully restore glucose and lipid metabolism. For example, to obtain adequate therapeutic effects in subjects with type 2 diabetes, fatty liver, polycystic ovarian syndrome, or obesity, it would be necessary to treat with a combination of drugs that regulate inflammation and metabolism together. This would not only correct dysglycemia and lipid homeostasis, but also reduce clinically relevant metabolic comorbidities. More importantly, this could reduce the severity of diabetes-related complications. Thus, subjects with metabolic syndrome should not be treated with only one drug, but with a combination of anti-inflammatory and anti-metabolic disease drugs. Summary of the Invention [Means for solving the problem]

[0009] The purpose of the present invention is to overcome the deficiencies of existing technology, i.e., existing drugs sold on the market cannot effectively treat or prevent metabolic syndrome caused by obesity, type 2 diabetes, and insulin resistance. Therefore, the present invention provides a pharmaceutical composition that not only corrects abnormalities in glucose homeostasis, but also reduces clinically relevant comorbidities, and more importantly, reduces the severity of diabetes-related complications.

[0010] To achieve the above objectives, in one aspect, the present invention provides a pharmaceutical composition comprising Therapeutic Agent A or a pharma- ceutically acceptable salt thereof, Therapeutic Agent B or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable excipient, wherein Therapeutic Agent A is a nonsteroidal anti-inflammatory drug, and Therapeutic Agent B is a fatty acid oxidation inhibitor.

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

[0012] In another embodiment, in the pharmaceutical composition, Therapeutic Agent A and Therapeutic Agent B are present in separate dosage forms.

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

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

[0015] In some embodiments, the present invention provides a pharmaceutical composition comprising Therapeutic Agent A, or a pharma- ceutically acceptable salt thereof, Therapeutic Agent B, or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable excipient, wherein Therapeutic Agent A is a salicylate and Therapeutic Agent B is trimetazidine.

[0016] In some embodiments, the present invention provides a pharmaceutical composition comprising Therapeutic Agent A or a pharma- ceutically acceptable salt thereof, Therapeutic Agent B or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable excipient, wherein Therapeutic Agent A is aspirin and Therapeutic Agent B is trimetazidine.

[0017] In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to 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 further relates to the treatment of type 1 diabetes, type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, postprandial hyperglycemia, overweight, obesity, and metabolic syndrome, or improving glycemic control and / or reducing fasting plasma glucose, postprandial plasma glucose, and / or HbA1c, or slowing, delaying, or reversing the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance, and / or metabolic syndrome to type 2 diabetes, or treating diabetic complications such as cataracts, and micro- 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 beta cell degeneration and / or reduced pancreatic beta cell function. and / or restoring pancreatic beta cell function and / or restoring pancreatic insulin secretion, or treating a disease or condition causing 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 complications of atherosclerosis, or treating glaucoma and complications of glaucoma, or treating dyslipidemia / hyperlipidemia and complications of dyslipidemia / hyperlipidemia, or treating a reproduction-related metabolic disease in a subject in need thereof, wherein Therapeutic A is a nonsteroidal anti-inflammatory drug and Therapeutic 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 diagnosed with the following condition: (a) fasting blood glucose or serum glucose concentration greater than 110 mg / dL, especially greater than 125 mg / dL; (b) a postprandial plasma glucose concentration of 140 mg / dL or greater, and (c) The individual is diagnosed with one or more of the following HbA1c values: HbA1c of 6.5% or more, particularly 8.0% or more.

[0022] In another embodiment, the subject is diagnosed with the following condition: (a) obesity, visceral obesity, and / or abdominal obesity, (b) blood triglyceride concentration of 150 mg / dL or more; (c) a blood HDL cholesterol concentration of less than 40 mg / dL in a female subject, or less than 50 mg / dL in a male subject; (d) systolic blood pressure of 130 mmHg or more, diastolic blood pressure of 85 mmHg or more, (e) fasting blood glucose level of 110 mg / dL or more, and (f) the individual has one or more of the following: a blood LDL cholesterol concentration of 130 mg / dL or greater.

[0023] In another embodiment, the subject is an individual who has contraindications to metformin monotherapy and / or is intolerant to therapeutic doses of metformin.

[0024] In another embodiment, the subject (a) biguanides, (b) sulfonylureas, (c) meglitinides, (d) thiazolidinediones, (e) α-glucosidase inhibitors, (f) insulin and insulin analogues; (g) dipeptidyl peptidase IV inhibitors, (h) SGLT2 inhibitors, (i) a PPARα modulator, (j) glucose-dependent insulinotropic polypeptide agonists, (k) beta-3 agonist, (l) GLP1 and GLP1 analogues, (m) PPARγ modulators, and (n) An individual having inadequate glycemic control after treatment with one or more antidiabetic drugs selected from the group consisting of HMG-CoA reductase inhibitors.

[0025] The pharmaceutical composition according to the present invention shows very good efficacy in glycemic control, especially in reducing fasting plasma glucose, postprandial plasma glucose, and / or glycosylated hemoglobin (HbA1c), and at the same time, can effectively treat or prevent obesity, non-alcoholic fatty liver disease, polycystic ovary syndrome, type 2 diabetes, and metabolic syndrome diseases caused by insulin resistance.

[0026] In some embodiments, therapeutic agent A is selected from a salicylate and therapeutic agent B is selected from trimetazidine.

[0027] In some embodiments, therapeutic agent A is selected from aspirin and therapeutic agent B is selected from trimetazidine.

[0028] In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0029] In some embodiments, Therapeutic Agent A and Therapeutic Agent B are administered simultaneously. In some embodiments, Therapeutic Agent A and Therapeutic Agent B are included in a single dosage form. In some embodiments, the single pharmaceutical dosage form is an oral dosage form.

[0030] In some embodiments, Therapeutic Agent A and Therapeutic Agent B are administered separately. In some embodiments, Therapeutic Agent A is administered before Therapeutic Agent B. In some embodiments, Therapeutic Agent A is administered after Therapeutic Agent B. In some embodiments, Therapeutic Agent A and Therapeutic Agent B are each administered orally. In some embodiments, Therapeutic Agent A and Therapeutic Agent B are administered separately by injection.

[0031] Other features and advantages of the present invention are explained in detail in the following description of specific embodiments. In certain embodiments, for example, the following items are provided: (Item 1) 1. A pharmaceutical composition comprising: a therapeutic agent A or a pharma- ceutical acceptable salt thereof, a therapeutic agent B or a pharma- ceutical acceptable salt thereof, and at least one pharma- ceutical acceptable excipient, wherein said therapeutic agent A is a nonsteroidal anti-inflammatory drug and said therapeutic agent B is a fatty acid oxidation inhibitor. (Item 2) 2. The pharmaceutical composition according to item 1, wherein the therapeutic agent A and the therapeutic agent B are contained in a single dosage form. (Item 3) 2. The pharmaceutical composition of claim 1, wherein the therapeutic agent A and the therapeutic agent B are present in separate dosage forms. (Item 4) The pharmaceutical composition according to any one of items 1 to 3, wherein the therapeutic agent A is at least one selected from salicylates, ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, and tolmetin. (Item 5) 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the therapeutic agent B is selected from trimetazidine, etomoxir, aminocarnitine, or a phosphonooxy derivative of carnitine. (Item 6) 6. The pharmaceutical composition according to any one of items 1 to 5, wherein said therapeutic agent A is selected as a salicylate and said therapeutic agent B is selected as trimetazidine. (Item 7) 7. The pharmaceutical composition according to any one of items 1 to 6, wherein said therapeutic agent A is selected as aspirin (acetylsalicylic acid). (Item 8) 8. The pharmaceutical composition according to any one of items 1 to 7, wherein the weight ratio of said therapeutic agent A to said therapeutic agent B is 1:1 to 10:1, preferably 6:1. (Item 9) 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, and metabolic syndrome, or a disease or condition that causes abnormal accumulation of fat in the liver, or atherosclerosis and atherosclerotic complications, or glaucoma and glaucoma complications, or dyslipidemia / hyperlipidemia and dyslipidemia / hyperlipidemia complications in a subject in need thereof, wherein Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. (Item 10) 10. The use of item 9, wherein the subject is an individual diagnosed with one or more conditions selected from the group consisting of overweight, obesity, visceral obesity, and abdominal obesity. (Item 11) 11. The use according to item 9 or 10, wherein the therapeutic agent A is selected as aspirin. (Item 12) 12. The use according to any one of items 9 to 11, wherein the weight ratio of said therapeutic agent A to said therapeutic agent B is 1:1 to 10:1, preferably 6:1. (Item 13) 13. The use according to any one of items 9 to 12, wherein the therapeutic agent A and the therapeutic agent B are administered simultaneously. (Item 14) 14. The use of item 13, wherein said therapeutic agent A and said therapeutic agent B are contained in a single dosage form. (Item 15) 13. The use according to any one of items 9 to 12, wherein the therapeutic agent A and the therapeutic agent B are administered separately. (Item 16) 16. The use of item 15, wherein said therapeutic agent A is administered before said therapeutic agent B. (Item 17) 16. The use of item 15, wherein said therapeutic agent A is administered after said therapeutic agent B. (Item 18) 18. The use according to any one of items 9 to 17, wherein the therapeutic agent A and the therapeutic agent B are administered orally or by injection.

