Treatment of tripeptides and metabolic, cardiovascular, and inflammatory disorders

Abstract

To provide a method for treating at least one of hyperlipidemia, fatty liver, steatohepatitis, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, hyperglycemia, metabolic syndrome, cardiovascular disease, and atherosclerosis in a mammalian subject.SOLUTION: The present disclosure provides a method comprising administering to a subject a therapeutical effective amount of glycine, a glycine-containing tripeptide molecule, or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Patent Application No. 62 / 717,546, filed on 10 August 2018. The entirety of the aforementioned application is incorporated herein by reference. [Background technology]

[0002] Non-alcoholic fatty liver disease (NAFLD) is becoming increasingly common worldwide, especially in Western countries. In the United States, it is the most common form of chronic liver disease, affecting an estimated 80 to 100 million people. NAFLD is a general term for various liver diseases that affect people who drink little to no alcohol. As the name suggests, the main characteristic of NAFLD is the accumulation of large amounts of fat in liver cells. It is normal for the liver to contain fat. However, if more than 5% to 10% of the liver's weight is fat, the condition is called fatty liver (or steatosis).

[0003] A more severe form of NAFLD is called non-alcoholic steatohepatitis (NASH). NASH causes the liver to swell and become damaged. NASH tends to develop in people who are overweight, i.e., obese, or who have diabetes, high cholesterol, high triglycerides, or inflammatory conditions. NASH, a potentially severe form of this disease, is characterized by ballooning of hepatocytes and inflammation of the liver, which can progress to scarring and irreversible damage. [Overview of the project]

[0004] 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 glycine-containing tripeptide molecules to a subject having one or more inflammatory diseases, metabolic diseases and / or cardiovascular diseases. In various embodiments, the glycine-containing tripeptide molecules may comprise one or more of DT-190 (Gly-Gly-Leu) and DT-110 (Gly-Gly-dLeu). In various embodiments, metabolic diseases refer to a group of identified disorders resulting in metabolic errors, metabolic imbalances, or suboptimal metabolism. Metabolic diseases described herein include diseases that can be treated through metabolic regulation, although the diseases themselves may or may not be caused by specific metabolic deficiencies. Such metabolic diseases may include, for example, glucose and fatty acid oxidation pathways. "Metabolic disorder" or "metabolic disease" refers to a pathological condition characterized by alteration or interference with metabolic function. "Metabolic" and "metabolism" are well-known terms in the art and generally encompass the full range of biochemical processes occurring in living organisms. Metabolic and cardiovascular diseases include, but are not limited to, obesity, diabetes mellitus, atherosclerosis, metabolic syndrome, dyslipidemia, coronary heart disease, coronary artery disease, arteriosclerosis, atherothrombotic stroke, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hyperlipidemia or metabolic syndrome, or a combination thereof. Dyslipidemia can be hyperlipidemia. Hyperlipidemia can be hypercholesterolemia, hypertriglyceridemia, or both. NAFLD can be fatty liver or steatohepatitis. Diabetes mellitus can be type 2 diabetes mellitus or type 2 diabetes mellitus with dyslipidemia.This specification presents methods applicable in various embodiments to metabolic diseases associated with glucose dysregulation and / or lipid accumulation in the body, in the circulation, or in various organs such as the liver, as well as to pathological complications arising therefrom, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hyperglycemia, prediabetes, diabetes mellitus (type I and type II), obesity, insulin resistance, metabolic syndrome, and diabetic dyslipidemia.

[0005] Metabolic diseases, disorders, or conditions may be characterized by a number of physical symptoms. Any symptoms known to those skilled in the art that are associated with metabolic diseases, disorders, or conditions may be prevented, treated, ameliorated, or otherwise regulated using glycine tripeptide molecules and the methods described herein. In certain embodiments, symptoms may be, but are not limited to, polyuria (excessive urination), polydipsia (excessive thirst and increased fluid intake), blurred vision, unexplained weight loss, and lethargy.

[0006] In certain embodiments, inflammatory diseases, disorders, or conditions include, but are not limited to, aortic stenosis, coronary artery disease (CAD), Alzheimer's disease, and thromboembolic diseases, disorders, or conditions. Specific thromboembolic diseases, disorders, or conditions include, but are not limited to, stroke, thrombosis, myocardial infarction, and peripheral vascular disease.

[0007] In certain embodiments, the use of glycine or glycine tripeptide molecules described herein modulates physiological markers or phenotypes of inflammatory diseases, disorders, or pathologies. For example, administration of the compounds to animals can reduce levels of inflammatory cytokines or other inflammatory markers in those animals compared to untreated animals. In certain embodiments, the modulation of physiological markers or phenotypes may be associated with inhibition of hepatic DAG, glucose reduction, and reduction of plasma LDL levels by the compounds.

[0008] In certain embodiments, physiological markers of inflammatory diseases, disorders, or pathologies can be quantified. For example, cytokine levels can be measured and quantified by standard tests known in the art. With respect to such markers, in certain embodiments, the markers can be reduced by a range defined 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.

[0009] Furthermore, this specification provides methods for preventing, treating, or improving symptoms associated with diseases, disorders, or conditions in subjects requiring such treatment. In certain embodiments, methods are provided for reducing the incidence of symptoms associated with inflammatory diseases, disorders, or conditions. In certain embodiments, methods are provided for reducing the severity of symptoms associated with inflammatory diseases, disorders, or conditions. In such embodiments, the method comprises administering a therapeutically effective amount of glycine or glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, to an individual requiring such treatment.

[0010] Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression of, and / or improving metabolic, cardiovascular, and inflammatory diseases, disorders, and conditions, particularly those related to cholesterol abnormalities, triglycerides, and glucose, in subjects requiring such treatment. In certain embodiments, such diseases, disorders, and conditions include inflammatory, cardiovascular, and / or metabolic diseases, disorders, and conditions. Specific such cardiovascular diseases, disorders, or conditions include, but are not limited to, aortic stenosis, aneurysms (e.g., abdominal aortic stenosis), angina, arrhythmias, atherosclerosis, cerebrovascular diseases, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular diseases (e.g., peripheral artery disease, peripheral artery occlusive disease), retinal vascular occlusion, or stroke. In certain embodiments, metabolic diseases, disorders, or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (types I and II), obesity, insulin resistance, metabolic syndrome, and diabetic dyslipidemia. Specific such inflammatory diseases, disorders, or conditions include, but are not limited to, aortic stenosis, coronary artery disease (CAD), Alzheimer's disease, and thromboembolic diseases, disorders, or conditions. Specific thromboembolic diseases, disorders, or conditions include, but are not limited to, stroke, thrombosis (e.g., venous thromboembolism), myocardial infarction, and peripheral vascular disease. Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression, and / or improving glucose sensitivity and systemic inflammation and fibrosis, particularly in relation to liver disease, as well as for preventing, treating, delaying, slowing the progression, and / or improving diet-induced hyperlipidemia and steatohepatitis with inhibitory mitochondrial / peroxisome fatty acid β-oxidation (FAO).

