Tripeptides and treatments for metabolic, cardiovascular, and inflammatory conditions.

Abstract

This disclosure provides a method for treating NAFLD, NASH, and atherosclerosis, the method comprising administering to a subject a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Serial No. 62 / 717,546, filed August 10, 2018. The entire contents of the aforementioned application are incorporated herein by reference. Background Technology

[0003] Nonalcoholic fatty liver disease (NAFLD) is a general term for a range of liver disorders that affect people who drink little or no alcohol. As the name suggests, the main characteristic of NAFLD is the storage of too much fat in liver cells. It is normal for the liver to contain some fat. However, if more than 5%-10% of the liver's weight is fat, then this condition is called fatty liver (steatohepatitis).

[0004] A more serious form of NAFLD is called nonalcoholic steatohepatitis (NASH). NASH causes the liver to swell and become damaged. NASH tends to develop in people who are overweight or obese, or who have diabetes, high cholesterol or high triglycerides, or inflammatory disorders. NASH is a potentially serious form of the disease, marked by hepatocellular ballooning degeneration and hepatitis, which can progress to scarring and irreversible damage. Summary of the Invention

[0005] A first aspect of this disclosure provides a method for treating inflammatory diseases, metabolic diseases, and / or cardiovascular diseases, the method comprising administering a therapeutically effective amount of glycine or a glycine-containing tripeptide molecule to a subject suffering from one or more of the inflammatory diseases, metabolic diseases, and / or cardiovascular diseases. In various embodiments, the glycine-containing tripeptide molecule may include one or more of DT-109 (Gly-Gly-Leu) and DT-110 (Gly-Gly-dLeu). In various embodiments, metabolic diseases refer to a defined group of conditions in which metabolic errors, metabolic imbalances, or metabolic deficiencies occur. Metabolic diseases as described herein also include diseases that can be treated by regulating metabolism, although the disease itself may or may not be caused by a specific metabolic defect. Such metabolic diseases may involve, for example, glucose and fatty acid oxidation pathways. As used herein, “metabolic disorder” or “metabolic disease” refers to a condition characterized by alterations or disturbances in metabolic function. “Metabolic” and “metabolism” are well-known terms in the art and generally encompass a whole range of biochemical processes occurring within a living organism. Metabolic and cardiovascular diseases include, but are not limited to, obesity, diabetes, atherosclerosis, metabolic syndrome, dyslipidemia, coronary heart disease, coronary artery disease, arteriosclerosis, atherosclerotic thrombotic stroke, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hyperfatty acidemia, or metabolic syndrome, or a combination thereof. Dyslipidemia may be hyperlipidemia. Hyperlipidemia may be hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemia and hypertriglyceridemia. NAFLD may be hepatic steatosis or steatohepatitis. Diabetes may be type 2 diabetes or type 2 diabetes with dyslipidemia. In various implementations, the methods presented herein are applicable to metabolic diseases associated with abnormal glucose regulation and / or lipid accumulation in the body, circulation, or various organs (e.g., the liver), and the pathological sequelae arising therefrom, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome, and diabetic dyslipidemia.

[0006] Metabolic diseases, conditions, or disorders can be characterized by a variety of physical symptoms. The glycine tripeptide molecules and methods described herein can be used to prevent, treat, improve, or otherwise modulate any symptoms known to those skilled in the art as being related to metabolic diseases, conditions, or disorders. In some embodiments, symptoms may be, but are not limited to, any of the following: excessive urine production (polyuria), excessive thirst and increased fluid intake (hypersomnia), blurred vision, unexplained weight loss, and lethargy.

[0007] In some implementations, inflammatory diseases, conditions, or disorders include, but are not limited to, aortic stenosis, coronary artery disease (CAD), Alzheimer's disease, and thromboembolic diseases, conditions, or disorders. Certain thromboembolic diseases, conditions, or disorders include, but are not limited to, stroke, thrombosis, myocardial infarction, and peripheral vascular disease.

[0008] In some embodiments, glycine or glycine tripeptide molecules described herein are used to modulate physiological markers or phenotypes of inflammatory diseases, conditions, or disorders. For example, administration of the compound to an animal reduces the levels of inflammatory cytokines or other inflammatory markers in the animal compared to an untreated animal. In some embodiments, modulation of physiological markers or phenotypes may be associated with inhibition of hepatic DAG, reduction of glucose, and reduction of plasma LDL levels by the compound.

[0009] In some embodiments, physiological markers of inflammatory diseases, conditions, or disorders may be quantifiable. For example, cytokine levels can be measured and quantified using standard tests known in the art. For such markers, in some embodiments, the marker may be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or any two of these values.

[0010] Additionally, this document provides methods for preventing, treating, or improving symptoms associated with inflammatory diseases, conditions, or disorders in subjects in need. In some embodiments, a method is provided for reducing the incidence of symptoms associated with inflammatory diseases, conditions, or disorders. In some embodiments, a method is provided for reducing the severity of symptoms associated with inflammatory diseases, conditions, or disorders. In such embodiments, the method comprises administering to the individual in need a therapeutically effective amount of glycine or a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof.

[0011] Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing, and / or improving the progression of metabolic, cardiovascular, and inflammatory diseases (particularly those involving abnormalities in cholesterol, triglycerides, and glucose) in subjects of need. Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing, and / or improving the progression of triglyceride and total cholesterol-related diseases, conditions, and disorders in subjects of need. In some embodiments, such diseases, conditions, and disorders include inflammatory, cardiovascular, and / or metabolic diseases, conditions, and disorders. Certain such cardiovascular diseases, conditions, or disorders include, but are not limited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), angina pectoris, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease, peripheral artery occlusion), retinal vascular occlusion, or stroke. Certain metabolic diseases, conditions, or disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome, and diabetic dyslipidemia. Certain inflammatory diseases, conditions, or disorders include, but are not limited to, aortic stenosis, coronary artery disease (CAD), Alzheimer's disease, and thromboembolic diseases, conditions, or disorders. Certain thromboembolic diseases, conditions, or disorders include, but are not limited to, stroke, thrombosis (e.g., venous thromboembolism), myocardial infarction, and peripheral vascular disease. Certain embodiments provide compositions and methods, particularly in the context of liver disease, for the prevention, treatment, delay, or slowing of the progression and / or improvement of diet-induced hyperlipidemia and the progression and / or improvement of steatohepatitis in cases of impaired mitochondrial / peroxisome fatty acid β-oxidation (FAO), in addition to preventing, treating, delaying, slowing the progression of glucose sensitivity and systemic inflammation and fibrosis.

[0012] One embodiment of the first aspect of this disclosure provides a method for treating non-alcoholic fatty liver disease (NAFLD), the method comprising administering glycine or DT-109 (Gly-Gly-Leu). Another embodiment of the first aspect of this disclosure provides a method for treating non-alcoholic steatohepatitis (NASH), the method comprising administering glycine or DT-109 (Gly-Gly-Leu). Another embodiment of the first aspect of this disclosure provides a method for treating non-alcoholic fatty liver disease (NAFLD), the method comprising administering DT-110 (Gly-Gly-dLeu). One embodiment of the first aspect of this disclosure provides a method for treating non-alcoholic steatohepatitis (NASH), the method comprising administering glycine or DT-110 (Gly-Gly-dLeu). Optionally, these tripeptides may be administered in combination with a second therapeutic agent as described herein.

[0013] A second aspect of this disclosure provides a method for reducing fibrosis in a patient, the method comprising administering a selected tripeptide or a pharmaceutically acceptable salt thereof. One embodiment of the second aspect of this disclosure provides a method for reducing fibrosis in a patient, the method comprising administering glycine or DT-109 (Gly-Gly-Leu). Another embodiment of the second aspect of this disclosure provides a method for reducing fibrosis in a patient, the method comprising administering glycine or DT-110 (Gly-Gly-dLeu). These tripeptides may optionally be administered in combination with statins.

[0014] A third aspect of this disclosure provides a method for treating hepatic steatosis, the method comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need. In one embodiment of the third aspect, the method reduces triglyceride levels or total cholesterol levels in liver lipids.

[0015] A fourth aspect of this disclosure provides a kit for treating subjects with NAFLD or NASH, the kit comprising a selected tripeptide, an optional statin, and instructions for use. In one embodiment of the kit, the kit comprises an effective amount of glycine or DT-109 (Gly-Gly-Leu), an optional statin, and instructions for use. In another embodiment, the kit comprises glycine or DT-110 (Gly-Gly-dLeu), an optional statin, and instructions for use. In yet another kit, the kit comprises glycine or DT-109 (Gly-Gly-Leu) and / or DT-110 (Gly-Gly-dLeu), an optional statin, and instructions for use. Attached Figure Description

[0016] Figures 1A to 1F Experimental design Figure 1A ), the average food intake throughout the study ( Figure 2B ), final weight ( Figure 1C ), total weight gain at the end point compared to baseline ( Figure 1D ()( Figures 1B to 1D (n=8-10), the overall morphology of the peritoneal cavity at the endpoint ( Figure 1E ), plasma glycine-containing tripeptide molecule levels ( Figure 1F (n = 6-8). *p < 0.05; **p < 0.01, compared to WD + H2O.

[0017] Figures 2A to 2E Acute OGTT (in mice loaded with glucose and water as treatment or control) Figure 2A ), chronic OGTT (glucose-loaded mice alone) Figure 2B Non-fasting blood glucose levels measured before (pre-gavage) and 30 minutes after (post-gavage) oral administration with water or medication. Figure 2C ), endpoint blood glucose level (6-hour fasting) ( Figure 2D qPCR analysis of liver gene expression regulating glucose uptake and gluconeogenesis ( Figure 2E (n=8-10)*p<0.05;**p<0.01,***p<0.001,compared to WD+H2O;#p<0.05,###p<0.001,compared to before gavage.

[0018] Figures 3A to 3C Representative H&E slides of liver tissue ( Figure 3A Microvesicular and macrovesicular steatosis were marked with red and yellow arrows, respectively. Liver lipids were extracted and then analyzed. Figure 3B )TG and ( Figure 3C The TC content was quantified, **p<0.01, ***p<0.001, compared to WD+H2O.

[0019] Figures 4A to 4E qPCR analysis of liver gene expression regulating lipid oxidation pathway ( Figure 4A and Figure 4B ), qPCR analysis of liver gene expression regulating cholesterol homeostasis ( Figure 4C (n=7-10). Western blot analysis and quantification of ABCG8 abundance for β-actin normalization ( Figure 4D and Figure 4E (n=4-6). *p<0.05;**p<0.01,***p<0.001, compared to WD+H2O.

[0020] Figures 5A to 5GPlasma TC levels at baseline (after 1 week of feeding with WD without treatment) and at the endpoint (after 12 weeks of feeding with WD in either treatment or water control conditions). Figure 5A ). ( Figure 5B )TC、( Figure 5C LDL, ( Figure 5D HDL and ( Figure 5E The endpoint plasma level of TG. Blood was collected after a 6-hour fast. Figure 5F Analysis of atherosclerotic plaques observed visually through Oil Red O staining, and ( Figure 5G Representative image of the stained aorta. (n = 6-10). *p < 0.05; **p < 0.01, ***p < 0.001, compared to WD+H2O.

[0021] Figures 6A to 6E IL-6 Figure 6A ), resistin ( Figure 6B ) and MCP1 ( Figure 6C The endpoint plasma level. Figure 6D In the epididymal adipose tissue (EAT) and ( Figure 6E qPCR analysis of inflammatory cytokines in subcutaneous adipose tissue (SAT). (n = 6-10). *p < 0.05; **p < 0.01, ***p < 0.001, compared to WD+H2O.

[0022] 7A to 7C qPCR analysis of inflammatory cytokines in the liver Figure 7A Representative F4 / 80 immunohistochemistry in the liver ( Figure 7B ), and quantitative ( Figure 7C (n=7-10). *p<0.05, compared to WD+H2O.

[0023] Figure 8. AGXT1 fed with NASH-diet - / - Plasma changes in mice. HepG2 cells were transfected with siCTL or siAGXT1: ( Figure 8C ) relative to GAPDH's AGXT1 mRNA (n=12), ( Figure 8D ) Using GAPDH as a loading control for AGXT1 protein (n=4), and ( Figure 8E Cellular TG (200 μM, n = 12) with or without PA loading. Figure 8A The guide RNA target site on exon 1 of the AGXT1 gene is underlined and confirmed by Sanger sequencing to be the deletion of the third base A starting from PAM. Figure 8BWestern blotting confirmed the absence of AGXT1 (n=7). + / + and AGXT1 - / - Mice fed a NASH-diet for 12 weeks (n=12): plasma ( Figure 8L )TG, ( Figure 8M )TC, ( Figure 8N )AST, ( Figure 8O ALT, and ( Figure 8P Glycine / oxalate ratio. Data are mean ± SD, showing all points and p-values. PA, palmitic acid.

[0024] Figures 8F to 8K When fed CD, AGXT1 + / + and AGXT1 - / - For example, AGXT1. + / + and AGXT1 - / - Mice were fed standard CD for 12 weeks (n=6): Figure 8F )weight,( Figure 8G The overall appearance of the peritoneal cavity and histology stained with H&E and ORO (scale bar: H&E 50 μm, ORO 100 μm), Figure 8H Liver weight, Figure 8I The ratio of liver weight (LW) to body weight (BW), Figure 8J ) plasma AST, and ( Figure 8K Plasma ALT. Data are mean ± SD, all points are shown.

[0025] Figures 9A to 9K Glycine-based compounds. Compounds structurally similar to glycine were selected to evaluate structural, conformational, electronic, and isosteric modifications to the glycine skeleton. Figure 9A glycine, Figure 9B N-methylglycine, ( Figure 9C N,N-dimethylglycine, ( Figure 9D N,N,N-trimethylglycine, ( Figure 9E Glycolic acid, ( Figure 9F Glycineamide, ( Figure 9G 2-Amino-N-methylacetamide, ( Figure 9H )ethanolamine, ( Figure 9I )2-Oxypiperazine, ( Figure 9J )morpholin-2-one, and ( Figure 9K )(1H-tetrazole-5-yl)methylamine.

[0026] Figures 10A to 10I , Figures 10K to 10NImpaired glycine biosynthesis in NAFLD. C57BL / 6J mice were fed CD or WD for 12 weeks (n=4-5): Figure 10A ) plasma TC, ( Figure 10B Liver histology (scale bar: H&E 50μm, ORO 100μm), Figure 10C Liver TG, ( Figure 10D )Liver TC, ( Figure 10E ) relative to CD plasma AA, ( Figure 10F (This refers to the liver expression of the glycine biosynthesis gene relative to GAPDH.) Figure 10G ) cell TG, and ( Figure 10H AGXT1 expression in HepG2 cells was loaded with 200 μM PA or ethanol for 24 h (n = 3-4). C57BL / 6J mice were fed a NASH-diet or CD diet for 24 weeks (n = 10): Figure 10I Liver morphology, H&E and Sirius Red histology (scale bar: 50 μm). Figure 10K RNA sequencing of livers from CD or NASH mice revealed a significant downregulation of glycine biosynthesis genes / pathways (green) (n=3, log2FC, log2 fold change). Figure 10L AGXT1 expression relative to GAPDH in mice with diet-induced NASH (n=8). Figure 10M According to a meta-analysis of liver microarray data from healthy and NASH patients, glycine metabolism genes were significantly downregulated (green) or upregulated (red). Figure 10N The correlation between AGXT1 expression and total liver fat in livers from transplant donors (n=206). Data are mean ± SD, showing all points and P values.

[0027] Figure 10J Pathway analysis from livers of mice with NASH. Pathway analysis after RNA sequencing from livers of mice fed a CD or NASH-diet for 24 weeks (n=3). Pathways rich in upregulated differentially expressed genes (DEGs) are plotted in red, while pathways rich in downregulated DEGs are plotted in green.

[0028] Figures 11A to 11L .AGXT1 - / - Accelerated diet-induced NASH in mice. (AGXT1) + / + and AGXT1 - / - Mice were fed a NASH-diet for 12 weeks (n=12): Figure 11A The overall appearance of the peritoneal cavity, and liver histology (scale bar: H&E and Sirius Red 50 μm, ORO 100 μm), Figure 11B Liver weight / body weight (LW / BW) ratio, Figure 11E Liver TG, ( Figure 11F )Liver TC, ( Figure 11G NAS, and ( Figure 11H Fibrosis score. Data are mean ± SD, showing all points and p-values. Figure 11J (From AGXT1) + / + and AGXT1 - / - Pathway analysis of mouse liver RNA after sequencing (n=4). Pathways rich in upregulated or downregulated DEG are plotted in red or green, respectively. Figure 11K Heatmaps of 25 NASH-related DEGs. Figure 11L FAO-related DEGs (n=10) confirmed by qPCR, and ( Figure 11M Western blot analysis (n=4). Proteins confirmed by qPCR ( Figure 11N Inflammation-related and ( Figure 11O Fibrosis-related DEG (n=10). Data are mean ± SE. *P<0.05, **P<0.01, ***P<0.001, compared to AGXT1 + / + .

[0029] Figure 11C , Figure 11D , Figure 11I AGXT1 fed with NASH diet - / - NAFLD-related parameters in mice. (AGXT1) + / + and AGXT1 - / - Mice were fed a NASH-diet for 12 weeks (n=12): Figure 11C )weight,( Figure 11D Liver weight, and ( Figure 11I Based on H&E scores for steatosis, hepatocellular ballooning degeneration, and lobular inflammation. Data are presented as mean ± SD.

[0030] Figures 12A to 12J Glycine deficiency exacerbates obesity induced by Wild-Dysplastic syndrome (WD). (ApoE) - / - Mice fed CD and WD AA +Gly or WD AA -Gly lasts for 10 weeks (n=6): ( Figure 12A Plasma glycine. Body composition analysis based on NMR: ( Figure 12B )weight,( Figure 12C Body fat percentage (%), and ( Figure 12D Lean body mass percentage (%). CLAMS analysis: ( Figure 12E Food intake, ( Figure 12F ) Total activities, ( Figure 12G) respiratory exchange rate (RER), and ( Figure 12H Energy consumption. Figure 12I ) plasma glycine tripeptide molecules, and ( Figure 12J H&E histology of the epididymis and subcutaneous adipose tissue (EAT and SAT, scale bar: 100 μm). Data are mean ± SD, showing all points and P values.

[0031] Figures 13A to 13K Glycine deficiency exacerbates hyperlipidemia and hepatitis B (HS) induced by Wild-Dysplastic syndrome (WD). (ApoE) - / - Mice fed CD and WD AA +Gly or WD AA -Gly lasts for 10 weeks (n=6): ( Figure 13A ) plasma TC, ( Figure 13B Plasma TG, ( Figure 13C plasma LDL, ( Figure 13D plasma HDL, ( Figure 13E plasma glucose, ( Figure 13F Liver histology stained with H&E and ORO (scale bar: H&E 50 μm, ORO 100 μm), Figure 13G ) liver TG, and ( Figure 13H Liver TC. Data are mean ± SD, showing all points and P values. Plasma glycine and ( Figure 13I ) plasma TC, ( Figure 13J ) plasma glucose and ( Figure 13K Spearman's correlation analysis of liver triglycerides.

[0032] Figures 14A to 14G Effects of glycine-based compounds on glucose tolerance. Oral glucose tolerance test (OGTT) was performed in C57BL / 6J mice (n = 6–8) after a 12-hour fast. Mice received oral glucose alone (2 mg / g body weight), glucose with 0.5 mg / g body weight of glycine, or with 0.5 mg / g body weight of a glycine-based compound. Figure 14A N-methylglycine, ( Figure 14B N,N-dimethylglycine, ( Figure 14C N,N,N-trimethylglycine, ( Figure 14D Glycolic acid, ( Figure 14E DT-110, and ( Figure 14F DT-109. Figure 14GMice were administered oral glucose alone (2 mg / g body weight), glucose with DT-109 (0.5 mg / g body weight), or equivalent levels of free leucine or glycine (0.17 or 0.33 mg / g body weight, respectively). Data are mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, compared to glucose; # P < 0.05, compared to leucine. ^P < 0.05, compared to glycine.

[0033] Figures 15A to 15L The lipid-lowering effect of DT-109. Figure 15A ) for ApoE - / - Mice were fed standard WD diets and received oral DT-109 (1 mg / g body weight daily), equivalent levels of free leucine or glycine (0.33 or 0.67 mg / g body weight daily), or H2O for 12 weeks (n = 8-10). Figure 15B At week 10, an oral glucose tolerance test (OGTT) was performed after a 12-hour fast. Mice received oral glucose alone (2 mg / g body weight), glucose with 1 mg / g body weight of DT-109, or equivalent levels of free leucine (0.33 mg / g body weight) or glycine (0.67 mg / g body weight). Data are presented as mean ± SE. **P < 0.01, ***P < 0.001, compared to H2O; # P < 0.05 ## P < 0.01, ### P < 0.01, compared to leucine. Figure 15C Non-fasting blood glucose was measured daily before and 30 minutes after gavage with DT-109, leucine, glycine, or H2O. Figure 15D Final weight. Figure 15E Average food intake. Figure 15F Baseline (before randomization to the experimental group) and endpoint plasma TC (data are mean ± SE). Endpoint plasma analysis (n = 6–8): Figure 15G )TC. ( Figure 15H LDL. Figure 15I HDL. Figure 15J )TG, and ( Figure 15K Glycine tripeptide molecule. Data are mean ± SD, showing all points and p-values. Figure 15L H&E histology of epididymis and subcutaneous adipose tissue (EAT and SAT, scale bar: 100 μm).

[0034] Figures 16A to 16D Glycine or DT-109 was used to prevent WD-induced HS. Endpoint liver analysis (n=8-10): Figure 16AThe overall appearance of the peritoneal cavity and histology using H&E and ORO (scale bar: 50 μm for H&E, 100 μm for ORO), Figure 16B ) liver TG, and ( Figure 16C Liver total cholesterol (TC). Data are mean ± SD, showing all points and p-values. Figure 16D qPCR analysis of key genes regulating FAO and inflammation relative to GAPDH. Data are mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, compared to WD+H2O.

[0035] Figures 17B to 17H NASH was confirmed before randomization to the experimental group. C57BL / 6J mice were fed either CD (n=11) or a NASH-diet (n=50) for 12 weeks. Figure 17B Fasting blood was collected from the submandibular vein for analysis of plasma glucose, TC, AST, and ALT (data are mean ± SD). A subset of mice (CD: n = 3, NASH-diet n = 5) were sacrificed to confirm liver lesions: Figure 17C Liver weight to body weight ratio (LW / BW). Figure 17D The overall appearance of the peritoneal cavity and histological examination using H&E, ORO, and Sirius Red (scale bar: 50 μm for H&E and Sirius Red, 100 μm for ORO), Figure 17E ) as ( Figure 17F NAS of the sum of scores for steatosis, hepatocellular ballooning degeneration, and lobular inflammation. Data are mean ± SD, showing all points and p-values. Figure 17G At week 18, oral glucose tolerance tests (OGTT) were performed after a 12-hour fast (n = 8–9). Mice received oral glucose alone (2 mg / g body weight), glucose with 0.5 mg / g body weight of DT-109, or equivalent levels of free leucine (0.17 mg / g body weight), glycine (0.33 mg / g body weight), or H2O. Data are presented as mean ± SE. *P < 0.05, **P < 0.01, compared to CD + H2O; ## P < 0.01, ### P < 0.001, compared to NASH + H₂O; ^^P < 0.01, compared to NASH + leucine. Figure 17H Non-fasting blood glucose levels were recorded daily before and 30 minutes after administration of DT-109, leucine, glycine, or H2O via gavage. Data are presented as mean ± SD, with all points and p-values ​​shown.

[0036] Figures 17L to 17RThe metabolic effects of DT-109 in C57BL / 6J mice with confirmed NASH. C57BL / 6J mice were fed CD or a NASH-based diet for 12 weeks. After NASH confirmation, mice were randomized to receive 0.125 or 0.5 mg DT-109 or equivalent levels of leucine or glycine (0.17 or 0.33 mg DT-109 per gram of body weight per day) or H2O orally via gavage for another 12 weeks under a NASH-based diet. Mice fed CD and administered H2O served as controls (n = 8–9). Figure 17L H&E histology of the epididymis and subcutaneous adipose tissue (EAT and SAT, scale bar: 100 μm). CLAMS analysis at weeks 22-23: ( Figure 17M Food intake, ( Figure 17N Fat oxidation, Figure 17O Glucose oxidation, Figure 17P Respiratory exchange rate (RER), Figure 17Q Energy consumption, and ( Figure 17R Total activity. Data is mean ± SD, showing all points and P-values.

[0037] Figure 17A , Figure 17I , Figure 17J , Figure 17K as well as Figures 18A to 18G , Figure 18I DT-109 prevents diet-induced NASH. Figure 17A C57BL / 6J mice were fed a CD or NASH-diet for 12 weeks. After NASH confirmation, mice were randomized to receive DT-109 or its equivalents (0.17 or 0.33 mg / g daily) or H2O via oral gavage for another 12 weeks on the NASH-diet. Mice fed CD and administered H2O served as controls (n = 8–9). Body composition analysis based on NMR at weeks 22–23: ( Figure 17I )weight,( Figure 17J Body fat percentage (%), and ( Figure 17K Lean body mass percentage (%). Endpoint plasma analysis: ( Figure 18A )AST, ( Figure 18B ALT, ( Figure 18C ALP, ( Figure 18D )TG, and ( Figure 18E )TC. ( Figure 18F Overall morphology and H&E histology (scale bar: 50 μm). Figure 18G LW / BW ratio. Figure 18I NAS. Data is the mean ± SD, showing all points and P-values.