[0032] The accompanying drawings are provided to facilitate a further understanding of the present invention and constitute a part of this specification. The drawings are used to explain the present invention with the following specific embodiments, but are not intended to be limiting of the present invention. In the drawings: [Brief description of the drawings]

[0033] [Figure 1] 1 shows the results of body weight change in diet-induced obese (DIO) mice after 10 days of treatment with pharmaceutical treatments A+B in the experimental group using a pharmaceutical composition according to an embodiment of the present invention compared to the control group. [Diagram 2] 1 shows the results of fat loss in DIO mice induced by treatment with pharmaceutical treatments A+B in the experimental group using a pharmaceutical composition according to an embodiment of the present invention compared to the control group. [Diagram 3] 1 shows the results of gonadal fat pad adipocyte morphology in experimental groups treated with pharmaceutical compositions according to embodiments of the present invention compared to a DIO control group. [Figure 4] 1 shows the results of hepatic steatosis and hepatocyte morphology in experimental groups treated with pharmaceutical compositions according to embodiments of the present invention compared to a DIO control group. [Diagram 5] 1 shows the results of cardiomyocyte morphology in experimental groups treated with pharmaceutical compositions according to embodiments of the present invention compared to a DIO control group. [Figure 6] 1 shows the results of skeletal muscle cell morphology in experimental groups treated with pharmaceutical compositions according to embodiments of the present invention compared to a DIO control group. [Figure 7] 1 shows the results of nephrotoxicity index serum creatinine (CREA) and blood urea nitrogen (BUN) in experimental groups treated with pharmaceutical compositions according to embodiments of the present invention compared to a DIO control group. [Figure 8] 1 shows the results of body weight change in diabetic DIO mice after 4 weeks of treatment with pharmaceutical treatment agents A+B in the experimental group treated with a pharmaceutical composition according to an embodiment of the present invention compared to the control group. [Figure 9]1 shows a comparison of the total serum cholesterol level, an index related to hyperlipidemia, between each group of DIO mice. [Figure 10] 1 shows a comparison of serum LDL cholesterol, a marker associated with hyperlipidemia, between each group of DIO mice. [Figure 11] FIG. 1 shows a comparison of serum alanine aminotransferase (ALT), an index related to fatty liver, in each group of DIO mice. [Figure 12] 1 shows a comparison of serum albumin / globulin ratio, an index related to nitrogen metabolic function, between each group of DIO mice. [Figure 13] 1 shows a comparison of serum total protein, an index related to nitrogen metabolic function, between each group of DIO mice. [Figure 14] 1 shows the results of glucose tolerance in diabetic DIO mice. [Figure 15] 1 shows the results of insulin sensitivity in diabetic DIO mice. [Figure 16] 1 shows a comparison of body weight in DIO mice after administration of various therapeutic combinations. [Figure 17] 1 shows a comparison of fasting blood glucose levels in DIO mice after administration of various therapeutic combinations. [Figure 18] 1 shows the results of body weight changes in normal wild-type mice induced by pharmaceutical treatments A+B in the experimental group treated with a pharmaceutical composition according to an embodiment of the present invention compared to the control group. [Figure 19] 1 shows the results of fat loss in normal wild-type mice induced by pharmaceutical treatments A+B in the experimental group treated with a pharmaceutical composition according to an embodiment of the present invention compared to the control group. [Figure 20] 1 shows the results of body weight change in rats with polycystic ovary syndrome induced by pharmaceutical treatment A+B in the experimental group treated with a pharmaceutical composition according to an embodiment of the present invention compared to the control group. [Figure 21] 1 shows a comparison of fasting blood glucose levels in each group of rats with polycystic ovary syndrome. [Figure 22]FIG. 1 shows a comparison of serum aspartate aminotransferase (AST) and total cholesterol, which are indicators related to fatty liver, in each group of rats with polycystic ovary syndrome. [Figure 23] 1 shows a comparison of serum total protein, an index related to the nitrogen metabolism function of the liver, between each group of rats with polycystic ovary syndrome. [Figure 24] 1 shows a comparison of serum creatinine, an index related to renal function, in each group of rats with polycystic ovary syndrome. [Diagram 25] This shows a comparison of serum lactate dehydrogenase (LDH), creatine kinase (CK), cardiac creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH), which are indices related to heart disease, in each group of rats with polycystic ovary syndrome. [Figure 26] 1 shows the results of insulin sensitivity in rats with polycystic ovary syndrome. [Figure 27] 1 shows the results of the estrous cycle in rats with polycystic ovary syndrome. [Figure 28] 1 shows the results of serum hormone ELISA in rats with polycystic ovary syndrome. [Figure 29] 1 shows the results of Western blot analysis of skeletal muscle proteins in rats with polycystic ovary syndrome. [Diagram 30] 1 shows the results of left ventricular wall thickness analysis in rats with polycystic ovary syndrome. [Diagram 31] FIG. 1 shows the results of myocardial fibrosis analysis in rats with polycystic ovary syndrome. [Diagram 32] 1 shows the results of routine blood analysis in rats with polycystic ovary syndrome. [Diagram 33] The results of pharmaceutical treatments A+B on body weight change in NASH mice are shown. [Diagram 34] 1 shows the results of a food intake study in NASH mice. [Diagram 35] 4 shows the results of liver weight studies in NASH mice. [Diagram 36] 1 shows the results of liver section analysis in NASH mice. [Figure 37] 1 shows the results of cardiac slice analysis in NASH mice. [Figure 38] A comparison of two indices related to liver injury in each group of mice is shown. [Figure 39] 1 shows a comparison of indices related to hyperlipidemia in each mouse group. [Diagram 40] 1 shows a comparison of indices related to organ damage in each group of mice. [Diagram 41] The results of fasting blood glucose in each group of mice are shown. [Diagram 42] 1 shows the results of glucose tolerance in NASH mice. [Diagram 43] 1 shows the results of insulin sensitivity in NASH mice. [Diagram 44] 1 shows the results of Western blot analysis of skeletal muscle proteins in NASH mice. [Diagram 45] 1 shows the results of transcriptome sequencing analysis in NASH mice. [Diagram 46] 1 shows the results of protein Western blot analysis of primary human skeletal muscle cells in vitro 24 hours after administration. [Figure 47] FIG. 1 shows the results of a protein Western blot analysis of primary human skeletal muscle cells with inducible insulin resistance in vitro 7 days after treatment. [Figure 48] 1 shows the results of a skeletal muscle transcriptome sequencing analysis induced by a high-fat and high-sugar diet in rats with PCOS polycystic ovary syndrome. [Figure 49] 1 shows the results of insulin ELISA and the insulin resistance index HOMA-IR of serum samples in NASH mice. [Figure 50] 1 shows the results of adiponectin ELISA of serum samples in NASH mice. [Figure 51] 1 shows comparative liquid mass spectrometry results of serum samples from mice in control group 1 and control group 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are merely used to illustrate and explain the present invention, and are not intended to limit the present invention.

[0035] The end points of ranges and any values ​​disclosed herein are not limited to exact ranges or values.It should be understood that these ranges or values ​​include the values ​​close to these ranges or values.For numerical ranges, the end points of each range, the end points of each range and each point value, and each point value can all be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein.

[0036] The present invention is mainly based on the unexpected results obtained by the inventors in the combined use of a nonsteroidal anti-inflammatory drug (therapeutic drug A) and a fatty acid oxidation inhibitor (therapeutic drug B). The inventors have surprisingly discovered that when a subject with diabetes, cardiovascular and cerebrovascular disease, fatty liver, or polycystic ovarian syndrome is administered both therapeutic drug A and therapeutic drug B, multiple indicators of the subject obtained are improved. In addition, the drug combination can also achieve the effect of reversing various symptoms. The inventors have also surprisingly discovered that the mechanism of action of the drug combination is not limited to regulating inflammatory response and metabolism. Daily administration of the drug combination can also repeatedly specifically activate the p38 and AMPK signaling pathways and related metabolic changes, forming a cycle of excitatory effects that function in the same way as mimicking exercise. After short-term administration (within 60 minutes), the pharmaceutical therapeutic agent A+B provided by the present invention can regulate the p38 and AMPK signaling pathway mechanism model together via fatty acid oxidation (FAO), fatty acid metabolites (acyl metabolites), and adenosine triphosphate (ATP) to simultaneously increase the p38 and AMPK signaling pathway, thereby promoting catabolism such as fat hydrolysis and oxidation of fatty acids. In addition, after long-term administration (3-24 hours), the pharmaceutical therapeutic agent A+B provided by the present invention can regulate the p38 and AMPK signaling pathway mechanism model together via inflammatory cytokines (Inf cytokines), fatty acid oxidation (FAO), mitochondrial reactive oxygen species (mtROS), and glycolysis (glycolysis), simultaneously plummeting the p38 and AMPK signaling pathway, thereby promoting anabolism and muscle repair. Thus, the present patent application provides a novel and effective pharmaceutical composition on the one hand, and a treatment method using the pharmaceutical composition on the other hand.

[0037] Pharmaceutical Compositions In one aspect, the invention provides a pharmaceutical composition comprising Therapeutic Agent A, or a pharma- ceutically acceptable salt thereof, Therapeutic Agent B, or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable excipient, wherein Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor.

[0038] The term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergy or other problems, or complications, and with a reasonable benefit / risk ratio. Therapeutic Agent A and Therapeutic Agent B herein may form stable pharma- ceutically acceptable acid or base salts, and in such cases, it may be appropriate to administer the compound as a salt. Examples of acid salts include acetate, adipate, ascorbate, benzoate, besylate, bicarbonate, bisulfate, butyrate, camphorate, camphorsulfonate, choline, citrate, cyclohexylsulfamate, diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-isethionate, heptanoate, caproate, hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate. , malate, maleate, mesylate, meglumine, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenyl acetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate, and undecylate. Examples of base salts include ammonium salts; alkali metal salts such as sodium salt, lithium salt, and potassium salt; alkaline earth metal salts such as aluminum salt, calcium salt, and magnesium salt; salts with organic bases such as dicyclohexylamine and N-methyl-D-glucosamine, and salts with amino acids such as arginine, lysine, and ornithine.

[0039] In one embodiment, the therapeutic agent A and the therapeutic agent B in the pharmaceutical composition may be present in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount of a compound or composition sufficient to significantly and positively alter the symptoms and / or condition being treated (e.g., provide a positive clinical response). The effective amount of the active ingredient used in the pharmaceutical composition may vary based on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of the combination therapy, the particular active ingredient used, the particular pharmaceutically acceptable excipient used, and similar factors within the knowledge and expertise of the attending physician.

[0040] As used herein, the term "treatment" refers to a method for obtaining beneficial or desired clinical results. The term "treat" refers to inhibiting, preventing, or arresting the onset or progression of a condition (disease, disorder, or condition), and / or alleviating, ameliorating, or regressing a condition. Those skilled in the art will understand that a variety of methods and assays can be used to assess the onset of a condition, and similarly, a variety of methods and assays can be used to assess the alleviation, amelioration, or regression of a condition.

[0041] As used herein, the term "prevention" refers to preventing the development of a disease, disorder, or condition in a subject who may be at risk of the disease but has not been diagnosed with the disease. Prevention (and the effective dose for prevention) can be demonstrated in population studies. For example, the amount effective for preventing a given disease or medical condition is the amount effective for reducing the incidence of the treated population compared to an untreated control population.

[0042] As used herein, the term "subject" may include a mammal, preferably a human of any age, having a pathological characteristic. Preferably, the term also includes an individual at risk of developing the pathological characteristic.

[0043] Treatment A and Treatment B According to the present invention, the therapeutic agent A is a nonsteroidal anti-inflammatory drug. As used herein, the term "nonsteroidal anti-inflammatory drug (NSAID)" is a class of anti-inflammatory drugs that do not contain a steroid structure. It can be divided into three main categories: acetylsalicylates, including aspirin (acetylsalicylic acid); non-acetylsalicylates, including magnesium salicylate, sodium salicylate, choline magnesium salicylate, diflunisal, and salsalate; and non-salicylates, including ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, tolmetin, etc. In addition, the therapeutic agent A of the present invention can be selected from any of these nonsteroidal anti-inflammatory drugs. Preferably, therapeutic agent A may be selected from at least one of salicylates, ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, and tolmetin. More preferably, therapeutic agent A may be a salicylate, such as aspirin or magnesium salicylate.