[0011] One embodiment of the first aspect of this disclosure provides a method for treating non-alcoholic fatty liver disease (NAFLD), comprising the administration of 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), comprising the administration of 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), comprising the administration of DT-110 (Gly-Gly-dLeu). One embodiment of the first aspect of this disclosure provides a method for treating non-alcoholic steatohepatitis (NASH), comprising the administration of glycine or DT-110 (Gly-Gly-dLeu). These tripeptides may optionally be administered in combination with second-line therapeutic agents as described herein.

[0012] A second aspect of the present disclosure provides a method for alleviating fibrosis in a patient, comprising administering a selected tripeptide or a pharmaceutically acceptable salt thereof. One embodiment of the second aspect of the present disclosure provides a method for alleviating fibrosis in a patient, comprising administering glycine or DT-109 (Gly-Gly-Leu). Another embodiment of the second aspect of the present disclosure provides a method for alleviating fibrosis in a patient, comprising administering glycine or DT-110 (Gly-Gly-dLeu). These tripeptides may optionally be administered in combination with statins.

[0013] A third aspect of this disclosure provides a method for treating fatty liver, comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need thereof. In one embodiment of the third aspect, the method reduces triglyceride levels in hepatic lipids or reduces total cholesterol levels in hepatic lipids.

[0014] A fourth aspect of the present disclosure provides a kit for treating a subject having NAFLD or NASH, comprising a selected tripeptide, optionally a 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), optionally a statin, and instructions for use. In another embodiment, the kit comprises glycine or (DT-110 (Gly-Gly-dLeu), optionally a 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), optionally a statin, and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] [Figure 1A] Experimental design (Figure 1A). [Figure 1B] Average food intake over the entire study (Figure 2B). [Figure 1C] Endpoint body weight (Figure 1C). [Figure 1D] Total increase in body weight from baseline to endpoint (Figure 1D) (n = 8-10 for B-D). [Figure 1E] Overall abdominal morphology at endpoint (Figure 1E). [Figure 1F] Plasma glycine-containing tripeptide molecule levels (Figure 1F) (n = 6-8). *p < 0.05; **p < 0.01 vs. WD + H2O.

[0016] [Figure 2A] Non-fasting blood glucose levels measured by acute OGTT (mice were given glucose and either the therapeutic agent or water as a control) (Figure 2A). [Figure 2B] Non-fasting blood glucose levels measured by chronic OGTT (mice were given only glucose) (Figure 2B). [Figure 2C] Non-fasting blood glucose levels measured before (before forced oral administration) and 30 minutes after (after forced oral administration) forced oral administration in the presence of water or the therapeutic agent (Figure 2C). [Figure 2D]Endpoint blood glucose level (after 6 hours of fasting) (Figure 2D). [Figure 2E] qPCR analysis of hepatic gene expression regulating glucose uptake and gluconeogenesis (Figure 2E). (n=8~10)*p<0.05;**p<0.01, ***p<0.001 vs WD+H2O;#p<0.05, ###p<0.001 vs Before forced oral administration.

[0017] [Figure 3A] This is a typical H&E slide of liver tissue (Figure 3A). [Figure 3B] Small droplet fatty liver and large droplet fatty liver are marked with red and yellow arrows, respectively. Liver lipids were extracted, and the TG content was then quantified (Figure 3B), with **p<0.01 and ***p<0.001 versus WD+H2O. [Figure 3C] Small droplet fatty liver and large droplet fatty liver are marked with red and yellow arrows, respectively. Liver lipids were extracted, and the TC content was then quantified (Figure 3C), with **p<0.01 and ***p<0.001 versus WD+H2O.

[0018] [Figure 4A] qPCR analysis of liver gene expression that regulates the lipid oxidation pathway (Figures 4A and 4B). [Figure 4B] qPCR analysis of liver gene expression that regulates the lipid oxidation pathway (Figures 4A and 4B). [Figure 4C] qPCR analysis of liver gene expression regulating cholesterol homeostasis (Figure 4C) (n=7-10). [Figure 4D] Western blot analysis and quantification of ABCG8 abundance normalized to β-actin (Figures 4D and 4E) (n=4-6). *p<0.05; **p<0.01, ***p<0.001 vs. WD+H2O. [Figure 4E] Western blot analysis and quantification of ABCG8 abundance normalized to β-actin (Figures 4D and 4E) (n=4-6). *p<0.05; **p<0.01, ***p<0.001 vs. WD+H2O.

[0019] [Figure 5A] Plasma TC levels at baseline (1 week of WD feeding without therapeutic agent) and endpoint (12 weeks after WD feeding with therapeutic agent or water control) (Figure 5A). [Figure 5B] (Figure 5B) Endpoint plasma levels of TC. Blood samples were taken after a 6-hour fast. [Figure 5C] Endpoint plasma levels (Figure 5C). Blood samples were taken after a 6-hour fast. [Figure 5D] (Figure 5D) Endpoint plasma levels of HDL. Blood samples were taken after a 6-hour fast. [Figure 5E] (Figure 5E) Endpoint plasma levels of TG. Blood samples were taken after a 6-hour fast. [Figure 5F] (Figure 5F) A representative image of the analysis of arteriosclerotic lesions visualized by oil red O staining. [Figure 5G] (Figure 5G) Stained aorta. (n=6~10). *p<0.05; **p<0.01, ***p<0.001 vs. WD+H2O.

[0020] [Figure 6A] Endpoint plasma levels of IL-6 (Figure 6A). [Figure 6B] End-point plasma levels of resistin (Figure 6B). [Figure 6C] Endpoint plasma levels of MCP1 (Figure 6C). [Figure 6D] (Figure 6D) qPCR analysis of inflammatory cytokines in epididymal adipose tissue (EAT). [Figure 6E] (Figure 6E) qPCR analysis of inflammatory cytokines in subcutaneous adipose tissue (SAT). (n=6-10). *p<0.05; **p<0.01, ***p<0.001 vs. WD+H2O.

[0021] [Figure 7A] qPCR analysis of inflammatory cytokines in the liver. [Figure 7B] Representative F4 / 80 immunohistochemistry in the liver. [Figure 7C] Quantification (n=7~10). *p<0.05 vs. WD+H2O.