[0038] Figure 18H , Figures 18J to 18M as well as Figures 20H to 20J DT-109 prevents diet-induced NASH. C57BL / 6J mice were fed a CD or NASH-diet for 12 weeks. After NASH confirmation, mice were randomized to receive 0.125 or 0.5 mg DT-109 or equivalent levels of leucine or glycine (0.17 or 0.33 mg DT-109 per gram of body weight per day) or H2O orally by gavage for another 12 weeks under NASH-diet conditions. Mice fed CD and administered H2O served as controls (n = 8–9). Figure 18H Liver weight, and ( Figure 18J Based on H&E scores for steatosis, hepatocellular ballooning degeneration, and lobular inflammation. Data are mean ± SD. NAS and ( Figure 18K ) plasma AST, ( Figure 18L ) plasma ALT and ( Figure 18M Spearman correlation analysis between plasma ALP levels. Liver fibrosis score and ( Figure 20H ) plasma AST, ( Figure 20I ) plasma ALT and ( Figure 20J Spearman correlation analysis between plasma ALPs.

[0039] Figure 19A , Figure 19B , Figures 19F to 19I , Figure 19K , Figure 19L , Figure 19N Glycine-based therapy corrects NASH-induced diet-induced impaired FAO and alleviates HS. RNA sequencing of the liver collected at the endpoint (n=4): Figure 19A PCA, ( Figure 19B Compared to CD, the volcano plots of DEG in each group (green: down-tone; red: up-tone) Figure 19F Comparison of NASH+H2O and NASH+ DT-109 pathways at 0.5 mg / g daily. Pathways rich in upregulated or downregulated DEG are plotted in red or green, respectively. Figure 19G Heatmaps of 50 NASH-related DEGs across all experimental groups (log2 fold change compared to the CD group). Figure 19H FAO-associated DEG was validated using qPCR (n=8-9). Data are mean ± SE, *P<0.05, **P<0.01, ***P<0.001, compared to CD; # P < 0.05 ## P < 0.01, ### P < 0.001, compared to NASH + H₂O, and ( Figure 19I Western blot analysis (n=4). Figure 19K ORO histology (scale bar: 100 μm), Figure 19L Liver TG. Figure 19N Liver DAG (n=8-9). Data are mean ± SD, showing all points and p-values.

[0040] Figure 19C DT-109 reverses NASH-diet-induced transcriptome alterations. A heatmap-based illustration of the top 50 DEGs across all experimental groups, as determined by log2 fold change compared to the CD group. Each row represents one gene, and each column represents one comparison to the CD group (n=4).

[0041] Figure 19D , Figure 19E , Figure 19J , Figure 19M Pathway analysis of liver tissue from mice fed CD and NASH diets. Figure 19D Paths rich in upregulating DEG are drawn in red, while paths rich in downregulating DEG are drawn in green. Figure 19E Changes in glycine biosynthesis genes / pathways analyzed by RNA sequencing and pathway analysis from livers of CD or NASH mice (n=4). Significantly downregulated genes / pathways are highlighted in green (log2FC, log2 fold change). Figure 19J qPCR validation of FAO-related DEG (n=8-9). Data are mean ± SE. *P<0.05, **P<0.01, ***P<0.001, compared to CD; # P < 0.05 ## P < 0.01, compared to NASH + H₂O. Figure 19M Liver total cholesterol (n = 8-9). Data are mean ± SD, showing all points and p-values.

[0042] Figures 20A to 20F , Figure 20K , Figure 20L Glycine-based therapy reduced NASH-induced diet-induced hepatitis and fibrosis. Figure 20A F4 / 80 immunohistochemistry and Sirius red histology (scale bar: 50 μm), Figure 20B F4 / 80 positive area, ( Figure 20C ) plasma MCP-1, and ( Figure 20D Resistance. Data are mean ± SD, showing all points and p-values ​​(n = 6–9). Figure 20EqPCR validation of inflammation-related DEG (data are mean ± SE, n = 8–9). *P < 0.05, **P < 0.01, ***P < 0.001, compared to CD; # P < 0.05 ## P < 0.01, ### P < 0.001, compared to NASH + H₂O. Figure 20F The red-positive area of ​​Sirius. Figure 20G Fibrosis score. Figure 20K Western blot analysis of phosphorylated SMAD2 (Ser465 / 467) and total SMAD2. Figure 20L qPCR validation of fibrosis-related DEG. Detailed Implementation

[0043] As used in this article, "fatty degeneration" can be used interchangeably with "fatty liver," which is the accumulation of fat in the liver.

[0044] Subjects may be mammals, and mammals may be, for example, laboratory animals or humans, and human subjects include adults, young adults and pediatric subjects.

[0045] The terms “fatty degeneration” and “hepatic steatosis” can be used interchangeably in this article.

[0046] The terms “blood plasma” and “plasma” are used interchangeably in this article.

[0047] "Blood" and "plasma" are used interchangeably in this article.

[0048] Western diets are referred to as "WD" in this article.

[0049] Facial veins are abbreviated as "FV" in this article.

[0050] "TG" is an abbreviation for triglycerides.

[0051] "TC" is an abbreviation for total cholesterol.

[0052] "OGTT" is an abbreviation for Oral Glucose Tolerance Test.

[0053] Unless the context clearly indicates otherwise, as used herein, the singular forms “a,” “an,” and “the” include the plural references.

[0054] In some cases, "or" can mean "in combination with". Therefore, applying A or B can mean applying A, applying B, or applying A and B.

[0055] With the exception of glycine, all common amino acids contain at least one chiral carbon atom. Therefore, these amino acids exist as multiple stereoisomers designated as L- and D-isomers. Most naturally occurring proteins and peptides are exclusively composed of L-isomers. D-isomers can affect the conformation of peptides or proteins and can produce changes in stability or activity.

[0056] As used herein, the term "pharmaceutically acceptable salt" means salts that, within the limits of precise medical judgment, are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and that meet a reasonable benefit / risk ratio. "Pharmaceutically acceptable salt" means any salt of the compounds of the present invention that, upon administration to a recipient, can directly or indirectly provide a non-toxic salt or an ester of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977).

[0057] Pharmaceutical compositions suitable for delivering the peptides of the present invention, and methods for their preparation, will readily become apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, The Science and Practice of Pharmacy, 20th edition, Lippincott Williams & White, Baltimore, Md. (2000). The peptides of the present invention can be formulated for immediate release and / or modified release.

[0058] The term “treatment” (or other forms of the term, such as “treatment” or “treat”) is used herein to refer to the application of the compositions of the present invention to alleviate a patient’s disease and / or reduce, suppress, or eliminate specific characteristics or events associated with the disease. Therefore, the term “treatment” includes preventing the occurrence of a patient’s disease, particularly when the patient is susceptible to said disease; reducing or suppressing the disease; and / or improving or reversing the disease. Within the scope of the methods of the present invention, which are directed at disease prevention, it should be understood that the term “prevention” does not require the complete prevention of the disease. Rather, as used herein, the term prevention refers to the ability of a skilled technician to identify a susceptible population so that the compositions of the present invention can be applied before the onset of the disease. The term does not imply that the disease must be completely avoided.

[0059] As used herein, "effective amount" refers to an amount of the glycine-containing tripeptide of the present invention sufficient to demonstrate a detectable therapeutic effect. This effect is detected, for example, by the improvement of a clinical symptom or the prevention, reduction, or improvement of complications. The precise effective amount for a patient will depend on the patient's weight, body type, and health; the nature and severity of the symptom; and the choice of therapeutic agent or combination of therapeutic agents for administration. The therapeutically effective amount for a given situation is determined through routine laboratory testing within the technical and judgmental scope of a clinician.

[0060] As used herein, “identifying” or “selecting subjects with metabolic and / or cardiovascular and / or inflammatory diseases” means identifying or selecting subjects who are predisposed to or have been diagnosed with metabolic disease, cardiovascular disease, systemic or localized inflammatory disease; or metabolic syndrome; or identifying or selecting subjects with any symptoms of metabolic disease, cardiovascular disease, or metabolic syndrome, including but not limited to hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, overweight and / or overweight or any combination thereof. Such identification may be achieved by any method, including but not limited to standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) glucose, measuring serum or circulating (plasma) triglycerides, measuring pro-inflammatory cytokines or cortisol, measuring blood pressure, measuring body fat percentage, measuring weight, etc.

[0061] As used in this article, "glucose" refers to a monosaccharide used by cells as an energy source and an inflammatory intermediate. "Plasma glucose" refers to glucose present in blood plasma.

[0062] As used herein, "high-density lipoprotein-C" or "HDL-C" refers to cholesterol associated with high-density lipoprotein particles. The concentration of HDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. "Serum HDL-C" and "Plasma HDL-C" refer to HDL-C in serum and plasma, respectively.

[0063] As used in this article, "HMG-CoA reductase inhibitor" refers to agents that work by inhibiting HMG-CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

[0064] As used in this article, according to the National Cholesterol Education Program (NCEP) guidelines for the detection and assessment of hypercholesterolemia in adults reported by the expert group (see Arch. Int. Med. (1988) 148, 36-39), "hypercholesterolemia" refers to a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol, and VLDL-cholesterol.

[0065] As used in this article, “hyperlipidemia” or “high lipid levels” is a disorder characterized by elevated serum or circulating (plasma) lipids. This disorder presents with abnormally high fat concentrations. Circulating lipids are classified into cholesterol, low-density lipoprotein, very low-density lipoprotein, chylomicrons, and triglycerides. The Fredrickson classification of hyperlipidemia is based on patterns of TG and cholesterol-rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation, and is often used to characterize the main causes of hyperlipidemia, such as hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965, 31:321-327; Fredrickson et al., New Eng J Med, 1967, 276(1):34-42).

[0066] As used in this article, “hypertriglyceridemia” refers to a condition characterized by elevated triglyceride levels. Its etiology includes primary (i.e., genetic causes) and secondary (other underlying causes, such as diabetes, metabolic syndrome / insulin resistance, obesity, physical inactivity, smoking, excessive alcohol consumption, and a diet with very high carbohydrate content) factors, or, most often, a combination of both (Yuan et al., CMAJ, 2007, 176:1113-1120).

[0067] Before describing the compositions and methods of the present invention, it should be understood that the invention is not limited to the specific processes, compositions, or methods described, as these can be modified. It should also be understood that the terminology used in this specification is for the purpose of describing particular types or embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. All disclosures mentioned herein are incorporated herein by reference in their entirety to the extent that they support the invention.

[0068] Glycine tripeptide molecules used to treat metabolic, cardiovascular, and inflammatory diseases.

[0069] The inventors of this disclosure have developed glycine tripeptide molecules or pharmaceutically acceptable salts thereof that exhibit preventative or therapeutic activity against metabolic, cardiovascular, and / or inflammatory diseases, including but not limited to obesity, diabetes, dyslipidemia, fatty liver, and insulin resistance syndrome. In some embodiments, metabolic or cardiovascular diseases include, but are not limited to, obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, coronary artery disease, non-alcoholic fatty liver disease (NAFLD), hyperfatty acidemia, or metabolic syndrome, or combinations thereof. Dyslipidemia may be hyperlipidemia. Hyperlipidemia may be hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemia and hypertriglyceridemia. NAFLD may be hepatic steatosis or steatohepatitis. Diabetes may be type 2 diabetes or type 2 diabetes with dyslipidemia.

[0070] In various embodiments, the amino acid sequence of the glycine-containing tripeptide molecule DT-109 is Gly-Gly-Leu (SEQ ID NO:1). The amino acid sequence of the glycine-containing tripeptide molecule DT-110 is Gly-Gly-dLeu (SEQ ID NO:2). The glycine tripeptide molecules of the present invention also include pharmaceutically acceptable salts of DT-109 and DT-110. Examples of such salts include metal salts, ammonium salts, salts in the case of organic bases, salts in the case of inorganic acids, salts in the case of organic acids, salts in the case of basic or acidic amino acids, etc. Preferred examples of metal salts include alkali metal salts, such as sodium salts, potassium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, barium salts, etc.; aluminum salts, etc. Preferred examples of salts in the case of organic bases include salts in the case of trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-diphenylmethylethylenediamine, etc. Preferred examples of salts in the inorganic acid case include those in the cases of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Preferred examples of salts in the organic acid case include those in the cases of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Among the above salts, pharmaceutically acceptable salts are preferred. For example, when the compound has an acidic functional group, inorganic salts such as alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and ammonium salts are preferred. When the compound has a basic functional group, salts such as those in the form of inorganic acids (e.g., hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.) or those in the form of organic acids (e.g., acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.) are preferred.

[0071] In various embodiments, the glycine tripeptide molecule may also be administered as a synthetic and / or as a prodrug in its original synthetic form. For example, the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof may be in prodrug form. A prodrug refers to a compound that is converted into a glycine-containing tripeptide molecule under physiological conditions in vivo, attributable to reactions caused by enzymes, gastric acid, etc., said compound being converted into a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof by enzymes under conditions of oxidation, reduction, hydrolysis, etc.; or a compound that is converted into a glycine-containing tripeptide molecule attributable to gastric acid through hydrolysis, etc., etc.

[0072] Examples of prodrugs containing glycine tripeptides or their pharmaceutically acceptable salts include compounds in which the amino group of the glycine tripeptide has been acylated, alkylated, or phosphorylated (e.g., compounds in which the amino group of the glycine tripeptide has undergone eicosanoylation, alaninoylation, pentylamino carbonylation, (5-methyl-2-oxo-1,3-dioxacyclopenten-4-yl)methoxycarbonylation, tetrahydrofuranylation, pyrrolidinylmethylation, neopentyloxymethylation, or tert-butylation, etc.); and compounds in which the hydroxyl group of the glycine tripeptide has been acylated, alkylated, phosphorylated, or borated (e.g., compounds in which the amino group of the glycine tripeptide has undergone acylation, alkylation, phosphorylation, or borate formation, etc.). Compounds in which the carboxyl group of a glycine-containing tripeptide molecule has undergone acetylation, palmitoylation, propionylation, neopentanoylation, succinylation, fumarylation, alanineylation, or dimethylaminomethyl carbonylation; and compounds in which the carboxyl group of a glycine-containing tripeptide molecule has undergone esterification or amidation (e.g., compounds in which the carboxyl group of a glycine-containing tripeptide molecule has undergone C1-6 alkyl esterification, phenyl esterification, carboxymethyl esterification, dimethylaminomethyl esterification, pivaloyloxymethyl esterification, ethoxycarbonyloxyethyl esterification, phthaloyl esterification, (5-methyl-2-oxo-1,3-dioxacyclopenten-4-yl)methyl esterification, cyclohexyloxycarbonylethyl esterification, or methylamidation). Among these, compounds in which the carboxyl group of a glycine-containing tripeptide molecule has been esterified with C1-6 alkyl groups such as methyl, ethyl, or tert-butyl are preferred. These compounds can be produced from glycine-containing tripeptide molecules by methods known per se.

[0073] Glycine-containing tripeptide molecules or their pharmaceutically acceptable salts as prodrugs can also be substances that are converted into glycine-containing tripeptide molecules under physiological conditions, such as those described in IYAKUHINno KAIHATSU (Development of Pharmaceuticals), Vol. 7, Design of Molecules, pp. 163-198, HIROKAWA SHOTEN (1990).

[0074] The exemplary glycine tripeptide molecule is a triamino acid polymer that can be produced according to peptide synthesis methods described herein and known to those skilled in the art. Peptide synthesis methods can employ currently known methods, such as solid-phase synthesis and liquid-phase synthesis. In other words, the peptide of the present invention (e.g., the glycine tripeptide molecule) or a pharmaceutically acceptable salt thereof can be produced by repeated condensation of a portion of the peptide or amino acid capable of constituting the glycine tripeptide molecule, the peptide to be synthesized, and the remaining portion (which may consist of two or more amino acids), depending on the desired sequence. When the product having the desired sequence has a protecting group, the peptide of the present invention can be produced by removing the protecting group. Examples of condensation methods and methods for removing protecting groups to be known include those described in (1)-(5) below.

[0075] (1) M.Bodanszky and MAOndetti: Peptide Synthesis, IntersciencePublishers, New York (1966)

[0076] (2)Schroeder and Luebke: The Peptide, Academic Press, New York (1965)

[0077] (3) Nobuo Izumiya et al.: Peptide Gosei-no-Kiso to Jikken (Basics and experiments of peptide synthesis), published by Maruzen Co. (1975)

[0078] (4) Haruaki Yajima and Shunpei Sakakibara: Seikagaku Jikken Koza (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of Proteins) IV, 205 (1977)

[0079] (5) Haruaki Yajima edited: Zoku Iyakuhin no Kaihatsu (A sequel to Development of Pharmaceuticals), Volume 14, Peptide Synthesis, published by Hirokawa Shoten.

[0080] The use of glycine tripeptide molecules for the treatment and prevention of metabolic, cardiovascular, and / or systemic inflammatory diseases.

[0081] Glycine or glycine-containing tripeptide molecules or pharmaceutically acceptable salts thereof, as described and exemplified herein, can be used to prevent and / or treat one or more metabolic, cardiovascular, and inflammatory diseases in subjects of need. For the purposes of this disclosure, “metabolic disease” refers to a broad range of diseases and conditions of the endocrine system, including, for example, insulin resistance, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, dyslipidemia and hyperlipidemia, and liver diseases such as NAFLD and NASH. Metabolic diseases as described herein also include diseases that can be treated by regulating metabolism, although the disease itself may or may not be caused by a specific metabolic defect. Such metabolic diseases may involve, for example, glucose and fatty acid oxidation pathways.

[0082] The subjects in need are mammals that may experience metabolic diseases and / or cardiovascular diseases and / or systemic inflammatory diseases or one or more symptoms associated with these diseases, preferably humans, or domesticated or laboratory mammals.

[0083] In some embodiments, it is not desired to be limited by any particular theory, but it is believed that administration of glycine or the glycine tripeptide molecule of the present invention or a pharmaceutically acceptable salt thereof to subjects suffering from metabolic diseases, cardiovascular diseases, and chronic systemic inflammatory diseases, or symptoms related to or associated with any of these general conditions, results in a reduction of lipid levels, including triglyceride levels, cholesterol levels, insulin resistance, glucose levels, or combinations thereof. One or more of these levels may be reduced independently by 5%, 10%, 20%, 30%, 35%, or 40% or more. Administration of the glycine tripeptide molecule of the present invention or a pharmaceutically acceptable salt thereof may result in improved insulin sensitivity or hepatic insulin sensitivity. Administration of the glycine tripeptide molecule of the present invention or a pharmaceutically acceptable salt thereof may result in reduced atherosclerotic plaques, obesity, glucose, lipids, glucose resistance, cholesterol, or improved insulin sensitivity, or any combination thereof.

[0084] Some embodiments provide for the use of glycine or a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament for the treatment, improvement, delay, or prevention of one or more metabolic or cardiovascular diseases.

[0085] Some embodiments provide a kit for treating, preventing, or improving one or more metabolic or cardiovascular diseases as described herein, wherein the kit comprises: a) a glycine tripeptide molecule as described herein or a pharmaceutically acceptable salt thereof; and optionally b) another second therapeutic agent or therapy as described herein. The kit may also include instructions or a label for using the kit to treat, prevent, or improve one or more metabolic or cardiovascular diseases using the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments of the invention, glycine or a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof has a potent glucose / lipid-lowering effect. In mice with confirmed NASH, the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof (e.g., DT-109) was able to alleviate steatohepatitis, improve body composition, reduce circulating lipids, and normalize or correct liver enzymes and steatohepatitis by stimulating the FAO pathway. It has been unexpectedly demonstrated that the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof reduces or alleviates lobular / systemic inflammation and liver fibrosis by inhibiting the NF-κB and TGFα / SMAD pathways.

[0087] As used herein, the term "dyslipidemia" refers to disorders of lipid abnormalities, including hyperlipidemia caused by abnormal lipoprotein metabolism, as well as hypercholesterolemia, hypertriglyceridemia, and hypoHDL-cholesterolemia attributed to increased levels of fat in the blood. As used herein, the term "fatty liver" refers to a disorder in which excessive fat accumulates in liver cells due to impaired lipid metabolism. It can cause various diseases such as angina, myocardial infarction, stroke, arteriosclerosis, and pancreatitis. As used herein, the term "diabetes" refers to a chronic disease characterized by a relative or absolute deficiency of insulin, leading to glucose intolerance. The term diabetes includes all types of diabetes, such as type 1 diabetes, type 2 diabetes, and hereditary diabetes. Type 1 diabetes is insulin-dependent diabetes, primarily caused by the destruction of β-cells. Type 2 diabetes is non-insulin-dependent diabetes, caused by insufficient postprandial insulin secretion or insulin resistance. As used herein, the term "insulin resistance" refers to a physiological condition in which insulin becomes less effective at lowering blood glucose and glucose is not efficiently consumed by cells. In cases of high insulin resistance, the body produces excessive insulin, leading to conditions such as high blood pressure, dyslipidemia, heart disease, and diabetes. Specifically, in type 2 diabetes, increased insulin levels are not observed in muscle and adipose tissue. As used herein, the term "insulin resistance syndrome" refers to a combination of symptoms caused by insulin resistance, characterized by cellular resistance to insulin action, hyperinsulinemia, increased very low-density lipoprotein (VLDL) and triglycerides, decreased high-density lipoprotein (HDL), and high blood pressure. It is considered a risk factor for cardiovascular disease and type 2 diabetes (Reaven G M., Diabetes, 37:1595-607 (1988)). In addition, insulin resistance is known to increase oxidative stress and, along with other risk factors such as hypertension, diabetes, and smoking, alter signal transduction systems in cells, thus inducing inflammatory responses and leading to atherosclerosis (Freeman B A et al., Lab.Invest.47:412-26 (1982); Kawamura M et al., J.Clin.Invest.94:771-8 (1994)).

[0088] As used in this article, the term "metabolic disease" refers to a group of diseases involving metabolic disorders that are risk factors for various cardiovascular diseases and type 2 diabetes. It includes insulin resistance and the complex and diverse metabolic symptoms associated with it. In 1988, Reaven proposed insulin resistance as a fundamental factor constituting these symptoms and named a series of abnormalities insulin resistance syndromes. However, in 1998, the World Health Organization (WHO) introduced the term metabolic syndrome or metabolic disease because insulin resistance alone cannot explain all aspects of the symptoms.

[0089] The compositions of this disclosure, comprising a tripeptide molecule of glycine or a pharmaceutically acceptable salt thereof as an active agent, tend to improve various metabolic diseases and / or their symptoms (e.g., obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver disease, systemic inflammation, and / or insulin resistance syndrome). The compositions of this disclosure can prevent or treat metabolic diseases with various activities.

[0090] As used herein, the term “hyperlipidemia” refers to a condition caused by elevated levels of blood lipids, attributed to poor lipid metabolism, such as triglycerides and cholesterol. More specifically, hyperlipidemia is characterized by elevated levels of lipids in the blood, such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids, including hypercholesterolemia and hypertriglyceridemia.

[0091] According to a preferred embodiment, the insulin resistance syndrome treated by the present invention includes obesity, hypertension, atherosclerosis, hyperlipidemia, hyperinsulinemia, non-alcoholic fatty liver disease, and type 2 diabetes.

[0092] According to a preferred embodiment, the compositions of the present invention reduce the levels of blood lipids, liver fat, or visceral fat. The terms "liver" or "viscera" are used to encompass organs, tissues, and cells.

[0093] According to the present invention, subjects fed a diet containing glycine or the glycine tripeptide molecule of the present invention or a pharmaceutically acceptable salt thereof experienced a significant reduction in liver weight and improved lipid concentrations of triglycerides and total cholesterol in the blood and liver tissue, and a significant reduction in total visceral fat weight.

[0094] According to a more preferred embodiment, the fat reduced by the present invention comprises triglycerides, cholesterol, and free fatty acids.

[0095] According to a more preferred embodiment, the visceral fat reduced by the present invention includes epididymal fat, perirenal fat, mesenteric fat, and / or retroperitoneal fat.

[0096] According to a preferred embodiment, the composition of the present invention reduces the activity of ALT (alanine aminotransferase) or AST (aspartate aminotransferase). ALT and AST, as indicators of liver function, are enzymes that show increased levels in the blood after liver injury.

[0097] Nonalcoholic fatty liver disease (NAFLD) is a general term for a range of liver disorders that affect people who drink little or no alcohol. As the name suggests, the main characteristic of NAFLD is the storage of too much fat in liver cells. It is normal for the liver to contain some fat. However, if more than 5%-10% of the liver's weight is fat, then this condition is called fatty liver (steatohepatitis).

[0098] NAFLD is strongly associated with features of metabolic syndrome, including obesity, insulin resistance, type 2 diabetes, and dyslipidemia; it is considered a hepatic manifestation of this syndrome.