[0044] 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 carnitine derivatives.Preferably, therapeutic agent B can be trimetazidine, aminocarnitine (e.g., as described in WO85 / 04396), phosphinyloxy derivatives of carnitine (e.g., as described in EP0574355B1), or conjugation products of carnitine and other compounds (e.g., as described in JP5127093B2 / US6369073B1).More preferably, therapeutic agent B can be trimetazidine.

[0045] According to the present invention, the combination of therapeutic agent A and therapeutic agent B in the pharmaceutical composition of the present invention is not particularly limited. That is, 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 be present in separate dosage forms. Thus, upon subsequent use of the pharmaceutical composition of the present invention, 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 (e.g., in the same dosage form). After administering therapeutic agent A to the subject, therapeutic agent B can be administered to the subject immediately, 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 administering therapeutic agent A to the subject. In some embodiments, therapeutic agent B can be administered to the subject and therapeutic agent A can be administered to the subject immediately after administering therapeutic agent B to the subject. Alternatively, 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 administering therapeutic agent B to the subject.

[0046] Method of administration The pharmaceutical compositions of the present invention are suitable for oral administration (e.g., tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups, or elixirs, etc.), topical administration (e.g., creams, ointments, gels, or aqueous or oily solutions or suspensions, etc.), inhalation administration (e.g., fine powders or liquid aerosols, etc.), insufflation administration (e.g., fine powders, etc.), or parenteral administration (e.g., sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, or intramuscular administration, or suppositories for rectal administration, etc.). In a preferred embodiment, the pharmaceutical compositions of the present invention are administered orally.

[0047] The pharmaceutical composition of the present invention can be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Pharmaceutically acceptable excipients suitable 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 paraben, and antioxidants such as ascorbic acid. Tablet formulations can be uncoated or coated. Coatings are either intended to modify disintegration in the gastrointestinal tract and subsequent absorption of active ingredients, or to improve stability and / or appearance. In either case, conventional coating agents and methods well known in the art can be used.

[0048] The pharmaceutical compositions of the present invention are not strictly limited with respect to dosage and frequency of administration. They may vary depending on many factors, such as age, body weight, general health, diet, sex, the drug administered, the route (or method) of administration, and the severity of the condition being treated, as well as the judgment of the attending physician. In general, the pharmaceutical compositions of the present invention can be administered once or more per day, such as once per day, twice per day, three or more times per day, and can also be administered every two days, every three days, once per week, or at other frequencies. In terms of daily dosage, the amount of therapeutic agent A can be administered at 0.1 mg to 5000 mg per day, or preferably 10 mg to 3000 mg, more preferably 80 mg to 2000 mg, or even more preferably 500 mg to 1500 mg. The amount of therapeutic agent B can be administered at 0.1 mg to 1000 mg per day, or preferably 10 mg to 1000 mg, more preferably 100 mg to 500 mg, or even more preferably 35 mg to 300 mg. Therefore, the application of the above dosages can be reasonably realized according to the above different application frequencies and methods, for example, administering 5 mg to 500 mg of therapeutic drug A once a day, or administering 10 mg to 250 mg of therapeutic drug A twice a day.

[0049] 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 an 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 salicylate (such as aspirin) to trimetazidine is 1:1 to 10:1, including 2:1, 3:1, 4:1, 5:1, 6:1 1, 7:1, 8:1, 9:1, or 10:1.

[0050] Use (or method) In another aspect, the present invention further relates to the treatment of type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, postprandial hyperglycemia, type 1 diabetes, overweight, obesity, and metabolic syndrome, or improving glycemic control and / or reducing fasting plasma glucose, postprandial plasma glucose, and / or HbA1c A1c, or slowing, delaying, or reversing the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance, and / or metabolic syndrome to type 2 diabetes, or treating diabetic complications such as cataracts, and micro- 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 beta cell degeneration and / or reduced pancreatic beta cell function, and / or restoring pancreatic beta cell function, and / or restoring pancreatic insulin secretion, or causing abnormal accumulation of fat in the liver. The present invention provides a method of use of Therapeutic Agents A and B in the preparation of a medicament for treating a disease or condition, or maintaining and / or improving insulin sensitivity and / or treating hyperinsulinemia and / or insulin resistance, or treating atherosclerosis and complications of atherosclerosis, or treating glaucoma and complications of glaucoma, or treating dyslipidemia / hyperlipidemia and complications of dyslipidemia / hyperlipidemia, or treating a reproduction-related metabolic disorder in a subject in need thereof, wherein Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor.

[0051] 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 2 diabetes, impaired glucose tolerance, impaired fasting glucose, type 1 diabetes, obesity, steatohepatitis, atherosclerosis, glaucoma, dyslipidemia / hyperlipidemia, and hyperglycemia in a subject in need thereof, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) for treating one or more of disorders including type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, type 1 diabetes, obesity, 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, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0052] In some embodiments, the present invention provides a 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, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for 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, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 therapeutic agent A (eg, a salicylate such as aspirin) to therapeutic agent B (eg, trimetazidine) is 6:1.

[0053] In some embodiments, the present invention provides a 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 hepatic fat in a subject in need thereof, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for treating a disease or condition causing abnormal accumulation of hepatic fat in a subject in need thereof, comprising administering Therapeutic Agent A and Therapeutic Agent B to an affected individual, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 therapeutic agent A (eg, a salicylate such as aspirin) to therapeutic agent B (eg, trimetazidine) is 6:1.

[0054] In some embodiments, the present invention provides a use (or method) of therapeutic agent A and therapeutic agent B in the preparation for treating a reproductive-related metabolic disease in a subject in need thereof, where therapeutic agent A is a nonsteroidal anti-inflammatory drug and therapeutic agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for treating a reproductive-related metabolic disease in a subject in need thereof, comprising administering therapeutic agent A and therapeutic agent B to an affected individual, where therapeutic agent A is a nonsteroidal anti-inflammatory drug and therapeutic agent B is a fatty acid oxidation inhibitor. Reproductive-related metabolic diseases herein include, but are not limited to, polycystic ovarian syndrome (PCOS), gestational diabetes, preeclampsia, recurrent spontaneous abortion, fetal growth restriction, ovarian insufficiency, premature ovarian failure, and male infertility. In some embodiments, therapeutic agent A is selected from salicylates (e.g., aspirin) and therapeutic agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0055] 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 an affected individual in need thereof, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a method of treating polycystic ovary syndrome (PCOS), comprising administering Therapeutic Agent A and Therapeutic Agent B to an affected individual, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 therapeutic agent A (eg, a salicylate such as aspirin) to therapeutic agent B (eg, trimetazidine) is 6:1.

[0056] In some embodiments, the invention provides for 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 an affected individual (such as a patient with polycystic ovary syndrome) in need thereof, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the invention provides for the use (or method) for restoring skeletal muscle insulin sensitivity and protein anabolism (or alleviating muscle inflammation, insulin resistance, sarcopenia, and / or metabolic syndrome) in an affected individual (such as a patient with polycystic ovary syndrome) in need thereof, comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0057] 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 disease such as myocardial fibrosis, ventricular hypertrophy, heart failure, etc. in an affected individual (such as a patient with polycystic ovary syndrome or NASH), where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) for improving (including reversing) cardiovascular disease such as myocardial fibrosis, ventricular hypertrophy, heart failure, etc. in an affected individual (such as a patient with polycystic ovary syndrome or NASH), where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor, comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0058] In some embodiments, the present invention provides a 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 an affected individual in need thereof (such as a patient with polycystic ovary syndrome or NASH), where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and 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 an affected individual in need thereof (such as a patient with polycystic ovary syndrome or NASH), comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0059] In some embodiments, the present invention provides a 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 an affected individual in need thereof, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and 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 an affected individual, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0060] In some embodiments, the invention provides a use (or method) of Therapeutic Agent A and Therapeutic Agent B in the preparation of a medicament for reducing weight gain (or reversing hyperlipidemia) in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the invention provides a use (or method) for reducing weight gain (or reversing hyperlipidemia) in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), comprising administering to the affected individual Therapeutic Agent A and Therapeutic Agent B, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0061] In some embodiments, the present invention provides a use (or method) of Therapeutic Agent A and Therapeutic Agent B in the preparation of a medicament for reversing hepatomegaly (or liver damage) in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for reversing hepatomegaly (or liver damage) in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0062] 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 hepatic insulin sensitivity, and reducing muscle inflammation) in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides the use (or method) for increasing glucose tolerance (or reversing insulin resistance, restoring skeletal muscle and hepatic insulin sensitivity, and reducing muscle inflammation) in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0063] In some embodiments, the present invention provides a 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 an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and 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 an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0064] In some embodiments, the present invention provides a use (or method) of Therapeutic Agent A and Therapeutic Agent B in the preparation of a medicament for modulating the p38 and AMPK signaling pathways in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, the present invention provides a use (or method) for modulating the p38 and AMPK signaling pathways in an affected individual in need thereof (such as a patient with non-alcoholic fatty liver disease (NASH)), comprising administering Therapeutic Agent A and Therapeutic Agent B to the affected individual, where Therapeutic Agent A is a non-steroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. In some embodiments, Therapeutic Agent A is selected from a salicylate (e.g., aspirin) and Therapeutic Agent B is selected as trimetazidine. In some embodiments, Therapeutic Agent A (e.g., a salicylate such as aspirin) and Therapeutic Agent B (e.g., trimetazidine) are present in a weight ratio of 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 Therapeutic Agent A (e.g., a salicylate such as aspirin) to Therapeutic Agent B (e.g., trimetazidine) is 6:1.

[0065] The definitions of therapeutic agent A and therapeutic agent B, as well as the administration frequency, mode, dosage, etc., may be the same as previously described.

[0066] In some embodiments, therapeutic agent A is selected from a salicylate and therapeutic agent B is selected as trimetazidine. In some embodiments, therapeutic agent A is selected as aspirin and therapeutic agent B is selected as trimetazidine. In some embodiments, the weight ratio of therapeutic agent A (e.g., a salicylate such as aspirin) to therapeutic agent B (e.g., trimetazidine) is 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 therapeutic agent A (e.g., a salicylate such as aspirin) to therapeutic agent B (e.g., trimetazidine) is 6:1. In some embodiments, therapeutic agent A and therapeutic agent B are administered simultaneously (e.g., orally). In some embodiments, therapeutic agent A and 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, therapeutic agent A and therapeutic agent B are administered separately (e.g., orally).

[0067] A course of treatment may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, Therapeutic Agent A may be administered once a day, twice a day, three times a day, etc., or may be administered once every 2 days, once every 3 days, once a week, or other frequency. In some embodiments, Therapeutic Agent B may be administered once a day, twice a day, three times a day, etc., or may be administered once every 2 days, once every 3 days, once a week, or other frequency. When Therapeutic Agent A and Therapeutic Agent B are administered separately, the administration frequencies of the two may or may not be the same.