[0022] [Figure 8A] Plasma changes in AGXT1- / - mice fed a NASH diet. (Figure 8A) The guide RNA target site on exon 1 of the AGXT1 gene is underlined, and an A deletion located 3 bases away from PAM was confirmed by Sanger sequencing. [Figure 8B] (Figure 8B) Deficiency of AGXT1 confirmed by Western blot (n=7). [Figure 8C] HepG2 cells were transfused with siCTL or siAGXT1: (Figure 8C) AGXT1 mRNA compared to GAPDH (n=12). [Figure 8D] (Figure 8D) AGXT1 protein containing GAPDH as a loading control (n=4). [Figure 8E] (Figure 8E) Cellular TG (200 μM, n=12) in the presence or absence of PA loading. [Figure 8F] Under CD feeding, AGXT1+ / + and AGXT1- / - are equivalent. AGXT1+ / + mice and AGXT1- / - mice were fed standard CD for 12 weeks (n=6): (Figure 8F) Body weight. [Figure 8G] (Figure 8G) Macroscopic appearance of the peritoneal cavity and histology using H&E and ORO staining (scale bars: 50 μm for H&E, 100 μm for ORO). [Figure 8H] (Figure 8H) Liver weight. [Figure 8I] (Figure 8I) Ratio of liver weight (LW) to body weight (BW). [Figure 8J] (Figure 8J) Plasma AST. [Figure 8K] (Figure 8K) Plasma ALT. Data are shown as mean ± SD, and all scores are displayed. [Figure 8L] AGXT1+ / + mice and AGXT1- / - mice were fed a NASH diet for 12 weeks (n=12): plasma (Figure 8L) TG. [Figure 8M] (Figure 8M)TC. [Figure 8N] (Figure 8N)AST. [Figure 8O] (Figure 8O) ALT. [Figure 8P] (Figure 8P) Glycine / oxalate ratio. Data are mean ± SD, and all scores and P values ​​are shown. PA, palmitic acid.

[0023] [Figure 9A] Glycine compounds. Compounds structurally similar to glycine were selected, and their structural, conformational, electronic, and steric equivalence modifications to the glycine skeleton were evaluated. (Figure 9A) Glycine. [Figure 9B] (Figure 9B) N-methylglycine. [Figure 9C] (Figure 9C) N,N-dimethylglycine. [Figure 9D] (Figure 9D) N,N,N-trimethylglycine. [Figure 9E] (Figure 9E) Glycolic acid. [Figure 9F] (Figure 9F) Glycineamide. [Figure 9G] (Figure 9G) 2-amino-N-methylacetamide. [Figure 9H] (Figure 9H) Ethanolamine. [Figure 9I] (Figure 9I) 2-oxopiperazine. [Figure 9J] (Figure 9J) Morpholine-2-one. [Figure 9K] (Figure 9K) (1H-tetrazole-5-yl)methanamine.

[0024] [Figure 10A] Impaired glycine biosynthesis in NAFLD. C57BL / 6J mice were given CD or WD for 12 weeks (n=4-5): (Figure 10A) Plasma TC. [Figure 10B] (Figure 10B) Liver histology (Scale bars: H&E 50 μm, ORO 100 μm). [Figure 10C] (Figure 10C) Liver triglycerides. [Figure 10D](Figure 10D) Liver TC. [Figure 10E] (Figure 10E) Plasma AA compared to CD. [Figure 10F] (Figure 10F) Liver expression of glycine biosynthesis genes compared with GAPDH. [Figure 10G] (Figure 10G) Cellular TG. [Figure 10H] (Figure 10H) AGXT1 expression in HepG2 cells loaded with 200 μM PA or ethanol for 24 hours (n=3-4). [Figure 10I] C57BL / 6J mice were fed either a NASH diet or CD for 24 weeks (n=10): (Figure 10I) Liver morphology, H&E and Sirius Red histology (scale bar: 50 μm). [Figure 10J] Pathway analysis of the liver of mice with NASH. Pathway analysis (n=3) after RNA sequencing of the livers of mice fed a CD or NASH diet for 24 weeks. Pathways rich in upregulated differentially expressed genes (DEGs) are plotted in red, while pathways rich in downregulated DEGs are plotted in green. [Figure 10K] (Figure 10K) Significant downregulation of glycine biosynthesis genes / pathways by RNA sequencing of livers from CD or NASH mice (green) (n=3, log2FC, log2 magnification change). [Figure 10L] (Figure 10L) AGXT1 expression compared to GAPDH in mice with diet-induced NASH (n=8). [Figure 10M] (Figure 10M) Meta-analysis of liver microarray data from healthy patients and NASH patients showing significant downregulation (green) or upregulation (red) in glycine metabolism genes. [Figure 10N](Figure 10N) Correlation between AGXT1 expression and total hepatic fat in transplant donor livers (n=206). Data are mean ± SD, and all scores and P values ​​are shown. Pathway analysis of livers in mice with NASH. Pathway analysis after RNA sequencing of livers in 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.

[0025] [Figure 11A] Accelerated diet-induced NASH in AGXT1- / - mice. AGXT1+ / + mice and AGXT1- / - mice were fed a NASH diet for 12 weeks (n=12): (Figure 11A) Macroscopic appearance of the abdominal cavity and liver histology (scale bars: H&E and Sirius Red 50 μm, ORO 100 μm). [Figure 11B] (Figure 11B) Liver weight / body weight (LW / BW) ratio. [Figure 11C] NAFLD-related parameters in AGXT1- / - mice fed a NASH diet. AGXT1+ / + mice and AGXT1- / - mice were fed a NASH diet for 12 weeks (n=12): (Figure 11C) Body weight. [Figure 11D] (Figure 11D) Liver weight [Figure 11E] (Figure 11E) Liver triglycerides. [Figure 11F] (Figure 11F) Liver TC. [Figure 11G] (Figure 11G) NAS. [Figure 11H] (Figure 11H) Fibrosis score. Data are mean ± SD, and all scores and P values ​​are shown. [Figure 11I] (Figure 11I) H&E-based scoring for fatty liver, hepatocyte ballooning, and intralobular inflammation. Data are mean ± SD. [Figure 11J] (Figure 11J) Pathway analysis after RNA sequencing of livers from AGXT1+ / + and AGXT1- / - mice (n=4). Upregulated or downregulated DEG-rich pathways are plotted in red or green, respectively. [Figure 11K] (Figure 11K) Heatmap of 25 NASH-related DEGs. [Figure 11L] (Figure 11L) FAO-related DEG confirmed by qPCR (n=10). [Figure 11M] (Figure 11M) Western blot (n=4). [Figure 11N] (Figure 11N) Inflammation. [Figure 11O] (Figure 110) Fibrosis-associated DEGs (n=10) confirmed by qPCR. Data are mean ± SE. *P<0.05, **P<0.01, ***P<0.001 vs. AGXT1+ / +.