[0099] Pediatric NAFLD is currently the leading form of liver disease in children. Studies have confirmed that abdominal obesity and insulin resistance are considered major contributors to the development of NAFLD. As obesity becomes increasingly prevalent worldwide, the incidence of NAFLD has also increased. Weight loss is the only treatment that has proven truly effective in pediatric NAFLD.

[0100] A more severe form of NAFLD is called nonalcoholic steatohepatitis (NASH). NASH causes the liver to swell and become damaged. NASH tends to develop in people who are overweight or obese, or who have diabetes, high cholesterol or high triglycerides, or inflammatory disorders. NASH is a potentially severe form of the disease, marked by hepatocellular ballooning degeneration and hepatitis, which can progress to scarring and irreversible damage. This damage is similar to that caused by alcoholism. Macroscopically and microscopically, NASH is characterized by inflammation of the lobules and / or portal areas, varying degrees of fibrosis, hepatocellular death, and pathological angiogenesis. In the most severe cases, NASH can progress to cirrhosis, hepatocellular carcinoma, and liver failure. Current treatments for NAFLD and NASH include, for example, diet, management of insulin resistance, or vitamin administration (such as vitamin E or D).

[0101] The NAFLD activity score (NAS) can be calculated according to Kleiner's guidelines (Kleiner DE. et al., Hepatology, 2005; 41:1313). A NAS score of 0-2 is not considered a diagnosis of NASH, a NAS score of 3-4 is considered not a diagnosis of NASH, borderline NASH, or NASH positive, while a NAS score of 5-8 is largely considered a diagnosis of NASH. Treatment efficacy for NASH includes regression, stabilization, or a reduced rate of disease progression. Changes in NAS scores can be assessed using sequential liver biopsies from patients who may have NASH and used as an indicator of disease status changes. An increase in score indicates progression, no change in score indicates stabilization, and a decrease in score indicates NASH regression. In controlled clinical trials, assessing the difference in NAS scores between the placebo and test article treatment groups, typically over a period of 6 months to 2 years, can indicate treatment efficacy, even if progression occurs in both groups. Regulatory agencies typically require specified point diffusion to demonstrate meaningful changes in NASH.

[0102] The present invention also provides a method for treating hepatic steatosis, the method comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need, wherein hepatic steatosis is treated. In one embodiment, the tripeptide molecule reduces triglyceride levels in liver lipids without significantly affecting a leucine-negative control. In another embodiment, the tripeptide molecule reduces total cholesterol levels in liver lipids without significantly affecting a leucine-negative control. In any of the above embodiments, the tripeptide molecule is Gly-Gly-Leu or Gly-Gly-dLeu.

[0103] The present invention also provides a method for increasing or enhancing hepatic lipid oxidation to reduce triglyceride levels or treat cholesterol accumulation, the method comprising administering glycine or one or more glycine tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need, and measuring its effect on mRNA levels, wherein hepatic lipid oxidation, triglyceride levels, cholesterol accumulation, or any combination thereof are enhanced or treated.

[0104] Methods for enhancing hepatic lipid oxidation, reducing triglyceride levels, or treating cholesterol accumulation include administering the glycine tripeptide Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof to a subject in need, and measuring its effect on mRNA levels, wherein hepatic lipid oxidation, triglyceride levels, cholesterol accumulation, or any combination thereof are enhanced or treated. In these methods, the glycine-containing tripeptide molecules Gly-Gly-Leu or Gly-Gly-dLeu significantly induce the expression of the regulators of hepatic lipid oxidation, AMPKα1 or PPARα, with no effect observed in the leucine negative control. In these methods, the tripeptide molecules Gly-Gly-Leu or Gly-Gly-dLeu regulate triglyceride hydrolysis by significantly upregulating CPT1a, CACT, or ACADI (mitochondrial β-oxidation) or PNPLA2. In these methods, the tripeptide molecules Gly-Gly-Leu or Gly-Gly-dLeu regulate triglyceride hydrolysis by significantly upregulating the mitochondrial anion carrier UCP2.

[0105] In this method, the tripeptide molecules Gly-Gly-Leu or Gly-Gly-dLeu regulate cholesterol homeostasis in the liver by significantly increasing the expression of ABCG5 and ABCG8.

[0106] One method of treating the plasma lipid profile of a subject involves administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need to reduce the subject's plasma triglyceride, plasma LDL levels, or atherosclerotic plaques.

[0107] A method for treating the plasma lipid profile of a subject includes administering the tripeptide molecule Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof to a subject in need to reduce plasma triglyceride, plasma LDL levels, or atherosclerotic plaques. In this method, Gly-Gly-Leu or Gly-Gly-dLeu reduces atherosclerotic plaques. The method for treating the plasma lipid profile of a subject includes administering the tripeptide molecule Gly-Gly-Leu to a subject in need to reduce plasma total cholesterol, plasma LDL, or a combination thereof.

[0108] In any embodiment of the method of the present invention, another lipid-lowering agent may be administered to the subject. This other lipid-lowering agent is a cholesterol absorption inhibitor, a PCSK9 inhibitor, a PPAR-α agonist, fenofibrate, an ACC inhibitor, an ApoC-III inhibitor, an ACL inhibitor, a prescription fish oil, or a CETP inhibitor. In some embodiments, the method includes administering another cholesterol-lowering agent that is a cholesterol absorption inhibitor. In this method, the cholesterol-lowering agent is a cholesterol absorption inhibitor, and the cholesterol absorption inhibitor is ezetimibe. In some embodiments, the cholesterol-lowering agent is a PCSKS9 inhibitor.

[0109] Atherosclerosis occurs when the blood vessels (arteries) that carry oxygen and nutrients from your heart to the rest of your body become thin and hard (atherosclerosis). Sometimes, this restricts blood flow to your organs and tissues. Atherosclerosis can cause many complications, including myocardial infarction, coronary artery disease, carotid artery disease, peripheral artery disease, aneurysms, and chronic kidney disease.

[0110] Myocardial infarction (heart attack) occurs when blood flow to a part of the heart decreases or stops, damaging the heart muscle. Common symptoms include pain in the middle or left side of the chest, shortness of breath, and nausea. It can also cause heart failure, irregular heartbeat, cardiogenic shock, or cardiac arrest.

[0111] Coronary artery disease occurs when atherosclerosis narrows the arteries near your heart, which can cause chest pain (angina), heart attacks, or heart failure.

[0112] Carotid artery disease occurs when atherosclerosis narrows the arteries near your brain, which can cause a transient ischemic attack (TIA) or stroke. Symptoms include sudden numbness or weakness in your arm or leg, temporary vision loss in one eye, or drooping facial muscles.

[0113] Peripheral artery disease occurs when atherosclerosis narrows the arteries in your arm or leg; these circulatory problems are called peripheral artery disease. This can make you less sensitive to heat and cold, increasing your risk of burns or frostbite. Occasionally, poor circulation in the arms and legs can cause tissue death (gangrene). Symptoms include leg pain when walking (claudication).

[0114] An aneurysm can occur when atherosclerosis causes serious complications that can happen anywhere in your body. An aneurysm is a bulge in the wall of your artery. Aneurysms can be a medical emergency, and if they rupture, it can be a life-threatening event.

[0115] Chronic kidney disease can occur when atherosclerosis narrows the arteries, preventing the kidneys from receiving oxygenated blood. Over time, this can affect the kidneys' ability to prevent waste products from leaving the body. Symptoms include high blood pressure or kidney failure.

[0116] This invention provides a method for treating atherosclerosis by administering one or more glycine tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need, wherein the administration of the glycine-containing tripeptide molecule treats atherosclerosis. In one embodiment, the method is for treating atherosclerosis by administering the glycine-containing tripeptide molecule Gly-Gly-Leu and / or Gly-Gly-dLeu to a subject. This invention also provides a method for treating complications of atherosclerosis by administering the glycine-containing tripeptide molecule to a subject suffering from said complications to treat myocardial infarction, coronary artery disease, carotid artery disease, peripheral artery disease, aneurysm, or chronic kidney disease. In one embodiment, the method for treating complications of atherosclerosis is to treat myocardial infarction, coronary artery disease, carotid artery disease, peripheral artery disease, aneurysm, or chronic kidney disease by administering the glycine-containing tripeptide molecule Gly-Gly-Leu or Gly-Gly-dLeu to a subject suffering from said complications.

[0117] One method for treating inflammation in adipose tissue and circulation involves administering one or more glycine tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need, wherein the administration of the tripeptide molecules reduces inflammation.

[0118] Methods for treating inflammation in adipose tissue and circulation include administering Gly-Gly-Leu and / or Gly-Gly-dLeu or pharmaceutically acceptable saline to subjects in need, wherein the inflammation in circulation is reduced by lowering plasma MCP1 levels. One method for treating inflammation in adipose tissue, wherein the inflammation is located in epididymal adipose tissue (EAT) or subcutaneous adipose tissue (SAT), includes administering Gly-Gly-Leu and / or Gly-Gly-dLeu to subjects in need, and a reduction in MCP1 mRNA levels.

[0119] A method of treating a subject to reduce plasma levels of glycine tripeptide molecules includes administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to the subject in need, wherein the administration of the tripeptide molecules reduces plasma levels of glycine tripeptide molecules.

[0120] One method of treating a subject to reduce plasma levels of glycine tripeptide molecules includes administering the tripeptide Gly-Gly-Leu or Gly-Gly-dLeu to the subject in need.

[0121] A method of treating a subject to lower post-meal glucose includes administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to the subject in need. The method of treating a subject to lower post-meal glucose includes administering the glycine tripeptide Gly-Gly-Leu and / or Gly-Gly-dLeu to the subject in need.

[0122] One method of treating a subject involves administering Gly-Gly-Leu and / or Gly-Gly-dLeu to a subject in need, wherein the subject has liver disease.

[0123] Methods of treating subjects include administering Gly-Gly-Leu and / or Gly-Gly-dLeu to subjects in need of treatment, wherein the subjects have liver disease, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) or alcoholic hepatic steatosis.

[0124] A method for stabilizing or reducing a subject's NAFDL activity score (NAS) includes administering to the subject a therapeutically effective amount of a composition comprising Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

[0125] Methods for stabilizing or reducing a subject's NAFDL activity score (NAS) include administering to the subject a therapeutically effective amount of a composition comprising Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof, wherein the method includes slowing the progression of the steatotic component of NAS, stabilizing or reducing the steatotic component of NAS. The method includes slowing the progression of the lobular inflammatory component of NAS, stabilizing or reducing the lobular inflammatory component of NAS. The method includes slowing the progression of the hepatocellular ballooning degeneration component of NAS, stabilizing or reducing the hepatocellular ballooning degeneration component of NAS.

[0126] In any of the methods for stabilizing or reducing NAFLD activity scores, the difference in NAS is no more than 1.5 points after 6 months of treatment with a therapeutically effective amount of a composition comprising Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

[0127] A method for alleviating liver fibrosis in a subject in need includes administering to the subject a therapeutically effective amount of a composition comprising Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

[0128] A method for reducing plasma fibrinogen levels in a subject in need includes administering to the subject a therapeutically effective amount of a composition comprising Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

[0129] In a method for reducing plasma fibrinogen levels in subjects in need, the subject's fibrinogen level is greater than 300 mg / dL before administering a therapeutically effective amount of a composition comprising Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

[0130] One embodiment of the present invention is a pillbox for treating NAFLD, the pillbox comprising DT-109, an optional statin, and instructions for use. Another embodiment of the present invention is a pillbox for treating NASH, the pillbox comprising DT-109, an optional statin, and instructions for use. One embodiment of the present invention is a pillbox for treating NAFLD, the pillbox comprising DT-110, an optional statin, and instructions for use. Another embodiment of the present invention is a pillbox for treating NASH, the pillbox comprising DT-110, an optional statin, and instructions for use.

[0131] This invention provides a method for alleviating liver fibrosis in a patient, the method comprising administering DT-109 or DT-110 or a pharmaceutically acceptable salt thereof. One embodiment is a method for alleviating liver fibrosis in a subject in need, the method comprising administering DT-109 or DT-110 to the subject. Another embodiment is a method for alleviating liver fibrosis in a subject in need, the method comprising administering DT-109 or DT-110 to the subject, wherein the subject suffers from NASH.

[0132] Fibrinogen (factor I) is a mammalian glycoprotein that plays a role in blood clot formation. During blood clot formation, fibrinogen is converted into fibrin by thrombin. Fibrinogen is synthesized in hepatocytes. Therefore, fibrinogen can be a prognostic indicator or blood marker for many diseases and can also be used to influence the onset and progression of disease states.

[0133] In various implementation schemes, a method for treating at least one of hyperlipidemia, fatty liver, steatohepatitis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, hyperglycemia, metabolic syndrome, cardiovascular disease, and atherosclerosis in mammalian subjects comprises administering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to the subject in need.

[0134] In each implementation scheme, the glycine-containing tripeptide molecule significantly reduced triglyceride levels in liver lipids, while having no significant effect on the leucine-negative control.

[0135] In all implementation schemes, the glycine-containing tripeptide molecule significantly reduced the total cholesterol level in liver lipids, while having no significant effect on the leucine negative control.

[0136] In various embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0137] This invention provides a method for enhancing hepatic lipid oxidation or utilization, reducing triglyceride levels, or treating hypercholesterolemia in a subject in need. The method comprises administering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to a subject in need, wherein hepatic lipid oxidation, triglyceride levels, hypercholesterolemia, or any combination thereof is improved as a result of treatment. In one relevant aspect of these embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0138] In one relevant aspect of these implementation schemes, the glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof significantly induced the expression of the regulators of hepatic lipid oxidation, AMPKα1 or PPARα, which had no effect on the leucine negative control.

[0139] In one relevant aspect of these implementations, the glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof regulate triglyceride hydrolysis by significantly upregulating CPT1a, CACT, or ACADI (mitochondrial β-oxidation) or PNPLA2.

[0140] In one relevant aspect of these implementations, the glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof regulate triglyceride hydrolysis by significantly upregulating the mitochondrial anion carrier UCP2.

[0141] In one relevant aspect of these implementation schemes, glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof regulate cholesterol homeostasis in the liver by significantly increasing the expression of ABCG5 and ABCG8.

[0142] In one relevant aspect of these implementation methods, plasma lipid profile analysis of the subject includes administering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to the subject in need to reduce plasma triglyceride, plasma LDL levels, or atherosclerotic plaques. In one relevant aspect of these implementation methods, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0143] In one relevant aspect of these implementation schemes, Gly-Gly-Leu, Gly-Gly-dLeu, glycine tripeptide molecules, or pharmaceutically acceptable salts thereof reduce atherosclerotic plaques.

[0144] In one relevant aspect of these implementation schemes, Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, reduces plasma total cholesterol, plasma LDL, or a combination thereof.

[0145] This disclosure provides a method for treating inflammation in adipose tissue and circulation in a subject requiring such treatment. The method includes administering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to the subject in need, wherein administration of the glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof reduces inflammation in the subject's adipose tissue and circulation. In one related aspect of these embodiments, inflammation in circulation is reduced by administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to the subject to lower plasma MCP1 levels. In one related aspect of these embodiments, the inflammation in adipose tissue is located in epididymal adipose tissue (EAT) or subcutaneous adipose tissue (SAT), and the level of MCP1 mRNA is reduced.

[0146] This disclosure provides a method for treating a subject to reduce plasma leptin levels in a subject in need. In some embodiments, the method includes administering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to the subject in need, wherein administration of the glycine-containing tripeptide molecule reduces plasma leptin levels. In a related aspect of these embodiments, the treatment uses Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0147] This disclosure provides a method for treating a subject to reduce post-dietary glucose in a subject in need. The method includes administering to the subject in need a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof.

[0148] In one relevant aspect of these implementation schemes, treatment of the subject includes administering a therapeutically effective amount of a glycine tripeptide molecule, such as Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0149] This disclosure provides a method for treating a subject who has or may develop liver disease (e.g., liver disease characterized by excessive cholesterol, triglycerides, or other lipids in the liver, such as NAFLD, NASH, or alcoholic steatosis, cirrhosis, or hepatitis). The method includes administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to the subject in need to treat or prevent liver disease.

[0150] In one relevant aspect of these implementation schemes, the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or alcoholic hepatic steatosis.

[0151] In one aspect of these embodiments, this disclosure provides a method for stabilizing or reducing a subject's NAFDL activity score (NAS), wherein the method comprises administering to the subject a therapeutically effective amount of a glycine tripeptide having the amino acid sequence Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. In one aspect of these embodiments, the method comprises slowing the progression of the steatotic component of NAS, stabilizing or reducing the steatotic component of NAS. In one aspect of these embodiments, the method comprises slowing the progression of the lobular inflammatory component of NAS, stabilizing or reducing the lobular inflammatory component of NAS. In one aspect of these embodiments, the method comprises slowing the progression of the hepatocellular ballooning component of NAS, stabilizing or reducing the hepatocellular ballooning component of NAS. In one aspect of these embodiments, after 6 months of treatment with Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, the NAS difference is no more than 1.5 points.

[0152] This disclosure provides a method for alleviating liver fibrosis in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a glycine tripeptide molecule: Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0153] This disclosure provides a method for treating atherosclerosis, the method comprising administering to a subject in need a therapeutically effective amount of a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof. In one relevant aspect of these embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0154] This disclosure provides a method for treating complications of atherosclerosis by administering to a subject suffering from the said complications a therapeutically effective amount of a glycine-containing tripeptide molecule for treating complications selected from the group consisting of myocardial infarction, arteriosclerosis, coronary artery disease, carotid artery disease, peripheral artery disease, atherosclerotic thrombotic stroke, aneurysm, or chronic kidney disease.

[0155] This disclosure also covers the treatment of such exemplary metabolic diseases, cardiovascular diseases, and inflammatory diseases by administering a combination of a glycine-containing tripeptide molecule with a second therapeutic agent used to treat metabolic diseases, cardiovascular diseases, and inflammatory diseases. In some embodiments, the methods disclosed above further include administering a second therapeutic agent selected from the following to a subject in need: cholesterol absorption inhibitors, PCSK9 inhibitors, PPAR-α agonists, ACE inhibitors, calcium channel blockers, ARBs, renin, GLP-1 or synthetic variants thereof, insulin or synthetic variants thereof, metformin, sulfonylurea compounds, thiazolidinediones (TZDs), PCSK9 inhibitors, SGLT2 inhibitors, DPP-IV inhibitors, HMGCoA reductase inhibitors, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, etimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gemcabene (CI-1027), benpodoic acid. Fish oil (ETC-1002), ACC inhibitors, ApoC-III inhibitors, ACL inhibitors, prescription fish oils, CETP inhibitors, antifibrotic agents, and combinations thereof.

[0156] Some embodiments provide a kit for treating, preventing, or improving one or more of the metabolic diseases and / or cardiovascular diseases and / or inflammatory diseases as described herein, wherein the kit comprises: a) a glycine-containing tripeptide molecule as described herein; and optionally b) another dose or therapy as described herein. The kit may also include instructions or labeling for using the kit to treat, prevent, or improve one or more of the metabolic diseases and / or cardiovascular diseases and / or inflammatory diseases. In one relevant aspect of these embodiments, the kit is for treating a subject with NAFLD or NASH, and the kit includes a selected tripeptide, optionally a statin, and instructions for use. In one relevant aspect of these embodiments, the kit includes DT-109 (Gly-Gly-Leu) and / or DT-110 (Gly-Gly-dLeu), optional statins, and instructions for use. In another relevant aspect of these embodiments, the kit may also optionally contain cholesterol absorption inhibitors, PCSK9 inhibitors, PPAR-α agonists, ACE inhibitors, calcium channel blockers, ARBs, renin, GLP-1 or synthetic variants thereof, insulin or synthetic variants thereof, metformin, sulfonylurea compounds, thiazolidinediones (TZDs), PCSK9 inhibitors, SGLT2 inhibitors, DPP-IV inhibitors, statins, HMGCoA reductase (proprotein convertase / kexin), and other enzymes. Inhibitors of type 9 (ApoC-III), etimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gecarbene (CI-1027), bepacidic acid (ETC-1002), ACC inhibitors, ApoC-III inhibitors, ACL inhibitors, prescription fish oils, CETP inhibitors, antifibrotic agents, and combinations thereof. In one relevant aspect of these embodiments, the kit optionally contains etimibe.

[0157] preparation

[0158] As used herein, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, such as humans. The term "carrier" refers to a diluent, adjuvant, excipient, stabilizer, or medium formulated with the said agent for administration. Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a typical carrier for intravenous administration of pharmaceutical compositions. Saline solutions and aqueous solutions of dextran and glycerol can be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, powder, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. The composition may also contain trace amounts of wetting agents, emulsifiers, or pH buffers, if desired. Pharmaceutical compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. They can also be formulated into suppositories using conventional binders and carriers (such as triglycerides).

[0159] The pharmaceutical compositions of the present invention comprise a tripeptide molecule of glycine or a pharmaceutically acceptable salt thereof, and a mixture of at least one pharmaceutically acceptable excipient, carrier, or diluent. The pharmaceutically acceptable compositions contain one or more formulation materials for modifying, maintaining, or retaining properties of the composition, such as pH, osmotic pressure, viscosity, clarity, color, isotropic properties, odor, sterility, stability, dissolution or release rate, adsorption, or penetration. Suitable formulation materials include, but are not limited to, amino acids (such as glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris...). HCl, citrates, phosphates, other organic acids); compatibilizers (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); compounding agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight peptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, lochcedin). Exidine, sorbic acid, or hydrogen peroxide; solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, dehydrated sorbitol esters, polysorbate esters (such as polysorbate ester 20, polysorbate ester 80), triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); tension enhancers (such as alkali metal halides (in one aspect, sodium chloride or potassium chloride, mannitol or sorbitol); delivery media; diluents; excipients and / or adjuvants. (Remington's Pharmaceutical Sciences, 18th edition, edited by ARGennaro, Mack Publishing Company, 1990).

[0160] In one aspect of the invention, glycine-containing tripeptide molecules are supplied in the form of a single-use glass vial containing a lyophilized cake, said lyophilized cake being prepared in a formulation buffer consisting of 10 mM glutamic acid, 2% glycine, 1% sucrose, and 0.01% polysorbate 20 at pH 4.25. After reconstitution with a volume of sterile diluent (e.g., sterile isotonic saline or water) (e.g., 0.5 mL to about 10 mL, e.g., 2.2 mL of sterile water), the cake yields glycine tripeptide molecules at a concentration of 1 g / mL to about 100 g / mL. The glycine-containing tripeptide molecules are stored in a safe location under controlled conditions. The drug is administered immediately after the single-use vial has been reconstituted (no more than three hours after reconstitution). The investigational drug is allowed to reach room temperature (15 to 30°C) before injection.

[0161] Dosage and administration

[0162] As used herein, a “dosage” is generally equal to a dose of the active ingredient that can be administered once daily or several times daily (e.g., a unit dose is a portion of the desired daily dose). In various embodiments, various dosages and administration regimens are suitable for glycine or glycine-containing tripeptide molecules disclosed herein. For example, dosages that can be used in this invention are calculated based on the subject’s weight as 5 mg / kg to 2,000 mg / kg, or 30 mg / kg to 1,500 mg / kg, or 40 mg / kg to 1,250 mg / kg, or 50 mg / kg to 1,000 mg / kg, or 60 mg / kg to 5,000 mg / kg. The term “unit dose” as used herein can be used to indicate a discrete amount of a therapeutic composition comprising a predetermined amount of the active compound. The amount of the active ingredient is generally equal to a dose of the active ingredient that can be administered once daily or several times daily (e.g., a unit dose is a portion of the desired daily dose). The unit dose can also be used to indicate the total daily dose, which can be administered once a day or in the form of a convenient portion of such a dose (e.g., the unit dose is the total daily dose that can be given in fractional increments such as half or one-third of the dose).

[0163] As used herein, the term "starting day dose" refers to the amount of glycine-containing tripeptide molecules administered or prescribed daily to a patient who has not previously undergone a glycine tripeptide molecule titration protocol and is beginning treatment with the glycine-containing tripeptide molecule. This amount may be administered in multiple unit doses or as a single unit dose, once or multiple times a day.

[0164] In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 1 mg / kg / day to about 10,000 mg / kg / day, from about 1 mg / kg / day to about 1,000 mg / kg / day, from about 0.1 mg / kg / day to about 1,000 mg / kg / day, from about 1 mg / kg / day to about 1,000 mg / kg / day, from about 1,000 mg / kg / day to about 10,000 mg / kg / day, or from about 1 mg / kg / day to about 500 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 3 mg / kg / day to about 70 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 7 mg / kg / day to about 40 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 3 mg / kg / day to about 50 mg / kg / day.

[0165] In some embodiments, the dosage may be from 1000 mg / day to 100 g / day, more preferably from 1000 mg / day to 75 g / day. The amount of the glycine-containing tripeptide molecule in the composition is preferably from about 50 mg to about 100 g, from about 100 mg to about 90 g, from about 500 mg to about 75 g, from about 600 mg to about 80 g, or from about 700 mg to about 75 g. In some embodiments, the amount of the glycine-containing tripeptide molecule in the composition may be from about 100 mg to about 1,000 g, from about 200 mg to about 500 g, from about 300 mg to about 100 g, from about 400 mg to about 75 g, from about 500 mg to about 50 g, from about 750 mg to about 25 g, from about 1,000 mg to about 50 g, or from about 1,000 mg to about 25 g.