[0068] According to the present invention, the subject in need thereof can be classified into many types, such as subjects diagnosed with or at risk of various metabolic syndromes, or subjects with various metabolic syndrome indicators outside normal range, or subjects who are intolerant to, unable to use, or subsequently ineffective against the use of certain antidiabetic drugs.Thus, in one embodiment, the subject can be an individual diagnosed with one or more conditions selected from the group consisting of 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, particularly greater than 125 mg / dL, (b) postprandial plasma glucose concentration greater than or equal to 140 mg / dL, and (c) HbA1c value greater than or equal to 6.5%, particularly greater than or equal to 8.0%. In another embodiment, the subject may be an individual with one or more of the following conditions: (a) obesity, visceral obesity, and / or abdominal obesity, (b) blood triglyceride concentration of 150 mg / dL or more, (c) blood HDL cholesterol concentration of less than 40 mg / dL in female subjects and less than 50 mg / dL in male subjects, (d) systolic blood pressure of 130 mmHg or more, diastolic blood pressure of 85 mmHg or more, (e) fasting blood glucose level of 110 mg / dL or more, and (f) blood LDL cholesterol concentration of 130 mg / dL or more. In another embodiment, the subject may be an individual with contraindications to metformin monotherapy and / or intolerance to therapeutic doses of metformin. In another embodiment, the subject may be an individual with inadequate glycemic control following treatment with one or more antidiabetic agents 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) beta-3 agonists, (l) GLP1 and GLP1 analogs, (m) PPARγ modulators, and (n) HMG-CoA reductase inhibitors.

[0069] In some embodiments, the subject is a human, such as an individual over 20, 30, 40, 50, 60, 70, or 80 years of age.

[0070] The invention is explained in detail below with reference to examples. EXAMPLES

[0071] In the following examples, all animal (e.g., mouse) procedures were performed in accordance with 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.

[0072] In addition, the control group and experimental group of the present invention all adopt intraperitoneal injection in the process. The injection volume starts from 4 μl / g body weight and can be adjusted according to the dose, and the therapeutic agent A (aspirin (trade name Aspirin) is used in the following examples) of the experimental group is diluted with PBS to a dose of 0.3 mg / kg to 120 mg / kg body weight, and the therapeutic agent B (trimetazidine (trade name Trimetazidine) is used in the following examples) is diluted with PBS to a dose of 0.05 mg / kg to 500 mg / kg body weight. In the control group, usually only the same amount of therapeutic agent A or the same amount of therapeutic agent B is used, or only the same amount of PBS solvent is used as a blank control.

[0073] For each graphed result, data are expressed as mean ± standard error of the mean (SEM) and p-values ​​were calculated using Student's t-test (two-tailed, two-sample unequal variance). Statistical significance is indicated as p>0.05 (not significant, ns), p<0.05 (one asterisk, i.e. *), or p<0.01 (two asterisks, i.e. **), or p<0.001 (three asterisks, i.e. ***). Tests were performed using Microsoft Excel and the test type was always set to two-sample equal variance.

[0074] Example 1 Twenty 6-week-old diet-induced obese (Jackson Lab's Diet-Induced Obese, DIO) male mice with similar physical conditions were selected and then divided into four groups, with five mice in each group. In the experimental group, each mouse was effectively administered 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B per day. In control group 1, each mouse was effectively administered 30 mg / kg of therapeutic agent A per day. In control group 2, each mouse was effectively administered 5 mg / kg of therapeutic agent B per day. In control group 3, each mouse was administered only the same amount of PBS solvent every day as a blank control. The experiment was performed for a total of 10 days. The weight of each mouse was recorded daily. A graph of the weight over time was plotted based on the weight of each mouse, as shown in Figure 1.

[0075] FIG. 1 shows the results of body weight change in obese DIO mice induced by pharmaceutical therapeutics A+B in the experimental group treated with the pharmaceutical composition according to an 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 DIO mice by more than about 10% within 10 days, while the control groups 1 to 3 had almost no significant effect on the body weight of the mice. Therefore, it can be considered that the therapeutics A and B provided by the present invention have a synergistic effect on the therapeutic effect of weight loss when used in combination.

[0076] Example 2 The mice in the experimental group and the control group 3 that had completed Example 1 were selected and dissected, and the fat accumulation around the mice in the experimental group and the control group 3 was observed and compared. The images capturing the fat deposition in various places after dissection are shown in FIG. 2.

[0077] Figure 2 shows the results of fat reduction in DIO mice induced by treatment with pharmaceutical treatment agents A+B in the experimental group using the pharmaceutical composition according to an embodiment of the present invention, compared with the control group. It can be seen that the mice in the control group 3 have obvious fat accumulation such as fatty liver, visceral fat, and subcutaneous fat, while the fat accumulation in the mice in the experimental group is greatly reduced. Therefore, it can be considered that the pharmaceutical composition provided by the present invention has the effect of greatly reducing the content of body fat.

[0078] Example 3 Similarly, mice from the experimental group that completed Example 1 and the control groups 1, 2, and 3 were selected, and the dissected mice were observed under a microscope to compare the shape and size of the fat granules in the gonadal fat pads. The morphology of the fat granules under a microscope is shown in Figure 3.

[0079] 3 shows the results of the morphology of adipocytes in gonadal fat pads in the experimental groups treated with pharmaceutical compositions according to embodiments of the present invention compared to the control group. It can be seen that the size of the adipocytes in the experimental groups was significantly reduced compared to the adipocytes in the control groups 1, 2, and 3, that is, the therapeutic agents A+B had a therapeutic effect of inducing fat loss.

[0080] Example 4 Similarly, mice from the experimental group that completed Example 1 and the control groups 1, 2, and 3 were selected, and the dissected mice were observed under a microscope to compare the morphology of hepatocytes and the size of fat granules. The morphology of hepatocytes under a microscope is shown in Figure 4.

[0081] Figure 4 shows the results of fatty liver and hepatocyte morphology in the experimental group treated with the pharmaceutical composition according to an embodiment of the present invention compared with the control group. It can be seen that the number of fat granules in the hepatocytes of the experimental group was reduced, their size was significantly reduced, and inflammatory cells were also reduced, compared with the hepatocytes of the control groups 1, 2, and 3. In other words, the therapeutic agent A+B had a therapeutic effect of inducing a reduction in fat and a reduction in inflammation in fatty liver.

[0082] Example 5 Similarly, mice from the experimental group that completed Example 1 and the control groups 1, 2, and 3 were selected, and the dissected mice were observed under a microscope to compare the cardiomyocyte morphology. The diagram of the cardiomyocyte morphology under a microscope is shown in FIG. 5.

[0083] Figure 5 shows the results of cardiomyocyte morphology in the experimental group treated with pharmaceutical compositions according to an embodiment of the present invention compared with the control group. It can be seen that the color and shape of muscle images of the experimental group and control groups 1, 2 and 3 are basically the same. That is, therapeutic drug A+B did not cause cardiotoxicity.

[0084] Example 6 Similarly, mice from the experimental group that completed Example 1 and the control groups 1, 2, and 3 were selected, and the dissected mice were observed under a microscope to compare the skeletal muscle cell morphology. The morphology of skeletal muscle cells under a microscope is shown in FIG. 6.

[0085] Figure 6 shows the results of skeletal muscle cell morphology in the experimental group treated with pharmaceutical compositions according to embodiments of the present invention compared with the control group. It can be seen that the color and shape of muscle images of the experimental group and control groups 1, 2 and 3 are basically the same. That is, therapeutic agent A+B did not cause muscle loss and muscle toxicity.

[0086] Example 7 Similarly, mice from the experimental group and control groups 1, 2 and 3 that completed Example 1 were selected, and the serum from the dissected mice was used for blood biochemistry testing to compare the serum creatinine (CREA) and blood urea nitrogen (BUN), which are nephrotoxicity indicators. The comparison of the two indicators is shown in Figure 7.

[0087] Figure 7 shows the results of serum creatinine (CREA) and blood urea nitrogen (BUN), which are nephrotoxicity indexes, in the experimental group treated with pharmaceutical compositions according to an embodiment of the present invention, compared with the control group. It can be seen that the serum creatinine (CREA) and blood urea nitrogen (BUN) levels of the experimental group and control groups 1, 2 and 3 are basically the same, and there is no significant difference between them. That is, therapeutic drug A+B did not cause nephrotoxicity.

[0088] Example 8 Diabetic DIO mice were tested for weight change in the same manner as in Example 1, except that each group contained 12 mice and the treatment period was extended to 4 weeks. The weight of each mouse was recorded daily and the weight of each mouse was plotted over time, as shown in Figure 8.

[0089] FIG. 8 shows the results of weight change in diabetic DIO mice after 4 weeks of treatment with pharmaceutical therapeutics A+B in the experimental group treated with the pharmaceutical composition according to an embodiment of the present invention compared to 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 the control groups 1 to 3 have almost no obvious effect on the weight of the mice. Therefore, it can be considered that the therapeutics A and B provided by the present invention have a synergistic effect on the treatment results of weight loss in diabetic patients when used in combination.

[0090] FIG. 9 shows a comparison of the total amount of serum cholesterol, an index related to hyperlipidemia, between each group of DIO mice.

[0091] FIG. 10 shows a comparison of serum LDL cholesterol, a marker associated with hyperlipidemia, in each group of DIO mice.

[0092] FIG. 11 shows a comparison of serum alanine aminotransferase (ALT), an index associated with fatty liver, in each group of DIO mice.

[0093] FIG. 12 shows a comparison of serum albumin / globulin ratio, an index related to nitrogen metabolic function, between each group of DIO mice.

[0094] FIG. 13 shows a comparison of serum total protein, an index related to nitrogen metabolic function, between each group of DIO mice.

[0095] Examples 9 to 13 Subsequent tests were performed on each group of mice that had completed the treatment of Example 8. Examples 9 and 10 detected two indicators related to obesity and hyperlipidemia, total cholesterol and LDL cholesterol. Example 11 detected alanine aminotransferase (ALT), an indicator related to fatty liver disease. Examples 12 and 13 detected two indicators related to nitrogen metabolism, albumin / globulin ratio and total protein. Comparisons of each indicator for each group are shown in Figures 9 to 13.

[0096] 9-13 show a comparison of various indicators related to metabolic syndrome, such as obesity, hyperlipidemia, fatty liver disease, and nitrogen metabolism function in each group of mice, respectively. Compared with the blank control group 3, after 4 weeks of administration of therapeutic drugs A+B, all the indicators of the experimental group mice showed a significant decrease, but neither therapeutic drug A in control group 1 nor therapeutic drug B in control group 2 used alone could achieve a significant therapeutic or recovery effect. It can be seen that the pharmaceutical composition provided by the present invention has obvious medical applications in the treatment or improvement of diseases related to obesity and hyperlipidemia, fatty liver disease, and diseases related to nitrogen metabolism function.