[0026] [Figure 12A] Glycine deficiency exacerbates WD-induced obesity. ApoE- / - mice were fed CD, WDAA+Gly, or WDAA-Gly for 10 weeks (n=6): (Figure 12A) Plasma glycine. [Figure 12B] NMR-based body composition analysis: (Figure 12B) Body weight. [Figure 12C] (Figure 12C) Body fat (%). [Figure 12D] (Figure 12D) Lean body mass (%). [Figure 12E] CLAMS analysis: (Figure 12E) Food intake. [Figure 12F] (Figure 12F) Total activity. [Figure 12G] (Figure 12G) Respiratory exchange rate (RER). [Figure 12H] (Figure 12H) Energy consumption. [Figure 12I] (Figure 12I) Plasma glycine tripeptide molecule. [Figure 12J] (Figure 12J) H&E histology of epididymal tissue and subcutaneous adipose tissue (EAT and SAT, scale bar: 100 μm). Data are mean ± SD, and all scores and P values ​​are shown.

[0027] [Figure 13A]Glycine deficiency exacerbates WD-induced hyperlipidemia and HS. ApoE- / - mice were fed CD, WDAA+Gly, or WDAA-Gly for 10 weeks (n=6): (Figure 13A) Plasma TC. [Figure 13B] (Figure 13B) Plasma triglycerides. [Figure 13C] (Figure 13C) Plasma LDL. [Figure 13D] (Figure 13D) Plasma HDL. [Figure 13E] (Figure 13E) Plasma glucose. [Figure 13F] (Figure 13F) Liver histology using H&E and ORO staining (scale bar: 50 μm for H&E, 100 μm for ORO). [Figure 13G] (Figure 13G) Liver triglycerides. [Figure 13H] (Figure 13H) Liver TC. Data are in mean ± SD, and all scores and P values ​​are shown. [Figure 13I] Spearman correlation analysis between plasma glycine and plasma TC (Figure 13I). [Figure 13J] (Figure 13J) Spearman correlation analysis between plasma glycine and plasma glucose. [Figure 13K] (Figure 13K) Spearman correlation analysis between plasma glycine and liver triglycerides.

[0028] [Figure 14A] Effects of glycine compounds on glucose tolerance. An oral glucose tolerance test (OGTT) was performed on C57BL / 6J mice after 12 hours of fasting (n=6-8). Mice were orally administered either glucose alone (2 mg / g body weight), glucose and 0.5 mg / g body weight of glycine, or 0.5 mg / g body weight of a glycine compound: (Figure 14A) N-methylglycine. [Figure 14B] (Figure 14B) N,N-dimethylglycine was administered orally. [Figure 14C] (Figure 14C) N,N,N-trimethylglycine was administered orally. [Figure 14D] (Figure 14D) Glycolic acid was administered orally. [Figure 14E](Figure 14E) DT-110 was administered orally. [Figure 14F] (Figure 14F) DT-109 was administered orally. [Figure 14G] (Figure 14G) Mice were orally administered glucose alone (2 mg / g body weight), glucose and DT-109 (0.5 mg / g body weight), or equivalent levels of free leucine or glycine (0.17 mg or 0.33 mg / g body weight, respectively). Data are mean ± SE. *P<0.05, **P<0.01, ***P<0.001 vs. glucose; #P<0.05 vs. leucine; ^P<0.05 vs. glycine.

[0029] [Figure 15A] Lipid-lowering effect of DT-109. (Figure 15A) ApoE- / - mice were fed standard WD and orally administered DT-109 (1 mg / g body weight / day), an equivalent level of free leucine or glycine (0.33 or 0.67 mg / g body weight), or H2O for 12 weeks (n=8-10). [Figure 15B] (Figure 15B) At week 10, an OGTT was performed after a 12-hour fast. Mice were orally administered either oral glucose alone (2 mg / g body weight), glucose and DT-109 at 1 mg / g body weight, or an equivalent level of free leucine (0.33 mg / g body weight) or glycine (0.67 mg / g body weight). Data are mean ± SE. **P<0.01, ***P<0.001 vs. H2O; #P<0.05, ##P<0.01, ###P<0.01 vs. leucine. [Figure 15C] (Figure 15C) Non-fasting blood glucose was measured 30 minutes before and 30 minutes after daily forced oral administration with DT-109, leucine, glycine, or H2O. [Figure 15D] (Figure 15D) Endpoint body weight. [Figure 15E] (Figure 15E) Average food intake. [Figure 15F] (Figure 15F) Plasma TC at baseline (before randomization to experimental group) and endpoint (data are mean ± SE). [Figure 15G] Endpoint plasma analysis (n=6-8): (Figure 15G) TC. [Figure 15H] (Figure 15H) LDL. [Figure 15I] (Figure 15I) HDL. [Figure 15J] (Figure 15J)TG. [Figure 15K] (Figure 15K) Glycine tripeptide molecule. Data are mean ± SD, and all scores and P values ​​are shown. [Figure 15L] (Figure 15L) H&E histology of epididymal tissue and subcutaneous adipose tissue (EAT and SAT, scale bar: 100 μm).

[0030] [Figure 16A] Glycine or DT-109 prevent WD-induced HS. Endpoint liver analysis (n=8-10): (Figure 16A) Macroscopic appearance of the abdominal cavity and histology using H&E and ORO (scale bar: 50 μm for H&E, 100 μm for ORO). [Figure 16B] (Figure 16B) Liver triglycerides. [Figure 16C] (Figure 16C) Liver TC. Data are in mean ± SD, and all scores and P values ​​are shown. [Figure 16D] (Figure 16D) qPCR analysis of key genes regulating FAO and inflammation compared to GAPDH. Data are mean ± SE. *P<0.05, **P<0.01, ***P<0.001 vs. WD+H2O.