[0166] In some embodiments, the therapeutically effective dose of the glycine-containing tripeptide molecule to be applied is from about 100 mg to about 200 g. This dose may be administered as a single daily dose or divided into several doses administered throughout the day, for example, 1 to 5 doses per day, preferably two or three doses. In some embodiments, the amount of the glycine-containing tripeptide molecule is from about 250 mg to about 100 g. In some embodiments, the amount of the glycine-containing tripeptide molecule is from about 500 mg to about 90 g. In some embodiments, the amount of the glycine-containing tripeptide molecule is from about 750 mg to about 75 g. In some embodiments, the amount of the glycine-containing tripeptide molecule is from about 1000 mg to about 50 g. In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is in a solid oral dosage form.

[0167] For glycine-containing tripeptide molecules, the composition may have a chiral purity of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity of the glycine-containing tripeptide molecule is 100%. In some embodiments, the composition has a chiral purity of 99.9% or greater for the glycine-containing tripeptide molecule. In some embodiments, the composition has a chiral purity of 99.95% or greater for the glycine-containing tripeptide molecule. In some embodiments, the composition has a chiral purity of 99.99% or greater for the glycine-containing tripeptide molecule.

[0168] In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the composition is a capsule. In some embodiments, the composition is a tablet. In some embodiments, the composition is formulated as an oral or parenteral solution.

[0169] For the sake of brevity, the embodiments concerning the amount of glycine-containing tripeptide molecules in the composition, chiral purity, and dosage form described separately herein can be combined in any suitable combination.

[0170] In another aspect, the present invention relates to compositions comprising glycine-containing tripeptide molecules that are chirally pure with respect to the glycine tripeptide molecule. In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 5 mg / kg / day to 5000 mg / kg / day, or from 5 mg / kg / day to about 2000 mg / kg / day, or from 30 mg / kg / day to 1500 mg / kg / day, or from 40 mg / kg / day to 1250 mg / kg / day, or from 50 mg / kg / day to 1000 mg / kg / day, or from 60 mg / kg / day to 500 mg / kg / day, calculated based on the subject's body weight. In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 10 mg / kg / day to about 1000 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule may be from about 7 mg / kg / day to about 900 mg / kg / day. In some embodiments, the amount of glycine-containing tripeptide molecules may be from about 5 mg / kg / day to about 800 mg / kg / day. In some embodiments, the dosage may be from 10 mg / day to 5,000 mg / day, more preferably from 100 mg / day to 2,000 mg / day. In some embodiments, the composition is administered at a dosage of about 500 mg to about 100 g, about 1,000 mg to about 75 g, about 1,500 mg to about 50 g, or about 2,000 mg to about 25 g of glycine tripeptide molecules. In some embodiments, the composition is administered at a dosage of about 250 mg to about 500 g, about 500 mg to about 250 g, about 750 mg to about 200 g, about 1,000 mg to about 100 g, about 1,250 mg to about 75 g, about 1,500 mg to about 50 g, about 2,000 mg to about 25 g, or about 2,500 mg to about 20 g. In some embodiments, the effective amount of a glycine-containing tripeptide molecule administered to a subject in need for the prevention or treatment of metabolic and / or cardiovascular and / or inflammatory diseases may range from about 500 mg to about 200 g. This dosage may be administered as a single daily dose or divided into several doses administered throughout the day, for example, 1 to 5 doses per day, preferably two to three doses per day. These doses of the glycine-containing tripeptide molecule are preferably contained in a formulation having a chemical purity of 97% or greater and a chiral purity of 99.6% or greater, 99.7% or greater, 99.8% or greater, 99.9% or greater, preferably 99.95% or greater, and more preferably 99.99% or greater for the glycine tripeptide molecule. In a preferred embodiment, the composition containing the glycine-containing tripeptide molecule may have 100% chiral purity for the glycine-containing tripeptide molecule. The composition may also contain a carrier. The compositions of the present invention are preferably administered orally as a solid oral dose and more preferably as a solid oral dose that may be in the form of capsules or tablets. In a preferred embodiment, the composition of the present invention can be formulated into tablets for oral administration.

[0171] In another aspect, the present invention also provides a composition comprising a therapeutically effective amount of a glycine tripeptide molecule. The composition may further comprise a pharmaceutically acceptable carrier.

[0172] In some implementations, the therapeutically effective dose of the glycine-containing tripeptide molecule may be about 1 mg / kg / day to about 10,000 mg / kg / day, about 5 mg / kg / day to about 5,000 mg / kg / day, about 20 mg / kg / day to about 1,000 mg / kg / day, about 30 mg / kg / day to about 1,000 mg / kg / day, about 50 mg / kg / day to about 10,000 mg / kg / day, or about 100 mg / kg / day to about 5,000 mg / kg / day.

[0173] In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule can be from about 5 mg / kg / day to about 5,000 mg / kg / day. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule can be from about 10 mg / kg / day to about 4,000 mg / kg / day. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule can be from about 25 mg / kg / day to about 2,000 mg / kg / day. In some embodiments, the dose can be from 10 mg / day to 1,000 g / day, more preferably from 500 mg / day to 100 g / day. The therapeutically effective amount of the glycine-containing tripeptide molecule in the composition is preferably from about 250 mg to about 500 g, from about 500 mg to about 400 g, from about 750 mg to about 200 g, or from about 1,000 mg to about 100 g. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule in the composition may be about 300 mg to about 1,000 g, about 500 mg to about 500 g, about 600 mg to about 400 g, about 700 mg to about 300 g, about 800 mg to about 200 g, about 900 mg to about 150 g, or about 1,000 mg to about 100 g. In some embodiments, the amount of the glycine-containing tripeptide molecule is about 600 mg to about 300 g. This dosage may be administered as a single daily dose or divided into several doses administered throughout the day, for example, 1 to 5 doses per day, preferably two to three doses per day. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule is about 500 mg to about 350 g. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule is about 750 mg to about 250 g. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule is about 1,000 mg to about 150 g. In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule is about 1,500 mg to about 100 g. In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the composition is a liquid oral dosage form. In some embodiments, the composition is a liquid parenteral dosage form.

[0174] In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the composition is a capsule. In some embodiments, the composition is a tablet.

[0175] In another aspect, (in one aspect) beneficial effects of glycine tripeptide molecules or their pharmaceutically acceptable salts on liver function were observed through favorable alterations in the expression of messenger RNA or proteins of hepatic lipid peroxidation, AMPKα1, glucoskinase, peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptor-γ, PPARγ coactivator 1, pyruvate kinase, sterol regulatory element-binding protein-1c, long-chain and very long-chain acyl-CoA dehydrogenases, or stearoyl-CoA desaturases. In another aspect, beneficial effects of glycine tripeptide molecules or their pharmaceutically acceptable salts on liver function were observed through improvements in the expression of messenger RNA or proteins of phosphoenolpyruvate kinase, microsomal transfer protein, arylacetamide deacetylase, apolipoprotein C2, carnitine palmitoyltransferase II, or phospholipase D1.

[0176] The glycine tripeptide molecule of the present invention or its pharmaceutically acceptable salt can be administered via any suitable route. For example, the compositions of the present invention can be administered orally, through the eyes, intradermally, intraperitoneally, intranasally, subcutaneously, intramuscularly, or intravenously.

[0177] Suitable formulations for oral administration include, for example, solid, semi-solid, and liquid systems, such as tablets; soft or hard capsules containing multiply or nanoparticles, liquids, or powders; sugar tablets (including liquid-filled tablets); chewables; gels; rapidly dispersible formulations; thin films; beads; and sprays. In some embodiments, the peptides of the present invention are formulated for oral administration using delivery media known in the art, including but not limited to microspheres, liposomes, enteric-coated dry emulsions, or nanoparticles.

[0178] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to active peptides, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0179] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active peptide is mixed with at least one inert, pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and / or the following: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) dissolution inhibitors, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. The active compound may also be in the form of microencapsulation using one or more excipients as mentioned above. Similar types of solid compositions may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose (or milksugar) and high molecular weight polyethylene glycol, poloxamer, etc. Solid dosage forms of tablets, sugar-coated pills, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings known in pharmaceutical formulation techniques. Injectable formulations may be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. Sterile injectable formulations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable media and solvents, water, Ringer's solution (USP), and isotonic sodium chloride solutions may be used. In addition, sterile, fixed oils are routinely used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid are used in the preparation of injectable formulations. Injectable formulations can be sterilized, for example, by filtration through a bacterial trap or by incorporating a sterilizing agent into a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0180] Those skilled in the art will understand that the amount of glycine-containing tripeptide molecules administered for therapeutic purposes varies. In one aspect, compositions containing glycine tripeptide molecules or pharmaceutically acceptable salts thereof are substantially free of contaminants, with a contamination level of less than 0.02% (w / w). For example, glycine-containing tripeptide molecule compositions suitable for injection into patients are prepared by reconstitution of a lyophilized sample comprising purified glycine-containing tripeptide molecules and a stable salt with a pharmaceutically acceptable diluent. As discussed in detail below, administration of glycine-containing tripeptide molecule compositions is alternatively systemic or local, and includes administration of a therapeutically effective amount of a glycine-containing tripeptide molecule protein composition.

[0181] Combination therapy and compositions

[0182] Combination therapies are specifically covered, in addition to therapies based solely on the delivery of compositions containing glycine tripeptide molecules or pharmaceutically acceptable salts thereof. In the context of this invention, it is contemplated that glycine-containing tripeptide molecule therapies be used in combination with other agents commonly used to treat metabolic diseases (e.g., fatty liver disease).

[0183] In various embodiments, one or more second therapeutic agents may be combined with a glycine tripeptide molecule, particularly for synergistic enhancement of activity. Administration of the active ingredients may be performed by administering the active ingredient alone to the patient or as a combination product in which multiple active ingredients are present in a pharmaceutical formulation. It is anticipated that when used as a single agent, the second therapeutic agent can be administered at an approved dose, or initially at a therapeutically effective concentration or at a subtherapeutically effective concentration, such that when administered in combination, the combination of the second therapeutic agent with the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof provides a therapeutically effective outcome in the patient being treated.

[0184] To achieve appropriate therapeutic outcomes using the methods and compositions of the present invention, compositions comprising a glycine-containing tripeptide molecule and at least one or more other therapeutic agents (one or more second therapeutic agents) may be provided. In the present invention, the second therapeutic agent is contemplated to be selected from pramlinitide, peptide YY (PYY), exanatide, or insulin sensitizers (including, but not limited to, thiazolidinediones or metformin), or glimepiride, and analogues of any of these compounds. Other second therapeutic agents for controlling dyslipidemia, diabetes, and cardiovascular diseases associated with atherosclerosis are well known in the art and can be used in combination with the glycine-containing tripeptide molecule of the present invention.

[0185] Combination therapy compositions are provided in combination amounts that are effective in producing desired therapeutic outcomes in the treatment of metabolic diseases, such as those associated with one or more of the following: glucose metabolism disorders; dyslipidemia, such as dyslipidemia associated with increased cholesterol (pure or independent hypercholesterolemia) and / or increased triglycerides (TG) only (pure or independent hypertriglyceridemia) and / or increased cholesterol and TG (mixed or combined hyperlipidemia); fatty liver disease, including NAFLD, NASH; steatosis, hepatitis, peripheral artery disease, coronary artery disease, atherosclerosis, systemic inflammation, and ischemic stroke associated with plaque-associated thrombosis. This method involves simultaneously administering to a subject in need a therapeutically effective amount of a first therapeutic agent of the invention (i.e., a composition comprising a therapeutically effective amount of a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof) and optionally one or more second therapeutic agents or factors. This is achieved by administering a single composition or pharmacological formulation comprising two therapeutic agents or by simultaneously administering two distinct compositions or formulations, wherein one composition comprises a glycine-containing tripeptide molecule therapeutic composition, and the other comprises a second therapeutic agent.

[0186] Antidiabetic drugs that can be used as a second therapeutic agent in combination with glycine-containing tripeptide molecules may include insulin and insulin derivatives (such as...) Or HMR 1964 or (Insulin detemir or those described in WO2005005477 (Novo Nordisk)), rapid-acting insulin (see U.S. Patent No. 6,221,633), inhalable insulin (such as...) Or oral insulin (such as IN-105 (Nobex) or Oral-lyn) TM(Generex Biotechnology), GLP-1 derivatives and GLP-1 agonists (such as exenatide, liraglutide or Novo Nordisk) Those disclosed in WO98 / 08871, WO2005027978, WO2006037811 or WO2006037810 of A / S, WO01 / 04156 of New Zealand or WO00 / 34331 of Beaufour-Ipsen, as well as those described in Symlin (Amylin Pharmaceuticals), BIM-51077, PC-DAC-exendin-4 (a sialin-4 analog covalently bonded to recombinant human albumin), such as those described in D. Chen et al., Proc. Natl. Acad. Sci. USA 104 (2007) 943, agonists such as those described in WO2006124529, and orally effective hypoglycemic active ingredients.

[0187] Antidiabetic drugs also include agonists of glucose-dependent insulinotropic peptide (GIP) receptors, such as those described in WO2006121860, as well as GLP-1 and GLP-2, and their synthetic variants and mutants, for controlling blood glucose and treating diabetes (including type II diabetes).

[0188] Exemplary second therapeutic agents that can be used in combination with the glycine-containing tripeptide molecules disclosed herein may include orally effective hypoglycemic active ingredients, preferably sulfonylureas, biguanides, meglitinide, oxadiazolidinediones, thiazolidinediones, glucosidase inhibitors, glycogen phosphorylation inhibitors, glucagon antagonists, glucoskinase activators, fructose-1,6-bisphosphatase inhibitors, glucose transporter 4 (GLUT4) regulators, glutamine-fructose-6-phosphatamidotransferase (GFAT) inhibitors, GLP-1 agonists, potassium channel openers (such as pinacidil, cromakalim, diazoxide, or RDCarr, etc., Diabetes 52, 2003, 2513, 2518, JB Hansen et al., Current Medicinal Chemistry), Those described in 11, 2004, 1595-1615, TMTagmose et al., J. Med. Chem. 47, 2004, 3202-3211, or MJ Coghlan et al., J. Med. Chem. 44, 2001, 1627-1653, or Novo Nordisk A / S WO 97 / 26265 and WO Those disclosed in 99 / 03861), inhibitors of dipeptidyl peptidase IV (DPP-IV), insulin sensitizers, inhibitors of liver enzymes involved in stimulating gluconeogenesis and / or glycogenolysis, regulators of glucose uptake, glucose transport and glucose reabsorption, inhibitors of 11β-HSD1, inhibitors of protein tyrosine phosphatase 1B (PTP1B), regulators of sodium-dependent glucose transporter 1 or 2 (SGLT1, SGLT2), compounds that alter lipid metabolism (such as anti-hyperlipidemic and lipid-lowering active ingredients), compounds that reduce food intake, compounds that increase thermogenesis, PPAR and RXR regulators, and active ingredients that act on ATP-dependent potassium channels in β cells.

[0189] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an HMGCoA reductase inhibitor such as simvastatin, fluvastatin, pravastatin, lovastatin, atorvastatin, cerivastatin, rovastatin, or L-659699.

[0190] In one embodiment of the invention, a glycine-containing tripeptide molecule is combined with cholesterol absorption inhibitors such as etimibe, tiqueside, pamaqueside, FM-VP4 (sitostanol / cream ester ascorbate phosphate; Forbes Medi-Tech, WO2005042692, WO2005005453), MD-0727 (Microbia Inc., WO2005021497, WO2005021495), or with cholesterol absorption inhibitors such as WO2002066464, WO2005000353 (Kotobuki Pharmaceutical Co. Ltd.), WO2005044256, WO2005062824 (Merck & Co.), or WO2005061451 and WO2005061452 (AstraZeneca). AB) and WO2006017257 (Phenomix) or WO2005033100 (Lipideon Biotechnology AG) as described or as WO2004097655, WO2004000805, WO2004000804, WO2004000803, WO2002050068, WO2002050060, WO2005047248, WO2006086562, WO2006102674, WO2006116499, WO2006121861, W The compounds described in O2006122186, WO2006122216, WO2006127893, WO2006137794, WO2006137796, WO2006137782, WO2006137793, WO2006137797, WO2006137795, WO2006137792, and WO2006138163 are applied in combination.

[0191] In one embodiment of the invention, a glycine-containing tripeptide molecule is combined with Vytorin. TM (A fixed combination of etimibe and simvastatin) is administered in combination.

[0192] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of etimibe and atorvastatin.

[0193] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of etimibe and fenofibrate.

[0194] In another embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of fenofibrate and rovastatin.

[0195] In one embodiment of the present invention, a glycine-containing tripeptide molecule is combined with... (A fixed combination of fenofibrate and metformin) is administered in combination.

[0196] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with ISIS-301012 (an antisense oligonucleotide capable of regulating the apolipoprotein B gene).

[0197] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a PPARγ agonist such as rosiglitazone, pioglitazone, JTT-501, GI 262570, R-483, CS-011 (rivoglitazone).

[0198] In one embodiment of the invention, a glycine-containing tripeptide molecule is combined with Competact TM (A fixed combination of pioglitazone hydrochloride and metformin hydrochloride) is administered in combination.

[0199] In one embodiment of the invention, a glycine-containing tripeptide molecule is combined with Tandemact. TM (A fixed combination of pioglitazone and glimepiride) is administered in combination.

[0200] In one embodiment of the invention, a fixed combination of a glycine-containing tripeptide molecule and pioglitazone hydrochloride and an angiotensin II receptor antagonist (such as TAK-536) is administered.

[0201] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with PPARα agonists such as GW9578, GW-590735, K-111, LY-674, KRP-101, DRF-10945, LY-518674, or those described in WO2001040207, WO2002096894, WO2005097076.

[0202] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a mixture of naveglitazar, LY-510929, ONO-5129, E-3030, AVE 8042, AVE 8134, AVE 0847, CKD-501 (rosiglitazone sulfate) or PPARα / γ agonists such as WO 00 / 64888, WO 00 / 64876, WO03 / 020269 or JPBerger, TRENDSin Pharmacological Sciences 28(5), 244-251, 2005.

[0203] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a PPARδ agonist such as GW-501516 or as described in WO2006059744, WO2006084176, WO2006029699, WO2007039172-WO2007039178.

[0204] In one implementation, the glycine-containing tripeptide molecule is administered in combination with metataglidasen or with MBX-2044 or other partial PPARγ agonists / antagonists.

[0205] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with fibrates such as fenofibrate, clofibrate, or bezafibrate.

[0206] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an MTP inhibitor such as impitapide, BMS-201038, R-103757, AS-1552133, or WO2005085226, WO2005121091, WO2006010423.

[0207] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with CETP inhibitors such as tocetrapib, JTT-705, or those described in WO2006002342, WO2006010422, WO2006012093, WO2006073973, WO2006072362, WO2006097169, and WO2007041494.

[0208] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with bile acid absorption inhibitors such as HMR 1741 or those described in DE 102005 033099.1 and DE 10 2005 033100.9, WO2007009655-56 (see, for example, U.S. Patent Nos. 6,245,744, 6,221,897, or WO00 / 61568).

[0209] In one embodiment of the invention, a glycine-containing tripeptide molecule is applied in combination with a polymeric cholic acid adsorbent such as cholestyramine or colesevelam.

[0210] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an LDL receptor inducer such as HMR1171, HMR1586, or those described in WO2005097738 (see U.S. Patent No. 6,342,512).

[0211] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an ABCA1 expression enhancer as described, for example, in WO2006072393.

[0212] In another embodiment of the invention, the glycine-containing tripeptide molecule is administered in combination with an inhibitor of PCSK9 (proprotein convertase subtilisin / kexin 9) (e.g., evolocumab, inclisiran) or an RNAi therapeutic agent targeting PCSK9. In another embodiment of the invention, the glycine-containing tripeptide molecule is administered in combination with an antibody targeting PCSK9, such as alirocumab (Praluent) administered at 75-150 mg every two weeks; and evolocumab (Repatha) administered at 140 mg every two weeks or 420 mg monthly.

[0213] In one embodiment, a glycine-containing tripeptide molecule is combined with... (A combination of highly concentrated ethyl esters of ω-3 fatty acids, eicosapentaenoic acid, and docosahexaenoic acid)

[0214] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an ACAT inhibitor such as avasimibe or SMP-797.

[0215] In one embodiment of the invention, a glycine-containing tripeptide molecule is applied in combination with an antioxidant such as OPC-14117, probucol, tocopherol, ascorbic acid, β-carotene, or selenium.

[0216] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a vitamin such as vitamin B6 or vitamin B12.

[0217] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a lipoprotein lipase regulator such as ibrolipim (NO-1886).

[0218] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an ATP citrate lyase inhibitor such as SB-204990.

[0219] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a squalene synthase inhibitor such as BMS-188494, TAK-475, or as described in WO2005077907, JP2007022943.

[0220] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a lipoprotein (a) antagonist such as gicocarbene (CI-1027).

[0221] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an agonist of GPR109A (HM74A receptor agonist; NAR agonist (nicotinic acid receptor agonist), such as nicotinic acid or extended-release nicotinic acid bound MK-0524A or compounds described in WO2006045565, WO2006045564, WO2006069242, WO2006124490, WO2006113150, WO2007017261, WO2007017262, WO2007017265, WO2007015744, WO2007027532.

[0222] In another embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with an agonist of GPR116, such as those described in WO2006067531 and WO2006067532.

[0223] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with a lipase inhibitor such as orlistat or cetilistat (ATL-962).

[0224] In one embodiment of the invention, a glycine-containing tripeptide molecule is administered in combination with insulin.

[0225] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a sulfonylurea such as tolbutamide, glibenclamide, glipizide, gliclazide, or glimepiride.

[0226] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with substances that enhance insulin secretion, such as KCP-265 (WO2003097064) or WO2007026761.

[0227] In one implementation, a glycine-containing tripeptide molecule is administered in combination with a glucose-dependent insulinotropic receptor (GDIR) agonist, such as APD-668.

[0228] In one implementation, a glycine-containing tripeptide molecule is administered in combination with a biguanide such as metformin.

[0229] In another embodiment, the glycine-containing tripeptide molecule is administered in combination with meglitinide, such as repaglinide, nateglinide, or mitiglinide.

[0230] In another embodiment, a tripeptide molecule containing glycine is administered in combination with miglitol and glitazone (e.g., pioglitazone hydrochloride) or rosiglitazone maleate, or glitazone in combination with glimepiride or metformin, or combinations thereof.

[0231] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with miglitol and an α-glucosidase inhibitor.

[0232] In one embodiment, a glycine-containing tripeptide molecule is applied in combination with a thiazolidinedione such as troglitazone, ciglitazone, pioglitazone, rosiglitazone, or a compound disclosed in WO 97 / 41097 of Dr. Reddy's Research Foundation (specifically, 5-[[4-[(3,4-dihydro-3-methyl-4-oxo-2-quinazolinylmethoxy]-phenyl]methyl]-2,4-thiazolidinedione).

[0233] In one implementation, a glycine-containing tripeptide molecule is administered in combination with an α-glucosidase inhibitor such as miglitol or acarbose.

[0234] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an active ingredient that acts on ATP-dependent potassium channels in β-cells (such as tolbutamide, glibenclamide, glipizide, glimepiride, or repaglinide).

[0235] In one embodiment, the glycine-containing tripeptide molecule is combined with more than one of the above-mentioned compounds, such as with sulfonylurea and metformin, sulfonylurea and acarbose, repaglinide and metformin, insulin and sulfonylurea, insulin and metformin, insulin and troglitazone, insulin and lovastatin, etc.

[0236] In one embodiment, the glycine-containing tripeptide molecule is administered in combination with glycogen phosphorylase inhibitors such as PSN-357 or FR-258900 or those described in WO2003084922, WO2004007455, WO2005073229-31 or WO2005067932.

[0237] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glucagon receptor antagonist such as A-770077, NNC-25-2504, or as described in WO2004100875 or WO2005065680.

[0238] In one embodiment, the glycine-containing tripeptide molecule is combined with, for example, LY-2121260 (WO2004063179), PSN-105, PSN-110, GKA-50, or, for example, WO2004072031, WO2004072066, WO2005080360, WO2005044801, WO2006016194, WO2006058923, WO2006112549, WO2006125972, WO2007017549, WO2007017649, WO2007007910, W The glucoskinase activators described in O2007007040-42, WO2007006760-61, WO2007006814, WO2007007886, WO2007028135, WO2007031739, WO2007041365, WO2007041366, WO2007037534, WO2007043638, WO2007053345, WO2007051846, WO2007051845, WO2007053765, and WO2007051847 are administered in combination.

[0239] In one implementation, a glycine-containing tripeptide molecule is administered in combination with a gluconeogenesis inhibitor such as FR-225654.

[0240] In one embodiment, the glycine-containing tripeptide molecule is administered in combination with those fructose-1,6-bisphosphatase (FBP) inhibitors described in CS-917 (MB-06322) or MB-07803 or WO2006023515, WO2006104030, WO2007014619.

[0241] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glucose transporter 4 (GLUT4) regulator such as KST-48 (D.-O. Lee et al.: Arzneim.-Forsch. Drug Res. 54(12), 835(2004)).