[0097] Example 14 Similarly, glucose tolerance tests were also performed on groups of mice that completed the treatment of Example 8. In the experimental and control groups 1-3, the experiments were performed by intraperitoneal glucose injection (2 mg / g body weight) on mice that had been fasted overnight. The glucose content (mM) of the mice was tested at each time point every 15-30 minutes with a Lifescan One Touch blood glucose meter, and a line graph was created according to the glucose content and time. The results are shown in Figure 14.

[0098] Figure 14 shows the results of glucose tolerance in diabetic DIO mice. It can be seen that compared with control groups 1 to 3, the glucose content of mice in the experimental group did not increase significantly to a very high level of glucose content, but rapidly decreased to a lower level of glucose content. Therefore, it can be seen that after 4 weeks of treatment with the pharmaceutical composition of the present invention, the mice showed excellent results in glucose tolerance.

[0099] Example 15 Similarly, insulin sensitivity tests were also performed on groups of mice that completed the treatment of Example 8. In experimental and control groups 1-3, 0.75 U insulin / kg body weight of insulin (humulin) was administered intraperitoneally to rats that had been fasted for 5 hours using a 27G needle. Every 15-30 minutes thereafter, the blood glucose levels (mM) of the mice were tested at each time point using a Lifescan One Touch blood glucose meter, and a line graph was plotted based on glucose content and time. The results are shown in Figure 15.

[0100] Figure 15 shows the results of insulin sensitivity in diabetic DIO mice. It can be seen that compared with control groups 1 to 3, the glucose content of mice in the experimental group was always kept at a lower level of glucose content, and changed relatively more significantly. Therefore, it can be seen that the mice after 4 weeks of treatment with the pharmaceutical composition of the present invention showed excellent results in terms of insulin sensitivity.

[0101] Example 16 DIO mice were treated for 10 days with different combinations of therapeutic drugs as follows: (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. At the end of the treatment, the body weight (%) of each combination of therapeutic drugs group was measured respectively. The results are shown in FIG. 16.

[0102] Figure 16 shows a comparison of body weight in DIO mice after administration of various combinations of therapeutic drugs. It can be seen that the expected therapeutic effect cannot be achieved when the normal dose is administered. In contrast, the combination of 30 mg / kg A + 5 mg / kg B in group (4) showed excellent results in weight loss.

[0103] Example 17 DIO mice were treated for 10 days with different combinations of therapeutic drugs as follows: (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. At the end of treatment, fasting blood glucose levels (mM) were measured for each therapeutic drug combination group and the results are shown in FIG.

[0104] Figure 17 shows a comparison of fasting blood glucose levels in DIO mice after administration of various therapeutic combinations. It can be seen that the expected therapeutic effect cannot be achieved when administered at normal doses. In contrast, the therapeutic combination of 30 mg / kg A + 5 mg / kg B in group (4) showed excellent results in reducing fasting blood glucose levels.

[0105] Example 18 Common wild-type C57BL / 6 mice with similar physical conditions in all respects were selected for the study. In the experimental group, each mouse was effectively administered with 30 mg / kg of therapeutic agent A and 5 mg / kg of therapeutic agent B per day. In the blank control group, each mouse was administered with only the same amount of PBS solvent per day. The experiment was carried out for a total of 13 days. The body weight of each mouse was recorded daily, and the body weight of each mouse was plotted over time, as shown in FIG. 18.

[0106] 18 shows the results of body weight change in normal wild-type C57BL / 6 mice induced by pharmaceutical therapeutics A+B in the experimental group treated with pharmaceutical compositions according to embodiments 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 normal wild-type C57BL / 6 mice by more than 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 therapeutics A and B provided by the present invention have a synergistic effect on the therapeutic effect of weight loss in normal wild-type C57BL / 6 mice when used in combination.

[0107] Example 19 Select the mice of the experimental group and the control group that have completed Example 18, and dissect them, and observe the fat accumulation in the mice of the experimental group and the control group.Then, compare them, and the images of fat deposition in various places after dissection are shown in FIG.

[0108] 19 shows the results of fat reduction in normal wild-type C57BL / 6 mice induced by pharmaceutical treatment A+B in the experimental group treated with a pharmaceutical composition according to an embodiment of the present invention, compared to the control group. It can be seen that the mice in the control group have a lot of obvious fat accumulation, such as visceral fat and gonadal fat, while the fat accumulation in the mice in the experimental group is significantly reduced. Therefore, it can be considered that the pharmaceutical composition provided by the present invention has the effect of significantly reducing body fat content.

[0109] Example 20 Twenty-four 8-week-old female Sprague-Dawley rats of similar physical condition were selected and divided into two groups. The number of rats in each group was 12. Each rat was fed a high-fat, high-sugar diet (HFHSD, D11092103; Research Diet Inc) daily and subcutaneously injected with 60 mg / kg dehydroepiandrosterone (DHEA; Sigma Aldrich) daily to mimic polycystic ovarian syndrome (PCOS) (Zhang et al.,Reproduction 2016). In the experimental group, each rat was effectively administered 30 mg / kg of treatment A and 5 mg / kg of treatment B per day. In the control group, each rat was effectively administered the same amount of PBS solvent daily as a blank control. The experiment was carried out for 3 weeks. The weight of each rat was recorded daily, and the weight of each rat was plotted over time, as shown in Figure 20.

[0110] Figure 20 shows the results of body weight change in rats with polycystic ovary syndrome induced by pharmaceutical therapeutics A+B in the experimental group treated with pharmaceutical compositions according to 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 prevent rats with polycystic ovary syndrome from gaining more than about 10% body weight within 3 weeks.Therefore, it can be considered that therapeutics A and B provided by the present invention have a synergistic effect on the prevention of obesity in patients with polycystic ovary syndrome when used in combination.

[0111] FIG. 21 shows a comparison of fasting blood glucose levels in each group of rats with polycystic ovary syndrome.

[0112] FIG. 22 shows a comparison of serum aspartate aminotransferase (AST) and total cholesterol, which are indices related to fatty liver, in each group of rats with polycystic ovary syndrome.

[0113] FIG. 23 shows a comparison of serum total protein, an index related to the nitrogen metabolic function of the liver, in each group of rats with polycystic ovary syndrome.

[0114] FIG. 24 shows a comparison of serum creatinine, an index related to renal function, in each group of rats with polycystic ovary syndrome.

[0115] FIG. 25 shows a comparison of serum lactate dehydrogenase (LDH), creatine kinase (CK), cardiac creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH), which are indices related to heart disease, in each group of rats with polycystic ovary syndrome.

[0116] Examples 21 to 25 Subsequent tests were conducted on each group of rats that had completed the treatment of Example 20. Example 21 detected fasting blood glucose, an index related to diabetes. Example 22 detected indexes related to fatty liver disease, serum aspartate aminotransferase (AST), and total cholesterol. Example 23 detected index serum total protein related to the nitrogen metabolism function of the liver. Example 24 detected serum creatinine (creatinine), an index related to renal function. Example 25 detected serum lactate dehydrogenase (LDH), creatine kinase (CK), cardiac creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH), which are indexes related to heart disease. Comparisons of the indexes of each group are shown in Figures 21 to 25, respectively.

[0117] 21-25 show the comparison of various indicators related to polycystic ovary syndrome, such as diabetes, obesity, hyperlipidemia, fatty liver disease, and heart disease, in each group of rats, respectively. It can be seen that, compared with the blank control group, all indicators in the rats of the experimental group showed a significant decrease after 3 weeks of administration of therapeutic drug A+B. It can be seen that the pharmaceutical composition provided by the present invention has obvious medical applications in the prevention or improvement of polycystic ovary syndrome and related diabetes, obesity, hyperlipidemia, fatty liver disease, and heart disease-related diseases.

[0118] Example 26 Similarly, insulin sensitivity tests were also performed on groups of rats that completed the treatment of Example 20. In the experimental and control groups, 0.75 U insulin / kg body weight of insulin (humulin) was administered intraperitoneally to rats that had been fasted for 5 hours using a 27G needle. Every 15-30 minutes thereafter, the rats' blood glucose levels (mM) were tested at each time point using a Lifescan One Touch blood glucose meter, and a line graph was plotted based on glucose content and time. The results are shown in Figure 26.

[0119] Figure 26 shows the results of insulin sensitivity in rats with polycystic ovary syndrome.It can be seen that compared with the control group, the glucose content of the rats in the experimental group is always kept at a lower level of glucose content, and changes relatively more significantly.Therefore, it can be seen that after 3 weeks of treatment with the pharmaceutical composition of the present invention, the rats show excellent results in terms of insulin sensitivity.

[0120] Example 27 Similarly, vaginal cytology analysis was also performed on each group of rats in Example 20. Evaluations were performed at all stages of the estrous cycle, including D estrus, P proestrus, E estrus, and M late estrus, for 11 consecutive days. Line graphs were generated according to estrous cycle stage and time, and the results are shown in Figure 27.

[0121] Figure 27 shows the results of estrous cycle in rats with polycystic ovary syndrome. It can be seen that compared with the normal control group (control), the estrous cycle of rats with polycystic ovary syndrome (DHEA+HFHSD) is abnormal, and 12 / 12 rats have non-cyclic problems. The estrous cycle of rats in the experimental group (DHEA+HFHSD+A+B) is relatively normal, and the estrous cycle of 4 / 12 rats has completely returned to normal. Therefore, it can be seen that after being treated with the pharmaceutical composition of the present invention for 3 weeks, some rats with polycystic ovary syndrome can completely recover normal estrous cycle.

[0122] Example 28 Similarly, ELISA analysis of serum hormones including androgen (T), estrogen (E2), and follicle stimulating hormone (FSH) was also performed on each group of rats in Example 20. The results are shown in FIG.

[0123] Figure 28 shows the results of serum hormone ELISA in rats with polycystic ovary syndrome. It can be seen that compared with the control group (control) of rats with polycystic ovary syndrome, the experimental group (A+B) had no significant changes in androgen (T) and estrogen (E2), and follicle-stimulating hormone (FSH) was significantly increased in 4 / 12 rats. Therefore, it can be seen that after 3 weeks of treatment with the pharmaceutical composition of the present invention, some rats with polycystic ovary syndrome can restore normal estrous cycle. This is due to the increase in follicle-stimulating hormone (FSH) rather than the regulation of androgen (T) and estrogen (E2).

[0124] Example 29 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 for each group of rats in Example 20. The results are shown in FIG.

[0125] Figure 29 shows the results of Western blot analysis of skeletal muscle proteins in rats with polycystic ovary syndrome. It can be seen that phospho-Akt (S473), Akt, myosin heavy chain (MHC) are significantly increased, while phospho-p38 is significantly decreased, in rats with polycystic ovary syndrome compared with the control group (PCOS). 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 and metabolism, and alleviate muscle inflammation, insulin resistance, muscle loss syndrome, and metabolic syndrome.