[0031] [Figure 17A] DT-109 protects against dietary NASH. (Figure 17A) C57BL / 6J mice were fed either a CD or NASH diet for 12 weeks. [Figure 17B] Confirmation of NASH before randomization to the experimental group. C57BL / 6J mice were fed either a CD diet (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). [Figure 17C]A subset of mice (CD: n=3, NASH diet n=5) were euthanized, and liver pathology was examined: (Figure 17C) Liver-to-body weight ratio (LW / BW). [Figure 17D] (Figure 17D) Macroscopic view of the abdominal cavity and histology using H&E, ORO, and Sirius Red (scale bar: 50 μm for H&E and Sirius Red, 100 μm for ORO). [Figure 17E] (Figure 17E) NAS as a total. [Figure 17F] (Figure 17F) Fatty liver, hepatocyte ballooning, and intralobular inflammation scores. Data are mean ± SD, and all scores and P values ​​are shown. [Figure 17G] (Figure 17G) At week 18, an OGTT was performed after a 12-hour fast (n=8-9). Mice were orally administered glucose alone (2 mg / g body weight), glucose and DT-109 at 0.5 mg / g body weight or equivalent levels of free leucine (0.17 mg / g body weight), glycine (0.33 mg / g body weight), or H2O. Data are mean ± SE. *P<0.05, **P<0.01 vs. CD+H2O;##P<0.01, ###P<0.001 vs. NASH+H2O;^^P<0.01 vs. NASH+Leucine. [Figure 17H] (Figure 17H) Non-fasting blood glucose 30 minutes before and 30 minutes after daily forced oral administration of DT-109, leucine, glycine, or H2O. Data are mean ± SD, and all scores and P values ​​are shown. [Figure 17I] After confirming NASH, mice were randomized and administered 0.125 or 0.5 mg / g / day of DT-109 or equivalent levels of leucine, glycine (0.17, 0.33 mg / g / day), or H2O, by forced oral administration under a NASH diet for an additional 12 weeks. Mice were fed CD and administered H2O as controls (n=8-9). NMR-based body composition analysis at 22-23 weeks: (Figure 17I) Body weight. [Figure 17J] (Figure 17J) Body fat (%). [Figure 17K] (Figure 17K) Lean body mass (%). [Figure 17L]Metabolic effects of DT-109 in C57BL / 6J mice with established NASH. C57BL / 6J mice were fed either a CD or NASH diet for 12 weeks. After confirmation of NASH, mice were randomized and administered 0.125 or 0.5 mg / g body weight / day of DT-109 or equivalent levels of leucine or glycine (0.17 or 0.33 mg / g body weight / day) or H2O by forced oral administration under a NASH diet for another 12 weeks. Mice fed CD and administered H2O served as controls (n=8-9). (Figure 17L) H&E histology of epididymal and subcutaneous adipose tissue (EAT and SAT, scale bar: 100 μm). [Figure 17M] CLAMS analysis at 22-23 weeks: (Figure 17M) Food intake. [Figure 17N] (Figure 17N) Oxidation of fat. [Figure 17O] (Figure 17O) Oxidation of glucose. [Figure 17P] (Figure 17P) Respiratory exchange rate (RER). [Figure 17Q] (Figure 17Q) Energy expenditure. [Figure 17R] (Figure 17R) Total activity. Data are in mean ± SD, and all scores and p-values ​​are shown.

[0032] [Figure 18A] Endpoint plasma analysis: (Figure 18A) AST. [Figure 18B] (Figure 18B) ALT. [Figure 18C] (Figure 18C) ALP. [Figure 18D] (Figure 18D)TG. [Figure 18E] (Figure 18E)TC. [Figure 18F] (Figure 18F) Macroscopic morphology and H&E histology (scale bar: 50 μm). [Figure 18G] (Figure 18G) LW / BW ratio. [Figure 18H]DT-109 protects against dietary NASH. C57BL / 6J mice were fed either a CD or NASH diet for 12 weeks. After confirming NASH, mice were randomized and administered 0.125 or 0.5 mg / g body weight / day of DT-109 or equivalent levels of leucine or glycine (0.17 or 0.33 mg / g body weight / day) or H2O by forced oral administration under a NASH diet for another 12 weeks. Mice fed CD and administered H2O served as controls (n=8-9). (Figure 18H) Liver weight. [Figure 18I] (Figure 18I) NAS. Data are mean ± SD, and all points and p-values ​​are shown. [Figure 18J] (Figure 18J) H&E-based scoring for steatosis, hepatocyte ballooning, and intralobular inflammation. Data are mean ± SD. [Figure 18K] Spearman correlation analysis between NAS and (Figure 18K) plasma AST. [Figure 18L] Spearman correlation analysis between NAS and (Figure 18L). [Figure 18M] Spearman correlation analysis between NAS and plasma ALP (Figure 18M).

[0033] [Figure 19A] Glycine-based therapies correct diet-induced FAO deficiency in NASH and reduce HS. RNA sequencing of liver samples taken at the endpoint (n=4): (Figure 19A) PCA. [Figure 19B] (Figure 19B) Volcano plots of DEG (green: downward control; red: upward control) in each group compared with CD. [Figure 19C] DT-109 reverses changes in the NASH dietary transcriptome. This is represented by a heatmap of the top 50 DEGs across all experimental groups, determined by log2 ratio changes compared to the CD group. Each row represents one gene, and each column represents one comparison with the CD group. (n=4). [Figure 19D]Liver pathway analysis from mice fed CD and mice fed a NASH diet (Figure 19D). Pathways enriched with upregulated DEG are plotted in red, while pathways enriched with downregulated DEG are plotted in green. [Figure 19E] (Figure 19E) Changes in glycine biosynthesis genes / pathways and pathway analysis (n=4) by RNA sequencing of livers from CD or NASH mice. Genes / pathways that were significantly downregulated are highlighted in green (log2FC, log2 magnification change). [Figure 19F] (Figure 19F) Pathway analysis comparing NASH + H2O and NASH + 0.5 mg / g / day DT-109. [Figure 19G] Upward-controlled or downward-controlled DEG-rich pathways are plotted in red or green, respectively, and a heatmap of 50 NASH-related DEGs across all experimental groups (log2 magnification change vs. CD group) is shown. (Figure 19G) [Figure 19H] (Figure 19H) Verification of FAO-associated DEG using qPCR (n=8-9), data are mean ± SE, *P<0.05, **P<0.01, ***P<0.001 vs CD; #P<0.05, ##P<0.01, ###P<0.001 vs NASH+H2O. [Figure 19I] (Figure 19I) Western blot (n=4). [Figure 19J] (Figure 19J) qPCR validation of FAO-associated DEG (n=8-9). Data are mean ± SE. *P<0.05, **P<0.01, ***P<0.001 vs. CD;#P<0.05, ##P<0.01 vs. NASH+H2O. [Figure 19K] (Figure 19K) ORO histology (scale bar: 100 μm). [Figure 19L] (Figure 19L) Liver triglycerides. [Figure 19M] (Figure 19M) Liver TC (n=8~9). Data are mean ± SD, and all scores and P values ​​are shown. [Figure 19N] (Figure 19N) Liver DAG (n=8~9). Data are mean ± SD, and all scores and P values ​​are shown.