[0242] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an inhibitor of glutamine-fructose-6-phosphoamyltransferase (GFAT) as described, for example, in WO2004101528.

[0243] In one embodiment, a glycine-containing tripeptide molecule is combined with, for example, vildagliptin (LAF-237), sitagliptin (MK-0431), sitagliptin phosphate, saxagliptin (BMS-477118), GSK-823093, PSN-9301, SYR-322, SYR-619, TA-6666. TS-021, GRC-8200, GW-825964X, KRP-104, DP-893, ABT-341, ABT-279 or another salt thereof, or dipeptidyl peptidase IV (DPP-IV) inhibitors such as WO2003074500, WO2003106456, WO2004037169, WO200450658, WO2005058901, WO20050123 12. WO2005 / 012308, WO2006039325, WO2006058064, WO2006015691, WO2006015701, WO20060156 99. WO2006015700, WO2006018117, WO2006099943, WO2006099941, JP2006160733, WO200607175 2. The compounds described in WO2006065826, WO2006078676, WO2006073167, WO2006068163, WO2006090915, WO2006104356, WO2006127530, WO2006111261, WO2007015767, WO2007024993, and WO2007029086 shall be used in combination.

[0244] In one embodiment, a glycine-containing tripeptide molecule is combined with Janumet TM (A fixed combination of sitagliptin phosphate and metformin hydrochloride) is administered in combination.

[0245] In one embodiment, the glycine-containing tripeptide molecule is combined with, for example, BVT-2733, JNJ-25918646, INCB-13739, or, for example, WO200190090-94, WO200343999, WO2004112782, WO200344000, WO200344009, WO2004112779, WO2004113310, WO2004103980, WO2004112784, WO2003065983, WO2003104207, WO2003 104208, WO2004106294, WO2004011410, WO2004033427, WO2004041264, WO2004037251, WO2004056744, WO2004058730, WO200 4065351, WO2004089367, WO2004089380, WO2004089470-71, WO2004089896, WO2005016877, WO2005097759, WO2006010546, W O2006012227, WO2006012173, WO2006017542, WO2006034804, WO2006040329, WO2006051662, WO2006048750, WO2006049952, WO2006048331, WO2006050908, WO2006024627, WO2006040329, WO2006066109, WO2006074244, WO2006078006, WO2006106423 The combination of 11-β-hydroxysterol dehydrogenase 1 (11β-HSD1) inhibitors described in WO2006132436, WO2006134481, WO2006134467, WO2006135795, WO2006136502, WO2006138695, WO2006133926, WO2007003521, WO2007007688, US2007066584, WO2007047625, WO2007051811, and WO2007051810.

[0246] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an inhibitor of protein tyrosine phosphatase 1B (PTP1B) as described in, for example, WO200119830-31, WO200117516, WO2004506446, WO2005012295, WO2005116003, WO2005116003, WO2006007959, DE 10 2004060542.4, WO2007009911, WO2007028145, and WO2007081755.

[0247] In one embodiment, a glycine-containing tripeptide molecule is combined with substances such as KGA-2727, T-1095, SGL-0010, AVE 2268, and SAR. 7226 and sergliflozin or, for example, WO2004007517, WO200452903, WO200452902, PCT / EP2005 / 005959, WO2005085237, JP2004359630, WO2005121161, WO2006018150, WO2006035796, WO2006062224, WO2006058597, WO2006073197, WO2006080577, WO2006087997, WO2006108842, WO2007000445, WO2007014895, WO2007080170 or derived from ALHandlon in Expert The combination of sodium-dependent glucose transporter 1 or 2 (SGLT1, SGLT2) modulators described in Opin. Ther. Patents (2005) 15(11), 1531-1540.

[0248] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a regulator of GPR40, such as those described in WO2007013689 and WO2007033002.

[0249] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a modulator of GPR119b, such as that described in WO2004041274.

[0250] In one embodiment, the glycine-containing tripeptide molecule is administered in combination with a modulator of GPR119 as described in, for example, WO2005061489 (PSN-632408), WO2004065380, WO2007003960-62 and WO2007003964.

[0251] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with a modulator of GPR120.

[0252] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an inhibitor of hormone-sensitive lipase (HSL) and / or phospholipase as described, for example, in WO2005073199, WO2006074957, WO2006087309, WO2006111321, and WO2007042178.

[0253] In one embodiment, the glycine-containing tripeptide molecule is administered in combination with acetyl-CoA carboxylase (ACC) inhibitors such as those described in WO199946262, WO200372197, WO2003072197, WO2005044814, WO2005108370, JP2006131559, WO2007011809, WO2007011811, and WO2007013691.

[0254] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with a regulator of xanthine oxidoreductase (XOR).

[0255] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, such as those described in WO2004074288.

[0256] In one embodiment, the glycine-containing tripeptide molecule is administered in combination with an inhibitor of glycogen synthase kinase 3β (GSK-3β) as described in, for example, US2005222220, WO2005085230, WO2005111018, WO2003078403, WO2004022544, WO2003106410, WO2005058908, US2005038023, WO2005009997, US2005026984, WO2005000836, WO2004106343, EP1460075, WO2004014910, WO2003076442, WO2005087727 or WO2004046117.

[0257] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a serum / glucocorticoid-regulated kinase (SGK) inhibitor, such as that described in WO2006072354.

[0258] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an RUP3 receptor agonist, such as that described in WO2007035355.

[0259] In one implementation, a glycine-containing tripeptide molecule is administered in combination with a protein kinase Cβ (PKCβ) inhibitor such as ruboxistaurin.

[0260] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with an activator of the gene encoding ataxia-telangiectasia mutant (ATM) protein kinase, such as chloroquine.

[0261] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an endothelin A receptor antagonist such as avosentan (SPP-301).

[0262] In one embodiment, the glycine-containing tripeptide molecule is administered in combination with an "I-κB kinase" inhibitor (IKK inhibitor) as described, for example, WO2001000610, WO2001030774, WO2004022553 or WO2005097129.

[0263] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glucocorticoid receptor (GR) modulator as described, for example, in WO2005090336, WO2006071609, and WO2006135826.

[0264] In another embodiment, the glycine-containing tripeptide molecule is administered in combination with a CART regulator (see "Cocaine-amphetamine-regulated transcript influences energy metabolism, anxiety and gastric emptying in mice" Asakawa, A. et al.: Hormone and Metabolic Research (2001), 33(9), 554-558).

[0265] In another embodiment, the glycine-containing tripeptide molecule is administered in combination with the following: an NPY antagonist, such as naphthalene-1-sulfonic acid {4-[(4-aminoquinazoline-2-ylamino)methyl]cyclohexylmethyl}amide hydrochloride (CGP 71683A); an NPY-5 receptor antagonist, such as L-152804 or as described, for example, in WO2006001318; an NPY-4 receptor antagonist, as described, for example, in WO2007038942; an NPY-2 receptor antagonist, as described, for example, in WO2007038943; and peptide YY. 3-36 (PYY3-36) or similar compounds, such as CJC-1682 (PYY3-36 conjugated to human serum albumin via Cys34), CJC-1643 (a PYY3-36 derivative conjugated to serum albumin in vivo), or those described in WO2005080424, WO2006095166; derivatives of obesitin, such as those described in WO2006096847; CB1R (cannabinoid receptor 1) antagonists (such as rimonabant, SR147778, SLV-319, AVE-1625, MK-0364 or their salts, or such as, for example, EP 0656354, WO00 / 15609, WO2001 / 64632-64634, WO 02 / 076949, WO2005080345, WO2005080328, WO2005080343, WO2005075450, WO2005080357, WO200170700, WO2003026647-48, WO200302776, WO2003040107, WO2003007887, WO2003027069, US Patent No. 6,509,367, WO200132663, WO2003086288, WO2003087037, WO2004048317, WO2004058145, WO2003084930, WO2003084 943. WO2004058744, WO2004013120, WO2004029204, WO2004035566, WO20040 58249, WO2004058255, WO2004058727, WO2004069838, US20040214837, US200 40214855, US20040214856, WO2004096209, WO2004096763, WO2004096794, WO 2005000809, WO2004099157, US20040266845, WO2004110453, WO2004108728,WO2004000817, WO2005000820, US20050009870, WO200500974, WO2004111033-34, WO200411038-39, WO2005016286 , WO2005007111, WO2005007628, US20050054679, WO2005027837, WO2005028456, WO2005063761-62, WO2005061509 , WO2005077897, WO2006047516, WO2006060461, WO2006067428, WO2006067443, WO2006087480, WO2006087476, WO2 006100208, WO2006106054, WO2006111849, WO2006113704, WO2007009705, WO2007017124, WO2007017126, WO200701 The terms described in 8459, WO2007016460, WO2007020502, WO2007026215, WO2007028849, WO2007031720, WO2007031721, WO2007036945, WO2007038045, WO2007039740, US20070015810, WO2007046548, WO2007047737, WO2007084319, and WO2007084450 are as follows: Those compounds described above); cannabinoid receptor 1 / cannabinoid receptor 2 (CB1 / CB2) modulating compounds, such as those described in WO2007001939, WO2007044215, WO2007047737; MC4 agonists (e.g., 1-amino-1,2,3,4-tetrahydronaphthyl-2-carboxylic acid [2-(3a-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydropyrazolo[4,3-c]pyridin-5-yl)-1-(4-chlorophenyl)-2-oxoethyl]amide; (WO 01 / 91752)) or LB53280, LB53279, LB53278 or THIQ, MB243, RY764, CHIR-785, PT-141 or WO2005060985, WO2005009950, WO2004087159, WO200407 8717, WO2004078716, WO2004024720, US20050124652, WO2005051391, WO2004112793, WOUS20050222014, US20050176728, US20050164914,US20050124636, US20050130988, US20040167201, WO2004005324, WO2004037 797, WO2005042516, WO2005040109, WO2005030797, US20040224901, WO200501 921. WO200509184, WO2005000339, EP1460069, WO2005047253, WO2005047251, WO2005118573, EP1538159, WO2004072076, WO2004072077, WO2006021655-57, Those described in WO2007009894, WO2007015162, WO2007041061, WO2007041052; orexin receptor antagonists (e.g., 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthidium-4-ylurea hydrochloride (SB-334867-A) or those described, for example, in WO200196302, WO200185693, WO2004085403, WO2005075458 or WO2006067224); histamine H3 receptor agonists (e.g., 3-cyclohexyl-1-(4,4-dimethyl-1,4,6,7-tetrahydroimidazo[4,5-c]pyridin-5-yl)prop-1-one oxalate (WO 00 / 63208) or as described in WO200064884, WO2005082893, WO2006107661, WO2007003804, WO2007016496, WO2007020213); histamine H1 / histamine H3 modulators, such as betahistine and its dihydrochloride; CRF antagonists (e.g., [2-methyl-9-(2,4,6-trimethylphenyl)-9H-1,3,9-triazafluorene-4-yl]dipropylamine (WO00 / 66585)); CRF BP antagonists (e.g., urocortin); urocortin agonists; β-3 adrenergic receptor agonists, such as 1-(4-chloro-3-methanesulfonylmethylphenyl)-2-[2-(2,3-dimethyl-1H-indol-6-yloxy)ethylamino]ethanol hydrochloride (WO 01 / 83451); or solabegron (GW-427353) or N-5984 (KRP-204) or those described in JP2006111553, WO2002038543, WO2007048840-843; MSH (melanocyte-stimulating hormone) agonists; MCH (melanin-concentrating hormone) receptor antagonists (such as NBI-845, A-761, A-665798, A-798).ATC-0175, T-226296, T-71, GW-803430, or models such as WO2005085200, WO2005019240, WO2004011438, WO2004012648, WO2003015769, WO2004072025, WO2005070898, WO2005070925, WO2004039780 , WO2004092181, WO2003033476, WO2002006245, WO2002089729, WO2002002744, WO2003004 027, FR2868780, WO2006010446, WO2006038680, WO2006044293, WO2006044174, JP20061764 43. WO2006018280, WO2006018279, WO2006118320, WO2006130075, WO2007018248, WO20070 12661, WO2007029847, WO2007024004, WO2007039462, WO2007042660, WO2007042668, WO200 Compounds described in WO2007093508, WO2007093509, WO2007048802, JP2007091649; CCK-A agonists (such as {2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexylethyl)thiazolyl-2-ylcarbamoyl]-5,7-dimethylindol-1-yl}acetic acid trifluoroacetate (WO 99 / 15525), SR-146131 (WO 0244150) or SSR-125180 or those described in WO2005116034); mixed serotonergic and norepinephrine-dependent compounds (e.g., WO 00 / 71549); 5-HT receptor agonists, such as 1-(3-ethylbenzofuran-7-yl)piperazine oxalate (WO 99 / 15525), SR-146131 (WO 0244150), ... 01 / 09111); mixed dopamine / norepinephrine / acetylcholine reuptake inhibitors (e.g., tesofensine); 5-HT2C receptor agonists (such as lorcaserin hydrochloride (APD-356), BVT-933, or WO200077010, WO20077001-02, WO2005019180, WO2003064423, WO200242304, WO2005035533, WO2005082859, WO2006077025, etc.).Those described in WO2006103511); 5-HT6 receptor antagonists, such as E-6837 or BVT-74316, or those described, for example, in WO2005058858, WO2007054257; bufotin receptor agonists (BRS-3 agonists); galopeptide receptor antagonists; growth hormone (e.g., human growth hormone or AOD-9604); growth hormone-releasing compounds (6-benzylmethyloxy-1-(2-diisopropyl-aminoethylcarbamoyl)-3,4-dihydro-1H-isoquinoline-2-carboxylic acid tert-butyl ester (WO01 / 85695)); growth hormone secretion receptor antagonists (ghrelin antagonists), such as A-778193, or those described, for example in WO2005030734; TRH agonists (see, for example, EP 0 462). 884); uncoupling protein 2 or 3 regulators; leptin agonists (see, for example, Lee, Daniel W.; Leinung, Matthew C.; Rozhayskaya-Arena, Marina; Grasso, Patricia. Leptinagonists as a potential approach to the treatment of obesity. Drugs of the Future (2001), 26(9), 873-881); DA agonists (bromocriptine or doprexin); lipase / amylase inhibitors (e.g., WO4). 00 / 40569); Inhibitors of diacylglycerol O-acyltransferase (DGAT), such as BAY-74-4113 or, for example, US2004 / 0224997, WO2004094618, WO200058491, WO2005044250, WO2005072740, JP2005206492, WO2005013907, WO20060 Inhibitors of fatty acid synthase (FAS), such as C75 or those described in WO2004005277, as described in WO2006019020, WO2006064189, WO2006082952, WO2006120125, WO2006113919, WO2006134317, and WO2007016538; stearoyl-CoA. Inhibitors of δ9 desaturase (SCD1), such as those described in WO2007009236, WO2007044085, WO2007046867, WO2007046868, and WO20070501124; gastrin; oleoyl-estrogens or thyroid hormone receptor agonists or partial agonists, such as KB-2115 or WO20058279.The ones described in WO200172692, WO200194293, WO2003084915, WO2004018421, WO2005092316, WO2007003419, WO2007009913, and WO2007039125.

[0266] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with varenicline tartrate (a partial agonist of the α4-β2 nicotinic acetylcholine receptor).

[0267] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with trodusquemine.

[0268] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with a regulator of the SIRT1 enzyme.

[0269] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with dexamphetamine or amphetamine.

[0270] In another embodiment, the glycine-containing tripeptide molecule is administered in combination with fenfluramine or dexfenfluramine.

[0271] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with sibutramine.

[0272] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with mazindole or phenylbutyramine.

[0273] Alternatively, the glycine-containing tripeptide molecule treatment may be administered before or after the second dose, with intervals ranging from minutes to weeks. In embodiments where the second dose and the glycine-containing tripeptide molecule are administered separately, it will generally be ensured that the interval between each delivery time does not exceed a significant time period, allowing the second dose and the glycine-containing tripeptide molecule to still exert a favorable combined effect. In such cases, it is anticipated that the two modalities will be administered approximately 12–24 hours apart and, in one aspect, approximately 6–12 hours apart, with a delay of only approximately 12 hours being most preferred. In some cases, a longer time period is required for significant treatment, however, in which several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) have elapsed between the corresponding administrations.

[0274] In some implementations, methods for treating metabolic diseases (such as fatty liver disease) may include administering a glycine-containing tripeptide molecule and a second therapeutic agent, such as an ACC inhibitor, an ApoC-III inhibitor, an ACL inhibitor, a prescription fish oil, or a CETP inhibitor.

[0275] Systemic delivery of glycine-containing tripeptide expression constructs or peptides to a patient is a highly efficient method for delivering therapeutically effective compositions to counteract the immediate clinical manifestations of disease. Alternatively, in certain circumstances, local delivery of glycine-containing tripeptide molecules and / or a second therapeutic agent may be appropriate.

[0276] The present invention also provides a method of administering the compositions of the invention to a mammal. In one aspect, the mammal is a human. The effective amount of the composition to be used for treatment will, for example, depend on the treatment context and objectives. Those skilled in the art will understand that the appropriate dose level for treatment will therefore vary in part depending on the molecules delivered, the indication for which the composition is used, the route of administration, and the patient's body size (weight, body surface or organ size) and condition (age and general health). Therefore, clinicians can titrate the dose and modify the route of administration to obtain the best therapeutic effect.

[0277] In one aspect, a regimen for delivering to mammals a composition comprising a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof will include administration of 5 mg / kg to 2000 mg / kg, or 30 mg / kg to 1500 mg / kg, or 40 mg / kg to 1250 mg / kg, or 50 mg / kg to 1000 mg / kg, or 60 mg / kg to 5000 mg / kg, at a daily dose or at longer or shorter intervals at equivalent doses, such as every other day, twice a week, once a week, once a month, once every six months, or even twice or three times a day. Administration may be by oral, intravenous, subcutaneous, intranasal, inhalation, transdermal, transmucosal, or any other route discussed herein.

[0278] In another aspect, the glycine-containing tripeptide molecule is administered subcutaneously at the dose specified herein. However, the glycine-containing tripeptide molecule can be administered by any of the methods discussed herein and as known in the art. In another aspect, a maximum permissible volume of 2.0 ml is injected at a single site. If a higher concentration of the glycine-containing tripeptide molecule is required, it is contemplated that the glycine-containing tripeptide molecule can be injected at multiple sites or at a larger dose. In another aspect, the glycine-containing tripeptide molecule is administered daily at approximately the same time. However, alternative delivery times are included in this invention.

[0279] Alternatively, the pharmaceutical composition may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or for delivery via the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the scope of the art.

[0280] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, a glycine-containing tripeptide molecular composition may be formulated as a dry powder for inhalation. The pharmaceutical composition inhalation solution may also be formulated together with a propellant for aerosol delivery. In another embodiment, the solution may be nebulized. Lung administration is further described in PCT application number PCT / US94 / 001875, which describes the lung delivery of chemically modified proteins.

[0281] It is also anticipated that some formulations can be administered orally. In one embodiment of the invention, the glycine-containing tripeptide molecular composition to be administered in this manner may be formulated with or without those carriers conventionally used in mixtures of solid dosage forms (such as tablets and capsules). For example, the capsule may be designed to contain the active portion of a point-release formulation that maximizes bioavailability in the gastrointestinal tract and minimizes pre-systemic degradation. Other agents may be included to aid in the absorption of the composition. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be employed.

[0282] Another glycine-containing tripeptide molecular composition may involve an effective amount of a glycine-containing tripeptide molecule mixed with a non-toxic excipient suitable for manufacturing tablets. The solution can be prepared into a unit dosage form by dissolving the tablet in sterile water or other suitable medium. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0283] Other glycine-containing tripeptide molecular compositions will be apparent to those skilled in the art, including formulations involving compositions in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery components, such as liposomes or micelle carriers, biodegradable microparticles or porous beads, and reservoir injections, are also known to those skilled in the art.

[0284] The frequency of administration depends on the pharmacokinetic parameters of the glycine-containing tripeptide molecular composition in the formulation used. Typically, clinicians will administer the composition up to a dose that achieves the desired effect. Therefore, the composition can be administered as a single dose, or over time in two or more doses (which may or may not contain the same amount of the desired molecule), or via an implanted device or catheter in a continuous infusion form. Further refinement of the appropriate dose is routinely performed by those skilled in the art and within the scope of their usual duties. The appropriate dose can be determined by using appropriate dose-response data.

[0285] In addition to the routes of administration disclosed herein, the compositions of the present invention may be introduced into mammals for treatment in any manner, such as, but not limited to, intravenous, intraperitoneal, intracranial (parenchymal), intraventricular, intramuscular, intraocular, intraarticular, intraportal, intralesional, intraarticular, intralesional, intraarticular, intratumoral, intracerebrospinal, intrarectal and intracolonic, via surface, subconjunctival, intrabladder, intravaginal, epidural, intracranial, intradermal, via inhalation, via dermis, via serous membrane, via buccal, via mouth, via nose, dissolved in the mouth or other body cavities, instilled into the airway, blown into the airway, injected into blood vessels, tumors, organs, etc., and injected or deposited into the body cavities of mammals.

[0286] Alternatively or additionally, the composition may be applied topically via an implantable membrane, sponge, or another suitable material that has absorbed or encapsulated the desired molecules. In the case of an implantable device, the device may be implanted into any suitable tissue or organ, and the desired molecules may be delivered via diffusion, delayed-release injection, or continuous administration.

[0287] In some cases, it may be necessary to use the composition ex vivo. In such cases, cells, tissues, or organs that have been removed from the patient are exposed to the composition, and then the cells, tissues, and / or organs are transplanted back into the patient.

[0288] The compositions of the present invention can be administered using any of several standard methods, including, for example, continuous infusion, rapid injection, intermittent infusion, inhalation, or a combination of these methods. For example, one possible administration mode involves continuous intravenous infusion. In such methods, the infusion rate of the compositions of the present invention can be, for example, 0.001-0.5 mg / kg body weight per hour, more preferably 0.01-0.2 mg / kg body weight per hour, and most preferably 0.03-0.1 mg / kg body weight per hour, over a period of, for example, 1-100, 10-100, or about 12, 24, 48, 72, 84, or 96 hours. If desired, the infusion of the compositions of the present invention can be preceded by a rapid injection. Such rapid injections are given at doses ranging from about 0.001 to about 10 mg / kg. The dosage and infusion time of the compositions of the present invention can vary and are also included in this invention.

[0289] A single rapid infusion can be administered via syringe through intravenous infusion, such as via a central access tube or peripheral venous tube, or by direct injection. Such administration may be necessary if the patient is only at risk of short-term exposure to endotoxins and therefore does not require prolonged drug duration. For example, this administration modality may be necessary in surgical patients, such as those undergoing cardiac surgery (e.g., coronary artery bypass grafting and / or valve replacement surgery), if appropriate. In these patients, a single rapid infusion of the drug may be administered over a four-hour period before and / or during surgery. (Note that the amount of drug administered is based on the patient's weight and condition and is determined by a skilled practitioner.) Shorter or longer administration periods may be used if determined appropriate by a person skilled in the art.

[0290] In cases where longer-term delivery of the glycine tripeptide molecule of the present invention or a pharmaceutically acceptable salt thereof is required, intermittent administration may be performed. In these methods, as determined by those skilled in the art to be appropriate, a loading dose is administered, followed by (i) a second loading dose and a maintenance dose (or multiple maintenance doses), or (ii) one or more maintenance doses without a second loading dose.

[0291] To achieve further delivery of the glycine tripeptide molecule or its pharmaceutically acceptable salt to a patient, one or more maintenance doses of the compound may be administered to maintain the compound level in the patient's blood. The maintenance dose may be less than the one or more loading doses, for example, about one-sixth of the loading dose. The specific amount administered in the maintenance dose can be determined by a medical professional, with the goal of at least maintaining the compound level. For example, the maintenance dose may be administered for about 2 hours every 12 hours starting from hour 24 and continued at, for example, hours 36, 48, 60, 72, 84, 96, 108, and 120. Of course, the maintenance dose may be discontinued at any point within this time frame as determined by a medical professional.

[0292] The infusion methods described above can be performed using catheters (e.g., peripheral venous, central venous, or pulmonary artery catheters) and related products widely available in the art (e.g., infusion pumps and tubing). An important criterion to consider when selecting catheters and / or tubing for these methods is the impact of the material of these products (or coatings on these products) on drug size.

[0293] Other catheter-related products that can be used in the methods of this invention can be identified by determining whether the material of the product alters the size of the compound under conditions consistent with those used in drug administration. Furthermore, in cases where the patient already has a catheter in a position that does not maintain optimal drug size, catheter inserts made of compatible materials (e.g., polyamide polymers) or including a compatible coating can be used, ensuring that the drug solution does not contact the surface of an incompatible catheter. Such inserts have an outer diameter small enough to allow easy insertion into an existing catheter while maintaining an inner diameter large enough to accommodate a flow of the compound solution; they are placed within the existing catheter and connected to a tube or syringe for drug delivery.

[0294] The appropriate frequency of application can be determined by those skilled in the art and can be applied once or several times a day. The compositions of the present invention can also be applied once a day or every other day. In the case of acute application, treatment typically lasts for several hours or days, while chronic treatment can last for weeks, months or even years.