[0126] Example 30 Similarly, cardiac section analysis including Masson's Trichrome staining for ventricular wall thickness and fibrosis was also performed for each group of rats in Example 20. The results are shown in Figures 30 and 31.

[0127] Figure 30 shows the analysis result of left ventricular wall thickness in rats with polycystic ovary syndrome. It can be seen that compared with the control group (control) of rats with polycystic ovary syndrome, the left ventricular wall thickness of experimental group (A+B) is significantly reduced (P<0.001). Therefore, it can be seen that rats with polycystic ovary syndrome treated with the pharmaceutical composition of the present invention for 3 weeks can significantly improve cardiovascular diseases such as ventricular hypertrophy and heart failure.

[0128] Figure 31 shows the results of myocardial fibrosis analysis in rats with polycystic ovary syndrome. It can be seen that compared with the control group (control) of rats with polycystic ovary syndrome, the myocardial fibrosis area of ​​rats in experimental group (A+B) is significantly reduced (P<0.05). Therefore, it can be seen that rats with polycystic ovary syndrome treated with the pharmaceutical composition of the present invention for 3 weeks can significantly improve cardiovascular diseases such as myocardial fibrosis, ventricular hypertrophy, and heart failure caused by myocardial infarction.

[0129] Example 32 Similarly, routine blood analyses were also performed on each group of rats in Example 20, and the results are shown in FIG.

[0130] Figure 32 shows the results of routine blood analysis of rats with polycystic ovary syndrome. It can be seen that compared with the control group (control) of rats with polycystic ovary syndrome, the blood routine indexes of rats in experimental group (A+B) are not significantly changed (P>0.05). Therefore, it can be seen that after being treated with the pharmaceutical composition of the present invention for 3 weeks, rats with polycystic ovary syndrome do not show significant toxic reaction.

[0131] Example 33 Twenty-six 6-week-old ob / ob obese (Jackson Lab B6.Cg-Lepob / J, Stock No: 000632) male mice of similar physical condition were selected from Jackson Laboratory. Each mouse was fed a high-fat and high-sugar diet (HFSD, D11092103; Research Diet Inc) daily for 45 days to mimic fatty liver disease (NASH) (Kristiansen et al., 2016; doi:10.4254 / wjh.v8.i16.673). After 45 days, the mice continued to feed on the high-fat and high-sugar diet and were divided into two groups. The number of mice in each group was 13. For a total of 37 days, in the experimental group, each mouse was effectively administered 30 mg / kg of treatment A and 5 mg / kg of treatment B per day, and in the control group, each mouse was administered only the same amount of PBS solvent daily as a blank control. The body weight of each mouse was recorded daily and plotted over time for each mouse, as shown in FIG.

[0132] Figure 33 shows the results of pharmaceutical treatment A+B on body weight change in NASH mice. 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% in 37 days (P=3.8e-10), while 1 to 3 had almost no significant effect on the body weight of the mice. Therefore, it can be considered that the pharmaceutical treatment A+B provided by the present invention can effectively suppress weight gain.

[0133] Example 34 Similarly, a group of mice completing the treatment of Example 33 was also tested for food intake over a 3 day period, and the results are shown in FIG.

[0134] Figure 34 shows the results of food intake test in NASH mice. It can be seen that compared with the NASH control group, the food intake of the mice in the experimental group not only does not decrease, but also increases slightly (P=0.109). Therefore, it can be seen that after being treated with the pharmaceutical composition of the present invention for 40 days, the appetite of NASH mice does not change significantly.

[0135] Example 35 Similarly, each group of mice that completed the treatment of Example 33 were also sacrificed and their livers were weighed, and the results are shown in FIG.

[0136] Figure 35 shows the results of liver weight test in NASH mice.It can be seen that compared with the NASH control group, the liver weight of the mice in the experimental group is significantly reduced (P<0.001), and the liver volume is also significantly reduced.Therefore, it can be seen that NASH mice can reverse hepatomegaly after being treated with the pharmaceutical composition of the present invention for 40 days.

[0137] Example 36 Similarly, groups of mice completing the treatment of Example 33 were also dissected, histologically fixed sections, stained with hematoxylin-eosin and picrosirius red (Sigma-Aldrich), microscopically examined, and expertly scored. The results of liver analysis slices are shown in Figure 36. The results of heart analysis slices are shown in Figure 37.

[0138] Figure 36 shows the results of liver slice analysis in NASH mice. It can be seen that the fatty liver grade and fatty liver activity score of the mice in the experimental group were significantly reduced compared with the NASH control group (P<0.01). That is, the therapeutic drug A+B has a therapeutic effect of inducing fat reduction and inflammation reduction in fatty liver. At the same time, it can be seen that the liver fibrosis area (%) and fibrosis stage of the mice in the experimental group were also significantly reduced compared with the NASH control group (P<0.01). That is, the therapeutic drug A+B has a therapeutic effect of inducing fibrosis reduction in fatty liver. After 40 days of treatment with the pharmaceutical composition of the present invention, the mice can reverse NASH fatty liver and liver fibrosis even if they continue to consume a large amount of high-fat and high-sugar diet.

[0139] Figure 37 shows the results of cardiac slice analysis in NASH mice. It can be seen that compared with the NASH control group, the cardiac fibrotic tissue area of ​​the mice in the experimental group was significantly reduced (P<0.01). In other words, therapeutic drug A+B has a therapeutic effect of inducing the alleviation of cardiac fibrosis. Even if the mice continue to consume a large amount of high-fat and high-sugar diet, cardiovascular diseases such as myocardial fibrosis and heart failure can be reversed in mice treated with the pharmaceutical composition of the present invention for 40 days.

[0140] Example 38 Similarly, blood biochemistry tests were performed on each group of mice that had completed the treatment of Example 33. Example 38 detected indicators related to liver damage: alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Example 39 examined indicators related to hyperlipidemia: triglycerides (TG), total cholesterol (CHOL), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C). Example 40 detected indicators related to various organ damage: albumin / globulin (albumin / globulin) ratio, urea (urea), creatinine (creatinine), lactate dehydrogenase (LDH), creatine kinase (CK), cardiac creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH). Comparisons of each indicator in each group are shown in Figures 38 to 40, respectively.

[0141] Figure 38 shows the comparison of two indicators related to liver damage in each group of mice.Compared with NASH control group, alanine aminotransferase (ALT, P<0.001) and aspartate aminotransferase (AST, P=0.02) of mice in experimental group are significantly decreased.After 40 days of treatment with pharmaceutical composition of the present invention, liver damage can be reversed even if mice continue to consume large amounts of high-fat and high-sugar diet.

[0142] Figure 39 shows the comparison of hyperlipidemia-related indexes in each group of mice.Compared with NASH control group, experimental group mice have lowered triglyceride (TG), total cholesterol (CHOL, P<0.01), HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C, P=0.016).After 40 days of treatment with the pharmaceutical composition of the present invention, mice can reverse hyperlipidemia even if they continue to consume a large amount of high-fat and high-sugar diet.

[0143] Figure 40 shows the comparison of the indexes related to organ damage in each group of mice.Compared with the NASH control group, there was no significant change in the ratio of albumin / globulin, urea, creatinine, lactate dehydrogenase (LDH), creatine kinase (CK), urea (urea), lactate dehydrogenase (LDH), creatine kinase (CK), cardiac creatine kinase isoenzyme MB (CKMB), and α-hydroxybutyrate dehydrogenase (HBDH) (P>0.05).After being treated with the pharmaceutical composition of the present invention for 40 days, various organs of mice did not show any toxic reaction.

[0144] Example 41 Similarly, each group of mice from Example 33 was tested for fasting blood glucose, with the glucose content (mM) of mice fasted overnight being tested every 3-7 days using a Lifescan One Touch blood glucose meter. A line graph was plotted based on glucose content and time, and the results are shown in FIG.

[0145] Figure 41 shows the results of fasting blood glucose in each group of mice. It can be seen that compared with the NASH control group, the glucose content of the mice in the A+B experimental group not only did not increase significantly, but actually decreased significantly to a lower glucose content level within 14 days (P<0.001). Therefore, it can be seen that the mice treated with the pharmaceutical composition of the present invention for 40 days show a significant improvement in glycemic control and can prevent the occurrence of type 2 diabetes.

[0146] Example 42 Similarly, each group of mice from Example 33 was also tested for glucose tolerance. In the experimental and control groups, mice were fasted overnight and intraperitoneally injected with the same amount of glucose (2 mg / g body weight). The glucose content (mM) of the mice was tested at each time point every 15-30 minutes with a Lifescan One Touch blood glucose meter, and a line graph was created according to glucose content and time. The results are shown in Figure 42.

[0147] Figure 42 shows the results of glucose tolerance in NASH mice.It can be seen that compared with the NASH control group, the glucose content of the mice in A+B experimental group increases to a not significantly high glucose content level, and then rapidly decreases to a lower glucose content level (P<0.001).Therefore, it can be seen that the NASH mice after being treated with the pharmaceutical composition of the present invention for 40 days have returned to normal in terms of glucose tolerance.

[0148] Example 43 Similarly, each group of mice from Example 33 was tested for insulin sensitivity. In the experimental and control groups, rats that had been fasted for 6 hours were given the same amount of insulin (humulin, 0.75 U / kg body weight) intraperitoneally using a 27G needle. The glucose content (mM) of the mice was tested at each time point every 15-30 minutes with a Lifescan One Touch blood glucose meter, and a line graph was created according to the glucose content and time. The results are shown in Figure 43.

[0149] Figure 43 shows the results of insulin sensitivity in NASH mice. It can be seen that compared with the control group, the glucose content of the mice in the experimental group is always kept at a relatively low level, and changes significantly after 60 minutes (P<0.01). Therefore, it can be seen that after 40 days of treatment with the pharmaceutical composition of the present invention, NASH mice return to normal in terms of insulin sensitivity, and insulin resistance is reversed.

[0150] Example 44 Similarly, the mice in each group of Example 33 were fasted for 6 hours and injected with insulin (humulin, 0.75U / kg body weight). After 15 minutes, skeletal muscle and liver samples were collected and protein Western blot analysis was performed to detect phospho-Irs1 (S307), phospho-Akt (S473), Akt, phospho-p38, and GAPDH. The results are shown in Figure 44.

[0151] Figure 44 shows the results of Western blot analysis of skeletal muscle proteins in NASH mice. It can be seen that compared with the NASH mouse control group (control), the skeletal muscle phospho-Irs1 (S307), phospho-Akt (S473), and Akt of the mice in the experimental group (A+B) are significantly increased, while p38 is significantly decreased. It can also be seen that compared with the NASH mouse control group (control), the liver phospho-Akt (S473) and phospho-Irs1 (S307) of the mice in the experimental group (A+B) are significantly increased. Therefore, it can be seen that NASH mice treated with the pharmaceutical composition of the present invention for 40 days can restore normal skeletal muscle and liver insulin sensitivity, and reduce muscle inflammation, whole-body insulin resistance, and metabolic syndrome.