[0034] [Figure 20A] Glycine-based therapies alleviate diet-induced hepatitis and fibrosis of NASH. (Figure 20A) F4 / 80 Immunohistochemistry and Sirius Red Histology (Scale bar: 50 μm). [Figure 20B] (Figure 20B) F4 / 80 positive area. [Figure 20C] (Figure 20C) Plasma MCP-1. [Figure 20D] (Figure 20D) Resistin. Data are shown as mean ± SD, with all scores and p-values ​​(n=6~9). [Figure 20E] (Figure 20E) qPCR validation of inflammation-related DEG (data are mean ± SE, n=8-9). *P<0.05, **P<0.01, ***P<0.001 vs. CD;#P<0.05, ##P<0.01, ###P<0.001 vs. NASH+H2O. [Figure 20F] (Figure 20F) Area positive for Sirius Red. [Figure 20G] (Figure 20G) Fibrosis score. [Figure 20H] Spearman correlation analysis between liver fibrosis score and plasma AST (Figure 20H). [Figure 20I] Spearman correlation analysis between liver fibrosis score and plasma ALT (Figure 20I). [Figure 20J] Spearman correlation analysis between liver fibrosis score and plasma ALP (Figure 20J). [Figure 20K] (Figure 20K) Western blot of phosphorylated SMAD2 (Ser465 / 467) and total SMAD2. [Figure 20L] (Figure 20L) qPCR validation of fibrosis-associated DEG. [Modes for carrying out the invention]

[0035] As used herein, “steatosis” is synonymous with “fatty liver,” which is the accumulation of fat in the liver.

[0036] The subjects may be mammals, which may be, for example, laboratory animals or humans, and human subjects include adults, adolescents, and children.

[0037] "Steatosis" and "hepatic steatosis" are used interchangeably in this specification.

[0038] "Blood plasma" and "plasma" are used interchangeably in this specification.

[0039] In this specification, "blood" and "plasma" are used interchangeably.

[0040] In this specification, Western food will be abbreviated as "WD".

[0041] In this specification, the facial vein will be abbreviated as "FV".

[0042] "TG" is an abbreviation for triglyceride.

[0043] "TC" is an abbreviation for total cholesterol.

[0044] "OGTT" is an abbreviation for oral glucose tolerance test.

[0045] In this specification, unless otherwise explicitly stated in the context, the singular forms "a," "an," and "the" refer to multiple objects.

[0046] Since "or" can mean "in combination with" in some cases, the administration of A or B could be the administration of A, the administration of B, or the administration of A and B.

[0047] All common amino acids, except glycine, contain at least one chiral carbon atom. Therefore, these amino acids exist as pairs of stereoisomers called L-isomers and D-isomers. Most natural proteins and peptides consist only of L-isomers. D-isomer amino acids can affect the conformation of peptides or proteins, potentially leading to improved stability or altered activity.

[0048] As used herein, the term “pharmaceutically acceptable salt” means a salt that is suitable for use in contact with human and lower animal tissues without causing excessive toxicity, irritation, allergic reactions, etc., within the normal range of reasonable medical judgment, and that has a reasonable benefit / risk ratio. “pharmaceutically acceptable salt” means any non-toxic salt or ester of the compound of the present invention that can directly or indirectly provide the compound of the present invention to the recipient upon administration. pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977).

[0049] Pharmaceutical compositions suitable for the delivery of the peptides of the present invention and methods for preparing them will be readily apparent to those skilled in the art. Such compositions and methods for preparing them 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 may be formulated to be immediate-release and / or release-controlled.

[0050] The term “to treat” (or other forms of the word such as “treat” or “to heal”) is used herein to mean that administration of the compositions of the present invention alleviates a patient’s condition and / or reduces, suppresses, or eliminates certain characteristics or events associated with the condition. Thus, the term “to treat” includes preventing the onset of a condition in a patient, particularly when the patient is predisposed to acquiring the condition; reducing or suppressing the condition; and / or improving or reversing the condition. To the extent that the methods of the present invention aim to prevent a condition, it is understood that the term “prevent” does not require the condition to be completely blocked. Rather, as used herein, the term “prevent” refers to the ability of those skilled in the art to identify a population susceptible to the condition so that administration of the compositions of the present invention can be performed before the onset of the condition. This term does not mean that the condition must be completely avoided.

[0051] As used herein, “effective dose” refers to the amount of the glycine-containing tripeptide of the present invention sufficient to exhibit a detectable therapeutic effect. This effect is detected, for example, by improvement of the clinical condition or by prevention, reduction, or improvement of complications. The exact effective dose for a patient will vary depending on the patient’s weight, size, and health condition; the nature and severity of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutic effective dose for a given situation is determined by routine experimentation within the scope of the clinician’s skill and judgment.

[0052] As used herein, “identifying” or “selecting” subjects having “metabolic and / or cardiovascular and / or inflammatory diseases” means identifying or selecting subjects who are likely to be diagnosed with or have been diagnosed with metabolic, cardiovascular, systemic or localized inflammatory diseases, or metabolic syndrome, including but not limited to hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, excess normal body weight, and / or excess normal body fat content, or any combination thereof; or identifying or selecting subjects who have symptoms of metabolic, cardiovascular, or metabolic syndrome. Such identification can be achieved by any method, including but not limited to standard clinical trials or assessments such as measuring serum or circulating (plasma) cholesterol, serum or circulating (plasma) blood glucose, serum or circulating (plasma) triglycerides, inflammatory cytokines or cortisol, blood pressure, body fat content, or weight.

[0053] As used herein, “glucose” refers to a monosaccharide used by cells as an energy source and inflammatory intermediate. “Plasma glucose” refers to glucose present in plasma.

[0054] As used herein, “high-density lipoprotein C” or “HDL-C” means 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” mean HDL-C in serum and plasma, respectively.

[0055] As used herein, "HMG-CoA reductase inhibitor" means a drug that acts by inhibiting the enzyme HMG-CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

[0056] As used herein, “hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol, and VLDL-cholesterol, as defined in the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see Arch.Int.Med. (1988) 148, 36-39).

[0057] As used herein, “hyperlipidemia” or “hyperlipidemia” refers to a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition is characterized by abnormally high lipid concentrations. The lipid fractions in circulating blood are cholesterol, low-density lipoproteins, very low-density lipoproteins, chylomicrons, and triglycerides. The Fredrickson classification of hyperlipidemia is based on the pattern of TG and cholesterol-rich lipoprotein particles measured by electrophoresis or ultracentrifugation and is commonly 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).