[0295] Both chronic and acute administration can be performed using standard hilar administration formulations, which can be made from formulations described elsewhere herein. Administration via this route offers several advantages, such as rapid action by delivering glycine tripeptide molecules or their pharmaceutically acceptable salts at a higher local concentration to the desired site of action. For a therapeutic agent to exert its desired effect, it needs physical contact with its physiological targets, such as receptors present on hepatocytes. Site-specific drug delivery ensures that such interactions occur only at the desired anatomical location in the liver; therefore, it must meet the following criteria: (i) it must be able to cross anatomical barriers, such as those of the stomach and intestines; (ii) it should be selectively recognized by receptors present on hepatocytes, such as desialyl glycoproteins; (iii) the exogenously delivered ligand for targeting should compete with endogenously produced ligands; (iv) the delivery system must be non-toxic, biocompatible, biodegradable, and physicochemically stable in hepatocytes, either in vivo or in vitro; (v) it should have a uniform sinusoidal capillary distribution; and (vi) it should... The drug release rate is controllable and predictable, such that only therapeutic amounts of the glycine tripeptide molecule or its pharmaceutically acceptable salt are released into hepatocytes; (vii) the drug release should not affect drug distribution; (viii) it should show minimal drug leakage during its passage through the stomach, intestines and other parts of the body; (ix) the carrier used to encapsulate the glycine tripeptide molecule must be eliminated from the body without producing any signs of toxicity and the carrier should not induce modulation of the disease state; and (x) finally, the preparation of the glycine tripeptide molecule or its pharmaceutically acceptable salt delivery system should be easy or appropriately simple, reproducible and cost-effective.

[0296] Glycine tripeptide molecules or pharmaceutically acceptable salts thereof may be administered as part of a drug delivery system for liver tissue delivery. Various methods for conjugating glycine-containing tripeptide molecules to liver-specific target moieties may include: (a) conjugating the target moieties to a pre-formed nanocarrier, (b) conjugating the target moieties by post-insertion, (c) conjugating the target moieties by an avidin / biotin complex, and (d) conjugating the target moieties prior to nanocarrier formulation. Exemplary target moieties and carriers for delivering compositions of the present invention to specific hepatocytes are described in Nidhi Mishra et al., (2013) “Efficient Hepatic Delivery of Drugs: Novel Strategies and Their Significance”, BioMed Research International, Volume 2013, the disclosure of which is incorporated herein by reference in its entirety.

[0297] While certain glycine tripeptide molecules and their pharmaceutically acceptable salts, compositions, and methods described herein have been specifically described according to certain embodiments, the following examples are for illustrative purposes only and are not intended to limit the scope of the compounds described herein. Each of the references recited in this application is incorporated herein by reference in its entirety.

[0298] Example

[0299] method

[0300] Animal program

[0301] All animal procedures were approved by the Institutional Animal Care & Use Committee of the University of Michigan (PRO00008239) and performed in accordance with institutional guidelines. Eight-week-old male apolipoprotein E-deficient (apoE- / -) mice (B6.129P2-Apoetm1Unc / J, Reserve No.: 002052) were obtained from Jackson Laboratories. Mice were fed a Western diet (WD, by weight, 42% of calories from fat and 0.2% cholesterol, Envigo TD.88137). After one week of WD feeding, blood was carefully collected from a facial vein to determine baseline plasma levels of total cholesterol (TC). Mice were then fed a WD diet and randomly assigned to five experimental groups. The following treatments were administered orally by gavage six times a week for 12 weeks: 1) WD + H2O (control group receiving water as a medium); 2) WD + Gly (0.67 mg / g glycine); 3) WD + Leu (0.33 mg / g leucine); 4) WD + DT-109 (1 mg / g DT-109 = Gly-Gly-Leu); 5) WD + DT-110 (1 mg / g DT-110 = Gly-Gly-dLeu). All mice maintained a 12-hour light / dark cycle and had unlimited access to food and water. Body weight and food intake were measured every week and every three weeks.

[0302] OGTT and non-fasting blood glucose

[0303] At week 10, an oral glucose tolerance test (OGTT) was performed after overnight fasting by oral gavage of 2 mg / g glucose. To assess the acute effect of treatment, glycine (0.67 mg / g), leucine (0.33 mg / g), or DT-109 or DT-110 (1 mg / g) were added to a glucose solution and administered orally by gavage. To assess the chronic effect of treatment, glucose was administered to mice alone. Baseline glucose levels were measured using a glucometer and test strips (NDC: 0193-7308-50, Contour Next) after blood was collected from the tail tip. Glucose was then administered to mice with or without (acute) treatment, and blood glucose levels were measured every 30 min for up to 120 min. To assess the effect of treatment on non-fasting blood glucose levels, blood was collected from the tail tip before (pre-gavage) and 30 min after (post-gavage) routine oral gavage administration of treatment. Glucose levels were measured using a blood glucose meter and test strips (NDC: 0193-7308-50, Contour Next).

[0304] Plasma lipids, adipokines, and cytokines

[0305] Plasma levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) were measured using a Cobas Mira chemical analyzer (Roche Diagnostics) with manufacturer-supplied analytical reagents and protocols in the chemistry laboratory of the Michigan Diabetes Research Center (MDRC). Plasma levels of glycine tripeptide, resistin, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP1) were measured using a Luminex 200 chemical analyzer (Luminex Corporation) with manufacturer-supplied analytical reagents and protocols (Millipore Multiplex, MMHMAG-44K) in the MDRC chemistry laboratory.

[0306] Histological analysis

[0307] Histological processing was performed at the In Vivo Animal Core (IVAC) Histology Laboratory within the Unit for Laboratory Animal Medicine (ULAM) at the University of Michigan. Formalin-fixed tissues were processed with graded ethanol and cleared with xylene, followed by infiltration with molten paraffin using an automated VIP5 or VIP6 tissue processor (TissueTek, Sakura-Americas). After paraffin embedding using a Histostar embedding station (ThermoFisher Scientific), the tissues were sectioned at a thickness of 4 μm on an M 355S rotary microtome (ThermoFisher Scientific) and mounted on glass slides. After dewaxing and hydration with xylene and fractionated ethanol, the slides were stained with Harris hematoxylin (ThermoFisher Scientific, catalog 842) for hematoxylin and eosin (H&E), cleared with a clearing agent (ThermoFisher Scientific, catalog 7401), blued with a bluing agent (ThermoFisher Scientific, catalog 7301), stained with alcoholic eosin Y (ThermoFisher Scientific, catalog 832), then dehydrated and cleared with fractionated ethanol and xylene, and covered with micromounts (Leica catalog 3801731) using a Leica CV5030 automatic coverslipper. Immunohistochemical staining was performed on an IntelliPATH FLX automated immunohistochemical stainer (Biocare Medical, catalog number IPS0001US), including blocking endogenous peroxidase and nonspecific binding, detection using a commercial detection system based on a horseradish peroxidase- and biotin-based polymer, revealing with diaminobenzidine chromogen, and nuclear counterstaining with hematoxylin. For F4 / 80 (Bio-Rad ABD Serotec, catalog number MCA497), rat monoclonal primary antibody (clone CI: A3-1) was diluted to 1:400 in DaVinci diluent (Biocare Medical, catalog number PD900) and incubated for 60 min, followed by detection using a rat-based mouse HRP-polymer (Biocare Medical, catalog number RT517) two-step probe polymer for 10 and 30 min, respectively.

[0308] RNA isolation, reverse transcription, and quantitative polymerase chain reaction (qPCR)

[0309] Total RNA was extracted from liver or adipose tissue samples using the QIAGEN RNeasy kit. RNA was reverse transcribed into cDNA using SuperScript III and random primers (Invitrogen). Specific transcript levels were assessed using the iQ SYBR Green Ultramix (Bio-Rad) real-time PCR system and the ΔΔCt threshold cycle normalization method. Gene expression levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer pairs for qPCR were obtained from IntegratedDNA Technologies and are listed below:

[0310]

[0311] Liver lipid extraction

[0312] Liver samples were rapidly removed from euthanized mice and maintained at -80°C. Frozen liver samples (approximately 100 mg) were homogenized in PBS and centrifuged (14,000 RPM, 20 min). The supernatant was collected and protein content was analyzed using the Bio-Rad Bradford assay. To assess liver lipid composition, lipids were extracted from the supernatant using a 3:2 (v:v) ratio of hexane (≥99%, 32293, Sigma-Aldrich) and isopropanol (≥99.5%, A426-4, Fisher Scientific), and the hexane phase was allowed to evaporate for 48 h. Liver TG or TC levels were determined spectrophotometrically using a commercially available kit (Wako Chemicals). Data were normalized for protein levels and presented as μg / mL TG or TC per mg of protein.

[0313] Liver protein extraction and Western blotting

[0314] Tissue extracts were prepared using radioimmunoprecipitation assay lysate buffer (RIPA buffer, Thermo Scientific) supplemented with a protease inhibitor mixture (Roche Applied Science). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1 hour at room temperature in Tris-buffered saline-Tween 20 (TBST) containing 5% skim milk and incubated overnight at 4°C with the primary antibody. The following primary antibodies were used: rabbit polyclonal anti-ABCG8 (Santa Cruz Biotechnology, sc-30111, working dilution 1:1000) and rabbit polyclonal anti-β-actin (Cell signaling, 4967S, working dilution 1:3000). After TBST washing, the membrane was incubated with the secondary antibody (LI-COR Biotechnology, donkey anti-rabbit IRDye 926-32213, 926-68073, working dilution 1:10000) at room temperature for 1 hour. After TBST washing, the bands were visually observed and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, version 2.1).

[0315] Analysis of atherosclerotic lesions

[0316] After blood collection, mice were perfused with 20 mL of saline solution via the heart, followed by 20 mL of 37% formalin. Mice were fixed in formalin and the entire aortic tree was dissected under a surgical microscope. Next, the aortic tree was stained with Oil Red O solution (3.5:1 methanol:1N NaOH containing 0.2% Oil Red O (w / v)) for 50 min, followed by 70% ethanol for 30 min. The aortic tree was then held in a DDW (distilled water bath). Adhering fat and connective tissue were then removed from the aortic tree, and it was longitudinally opened with a Venus scissor to expose atherosclerotic lesions. Images of the entire aortic tree were acquired using a digital camera, and the percentage of plaque area stained with Oil Red O relative to the total luminal surface area was quantified using ImageJ analysis software (http: / / imagej.nih.gov / ij / ).

[0317] Statistical analysis

[0318] Data analysis was performed using SPSS 24.0 software (SPSS Inc., IBM). Data were presented as box-and-whisker plots or mean ± SEM. For each FIGIG. legend, the number of animals used in each study was specified. One-way ANOVA was followed by Tukey post-hoc test for data analysis. Differences were considered statistically significant at p < 0.05.

[0319] result

[0320] Effects of Experiment 1 on body weight and obesity

[0321] Figure 1A The experimental design was presented. After one week of Western diet (WD) feeding, blood was collected from the facial vein (FV) to determine baseline plasma TC levels. Mice were then maintained on a WD diet and randomly assigned to five experimental groups, receiving the following treatments orally by gavage six times a week for 12 weeks: 1) WD + H2O; 2) WD + Gly (0.67 mg / g glycine); 3) WD + Leu (0.33 mg / g leucine); 4) WD + DT-109 (1 mg / g DT-109); 5) WD + DT-110 (1 mg / g DT-110). No significant differences in food intake were noted throughout the study. Figure 1B Endpoint measurements revealed significantly lower body weight in mice administered glycine (12% lower, p < 0.05). Figure 1C In mice treated with glycine or DT-109, the increase in total body weight at the endpoint compared to baseline was significantly lower (51% lower, p<0.01, and 28% lower, p<0.05, respectively). Figure 1D The overall appearance of the peritoneal cavity at the endpoint revealed pale yellow staining of the liver and enlarged visceral fat pads in both control mice and mice treated with leucine, which were reduced by treatment with glycine, DT-109, or DT-110. Figure 1E Therefore, in mice administered glycine, DT-109, or DT-110, plasma levels of glycine-containing tripeptides were significantly lower (53%, 62%, or 56% lower, p < 0.05). Figure 1F In summary, these results indicate the protective effects of glycine, DT-109, and to a lesser extent DT-110 against WD-induced obesity.

[0322] Effect of Experiment 2 on glucose homeostasis

[0323] An oral glucose tolerance test (OGTT) was performed on week 10 of WD feeding after overnight fasting. To determine the acute effect of treatment on blood glucose levels, glycine (0.67 mg / g), leucine (0.33 mg / g), and DT-109 or DT-110 (1 mg / g) were added to a glucose solution (2 mg / g) and administered orally by gavage. Figure 2A As shown, no significant differences in blood glucose levels were observed between groups at baseline. Blood glucose levels significantly decreased with all treatments 30 minutes after glucose administration, with glycine or DT-109 showing the most significant effects. At subsequent time points (60–120 min), blood glucose levels significantly decreased in mice treated with glycine, DT-109, and DT-110, but not with leucine. To assess the chronic effects of treatment, the OGTT was repeated at week 11 of WD feeding, this time without adding the treatment to the glucose solution. No significant differences were observed between groups after glucose loading in the absence of treatment. Figure 2B To assess the effect of treatment on non-fasting blood glucose levels, blood samples were collected before (pre-gavage) and 30 min after (post-gavage) administration via oral gavage, as was routinely performed. Pre-gavage glucose levels were significantly lower in all groups compared to control mice. However, while oral gavage with water or leucine tended to increase blood glucose, oral gavage with glycine, DT-109, or DT-110 significantly reduced blood glucose levels (10%, p<0.05, 21%, p<0.01, and 13%, p<0.05, respectively). Figure 2C Finally, in mice treated with glycine or DT-109, the endpoint glucose level (after 6 hours of fasting) was significantly lower (41% lower, p < 0.01, and 30% lower, %, p < 0.05, respectively). Figure 2D Analysis of liver gene expression showed that glycine treatment upregulated the expression of genes regulating glucose uptake (GLUT1, GLUT2, and GLUT4), while the expression of genes regulating gluconeogenesis (G6pc, PCK1, and FBP1) remained largely unchanged. GLUT1 expression was also significantly induced in the livers of mice treated with DT-110. Figure 2E Overall, these results indicate significant postprandial glucose-lowering properties of glycine, DT-109, and DT-110.

[0324] Effects of Experiment 3 on Hepatic Lipid Metabolism

[0325] H&E staining of liver samples revealed significant microvesicular and macrovesicular steatosis in both control and leucine-treated mice. Significantly, treatment with glycine, DT-109, or DT-110 eliminated WD-induced hepatic steatosis. Figure 3ATherefore, the extraction of liver lipids followed by quantification of TG and TC content showed that treatment with glycine, DT-109, or DT-110 reduced liver TG by 74%, 73%, or 68%, respectively (p<0.01). Figure 3B ) and TC (76% and 71%, respectively, p<0.01, or 63%, p<0.01, respectively). Figure 3C The levels of leucine were significantly reduced, while there was no significant effect when leucine was applied.

[0326] Experiment 4 next assessed the expression of genes regulating TG, fatty acid, and cholesterol accumulation in the liver to explain the observed effects on hepatic steatosis. Interestingly, treatment with glycine, DT-109, or DT-110 significantly induced the expression of the main regulators of hepatic lipid oxidation (AMPKα1 and PPARα), but did not significantly induce it in the presence of leucine. Figure 4A Therefore, in the livers of mice treated with glycine, DT-109, or DT-110, the major target genes regulating TG hydrolysis (PNPLA2), mitochondrial β-oxidation (CPT1a, CACT, ACAD1), and the mitochondrial anion carrier UCP2 were significantly upregulated. Figure 4A , Figure 4B Regarding genes regulating cholesterol homeostasis in the liver, treatment with glycine, DT-109, or DT-110 significantly increased the expression of ABCG5 and ABCG8 (key regulators of cholesterol excretion into bile). Significant upregulation of LDLR was observed in the livers of mice treated with DT-110, with similar trends observed with glycine and DT-109 treatment. Figure 4C Western blotting confirmed the overexpression of ABCG8 in the livers of mice treated with glycine, DT-109, or DT-110. Figure 4D and Figure 4E In summary, these results highlight the effective protective effects of glycine, DT-109, and DT-110 against WD-induced hepatic steatosis associated with the overexpression of genes regulating lipid oxidation and cholesterol extraction into bile.

[0327] Effects of Experiment 5 on plasma lipid profiles and atherosclerosis

[0328] Blood was collected from mice after one week of WD feeding to determine baseline plasma TC levels before randomization to the experimental groups. Plasma TC was measured again at the endpoint after 12 weeks of WD feeding, in conjunction with treatment or water control. Figure 5AAs shown, treatment with glycine or DT-109 significantly reduced the time-dependent increase in plasma TC, and the trend was similar in DT-110 treatment, but not in leucine treatment. Therefore, in mice treated with glycine or DT-109, the endpoint plasma levels of TC and LDL were significantly reduced (TC: 19% reduction, p<0.05 or 25%, p<0.01, respectively; LDL: 30% reduction, p<0.01 or 36%, respectively, p<0.01), and a similar trend was observed in DT-110 treatment. Figure 5B , Figure 5C Interestingly, plasma HDL levels were significantly lower in mice treated with glycine (35% lower, p<0.05), but were maintained after treatment with DT-109. Figure 5D It was noted that there was no significant change in plasma TG levels. Figure 5E Finally, analysis of the aortic lesions by Oil Red O staining revealed a significant reduction in total atherosclerotic plaques in mice treated with glycine, DT-109, or DT-110 (48%, p<0.01, 62%, p<0.001, and 49%, p<0.01, respectively). Figure 5F , Figure 5G However, it was not significantly reduced in mice treated with leucine.

[0329] Effects of Experiment 6 on systemic inflammation, hepatitis, and adipose tissue inflammation

[0330] Since inflammation plays a major role in the pathogenesis of NAFLD and atherosclerosis, plasma levels of pro-inflammatory cytokines and adipokines were subsequently assessed. Although no significant changes were observed in plasma levels of IL-6 and resistin between groups (…),… Figure 6A , Figure 6B However, MCP1 levels were significantly reduced in mice treated with glycine, DT-109, or DT-110. Figure 6C Analysis of gene expression in epididymal adipose tissue (EAT) and subcutaneous adipose tissue (SAT) revealed that TNFα was significantly downregulated in EAT by all treatments, with DT-110 showing the most significant effect. Figure 6D Based on lower MCP1 plasma levels, treatment with glycine, DT-109, or DT-110 significantly inhibited MCP1 expression in EAR and SAT, but not significantly inhibited in the presence of leucine. Figure 6D , Figure 6E Finally, although glycine inhibited MCP1 expression in the liver (DT-109 showed a similar trend), Figure 7A However, it was noted that no significant changes were observed in tissue F4 / 80 as determined by immunohistochemistry. Figure 7B , Figure 7CIn summary, these results indicate that some of the anti-inflammatory effects of glycine, DT-109, and DT-110 are primarily associated with the inhibition of MCP1 in adipose tissue and circulation.

[0331] Example x: Glycine-based treatment for NAFLD

[0332] A. Introduction

[0333] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, affecting 25% of the world's population. NAFLD encompasses a range of liver lesions, including hepatic steatosis (HS), NASH characterized by hepatocellular damage and lobular inflammation combined with fibrosis progression, and cirrhosis that can lead to liver failure or hepatocellular carcinoma. Cardiovascular and metabolic comorbidities, including obesity, type 2 diabetes (T2D), metabolic syndrome (MetS), and dyslipidemia, are prevalent in NAFLD patients. Besides liver-specific causes of death, cardiovascular disease is the leading cause of death in NAFLD patients, particularly in cases of NASH. Despite the extremely heavy global burden of NAFLD and significant efforts in drug development, no therapy has been approved to date.

[0334] Recent developments in metabolomics and the gut microbiota, in particular, have deepened our understanding of the pathogenesis of NAFLD, leading to the identification of new therapeutic targets. While lipid and carbohydrate metabolism abnormalities are known to be associated with NAFLD, current metabolomics-based research suggests that dysregulation of specific amino acid (AA) metabolism plays a role in the pathogenesis of NAFLD. Specifically, while most circulating AAs are increased in NAFLD patients, glycine levels are decreased. Circulating glycine levels are negatively correlated with hepatic sclerosis (HS), hepatocellular ballooning degeneration, and lobular inflammation. Plasma glycine has recently been included in models to predict NASH along with known biomarkers such as aspartate aminotransferase (AST) and PNPLA3 genotype. Lower plasma glycine is associated with higher rates of obesity, type 2 diabetes (T2D), metastatic hypertension (MetS), coronary artery disease, and myocardial infarction, while higher plasma glycine is associated with favorable lipid profiles (summarized in Table S1).

[0335] Table S1. Previous clinical evidence linking lower circulating glycine levels to NAFLD and cardiovascular metabolic disease.

[0336]

[0337] As a non-essential amino acid, glycine is synthesized from several precursors, primarily in the liver. These reactions are catalyzed by key enzymes driving the formation of glycine from serine (serine hydroxymethyltransferase, SHMT), threonine (threonine deaminase, TDH), choline via sarcosine (choline dehydrogenase, CHDH; sarcosine dehydrogenase, SARDH), and from alanine to glyoxylate (AGXT). Glycine is used in multiple pathways to produce essential molecules, including nucleic acids, heme, and glutathione. In NAFLD or T2D, glutathione synthesis is reduced due to limited glycine availability and recovers after dietary supplementation. In addition to this antioxidant role, glycine also has dual benefits in lipid and glucose metabolism. Glycine uptake partially lowers blood glucose by stimulating insulin secretion from pancreatic β-cells. In genogenic obesity / diabetes KK-A y In mice, dietary glycine improves glucose tolerance, reduces circulating triglycerides (TG) and alanine aminotransferase (ALT), and alleviates hepatic sclerosis (HS) and inflammatory infiltration. In sucrose-fed rats, glycine reduces intraperitoneal fat and plasma TG, and increases FAO markers in liver mitochondria. However, a comprehensive study of the role of glycine in NAFLD using a fully simulated human disease model with precise dosing has not yet been conducted.

[0338] Given the burden of NAFLD, the lack of available therapies, and consistent reports linking lower circulating glycine levels to NAFLD severity, there are strong reasons to better understand glycine metabolism in NAFLD, which could lead to novel therapeutics. In this paper, we identified the inhibition of glycine biosynthesis genes (primarily AGXT1) in human and mouse NAFLD. The gene (AGXT1) was applied... - / - By restricting glycine availability through dietary methods, we found that hyperlipidemia and steatohepatitis were exacerbated. Investigating glycine-based compounds with dual lipid-lowering / glucose-lowering properties, we determined that DT-109 effectively prevented diet-induced NASH in mice by modulating hepatic FAO.

[0339] B. Abbreviation:

[0340] AA, amino acid; ACAA, acetyl-CoA acyltransferase; ACAD, acyl-CoA dehydrogenase; ACOT, acyl-CoA thioesterase; ACOX, acyl-CoA oxidase; ACSL, acyl-CoA synthase (long chain); ACSM, acyl-CoA synthase (medium chain); AGXT, alanine-glyoxylate transaminase; ALP, alkaline phosphatase; ALT, alanine transaminase; ApoE, apolipoprotein E; AST, aspartate transaminase; CACT, botulinum toxin. Base / acylcarnitine translocase; Cas9, CRISPR-associated protein 9; CCL, CC motif chemokine ligand; CCR, CC motif chemokine receptor; CD, normal diet; CHD, coronary heart disease; CLAMS, integrated laboratory animal monitoring system; CPT, carnitine palmitoyltransferase; CRISPR, clustered regular short palindromic repeats; DAG, diacylglycerol; DAO, D-amino acid oxidase; DEG, differentially expressed gene; ECI, olefins. Acyl-CoAδ isomerase; FA, fatty acid; FAO, fatty acid β-oxidation; HADH, hydroxyacyl-CoA dehydrogenase; HS, hepatic steatosis; LDA, linear discriminant analysis; LEfSe, linear discriminant analysis effect size; MCP-1, monocyte chemoattractant protein-1; MetS, metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NF-κB, nuclear factor κ-light chain enhancer that activates B cells; NMR, nuclear magnetic resonance; OGTT, oral glucose tolerance test; ORO, Oil Red O; PGC1α, PPARG-coactivator-1α (PPARGC1A); PNPLA, protein containing a patatin-like phospholipase domain; PPAR, peroxisomal proliferator-activated receptor; RER, respiratory exchange rate; SARDH, sarcosine dehydrogenase; SERPINE, serine peptidase inhibitor E branch (serinepeptidase) Inhibitor clade E); SHMT, serine hydroxymethyltransferase; T2D, type 2 diabetes; TC, total cholesterol; TDH, threonine deaminase; TG, triglycerides; TGFBR, transforming growth factor β receptor; TGFβ, transforming growth factor-β; TIMP, tissue inhibitor of metalloproteinases; TLR, clock-like receptor; TNF, tumor necrosis factor; TNFRSF, TNF receptor superfamily; WD, Western diet.

[0341] C. Transcriptional expression profile:

[0342] RNA sequencing data have been deposited in NCBI's SRA or GEO (accession number: PRJNA556537 or GSE126204). Metagenomic sequencing data have been deposited in NCBI's SRA (accession number: PRJNA544728).