[0152] Example 45 Six 6-week-old ob / ob obese (Jackson Lab B6.Cg / J, Stock No: 000632) male mice with similar physical condition were selected from Jackson Laboratory. Each mouse was fed a high-fat, high-sugar diet (HFSD, D11092103; Research Diet Inc) daily for 45 days to mimic fatty liver disease (NASH) (Kristiansen et al., 2016; doi:10.4254 / wjh.v8.i16.673). After 45 days, the mice continued to feed on the high-fat, high-sugar diet and were divided into two groups. The number of mice in each group was three. In the experimental group, each mouse was effectively administered with 30 mg / kg of treatment A and 5 mg / kg of treatment B per day. In the control group, each mouse was administered only the same amount of PBS solvent daily as a blank control. The experiment was performed for a total of 7 days. After the experiment, skeletal muscle and liver samples were collected and subjected to RNAseq transcriptome sequencing analysis and GSEA (Gene Set Enrichment Analysis) analysis to extract the most significant gene tags and markers, as shown in FIG. 45.

[0153] Figure 45 shows the results of transcriptome sequencing analysis in NASH mice. Compared with the NASH control group (C), it can be seen that the pharmaceutical composition of the present invention (F) can effectively upregulate the VEGF signaling pathway in skeletal muscle and liver (P<0.001, FDR<0.001), bone muscle axon guidance (neural guidance, P<0.001, FDR<0.001), ephrin signaling pathway (P<0.001, FDR=0.0068), and hepatic IGF (insulin-like) signaling pathway (P=0.0039, FDR=0.07) in NASH mice within 7 days. Therefore, it can be considered that the pharmaceutical therapeutic agent A+B provided by the present invention can effectively promote angiogenesis, neuromuscular tissue, and insulin sensitivity in the same way as 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).

[0154] Example 46 The study used in vitro human primary skeletal muscle cells, and the cells were divided into four groups, with six time points for each group. In the experimental group, 100 mg / L of therapeutic agent A and 2 mg / L of therapeutic agent B were administered. In control group 1, 100 mg / L of therapeutic agent A was administered. In control group 2, 2 mg / L of therapeutic agent B was administered. In control group 3, the same amount of DMSO solvent was used as a blank control. The experiment was performed for a total of 24 hours. Then, samples from each time point were subjected to protein Western blot analysis, as shown in Figure 46.

[0155] FIG. 46 shows the results of protein Western blot analysis of primary human skeletal muscle cells in vitro.

[0156] FIG. 46A shows that in the experimental group using the pharmaceutical composition according to some embodiments of the present invention, compared with the control groups 1 to 3, after short-term administration (within 60 minutes), the pharmaceutical therapeutics A+B can rapidly and significantly upregulate phospho-p38, phospho-AMPK, phospho-ACC, and PGC1a. Compared with the control group 3, the control groups 1 and 2 can also upregulate phospho-p38, phospho-AMPK, phospho-ACC, and PGC1a with only treatment A or treatment B, but their effects were weak or slow and not uniform in time. It can be seen that after short-term administration, the pharmaceutical therapeutics A+B provided by the present invention can more effectively promote the p38 and AMPK signaling pathways and their downstream targets than the single use of therapeutics A or the single use of therapeutics B, thereby promoting catabolic actions such as fat hydrolysis and fatty acid oxidation. After long-term administration (3-24 hours), pharmaceutical therapeutics A+B can significantly down-regulate phospho-p38, phospho-AMPK, phospho-ACC, and PGC1a in the experimental group using the pharmaceutical composition according to some embodiments of the present invention, compared with control groups 1-3. Compared with control group 3, control groups 1 and 2 could also down-regulate phospho-p38, phospho-ACC, and PGC1a within 24 hours, either with treatment A or treatment B alone, but the effect was weaker or slower, and the time was not uniform. It can be seen that after long-term administration, the pharmaceutical therapeutics A+B provided by the present invention can more effectively inhibit the p38 and AMPK signaling pathways and their downstream targets than only treatment A or treatment B, thereby promoting anabolism.

[0157] Figure 46B shows that after short-term administration (within 60 minutes), pharmaceutical therapeutic A+B provided by the present invention can regulate the mechanism model of p38 and AMPK signaling pathways together through fatty acid oxidation (FAO), fatty acid metabolites (acyl metabolites) and adenosine triphosphate (ATP).It can be seen that pharmaceutical therapeutic A+B can simultaneously enhance the signaling pathways of p38 and AMPK after short-term administration, thereby promoting catabolic effects such as fat hydrolysis and fatty acid oxidation.

[0158] Figure 46C shows that after long-term administration (3-24 hours), the pharmaceutical therapeutics A+B provided by the present invention can regulate the mechanistic model of p38 and AMPK signaling pathways together through inflammatory cytokines (Inf cytokines), fatty acid oxidation (FAO), mitochondrial reactive oxygen species (mtROS), and glycolysis. Thus, the pharmaceutical therapeutics A+B can simultaneously sharply reduce the signaling pathways of p38 and AMPK after long-term administration, thereby promoting anabolism and muscle repair.

[0159] Thus, daily administration of pharmaceutical treatments A+B can specifically recapitulate technetium p38 and AMPK signaling pathways and associated metabolic changes, creating a cycle of excitatory effects in a manner similar to mimicking exercise.

[0160] Example 47 The experiment used in vitro human primary skeletal muscle cells, and the cells were divided into six groups. In control group 1, the same amount of DMSO solvent was administered for 7 days as a blank control. In control group 2, the same amount of bovine serum albumin (BSA) was administered for 7 days as a fat solvent control. In control group 3, palmitic acid (Pal) and TNFα were administered for 7 days as a high fat and inflammation-induced insulin resistance control. In control group 4, palmitic acid (Pal) and TNFα were administered for 7 days, and 100 mg / L of treatment A was administered on the 4th day. In control group 5, palmitic acid (Pal) and TNFα were administered for 7 days, and 2 mg / L of treatment B was administered on the 4th day. In the experimental group, palmitic acid (Pal) and TNFα were administered for 7 days, and 100 mg / L of treatment A and 2 mg / L of treatment B were administered on the 4th day. Each group of samples was then subjected to protein Western blot analysis, as shown in FIG.

[0161] FIG. 47 shows the results of protein Western blot analysis of primary human skeletal muscle cells of induced insulin resistance in vitro after 7 days of administration. After 7 days of testing, in the experimental group treated with pharmaceutical compositions according to some embodiments of the present invention, pharmaceutical therapeutics A+B can more significantly upregulate insulin sensitivity indicators phospho-Akt, phospho-S6, and myosin heavy chain (MHC), and downregulate cellular senescence indicator H3K9me3, restoring them to the levels of control groups 1 and 2. Compared with control group 3, control groups 4 and 5 can also upregulate insulin sensitivity indicators phospho-Akt, phospho-S6, and myosin heavy chain by treating with therapeutics A or therapeutics B alone, but the effect is relatively weak. Compared with control group 3, only therapeutics A in control group 4 can downregulate cellular senescence indicator H3K9me3 and restore it to the level of control group 1, but only therapeutics B in control group 5 cannot achieve the same effect. It can be seen that pharmaceutical treatments A+B provided by the present invention can promote insulin sensitivity and reverse cellular senescence more effectively under conditions of insulin resistance than treatments A or B alone.

[0162] Example 48 Similarly, skeletal muscle RNAseq transcriptome sequencing analysis and GSEA (gene set enrichment analysis) analysis were also performed on each group of rats in Example 20 to extract the most significant gene tags and markers, as shown in FIG. 48.

[0163] Figure 48 shows the results of skeletal muscle transcriptome sequencing analysis induced by high-fat and high-sugar diet in rats with PCOS polycystic ovary syndrome.Compared with the PCOS rat control group (M2), it can be seen that 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 mitochondrial genes in rats of the A+B experimental group (M1) were significantly increased. It can be seen that compared with the control group (M2) of rats with polycystic ovary syndrome, the inflammation-related interferon targets (P<0.0001, FDR<0.0001), fibrosis-related collagen pathway targets (P<0.0001, FDR=3.5e-5), and mesenchymal cell division targets (P<0.0001, FDR<0.0001) in rats of the A+B experimental group (M1) were significantly decreased. Therefore, it can be seen that rats with polycystic ovary syndrome treated with the pharmaceutical composition of the present invention can restore normal insulin (insulin / IGF-IRS-PI3K-mTOR) sensitivity and mitochondrial metabolism, reduce inflammation and fibrosis, and thereby reverse insulin resistance, sarcopenic syndrome, and metabolic syndrome.

[0164] Example 49 Similarly, fasting insulin enzyme-linked immunosorbent assay (ELISA, Abcam) was performed on serum samples from mice in each group of Example 33, and the insulin resistance index HOMA-IR was calculated, as shown in FIG.

[0165] Figure 49 shows the results of insulin ELISA and insulin resistance index HOMA-IR of serum samples in NASH mice. It can be seen that, compared with the NASH control group, the pharmaceutical composition A+B of the present invention can effectively reduce serum fasting insulin (P<0.05) and insulin resistance index (P<0.05). Therefore, it can be considered that the pharmaceutical treatment drug A+B provided by the present invention can effectively promote insulin sensitivity and reduce insulin resistance and hyperinsulinemia in NASH patients, just like mimicking exercise (van der Windt et al. 2018, doi:10.3727 / 105221617X15124844266408).

[0166] Example 50 Similarly, as shown in FIG. 50, adiponectin enzyme-linked immunosorbent assay (ELISA, Abcam) was performed on serum samples from mice in each group of Example 33.

[0167] Figure 50 shows the results of adiponectin ELISA of serum samples in NASH mice. It can be seen that, compared with the NASH control group, the pharmaceutical composition A+B of the present invention can effectively upregulate serum adiponectin (P<0.001). Therefore, it can be considered that the pharmaceutical treatment A+B provided by the present invention can effectively promote glucose and lipid metabolism and inhibit inflammation through adiponectin, in the same way as mimicking exercise (Simpson & Singh 2008,doi:10.1038 / oby.2007.53).

[0168] Example 51 Mice were grouped and administered in the same manner as in Example 8, except that serum samples from each group of mice were subjected to liquid phase mass spectrometry (LC-MS, Waters XBridge C18 column and Xevo G2-XS) analysis after 1 hour to extract the most prominent small molecule markers, as shown in Figure 51.