[0058] As used herein, “hypertriglyceridemia” means a condition characterized by elevated triglyceride levels. Its etiology may include primary factors (i.e., genetic predisposition) and secondary factors (other underlying causes, e.g., diabetes mellitus, metabolic syndrome / insulin resistance, obesity, physical inactivity, smoking, excessive alcohol and carbohydrate-heavy diet), or most often, a combination of both (Yuan et al., CMAJ, 2007, 176:1113-1120).

[0059] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific processes, compositions, or methodologies described, as they may vary considerably. It should also be understood that the technical terms used herein are merely for the purpose of describing specific versions or embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. All publications referenced herein are incorporated by reference in their entirety to the extent that they support the present invention.

[0060] Glycine tripeptide molecules for the treatment of metabolic, cardiovascular, and inflammatory diseases

[0061] The inventors of this disclosure have developed glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, that exhibit prophylactic or therapeutic activity for metabolic, cardiovascular, and / or inflammatory diseases, including but not limited to obesity, diabetes mellitus, dyslipidemia, fatty liver, and insulin resistance syndrome. In certain embodiments, metabolic or cardiovascular diseases include, but are not limited to, obesity, diabetes mellitus, atherosclerosis, dyslipidemia, coronary heart disease, coronary artery disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or metabolic syndrome, or combinations thereof. Dyslipidemia may be hyperlipidemia. Hyperlipidemia may be hypercholesterolemia, hypertriglyceridemia, or both. NAFLD may be fatty liver or steatohepatitis. Diabetes mellitus may be type 2 diabetes mellitus or type 2 diabetes mellitus with dyslipidemia.

[0062] In various embodiments, the amino acid sequence of the DT-190 glycine-containing tripeptide molecule is Gly-Gly-Leu-SEQ ID NO: 1. The amino acid sequence of the DT-110 glycine-containing tripeptide molecule 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 include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Preferred examples of metal salts include alkali metal salts, e.g., sodium salts, potassium salts; alkaline earth metal salts, e.g., calcium salts, magnesium salts, barium salts; and aluminum salts. Preferred examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine. Preferred examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Preferred examples of salts with organic acids include salts with 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. Of the above salts, pharmaceutically acceptable salts are preferred. For example, if the compound has an acidic functional group, an inorganic salt, such as an alkali metal salt (e.g., sodium salt, potassium salt, etc.), an alkaline earth metal salt (e.g., calcium salt, magnesium salt, barium salt, etc.), or an ammonium salt, is preferred. If the compound has a basic functional group, a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, or phosphoric acid, or a salt with an organic acid such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, or p-toluenesulfonic acid is preferred.

[0063] In various embodiments, glycine tripeptide molecules may also be synthesized and / or administered as prodrugs in their original synthetic form. For example, a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, may be in prodrug form. A prodrug is a compound that is converted into a glycine-containing tripeptide molecule by reaction with enzymes or gastric acid under physiological conditions in the body; that is, a compound that is converted into a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof by enzymatic oxidation, reduction, hydrolysis, etc.; a compound that is converted into a glycine-containing tripeptide molecule by hydrolysis, etc., by gastric acid, etc.

[0064] Examples of prodrugs of glycine-containing tripeptide molecules or their pharmaceutically acceptable salts include compounds in which the amino group of the glycine-containing tripeptide molecule is acylated, alkylated, or phosphorylated (e.g., compounds in which the amino of the glycine-containing tripeptide molecule is eicosanoylated, alanylated, pentylaminocarbonylated, (5-methyl-2-oxo-1,3-dioxolene-4-yl)methoxycarbonylated, tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated, or tert-butylated); and compounds in which the hydroxyl of the glycine-containing tripeptide molecule is acylated, alkylated, phosphorylated, or borated (e.g., compounds in which the hydroxyl of the glycine-containing tripeptide molecule is acetylated, palmitic...). Examples include compounds that have been ylated, propanoylated, pivaloylated, succinylated, fumalylated, alanylated, or dimethylaminomethylcarbonylated); and compounds in which the carboxyl group of a glycine-containing tripeptide molecule is esterified or amidated (for example, compounds in which the carboxyl group of a glycine-containing tripeptide molecule is C1-6 alkyl esterified, phenyl esterified, carboxymethyl esterified, dimethylaminomethyl esterified, pivaloyloxymethyl esterified, ethoxycarbonyloxyethyl esterified, phthalidyl esterified, (5-methyl-2-oxo-1,3-dioxolenn-4-yl)methyl esterified, cyclohexyloxycarbonylethyl esterified, or methyl amidated). In particular, compounds in which the carboxyl group of a glycine-containing tripeptide molecule is esterified with C1-6 alkyl groups such as methyl, ethyl, or tert-butyl are preferably used. These compounds can themselves be produced from glycine-containing tripeptide molecules by known methods.

[0065] A prodrug of a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof may also be a prodrug that is converted to a glycine-containing tripeptide molecule under physiological conditions, as described, for example, in IYAKUHIN no KAIHATSU (Development of Pharmaceuticals), Vol. 7, Design of Molecules, pp. 163-198, Published by HIROKAWA SHOTEN (1990).

[0066] Exemplary glycine tripeptide molecules are three-residue amino acid polymers that can be produced according to peptide synthesis methods described herein and known to those skilled in the art. Peptide synthesis methods may include currently known methods, such as solid-phase and liquid-phase synthesis processes. That is, the peptide of interest, e.g., a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, can be produced by repeated condensation of a partial peptide or amino acid that can constitute the glycine tripeptide molecule according to the desired sequence, i.e., the peptide to be synthesized, and the remaining portion (which may consist of two or more amino acid residues). If the product having the desired sequence has a protecting group, the peptide of interest can be produced by removing the protecting group. Examples of known methods for condensing and removing protecting groups include those described in (1) to (5) below.

[0067] (1) M. Bodanszky and MA Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966)

[0068] (2) Schroeder and Luebke: The Peptide, Academic Press, New York (1965)

[0069] (3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics and experiments of peptide synthesis), published by Maruzen Co. (1975)

[0070] (4) Haruaki Yajima and Shunpei Sakakibara: Biochemical Experiment 1, Chemistry of Proteins IV, 205 (1977)

[0071] (5) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu (A sequel to Development of Pharmaceuticals), Vol. 14, Peptide Synthesis, published by Hirokawa Shoten.