[0343] D. Background and Objectives:

[0344] The incidence of nonalcoholic fatty liver disease (NAFLD), including steatohepatitis (NASH), is increasing worldwide, and there are no approved pharmacological therapies. Lower circulating glycine levels have been consistently reported in NAFLD patients, but the inducing role of glycine and its therapeutic potential remain unclear. We applied genetic and dietary strategies to investigate glycine metabolism in NAFLD and evaluated glycine-based therapies.

[0345] E. Method:

[0346] We performed transcriptomic analysis of the livers of humans and mice with NAFLD, revealing the inhibition of glycine biosynthesis genes, primarily alanine-glyoxylate transaminase-1 (AGXT1). AGXT1 was generated using CRISPR / Cas9. - / - We developed a glycine-modified diet for mice. We investigated glycine-based compounds with glucose / lipid-lowering properties and tested their therapeutic use in a mouse model of hyperlipidemia / NAFLD. Plasma lipids, liver enzymes, and cytokines were analyzed. The liver was studied using histology, lipid quantification, RNA sequencing, qPCR, and Western blotting. Indirect calorimetry was used to assess NMR-based body composition and energy metabolism. The gut microbiota was investigated using 16S metagenomic sequencing.

[0347] F. Results:

[0348] Genes used to restrict glycine availability (AGXT1) - / - Dietary approaches and lifestyle choices exacerbate diet-induced hyperlipidemia and steatohepatitis with suppressed mitochondrial / peroxisome fatty acid β-oxidation (FAO) and enhanced inflammation as potential pathways. We identified a glycine-based tripeptide (gly-gly-L-leu / DT-109) with potent glucose / lipid-lowering effects. In mice with confirmed NASH, DT-109 improved body composition by reducing circulating lipids, liver enzymes, and steatohepatitis through stimulation of the FAO pathway. DT-109 alleviated lobular / systemic inflammation and fibrosis by inhibiting the NF-κB and TGFβ / SMAD pathways. The bacterial genus *Clostridium sensu stricto* was positively correlated with NAFLD severity and decreased by DT-109, while *Alistipes* showed an inverse correlation and increased by DT-109.

[0349] G. Method

[0350] (i) Animal program

[0351] Animal procedures were approved by the University of Michigan (UM) Institutional Animal Care and Use Committee (PRO00008239) and performed in accordance with institutional guidelines. Seven-week-old C57BL / 6J or apoE- / - mice (B6.129P2-Apoetm1Unc / J, Reserve: 002052) were obtained from Jackson Laboratories. AGXT1- / - mice based on the C57BL / 6J background were generated using CRISPR / Cas9, with guide RNA targeting exon 1 of AGXT1: 5'-GGGTCCGGGGCCCTCCAACC-3'. Eight-week-old male C57BL / 6J, apoE- / -, or AGXT1- / - mice were used throughout the process and fed unlimited diets: standard normal diet (CD, LabDiet5L0D, 13% calories from fat); high-fat, high-cholesterol Western diet (WD, Envigo TD.88137, 42% fat); AA-determined WD with or without glycine (developed using Envigo-Teklad custom diets, Table S2): WDAA+Gly (TD.170525) or WDAA-Gly (TD.170526); and high-fat, high-cholesterol, high-fructose NASH diet (research diet D17010103, 40% fat). We confirmed that the diets effectively induced NASH in mice. When indicated, mice were administered 0.125–1 mg / g of glycine (Sigma-Aldrich G5417), leucine (Sigma-Aldrich L8912), DT-109 (CSBio), or H2O orally via gavage daily as a control.

[0352] Table S2. WD (Wt) determined by amino acids containing or without glycine. AA Composition of + / -Gly)

[0353]

[0354]

[0355] (ii) Human Data

[0356] We analyzed differentially expressed genes (DEGs) driving glycine biosynthesis using two public liver microarray datasets: 1) GSE83452, from 104 NASH patients and 44 healthy controls, and 2) GSE61260, from 24 NASH patients and 24 healthy obese controls. We used a linear regression model with age, sex, and BMI as covariates to identify significant glycine biosynthesis genes associated with NASH. To enhance statistical power, we also performed a meta-analysis of the two studies with a fixed-effects model. We used our previously published microarray data (GSE26106, n = 206 liver transplant donors) to test the correlation between gene expression levels and liver fat content.

[0357] (iii) Histology and Immunohistochemistry

[0358] All histological procedures were performed by the UM In Vivo Animal Core, Histology Laboratory. Technicians were unaware of the experimental group's findings. NAFLD activity (NAS) or fibrosis was scored using H&E or Sirius Red staining.

[0359] (iv) Identification of compounds based on glycine

[0360] Compounds structurally similar to glycine were selected to evaluate structural, conformational, electronic, and isosteric modifications to the glycine skeleton. (via...) A search was conducted to determine commercial availability. The oral LD50 was determined. 50 Water-soluble compounds at concentrations above 1 mg / g are specified as suitable for oral administration to mice.

[0361] (v) RNA sequencing

[0362] Library preparation and sequencing were performed by the UM DNA Sequencing Core on an Illumina NovaSeq 6000 sequencing system. RNA sequencing and pathway analysis were performed as described.

[0363] (vi) Integrated Laboratory Animal Monitoring System (CLAMS)

[0364] Body composition was assessed at the UM Animal Phenotyping Core using a nuclear magnetic resonance (NMR)-based analyzer (LF90II; Bruker Optics). Oxygen consumption (VO2), carbon dioxide production (VCO2), and kinetic activity were measured using CLAMS (Columbus Instruments). Respiratory exchange rate (RER) was calculated as VCO2 / VO2.

[0365] (vii) Fecal microbiome analysis

[0366] Fecal DNA was extracted as previously described, amplified using primers specific to the V4 region of 16S rRNA, the gut microbiota was characterized using linear discriminant analysis (LDA) effect size (LEfSe) method, and correlation analysis was performed under disease parameter conditions.

[0367] (viii) Statistical Analysis

[0368] Statistical analysis was performed using GraphPad Prism 7.0. Unless otherwise specified, values ​​are presented as mean ± SD, showing all points. Normality and isosquaricity were tested for all data. If normality and isosquaricity were found, a Student's t-test was used to compare two groups, or a one-way ANOVA followed by a Bonferroni post-hoc test was used to compare >2 groups. Otherwise, nonparametric tests (Mann-Whitney U or Kruskal-Wallis) were used. A p-value < 0.05 was considered statistically significant.

[0369] (ix) Generation of AGXT1- / - mice using CRISPR / Cas9

[0370] AGXT1- / - mice under the C57BL / 6J background were generated using CRISPR / Cas9. The guide RNA target site on exon 1 of the AGXT1 gene is 5'-GGGTCCGGGGCCCTCCAACC-3'. Genotyping was performed using the following primers: forward: 5'-ACACCTCCACTGTCCTGTCC-3', reverse: 5'-GGTCAGATCTGCCTGCTACC-3'. Sanger sequencing using the following primer: 5'-GCAGAGCTAGCTGGGAAATG-3' confirmed the presence of the prespacer adjacent motif (PAM). Figure 8AThe third base A is deleted. A frameshift mutation after AA 53 in the ORF introduces a premature stopping codon, and Western blotting confirms the absence of AGXT1. Figure 8B As assessed using CRISPOR, no off-target effects of CRISPR were detected.

[0371] (x) Person Data

[0372] Differentially expressed glycine biosynthesis genes in NASH patients were analyzed using two public datasets. The first study consisted of liver microarray data (GSE83452) collected from 104 NASH patients and 44 healthy controls. Linear regression was used to test the association between gene expression levels and NASH. Age and sex were adjusted as covariates. The second dataset included liver microarray data (GSE61260) collected from 24 NASH patients and 24 healthy obese controls. Similarly, we used a linear regression model with age, sex, and BMI as covariates to identify significant glycine biosynthesis genes associated with NASH. To enhance statistical power and comparability, we also performed a meta-analysis of the two studies using the metafor R package in a fixed-effects model. Genes with a Benjamini-Hochberg adjusted p-value < 0.05 and a Cochran's Qheterogeneity test p-value > 0.05 were considered significant.

[0373] The correlation between gene expression levels and hepatic fat content was tested using our previously published microarray data (GSE26106) collected from liver transplant donors (n=206). Histological dissection, sample exclusion, and microarray data generation were performed as previously described. Hepatic fat content in the donor livers was quantified using an organic solvent (hexane / isopropane 3:2), as previously described. The extracted total fat content was normalized to total protein concentration and converted to a log10 scale for subsequent analysis. Pearson correlation was used to determine the significance of the association between hepatic fat and the expression of genes involved in glycine biosynthesis.

[0374] (xi) Histological, immunohistochemical, and NAFLD activity and fibrosis scores

[0375] Histological procedures were performed by technicians unaware of the experimental group's findings at the In Vivo Animal Center (IVAC) Histology Laboratory at the University of Michigan. Formalin-fixed tissues were processed with graded ethanol and cleared with xylene, followed by infiltration with molten paraffin using an automated VIP5 or VIP6 tissue processor (TissueTek, Sakura-Americas). The tissues were then sectioned at a thickness of 4 μm using a Histostar embedding station (ThermoFisher Scientific) and mounted on slides on an M355S rotary microtome (ThermoFisher Scientific). The slides were stained with hematoxylin and eosin (H&E, ThermoFisher Scientific). For Sirius red staining, slides were treated with 0.2% phosphomolybdic acid for 3 min and then transferred to 0.1% Sirius red saturated in picric acid for 90 min, followed by transfer to 0.01N hydrochloric acid for 3 min.

[0376] NAFLD activity was scored using H&E staining. Steroidosis was scored from 0 to 3 (0: <5% steatosis; 1: 5-33%; 2: 34-66%; 3: >67%). Hepatocellular ballooning was scored from 0 to 2 (0: normal hepatocytes; 1: normal size with pale cytoplasm; 2: pale and enlarged hepatocytes, at least 2-fold). Lobular inflammation was scored from 0 to 2 based on the number of inflammatory lesions counted at 20X (0: none; 1: <2 lesions; 2: ≥2 lesions). The NAFLD activity score (NAS) was calculated as the sum of the scores for steatosis, hepatocellular ballooning, and lobular inflammation. Liver fibrosis was scored from 0 to 4 using Sirius red staining (0: no fibrosis; 1: perisinusoidal or portal fibrosis; 2: perisinusoidal and portal fibrosis; 3: bridging fibrosis; 4: cirrhosis).

[0377] Oil Red O (ORO) staining was performed using frozen sections. Formalin-fixed liver samples were cryoprotected overnight at 4°C in 20% sucrose to form blots, then rapidly frozen in liquid nitrogen in an OCTCompound (Tissue-Tek, catalog 4583) and stored at -80°C until ready for cryosectioning. Before sectioning, the frozen blocks were thawed to approximately -20°C and sectioned at 5 μm on a Cryptometa SME (Thermo-Shandon, catalog 77200227). Slides were stored at -80°C until staining. Before staining, liver slides were thawed to room temperature for 30 min. Slides were then fixed in 10% neutral buffered formalin for 20 min, rinsed in DDW, followed by rinsing in 60% isopropanol, and then placed in working ORO-isopropanol staining solution (Rowley Biochemical Inc., H-503-1B) for 5 min. The slides were then rinsed in 60% isopropanol, followed by three DDW replacements. The slides were then nuclear counterstained in Harris Hematoxylin and mounted on Aqua-Mount (Lerner Laboratories, catalog 13800) aqueous fixative.

[0378] Immunohistochemical staining was performed on an IntelliPATH FLX automated immunohistochemical stainer (Biocare Medical) to block endogenous peroxidase and nonspecific binding. Detection was then performed using a commercial detection system based on a horseradish peroxidase- and biotin-based polymer, revealed using diaminobenzidine chromogen, and counterstained with hematoxylin. Specific to F4 / 80 (Bio-Rad ABD Serotec, catalog number MCA497), rat monoclonal primary antibody (clone CI: A3-1) was diluted 1:400 in DaVinci diluent (Biocare Medical, catalog number PD900) and incubated for 60 min, followed by incubation for 10 and 30 min respectively using a rat-based mouse HRP-polymer (Biocare Medical, catalog number RT517) two-step probe polymer.

[0379] (xii) Plasma analysis

[0380] Intact plasma lipid profiles (TC, TG, LDL, and HDL) were measured using the Cobas Mira Chemical Analyzer (Roche Diagnostics) in the chemistry lab at the Michigan Diabetes Research Center (MDRC) using manufacturer-supplied reagents and protocols, or in our lab using commercially available kits (WakoChemicals 999-02601 and 994-02891). Plasma glycine-containing tripeptides and resistin were measured at MDRC using a Multiplex Assay (MMHMAG-44K, Millipore) on a Luminex 200 platform (Luminex). Plasma MCP-1 was measured using the Mouse CCL2 / JE / MCP-1 Quantikine ELISA Kit (R&D Systems). Clinical chemical analyses of ALT, AST, and ALP were performed by the University of Michigan IVAC on a Liasys 330 Chemical Analyzer (AMSDiagnostics) using manufacturer-supplied reagents and protocols. Plasma oxalate was measured using an oxalate analysis kit (Abcam, ab196990). Plasma glucose was measured using a glucometer and test strips (NDC: 0193-7308-50, Contour Next). Plasma amino acid analysis was performed by the University of Michigan Metabolomics Core. Twenty μL of plasma was derivatized and prepared for GC-MS analysis according to the instructions of the EZ Faast Amino Acid Analysis Kit (Phenomenex). In short, the sample was column-purified and transferred to a GC autosampler vial. The sample was then derivatized, dried at room temperature under a mild nitrogen flow, and resuspended for GC analysis on an Agilent 69890N GC-5975MS detector using the following parameters: 1 μL of sample was injected at a 1:15 split onto a ZB-AAA 10m column (Phenomenex), and the He gas flow rate was 1.1 mL / min. The GC oven was initially set at 110°C and increased to 320°C at 30°C per minute. The inlet temperature was 250°C, and the MS ion source and quadrupole temperatures were 230°C and 150°C, respectively. Data were processed using MassHunter Quantitative Analysis version B.07.00. Metabolites were normalized using the nearest isotopically labeled internal standard, and quantification was performed using five standards injected twice to generate linear calibration curves with accuracy better than 80% for each standard. Intergroup differences were analyzed using peak area.

[0381] (xiii) Quantitative real-time PCR analysis

[0382] Total RNA was extracted from mouse liver samples using the QIAGEN RNeasy kit. RNA was reverse transcribed into cDNA using SuperScript III and random primers (Invitrogen). Specific transcript levels were assessed using the iQ SYBR Green Ultramix (Bio-Rad) real-time PCR system and the ΔΔCt threshold cycle normalization method. Gene expression levels were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer pairs for qPCR were obtained from Integrated DNA Technologies and are listed in Table S3.

[0383] Table S3. Primers used for qPCR analysis

[0384]

[0385]

[0386]

[0387]

[0388] (xiv) RNA sequencing and data analysis

[0389] Total RNA was extracted from mouse liver samples as described above. Library preparation and sequencing were performed at the University of Michigan DNA Sequencing Core. RNA quality was assessed using TapeStation (Agilent, Santa Clara, CA). All samples had an RNA integrity index (RIN) >8.5. Samples were prepared using the NEBNext Ultra II Directed RNA Library Preparation Kit for Illumina (NEB, E7760L) with a Poly(A) mRNA magnetic separation module (NEB, E7490L) and NEBNext multiple oligonucleotides for Illumina Unique dual (NEB, E6440L), where 10 ng–1 μg of total RNA was purified using polyA. The mRNA was then fragmented and copied into the first cDNA strand using a mixture of reverse transcriptase and dUTP. Samples underwent end repair and dA tailing, followed by ligation with NEBNext adaptors. The products were purified and enriched by PCR to form the final cDNA library. The final library was validated for quantity and quality using Kapa's Library Quantification Kit for Illumina Sequencing Platform (Kapa Biosystems KK4835) via TapeStation (Agilent) and qPCR. Paired-end sequencing of the library was performed on a NovaSeq 6000 sequencing system (Illumina).

[0390] The quality of the original FastQ files was verified using FastQC v0.11.8 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Low-quality reads were trimmed using Trimmomatic v.0.35 with the following parameters: SLIDINGWINDOW:4:20MINLEN:25.12 Then, the obtained high-quality reads were mapped to the mouse reference genome (GRCm38.90) using HISAT2 v.2.1.0.13 Gene-level quantification was performed using HTSeq-counts v0.6.0 based on GRCm38.90 genome annotation.14 Then, the R package DESeq2 was used to identify differentially expressed genes.15 Genes with an adjusted p-value less than 0.05 and an absolute fold change greater than 2 were considered differentially expressed genes (DEGs). Then, the clusterProfiler package was used to analyze upregulated and downregulated DEGs for significantly enriched KEGG pathways. 16. Enrichment significance was determined by right-tailed Fisher's exact test followed by Benjamini-Hochberg multiple testing adjustment.

[0391] (xv) Liver lipid analysis

[0392] Liver samples were rapidly removed from euthanized mice, rapidly frozen in liquid nitrogen, and maintained at -80°C. Frozen liver samples (100 mg) were homogenized in PBS and centrifuged (14,000 RPM, 20 min). The supernatant was collected and protein content was analyzed using a Bio-Rad Bradford assay. To assess liver lipid composition, lipids were extracted from the supernatant using a 3:2 (v:v) ratio of hexane (≥99%, Sigma-Aldrich 32293) and isopropanol (≥99.5%, Fisher Scientific A426-4), and the hexane phase was allowed to evaporate for 48 h. Liver TG or TC levels were determined spectrophotometrically using commercially available kits (Wako Chemicals 999-02601 and 994-02891). Liver diacylglycerol (DAG) was determined using an ELISA kit (Aviva Systems Biology, OKEH02607) according to the manufacturer's instructions. Standardize liver TG, TC, and DAG data based on protein levels.

[0393] (xvi) Western blot analysis

[0394] Liver samples were dissolved in RIPA buffer (ThermoScientific) supplemented with a protease / phosphatase inhibitor mixture (Roche Applied Science). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked for 1 hour at room temperature in Tris-buffered saline-Tween 20 (TBST) containing 5% skim milk and incubated overnight at 4°C with the primary antibody. The following primary antibodies were used: AGXT1 (Santa Cruz Biotechnology sc-517388, 1:500), HADHA (Proteintech 10758-1-AP, 1:1000), ACAA2 (ABclonal A15778, 1:500), phosphate-SMAD2 (Ser465 / 467, Cell Signaling, #3108S; 1:1000), SMAD2 (Cell Signaling Technology, #5339S; 1:1000), GAPDH (Santa Cruz Biotechnology sc-365062; 1:2000), or β-actin (Cell Signaling Technology #3700). The membrane was washed with TBST and then incubated with the IRDye-conjugated secondary antibody (LI-COR Biosciences, 1:10000) at room temperature for 1 h. After TBST washing, the bands were visually observed and quantified using the Odyssey infrared imaging system (LI-COR Biosciences, version 2.1).

[0395] (xvii) Identification of compounds based on glycine

[0396] Compounds structurally similar to glycine were selected to evaluate the structural, conformational, electronic, and isosteric modifications to the glycine skeleton. Figure 9A ).via A search was conducted to determine commercial availability. N-methylglycine ( Figure 9B ), N,N-dimethylglycine ( Figure 9C ) and N,N,N-trimethylglycine ( Figure 9D The study explored various degrees of methylation on the glycine amine moiety, and glycolic acid ( Figure 9E The substitution of amines for ethanol was evaluated. Modification of the acid region Wx was also assessed.

[0397] Through glycine ( Figure 9F ), 2-amino-N-methylacetamide ( Figure 9G ) and ethanolamine ( Figure 9H ) to explore, while 2-oxopiperazine ( Figure 9I ) and morpholin-2-one ( Figure 9J Conformationally restricted analogues were explored. (1H-tetrazol-5-yl)methylamine Figure 9K The isochoric displacement of acids was explored.

[0398] (xviii) Oral glucose tolerance test (OGTT)

[0399] An oral glucose tolerance test (OGTT) was performed after a 12-hour fast. Blood samples were collected from the tail end at 0, 15, 30, 60, and 120 minutes after oral gavage of glucose (2 mg / g body weight), with or without an indicated dose (0.17–1 mg / g body weight). Blood glucose concentrations were measured using a blood glucose meter and a test strip (Contour Next).

[0400] (xix) Body Composition and Comprehensive Laboratory Animal Monitoring System (CLAMS)

[0401] All measurements were performed by the University of Michigan Animal Phenotyping Core. Body fat, lean body mass, and free fluid were measured using a nuclear magnetic resonance (NMR) based analyzer (Minispec LF90II; Bruker Optics). The analyzer was tested daily with reference samples as recommended by the manufacturer. Mice were individually placed in measurement tubes with minimal restrictions. Oxygen consumption (VO2), carbon dioxide production (VCO2), and kinetic activity were measured using CLAMS (Columbus Instruments) (an integrated open-circuit calorimeter equipped with a beam activity monitoring device). Mice were weighed prior to measurement and individually placed in sealed chambers (7.9" x 4" x 5") with free access to food and water. The study was conducted in a laboratory at 20-23°C with a 12-12 h dark-light cycle (6:00 PM to 6:00 AM). Measurements were performed continuously for 48 h. During this period, animals were provided with food and water via feeding and drinking devices located inside the chambers. VO2 and VCO2 in each chamber were sampled sequentially for 5 seconds at 10-minute intervals, and movement was recorded once per second along the X and Z dimensions. The airflow through the chambers was adjusted to maintain an oxygen difference of approximately 0.3% under resting conditions. The respiratory exchange rate (RER) was calculated as VCO2 / VO2. Total energy expenditure, glucose oxidation, and lipid oxidation were calculated based on the values ​​of VO2, VCO2, and protein breakdown.

[0402] (xx) Fecal microbiome analysis

[0403] Total genomic DNA of the gut microbiota was extracted from fecal samples using the QIAamp DNA Fecal Microkit (51540, Qiagen) according to the manufacturer's instructions. DNA was prepared for community analysis as previously described.17 In short, the DNA was amplified using barcoded double-index primers specific to the V4 region of the 16S rRNA gene.18 The PCR reaction consisted of 5 μL of a 4 μM equimolar primer set, 0.15 μL of AccuPrime Taq DNA high-fidelity polymerase, 2 μL of 10x AccuPrime PCR Buffer II (Thermo Fisher Scientific 12346094), 11.85 μL of PCR-grade water, and 1 μL of DNA template. PCR conditions were: 95°C for 2 min, followed by 30 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 5 min, followed by 72°C for 10 min. Each PCR reaction was normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific A1051001). The normalized reactions were pooled and quantified using the Kapa Biosystems Library qPCR Premix (ROX Low) Quantification Kit (KK4873) for the Illumina platform. The size of the amplicon library (approximately 399 bp) was confirmed using an Agilent Bioanalyzer with a High Sensitivity DNA Analysis Kit (5067-4626). The pooled amplicon library was then sequenced according to standard protocols on an Illumina MiSeq NANO platform (Microbial Systems Molecular Biology Laboratory, University of Michigan).

[0404] DNA sequencing data were processed using Mothur according to standard operating procedures (SOPs), primarily focusing on analyses based on α- and β-diversity. Sequences were trimmed as previously described to remove primers and barcodes, quality filtered, and chimerism tests performed. A total of 3565 sequences from all samples were used for further statistical analysis. The trimmed DNA sequences were clustered using the average neighbor approach to form operational taxonomic units (OTUs) at a 97% sequence similarity cutoff (3% sequence divergence). Phylogenetic trees were constructed using the Clearcut procedure. β-diversity was measured using an OTU-based approach. A heatmap of the relative abundance of each OTU across all samples was generated using log2 conversion of the relative abundance values ​​of the top 107 OTUs (>1%). Molecular AMOVA statistical analysis was performed to determine the significance of structural similarity among communities in each sampling group. UniFrac analysis was used to estimate weighted (WUnF) and unweighted (UWUnF) UniFrac measures. Constrained ordination RDA (redundancy analysis) was calculated, and the significance of environmental variables was tested by forward selection analysis. Microbial characteristics distinguishing the gut microbiota were characterized using the Linear Discriminant Analysis (LDA) effect size (LEfSe) method for discovering biomarkers (http: / / huttenhower.sph.harvard.edu / lefse / ), which emphasizes both statistical significance and biological association. Principal component analysis (PCA) was plotted accordingly using the Phyloseq package in R. The nonparametric Spearman's test was used to calculate the correlation between changes in altered bacterial genera and NAFLD-related parameters in the liver or plasma.