[0169] Figure 51 shows the comparative results of liquid mass spectrometry of serum samples from mice in control group 1 and control group 3. Compared with control group 3 (blank control), it can be seen that drug A (control group 1) can effectively form various salicylic acid derivatives in serum, most of which have retention times of 5 to 5.5 minutes, similar to acetylsalicylic acid (https: / / mona.fiehnlab.ucdavis.edu / spectra / display / EQ357853). Therefore, it can be considered that any salicylic acid derivative can mimic the effectiveness of drug A in obese bodies.

[0170] In light of the above examples, it is easy to see that the pharmaceutical composition according to the present invention shows a very good synergistic effect in glycemic control, especially in terms of lowering fasting plasma glucose and postprandial plasma glucose, compared with the use of only individual components in the pharmaceutical composition of the present invention.At the same time, the present invention can effectively treat or prevent obesity, fatty liver, type 2 diabetes, and metabolic syndrome diseases caused by insulin resistance.

[0171] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details mentioned in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention. These simple modifications belong to the protection scope of the present invention.

[0172] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner under circumstances that are not inconsistent. In order to avoid unnecessary repetition, the present invention does not further describe various possible combinations.

[0173] In addition, the various embodiments of the present invention can also be arbitrarily combined unless such combination goes against the spirit of the present invention, and this should also be considered as the disclosure of the present invention. In certain embodiments, for example, the following items are provided: (Item 1) 1. A pharmaceutical composition comprising: a therapeutic agent A or a pharma- ceutical acceptable salt thereof, a therapeutic agent B or a pharma- ceutical acceptable salt thereof, and at least one pharma- ceutical acceptable excipient, wherein said therapeutic agent A is a nonsteroidal anti-inflammatory drug and said therapeutic agent B is a fatty acid oxidation inhibitor. (Item 2) 2. The pharmaceutical composition according to item 1, wherein the therapeutic agent A and the therapeutic agent B are contained in a single dosage form. (Item 3) 2. The pharmaceutical composition of claim 1, wherein the therapeutic agent A and the therapeutic agent B are present in separate dosage forms. (Item 4) The pharmaceutical composition according to any one of items 1 to 3, wherein the therapeutic agent A is at least one selected from salicylates, ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, and tolmetin. (Item 5) 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the therapeutic agent B is selected from trimetazidine, etomoxir, aminocarnitine, or a phosphonooxy derivative of carnitine. (Item 6) 6. The pharmaceutical composition according to any one of items 1 to 5, wherein said therapeutic agent A is selected as a salicylate and said therapeutic agent B is selected as trimetazidine. (Item 7) 7. The pharmaceutical composition according to any one of items 1 to 6, wherein said therapeutic agent A is selected as aspirin (acetylsalicylic acid). (Item 8) 8. The pharmaceutical composition according to any one of items 1 to 7, wherein the weight ratio of said therapeutic agent A to said therapeutic agent B is 1:1 to 10:1, preferably 6:1. (Item 9) or for the treatment of type 2 diabetes mellitus, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, postprandial hyperglycemia, type 1 diabetes mellitus, overweight, obesity, nonalcoholic fatty liver, nonalcoholic steatohepatitis, and metabolic syndrome, or for improving glycemic control and / or reducing fasting plasma glucose, postprandial plasma glucose, and / or glycosylated hemoglobin HbA1c, or for slowing, delaying, or reversing the progression of impaired glucose tolerance, impaired fasting glucose, insulin resistance, and / or metabolic syndrome to type 2 diabetes, or for the treatment of diabetic complications such as cataracts, and micro- and macrovascular diseases such as nephropathy, retinopathy, neuropathy, tissue ischemia, arteriosclerosis, myocardial infarction, stroke, and peripheral arterial occlusive disease, or for reducing or promoting weight loss, or for preventing pancreatic beta cell degeneration and / or pancreatic beta cell 20. The use (or method) of Therapeutic Agents A and B in the preparation of a medicament for treating impaired pancreatic beta cell function and / or restoring pancreatic beta cell function and / or restoring pancreatic insulin secretion, or treating a disease or condition causing 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 complications of atherosclerosis, or treating glaucoma and complications of glaucoma, or treating dyslipidemia / hyperlipidemia and complications of dyslipidemia / hyperlipidemia, or treating a reproductive-related metabolic disease in a subject in need thereof, wherein Therapeutic Agent A is a nonsteroidal anti-inflammatory drug and Therapeutic Agent B is a fatty acid oxidation inhibitor. (Item 10) 10. The use (or method) according to item 9, wherein the subject is an individual diagnosed with one or more conditions selected from the group consisting of overweight, obesity, visceral obesity, and abdominal obesity. (Item 11) The subject has the following condition: (a) a fasting blood glucose or serum glucose concentration greater than 110 mg / dL, especially greater than 125 mg / dL; (b) a postprandial plasma glucose concentration of 140 mg / dL or greater, and (c) The use (or method) according to item 9 or 10, wherein the individual is diagnosed with one or more of the following HbA1c values: HbA1c value of 6.5% or more, in particular 8.0% or more. (Item 12) The subject has the following condition: (a) obesity, visceral obesity, and / or abdominal obesity, (b) blood triglyceride concentration of 150 mg / dL or more; (c) a blood HDL cholesterol concentration of less than 40 mg / dL in a female subject, or less than 50 mg / dL in a male subject; (d) systolic blood pressure of 130 mmHg or more, diastolic blood pressure of 85 mmHg or more, (e) fasting blood glucose level of 110 mg / dL or more, and (f) The use (or method) according to item 6, wherein the individual has one or more of the following blood LDL cholesterol concentrations of 130 mg / dL or more. (Item 13) 12. The use (or method) according to any one of items 9 to 11, wherein the subject is an individual for whom metformin monotherapy is contraindicated and / or who is intolerant to therapeutic doses of metformin. (Item 14) The object is (a) biguanides, (b) sulfonylureas, (c) meglitinides, (d) thiazolidinediones, (e) α-glucosidase inhibitors, (f) insulin and insulin analogues; (g) dipeptidyl peptidase IV inhibitors, (h) SGLT2 inhibitors, (i) a PPARα modulator, (j) glucose-dependent insulinotropic polypeptide agonists, (k) beta-3 agonist, (l) GLP1 and GLP1 analogues, (m) PPARγ modulators, and (n) The use (or method) according to any one of items 9 to 12, wherein the individual has inadequate glycemic control after treatment with one or more antidiabetic drugs selected from the group consisting of HMG-CoA reductase inhibitors. (Item 15) 14. The use (or method) according to any one of items 9 to 13, wherein said therapeutic agent A is selected as a salicylate and said therapeutic agent B is selected as trimetazidine. (Item 16) The use (or method) according to any one of items 9 to 14, wherein said therapeutic agent A is selected as aspirin. (Item 17) 16. The use (or method) according to any one of items 9 to 15, wherein the weight ratio of said therapeutic agent A to said therapeutic agent B is from 1:1 to 10:1, preferably 6:1. (Item 18) 17. The use (or method) according to any one of items 9 to 16, wherein said therapeutic agent A and said therapeutic agent B are administered simultaneously. (Item 19) 18. The use (or method) of item 17, wherein said therapeutic agent A and said therapeutic agent B are contained in a single dosage form. (Item 20) 17. The use (or method) according to any one of items 9 to 16, wherein said therapeutic agent A and said therapeutic agent B are administered separately. (Item 21) 21. The use (or method) of item 20, wherein said therapeutic agent A is administered before said therapeutic agent B. (Item 22) 21. The use (or method) of item 20, wherein said therapeutic agent A is administered after said therapeutic agent B. (Item 23) 23. The use (or method) according to any one of items 9 to 22, wherein said therapeutic agent A and said therapeutic agent B are administered orally or by injection.

Claims

1. 1. A pharmaceutical composition comprising Therapeutic Agent A or Therapeutic Agent B for the treatment of one or more of overweight, obesity, visceral obesity, abdominal obesity, nonalcoholic steatohepatitis (NASH), and polycystic ovary syndrome (PCOS) in a subject, wherein Therapeutic Agent A and Therapeutic Agent B are for administration one or more times per day, wherein Therapeutic Agent A is a salicylate and Therapeutic Agent B is trimetazidine or a pharma- ceutical acceptable salt thereof, and wherein the pharmaceutical composition is used in combination therapy with Therapeutic Agent A and Therapeutic Agent B.

2. The pharmaceutical composition of claim 1 , wherein the subject has obesity, visceral obesity, and / or abdominal obesity.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the subject has a fasting blood glucose or serum glucose concentration greater than 110 mg / dL.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the subject has a postprandial plasma glucose concentration of 140 mg / dL or greater.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject has an HbA1c value of 6.5% or more, optionally 8.0% or more.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the subject has a blood triglyceride concentration of 150 mg / dL or greater.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the subject has a blood HDL cholesterol concentration of less than 40 mg / dL in a female subject, or less than 50 mg / dL in a male subject.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the subject has a systolic blood pressure of 130 mmHg or greater and a diastolic blood pressure of 85 mmHg or greater.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the subject has a blood LDL cholesterol concentration of 130 mg / dL or greater.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject has contraindications to metformin monotherapy and / or is intolerant to therapeutic doses of metformin.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the subject is an individual with inadequate glycemic control after treatment with one or more antidiabetic drugs.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the subject is a human.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the therapeutic agent A is aspirin or a pharma- ceutically acceptable salt thereof.

14. i) the weight ratio of said therapeutic agent A to said therapeutic agent B is from 1:1 to 10:1 per day; and / or ii) said therapeutic agent A is at a daily dosage of 0.3 mg / kg to 120 mg / kg of body weight and said therapeutic agent B is at a daily dosage of 0.05 mg / kg to 500 mg / kg of body weight; A pharmaceutical composition according to any one of claims 1 to 13.

15. 15. The pharmaceutical composition of claim 14, wherein the weight ratio of therapeutic agent A to therapeutic agent B is 6:1 per day.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein said therapeutic agent A and said therapeutic agent B are for simultaneous administration.

17. 17. The pharmaceutical composition of claim 16, wherein said therapeutic agent A and said therapeutic agent B are contained in a single dosage form.

18. The pharmaceutical composition of any one of claims 1 to 15, wherein said therapeutic agent A and said therapeutic agent B are for separate administration.

19. 20. The pharmaceutical composition of claim 18, wherein said therapeutic agent A is administered before said therapeutic agent B.

20. The pharmaceutical composition of claim 18, wherein said therapeutic agent A is administered after said therapeutic agent B.

21. The pharmaceutical composition according to any one of claims 1 to 20, which is for oral or injectable administration.

22. The pharmaceutical composition according to any one of claims 1 to 21, for use in the treatment of obesity.

23. A pharmaceutical composition described in any one of claims 1 to 22, for use in the treatment of PCOS.

24. A pharmaceutical composition described in any one of claims 1 to 23 for the treatment of NASH.

25. The pharmaceutical composition described in any one of claims 1 to 24, wherein the therapeutic agent A and the therapeutic agent B are for administration once a day.

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