[0405] (xxi) In vitro studies

[0406] HepG2 human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO2 in Durbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin (Pen-Strep, Gibco). In some experiments, cells were loaded with palmitic acid (PA, 200 μM, Sigma-Aldrich P0500) in DMEM supplemented with 0.1% BSA but without FBS. AGXT1-targeting siRNA (siAGXT1: GCAAGGAUAUGUACCAGAUtt, siRNA number s1190) and a non-targeting siRNA control (siCTL, siRNA number AM4611) were obtained from Ambion. HepG2 cells were transfected with 20 nM siAGXT1 or siCTL using liposome RNAiMAX (Invitrogen) in Opti-MEM serum-depleted medium (Gibco) according to the manufacturer's protocol. RNA isolation, protein, or lipid extraction were performed 48 h post-transfection. Total RNA was purified from HepG2 cells using the QIAGEN RNeasy kit (QIAGEN). qPCR analysis was performed using the human primer pairs listed in Table S3 as described above. Cells were lysed using RIPA buffer (Thermo Scientific) supplemented with a protease / phosphatase inhibitor mixture (Roche Applied Science). AGXT1 protein abundance was assessed using Western blotting as described above. Cell lipids were extracted from cells using a 3:2 hexane:isopropanol ratio, and the hexane phase was evaporated for 48 h. Remaining cells in the plate were lysed in 0.1 M NaOH for 24 h, and aliquots were collected for cellular protein measurements using a Bradford protein analyzer (Bio-Rad). Cellular TG or TC levels were determined spectrophotometrically using a commercially available kit (Wako Chemicals). Cellular TG and TC data were normalized to protein levels and presented as micrograms of TG or TC per milligram of protein.

[0407] H. Results

[0408] (i) Impaired glycine biosynthesis in mouse and human NAFLD

[0409] To test whether altered glycine metabolism promotes the development of NAFLD, we first investigated C57BL / 6J mice with WD-induced HS. After 12 weeks of WD feeding, hypercholesterolemia ( Figure 10A ) and HS was clearly confirmed by H&E and Oil Red O (ORO) staining and quantitative analysis of liver TG and total cholesterol (TC). Figures 10B to 10D Targeted metabolomics revealed that among all amino acids (AAs), plasma glycine was most significantly decreased, while its precursors serine, threonine, and alanine were significantly increased. Figure 10E This indicates impaired glycine biosynthesis. Therefore, we next investigated the expression of genes driving glycine formation. We found that major glycine biosynthesis genes were downregulated in mice with HS, among which AGXT1 was most significantly suppressed ( Figure 10F Additionally, AGXT1 was significantly downregulated in HepG2 cells with palmitic acid (PA)-induced TG accumulation. Figure 10G , Figure 10H ).

[0410] To test whether a similar pattern is evident in more severe NAFLD, we performed RNA sequencing on the livers of mice with advanced NASH and fibrosis induced by 24 weeks of NASH-dietary feeding. Figure 10I Pathway analysis revealed alterations in known pathways involved in NASH, including upregulation of chemokine, NF-κB, bell-like receptor (TLR), and transforming growth factor-β (TGFβ) signaling, and downregulation of FA degradation and peroxisomal proliferator-activated receptor (PPAR) signaling. Figure 10J Interestingly, in NASH, the pathways regulating AA biosynthesis, including the metabolism of glycine, serine, threonine, and glyoxylate, were significantly downregulated, including AGXT1 inhibition (P = 0.0009). Figure 10K Using an independent cohort of mice with diet-induced NASH, AGXT1 inhibition was confirmed by qPCR. Figure 10L ).

[0411] To test whether glycine biosynthesis genes are similarly suppressed in human NAFLD, we performed a meta-analysis of transcriptomics from livers of NASH patients. We found that AGXT1 (β = -0.141, P = 0.0041) and AGXT2 (β = -0.134, P = 0.0135) were significantly downregulated in NASH, while D-amino acid oxidase (DAO), which catalyzes the degradation of glycine to glyoxylate, was significantly upregulated (β = 0.216, P = 0.0025). Figure 10MIn 206 samples obtained from liver transplant donors, we found that AGXT1 expression was inversely correlated with liver fat content (r = -0.199, P = 0.0044). Figure 10N Interestingly, a similar inverse correlation was found between the expression of PPARα (the master regulator of liver FAO) and liver fat content (r = -0.154, P = 0.0275), while the expression of CC motif chemokine ligand 5 (CCL5) and TGFβ (key players in steatohepatitis and fibrosis) was positively correlated with liver fat (r = 0.185, P = 0.0078 and r = 0.284, P < 0.0001, respectively, not shown). Regarding the inhibition of glycine biosynthesis genes, these results are consistent with current reports of low circulating glycine levels in NAFLD and suggest a role for AGXT1 in NAFLD.

[0412] (ii) AGXT1 loss exacerbates diet-induced hyperlipidemia and NASH

[0413] To investigate the potential role of the liver-specific gene AGXT1, localized to hepatocyte peroxisomes or mitochondria, in cellular lipid accumulation, we knocked out AGXT1 in HepG2 cells, which enhanced PA-induced TG accumulation. Figures 8C to 8E To investigate the effects of AGXT1 loss in vivo, we generated AGXT1 using CRISPR / Cas9. - / - mice ( Figure 8A , Figure 8B In CD feeding conditions, as previously indicated, AGXT1 - / - Liver histology and AGXT1 + / + Similarly, and we also found that plasma liver enzymes were similar ( Figures 8F to 8K However, after 12 weeks of the NASH-diet, AGXT1 - / - Mice showed increased plasma TG, TC, AST, and ALT levels. Figures 8L to 8O Meanwhile, the ratio of glycine to oxalate decreased significantly. Figure 8P The overall appearance of the peritoneal cavity revealed in AGXT1 - / - Larger livers in mice with enhanced pale yellow staining ( Figure 11A Although no significant difference was observed in body weight, liver weight increased significantly. Figure 11B , Figure 11C , Figure 11D The combination of H&E and ORO staining with liver lipid quantification revealed in AGXT1 - / - Increased HS in mice ( Figure 11A , Figure 11E , Figure 11F Furthermore, Sirius red staining revealed increased fibrosis. Figure 11AFurther histological analysis confirmed higher NAS and fibrosis scores in AGXT1- / - mice. Figure 11G , Figure 11H , Figure 11I ).

[0414] To understand AGXT1 - / - We investigated the potential mechanisms underlying diet-induced accelerated NASH in mice using AGXT1. + / + and AGXT1 - / - RNA was sequenced from mouse livers and subsequently validated by qPCR. Pathway analysis revealed AGXT1. - / - In mice, energy metabolism and the FAO pathway were inhibited, while pro-inflammatory pathways were upregulated. Figure 11J ). In AGXT1 - / - In mice, genes regulating peroxisomes (acyl-CoA thioesterase-3, ACOT3; acyl-CoA synthase medium chain-5, ACSM5; acyl-CoA synthase long chain-1, ACSL1) and mitochondrial FAO (hydroxyacyl-CoA dehydrogenase-α, HADHA; and acetyl-CoA acyltransferase-2, ACAA2) were significantly downregulated. Figures 11K to 11M However, the effect was slight for PPARα (P = 0.0557), while genes encoding regulators of pro-inflammatory signaling (NFKB1 / 2, RELB, CCR2 / 5, and TLR2 / 4), cytokines (TNFα and CCL2), fibrosis (TGFB1 / 2 and TGFBR2), and extracellular matrix (ECM) remodeling (COL1A2, COL4A2, and TIMP1) were significantly upregulated. Figure 11N , Figure 11O Therefore, the liver-specific glycine biosynthesis gene AGXT1 is suppressed in NAFLD, and its deficiency accelerates diet-induced NASH.

[0415] (iii) Glycine deficiency exacerbates diet-induced hyperlipidemia and HS

[0416] Lower plasma glycine levels are associated with NAFLD and cardiovascular metabolic disease, while higher levels are associated with favorable lipid profiles. To investigate the role of dietary glycine in dyslipidemia, we developed AA-modified WD (WD) with or without glycine. AA +Gly or WD AA -Gly, Table S2) and for hyperlipidemia apoE - / - Mice fed CD and WD AA +Gly or WD AA -Gly lasts for 10 weeks. (While feeding WD) AA A significant decrease in plasma glycine was found in mice with -Gly. Figure 12ANMR-based body composition and CLAMS analysis indicated the need for feeding WD. AA -Gly mice showed increased body weight and fat, but when fed WD AA No increase was observed in mice with +Gly ( Figures 12B to 12D Food intake, activity, or energy metabolism did not change significantly. Figures 12E to 12H Therefore, when feeding WD AA In A-Gly mice, the plasma glycine-containing tripeptide molecule content increased ( Figure 12I ), and noted in the epididymis and subcutaneous adipose tissue (EAT and SAT, ), Figure 12J Increased hypertrophy of adipocytes in the lesions. (This is in contrast to WD.) AA Compared to +Gly, feeding WD AA -Gly mice showed increased plasma TC and TG ( Figures 13A to 13D ). In feeding WD AA Increased plasma glucose was observed in -Gly mice, but in mice fed WD AA No observation was made in +Gly mice ( Figure 13E Histological and lipid quantification revealed in WD AA- -Gly feeding increases HS ( Figures 13F to 13H Linear regression analysis indicated a significant negative correlation between plasma glycine and individual levels of plasma TC, glucose, or liver TG. Figures 13I to 13K Therefore, dietary glycine deficiency exacerbates hyperlipidemia, hyperglycemia, and hemorrhage in hyperlipidemic mice.

[0417] (iv) DT-109: A glycine-based tripeptide with dual glucose-lowering / lipid-lowering properties

[0418] Considering the above findings, we infer that glycine-based compounds possess therapeutic potential through a dual glucose / lipid-lowering effect. Therefore, we investigated compounds with structures similar to glycine by applying various chemical modifications to the amine or carboxyl groups. Ten potential compounds were identified. Figures 9A to 9K Four of these are suitable for oral administration. Chronic glycine supplementation (1 mg / g / day) has been shown to reduce apoE levels. - / - Decreased circulating triglycerides (TG) in mice restored glucose tolerance and accelerated fat loss in obese C57BL / 6 mice. In humans, glycine intake with glucose reduced the increase in plasma glucose by >50%. To test the hypoglycemic effect, we conducted an oral glucose tolerance test (OGTT) in C57BL / 6J mice administered glycine or a glycine-based compound (0.5 mg / g). None of the compounds tested reduced the increase in blood glucose more efficiently than glycine. Figures 14A to 14DAnother amino acid (AA) reported to lower glucose in humans and mice is leucine. Previous research in our lab revealed the significant hypoglycemic effect of DT-109 (a tripeptide combining glycine and leucine, gly-gly-L-leu). We tested DT-109 or its D-isomer (gly-gly-D-leu, DT-110) using OGTT. Although the hypoglycemic effect of DT-110 is similar to that of glycine, DT-109 is the only compound that lowers glucose more efficiently than glycine. Figure 14E , Figure 14F ), especially when compared with its individual AA at the equivalent level ( Figure 14G ).

[0419] To test the lipid-lowering effect, we administered DT-109 (1 mg / g / day), equivalent levels of leucine, glycine, or H2O to patients with hyperlipidemia using apoE. - / - Mice fed WD ( Figure 15A After 10 weeks, DT-109 showed the most effective glucose-lowering effect in both OGTT and non-fasting blood glucose tests. Figure 15B , Figure 15C No significant differences were observed in food intake or body weight. Figure 15D , Figure 15E Lipid pattern analysis showed that mice treated with DT-109 had the lowest TC and LDL, and no decrease in HDL was observed in mice treated with glycine. Figures 15F to 15I And there were significant differences in plasma TG levels. Figure 15J Glycine or DT-109 reduces glycine-containing tripeptide molecules and alleviates adipocyte hypertrophy in EAT and SAT. Figure 15K , Figure 15L Histological and lipid quantification revealed decreased HS in the livers of mice treated with glycine or DT-109, but not in the presence of leucine. 16A to 16C qPCR analysis revealed that in the livers of mice treated with glycine or DT-109, key genes regulating FAO (PPARα, PNPLA2, carnitine / acylcarnitine translocase CACT, carnitine palmitoyltransferase 1a CPT1a, and acyl-CoA dehydrogenase long chain ACADL) were upregulated and CCL2 was downregulated, while TNFα showed no significant change. Figure 16D Therefore, DT-109 has dual glucose / lipid-lowering properties and prevents WD-induced HS in hyperlipidemic mice.

[0420] (v)DT-109 improves body composition and prevents diet-induced NASH.

[0421] To further explore the therapeutic potential of DT-109 for NASH, we designed an experimental method to model advanced NAFLD. Figure 17A Compared to CD-fed mice, C57BL / 6J mice fed a NASH-diet for 12 weeks showed increased plasma glucose, TC, AST, and ALT levels. Figure 17B A subset of mice were sacrificed, confirming an increase in liver weight after feeding a NASH-diet. Figure 17C H&E and ORO histology revealed HS, hepatocellular ballooning degeneration, and inflammatory cell infiltration, while Sirius red staining confirmed early fibrosis. Figure 17D After confirming NASH ( Figures 17D to 17F The remaining mice were randomized to receive 0.125 or 0.5 mg of DT-109, equivalent levels of leucine, glycine, or H2O daily via re-oral gavage for 12 weeks under NASH-diet conditions. Mice fed CD and administered H2O served as controls.

[0422] At week 18, OGTT and non-fasting blood glucose measurements confirmed that 0.5 mg DT-109 per gram daily was the most effective in lowering glucose. Figure 17G , Figure 17H Body composition analysis revealed weight gain in all mice fed the NASH diet. Figure 17I However, compared to the H2O control, mice treated with 0.5 mg DT-109 daily showed reduced body fat while retaining muscle mass. Figure 17J , Figure 17K Furthermore, the hypertrophy of adipocytes in EAT and SAT was reduced. Figure 17L ), while food intake did not differ significantly. Figure 17M The metabolic response to diet-induced obesity involves a shift to higher fat utilization relative to lower carbohydrate utilization, reflecting a decreased renal efficiency (RER). CLAMS analysis revealed a decreased RER in all groups fed the NASH-diet, but no significant difference was observed in mice treated with 0.5 mg DT-109 per gram daily. Figures 17N to 17P No significant differences were observed in energy consumption or activity. Figure 17Q , Figure 17R ).

[0423] At the endpoint, the increases in plasma AST, ALT, and alkaline phosphatase (ALP) observed in NASH-fed mice were reduced by treatment with glycine or DT-109. Figures 18A to 18C In mice treated with glycine or DT-109, plasma TG levels decreased ( Figure 18D ), and TC decreased with 0.5 mg DT-109 per gram per day ( Figure 18ETherefore, treatment with glycine or DT-109 significantly reduced NASH-induced hepatomegaly and pale yellow discoloration, but did not significantly reduce them with leucine. Figure 18F , Figure 18G , Figure 18H ), and NAS was significantly reduced by 0.5 mg DT-109 per gram per day ( Figure 18I , Figure 18J Linear regression analysis indicated a highly significant positive correlation between individual AST, ALT, or ALP levels and NAS. Figures 18K to 18M ).

[0424] (vi) DT-109 reverses NASH-diet-induced transcriptome changes: its key role in FAO

[0425] To explore the mechanism by which glycine-based therapy prevents diet-induced NASH, we performed RNA sequencing on liver samples collected at the endpoint. Principal component analysis (PCA) showed that, in the intermediate glycine pattern and at 0.125 mg / g / day, the gene expression patterns of mice on a NASH-treated diet based on leucine clustered with H2O controls, while those receiving 0.5 mg / g DT-109 daily clustered more closely with CD mice. Figure 19A Volcano plot analysis confirmed that, compared to CD, large DEG changes (3606 or 3145 DEGs, respectively) in mice fed a NASH-diet and treated with H2O or leucine were significantly reduced by treatment with glycine or DT-109 at 0.125 or 0.5 mg / day (1300, 1093, or 642 DEGs, respectively). Figure 19B The analysis of the first 50 DEGs further highlighted the similarity between DT-109 (0.5 mg / g / day) and the CD group. Figure 19C ). Pathway analysis comparing NASH-dietary H2O controls with CD confirmed the inhibition of pathways regulating glycine biosynthesis and glyoxylate metabolism, as well as energy metabolism and FAO pathways, under conditions of downregulation of AGXT1, SHMT1, and SARDH. In contrast, known NASH-related pro-inflammatory / fibrotic pathways are upregulated ( Figure 19D , Figure 19E ). Pathway analysis comparing NASH-dietary H2O controls with 0.5 mg / day DT-109 showed a similar pattern to CD, indicating that DT-109 reverses the underlying pathway changes induced by NASH-dietary intake. Figure 19F Analysis of 50 genes involved in the main aspects of NASH pathogenesis (). Figure 19GThis study showed that key genes regulating FAO (PPARα, PPARG-coactivator-1α (PPARGC1A / PGC1α), acyl-CoA oxidase-1 (ACOX1), CPT2, ACADS / M / L, HADHA / B, and ACOT3 / 4) were overpresented in CD mice but suppressed in NASH mice treated with H2O or leucine. This suppression was reversed by treatment with glycine or DT-109 (specifically 0.5 mg / g / day), as confirmed by qPCR and Western blot analysis. Figure 19H , Figure 19I , Figure 19J Therefore, ORO and lipid quantification confirmed that significant HS in the livers of NASH mice treated with H2O or leucine was significantly reduced by glycine or DT-109. Figures 19K to 19L , Figure 19M In particular, diacylglycerols (DAG), known to promote liver damage and NASH, are significantly reduced with DT-109 at a dose of 0.5 mg per gram daily. Figure 19N Therefore, glycine-based treatment corrects impaired FAO, alleviates NASH-diet-induced HS, and reduces lipotoxic lipids.

[0426] (vii) DT-109 reduces NASH-induced diet-induced hepatitis and fibrosis.

[0427] RNA sequencing analysis revealed the inhibitory effect of glycine-based therapy on major inflammatory pathways / genes. Figure 19F , Figure 19G This indicates an anti-inflammatory effect. In fact, in the livers of mice treated with H2O or leucine under NASH-diet conditions, immunostaining for F4 / 80 (a well-established marker for hepatic macrophages) was significantly increased, but this was mitigated by glycine or DT-109. Figure 20A , Figure 20B In mice treated with glycine or DT-109, plasma levels of monocyte chemoattractant protein-1 (MCP-1 / CCL2) and resistin (a known inflammatory marker in NASH patients) were lower. Figure 20C , Figure 20D Therefore, RNA sequencing showed that in mice fed a NASH diet and treated with H2O or leucine, genes encoding pro-inflammatory signaling regulators (NFKB1 / 2, RELB, CCR1 / 2 / 5, TLR1 / 2 / 4, and TNFRSF1A / 9 / 12) and cytokines (TNFα and CCL2 / 5) were upregulated, and these upregulations were alleviated by glycine or DT-109. Figure 19GThis was confirmed by qPCR analysis, in which NFKB2, RELB, and TNFα were significantly downregulated by glycine or DT-109, while CCL2, CCR2, and CCR5 were only downregulated by 0.5 mg / day of DT-109. Figure 20E ).

[0428] RNA sequencing also confirmed that pathways / genes associated with TGFβ signaling (TGFB1 / 2 and TGFBR1 / 2) and ECM remodeling (COL1A1 / 1A2 / 3A1 / 4A1 / 4A2, TIMP1 / 2, and SERPINE1) were upregulated by the NASH diet and mitigated by glycine or DT-109. Figure 19F , Figure 19G In fact, histological analysis and fibrosis scoring based on Sirius red revealed the protective effect of glycine or DT-109 (rather than leucine) against NASH-diet-induced liver fibrosis. Figure 20A , Figure 20F , Figure 20G Linear regression analysis confirmed a highly significant positive correlation between individual AST, ALT, or ALP levels and fibrosis scores, indicating that glycine or DT-109 reduced NASH-induced liver damage. Figure 20H , Figure 20I , Figure 20J To test whether glycine-based treatment alleviated TGFβ-mediated liver fibrosis, we next analyzed SMAD signaling and found that SMAD2 Ser465 / 467 phosphorylation was mainly reduced by DT-109. Figure 20K qPCR analysis confirmed that TGFβ-related genes were significantly upregulated in the livers of NASH mice administered H2O or leucine, which was mitigated by DT-109. Figure 20L Therefore, consistent with reduced HS and lipotoxicity, the two glycine-based treatments reduced the degree of NASH-diet-induced steatohepatitis and fibrosis.

[0429] I. Discussion

[0430] Although lipid and glucose metabolism abnormalities are known features of NAFLD, perturbations in AA metabolism have been proposed in NASH. In particular, lower circulating glycine levels have been consistently reported in NAFLD patients, but the reasons for this reduction and its therapeutic potential remain unclear. In this paper, using genetic and dietary approaches to restrict glycine availability, we provide evidence of the induced role of glycine in the development of NAFLD. In an investigation of potential glycine-based therapies for NAFLD, we identified DT-109 as having a dual glucose-lowering / lipid-lowering effect and effectively preventing diet-induced NASH in mice.

[0431] The results presented in this article demonstrate that the lower glycine levels observed in NAFLD patients are associated with suppressed hepatic glycine biosynthesis genes. Specifically, in both mouse and human NASH, we found significant inhibition of AGXT1, which catalyzes the conversion of glyoxylate to glycine, and confirmed that AGXT1 expression is inversely correlated with hepatic steatosis in humans. While others have reported suppression of AGXT1 in NASH patients or mouse models, we report for the first time the induced role of AGXT1 in NAFLD. Mutations in AGXT1 cause primary hyperoxaluria type 1, which is caused by impaired glyoxylate-to-glycine conversion and the production of excessive hepatic oxalate, leading to renal failure. Interestingly, although AGXT1… - / - Proteomics analysis of mouse livers indicates significant alterations in glucose and lipid metabolic pathways, but the role of AGXT1 in NASH has not been previously evaluated. Using CRISPR / Cas9, we generated AGXT1... - / - Mice that had developed worsened hyperlipidemia and NASH after 12 weeks of NASH-dietary administration were identified in AGXT1 mice. - / - Inhibition of the FAO pathway in mice promotes fatty liver disease and fibrosis.

[0432] We also applied dietary approaches to restrict glycine availability and compared lipid profiles and hepatic steatohepatitis (HS) in hyperlipidemic mice fed glycine-containing or glycine-free WD. The enhanced obesity, hyperlipidemia, and HS observed in glycine-deficient WD mice were consistent with previous reports, where dietary glycine accelerated fat loss, improved glucose tolerance, reduced plasma lipids, or alleviated HS in various rodent models. Interestingly, despite small sample size and short treatment duration, HS reduction and decreased plasma liver enzymes were observed in NAFLD patients following supplementation with the glycine precursor serine. In a thorough investigation using a late-stage NAFLD model characterized by coexisting steatohepatitis and fibrosis, we report for the first time the protective effect of glycine treatment at a relatively low dose of 0.33 mg / g / day in mice.

[0433] During our investigation of glycine-based compounds, none of the compounds identified lowered plasma glucose more efficiently than glycine. Therefore, we tested the combination of glycine with leucine, another amino acid (AA) reported to lower glucose in humans and alleviate hemolytic disease (HS) in mice. Specifically, using various T2D models, our laboratory revealed that the tripeptide gly-gly-L-leu had a more potent glucose-lowering effect than free glycine, leucine, or combinations thereof. Using genetic and dietary models, we demonstrated for the first time that DT-109 also improves lipid profile, HS, and NASH. While no significant effect was observed in mice treated with equivalent leucine levels, metabolic benefits were evident after glycine treatment. However, some results were only evident at higher doses of DT-109, including robust glucose-lowering effects, maintenance of HDL levels in hyperlipidemic mice, prevention of NASH-diet-induced changes in body composition, reduction of hepatic DAG, and a significant reduction in NAS. It should be noted that significant benefits were also observed at lower doses of 0.125 mg / g DT-109 daily.

[0434] Lipid overload is crucial to the pathogenesis of NASH. When free fatty acids are excessively supplied to the liver and / or impaired via FAO treatment, they act as substrates for lipotoxic substances that induce oxidative stress and pro-inflammatory / fibrotic pathways, thereby promoting steatohepatitis and fibrosis. Using unbiased transcriptomics, we identified that the major FAO pathway, which was suppressed in the liver of NASH mice, was reversed by DT-109, followed by a reduction in hepatic toxicity (HS) and lipotoxic DAG. This suggests that glycine-based treatment normalizes impaired hepatic FAO and reduces the levels of HS and lipotoxicity, which in turn slows the progression of NASH. Indeed, using our model characterized by steatohepatitis and fibrosis, we found, as evident by tissue, transcriptomics, and plasma analyses, that glycine or DT-109 reduces NASH-diet-induced liver / systemic inflammation and fibrosis. Consistent with previous studies reporting anti-inflammatory and hepatoprotective effects of glycine in mice with endotoxemia. In patients with type 2 diabetes, glycine treatment (5 g / day) for 3 months reduced hemoglobin-A1c and plasma TNFR1.

[0435] In summary, the identification of impaired glycine metabolism in NAFLD led the inventors to determine a glycine-based treatment that has been shown to be effective in experimental NAFLD by modulating hepatic FAO.

Claims

1. Use of a therapeutically effective amount of Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver disease, wherein the liver disease is non-alcoholic fatty liver disease.

2. Use of a therapeutically effective amount of Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver disease, wherein the liver disease is non-alcoholic steatohepatitis.

3. Use of Gly-Gly-Leu, Gly-Gly-dLeu or pharmaceutically acceptable salts thereof in the preparation of a medicament for alleviating liver fibrosis in subjects in need.

4. The use as described in any one of claims 1 to 3, wherein the use further comprises administering a second therapeutic agent to the subject in need, the second therapeutic agent comprising etimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gecarbene (CI-1027), thiazolidinedione (TZD), GLP-1, insulin, metformin, bepadicarboxylic acid (ETC-1002), and combinations thereof.

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