[0072] Treatment and prevention of metabolic, cardiovascular, and / or systemic inflammatory diseases using glycine tripeptide molecules

[0073] Glycine, or glycine containing tripeptide molecules, or pharmaceutically acceptable salts thereof, as described and illustrated herein, can be used to prevent and / or treat one or more metabolic, cardiovascular, and inflammatory diseases in subjects requiring such treatment. For the purposes of this disclosure, “metabolic disease” means a wide range of endocrine disorders and disorders, including, for example, insulin resistance, diabetes mellitus, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, dyslipidemia, and hyperlipidemia, as well as liver diseases, such as NAFLD and NASH. Metabolic diseases described herein include diseases that can be treated through metabolic regulation, although the disease itself may or may not be caused by a specific metabolic deficiency. Such metabolic diseases may include, for example, glucose and fatty acid oxidation pathways.

[0074] The subjects requiring this are mammals, preferably humans, or domesticated or laboratory mammals, that may be experiencing metabolic diseases and / or cardiovascular diseases and / or systemic inflammatory diseases, or one or more symptoms associated with these diseases.

[0075] In certain embodiments, while we do not wish to adhere to any particular theory, administration of the glycine or glycine tripeptide molecules of the present invention, or pharmaceutically acceptable salts thereof, to subjects having symptoms related to or relating to any of the metabolic diseases, cardiovascular diseases, and chronic systemic inflammatory diseases or common conditions thereof is thought to result in a reduction in lipid levels, including triglyceride levels, cholesterol levels, insulin resistance, glucose levels, or combinations thereof. One or more levels can be independently reduced by 5%, 10%, 20%, 30%, 35%, or 40% or more. Administration of the glycine tripeptide molecules of the present invention or pharmaceutically acceptable salts thereof can improve insulin sensitivity or hepatic insulin sensitivity. Administration of the glycine tripeptide molecules of the present invention or pharmaceutically acceptable salts thereof can result in atherosclerotic plaques, obesity, reduced glucose, lipids, glucose tolerance, cholesterol, or improved insulin sensitivity, or any combination thereof.

[0076] Certain embodiments provide the use of glycine or glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, in the manufacture of agents for treating, improving, delaying, or preventing one or more metabolic or cardiovascular diseases, as described herein.

[0077] Certain embodiments provide a kit for treating, preventing, or improving one or more metabolic or cardiovascular diseases described herein, the kit comprising: a) a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof as described herein; and optionally b) a further second therapeutic agent or therapy as described herein. The kit may further include instructions or labels for using the kit with the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to treat, prevent, or improve one or more metabolic or cardiovascular diseases.

[0078] In some embodiments of the present invention, glycine or glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, have potent glucose / lipid-lowering effects. In established NASH mice, glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, for example, DT-109 can alleviate steatohepatitis, improve body composition, lower circulating lipids, and normalize or correct liver enzymes and steatohepatitis by stimulating the FAO pathway. Glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, have been shown to unexpectedly reduce or alleviate lobular / systemic inflammation and hepatic fibrosis by inhibiting inhibitory NF-κB and TGFα / SMAD pathways.

[0079] As used herein, the term “dyslipidemia” refers to abnormal lipid conditions, including hyperlipidemia caused by abnormal lipoprotein metabolism, as well as hypercholesterolemia, hypertriglyceridemia, and hypohDL cholesterolemia, resulting from elevated blood lipid levels. As used herein, the term “fatty liver” refers to a condition in which fat accumulates excessively in liver cells due to impaired lipid metabolism. Fatty liver can lead to a variety of diseases, including angina pectoris, 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, resulting in impaired glucose tolerance. The term diabetes includes all types of diabetes, such as type 1 diabetes, type 2 diabetes, and hereditary diabetes. Type 1 diabetes, which is insulin-dependent diabetes, is primarily caused by the destruction of beta cells. Type 2 diabetes, which is insulin-independent diabetes, is caused by insufficient insulin secretion or insulin resistance after meals. As used herein, the term “insulin resistance” refers to a physiological condition in which insulin has a reduced effect in lowering blood glucose and glucose is not effectively burned by cells. High insulin resistance can lead to the body overproducing insulin, potentially causing hypertension or dyslipidemia, as well as heart disease, diabetes, and other conditions. In particular, in type 2 diabetes, muscle and adipose tissue do not recognize the increase in insulin. As used herein, the term “insulin resistance syndrome” refers to a combination of disorders caused by insulin resistance, characterized by cellular resistance to the action of insulin, hyperinsulinemia, increased very low-density lipoprotein (VLDL) and triglycerides, decreased high-density lipoprotein (HDL), and hypertension. It is recognized as a risk factor for cardiovascular disease and type 2 diabetes (Reaven G M., Diabetes, 37:1595-607 (1988)).Furthermore, insulin resistance, along with other risk factors such as hypertension, diabetes, and smoking, is known to increase oxidative stress, alter intracellular signaling systems, induce inflammatory responses, and lead to atherosclerosis (Freeman BA et al., Lab.Invest. 47:412-26 (1982), Kawamura M et al., J.Clin.Invest. 94:771-8 (1994)).

[0080] As used herein, the term “metabolic disorder” refers to a group of diseases that include metabolic disorders, which are risk factors for various cardiovascular diseases and type 2 diabetes. This includes insulin resistance and the complex and diverse metabolic disorders associated with it. In 1988, Reaven proposed insulin resistance as the underlying factor for these disorders and named the group of abnormalities insulin resistance syndrome. However, in 1998, the World Health Organization (WHO) introduced the term metabolic syndrome or metabolic disorder, as not all aspects of the symptoms can be explained by insulin resistance.

[0081] Compositions of the present disclosure, comprising a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof as an active agent, tend to improve various metabolic diseases and / or their symptoms, such as obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver, systemic inflammation, and / or insulin resistance syndrome. Compositions of the present disclosure can prevent or treat various metabolic diseases with diverse activities.

[0082] As used herein, the term “hyperlipidemia” refers to a disorder caused by high levels of blood lipids resulting from insufficient metabolism of lipids such as triglycerides and cholesterol. More specifically, hyperlipidemia is characterized by elevated levels of lipids in the blood, including triglycerides, LDL cholesterol, phospholipids, and free fatty acids, and includes hypercholesterolemia and hypertriglyceridemia.

[0083] According to a preferred embodiment, insulin resistance syndromes treated by the present invention include obesity, hypertension, atherosclerosis, hyperlipidemia, hyperinsulinemia, non-alcoholic fatty liver disease, and type 2 diabetes.

[0084] According to a preferred embodiment, the composition of the present invention reduces levels of blood fat, liver fat, or visceral fat. The terms “liver” or “visceral” are used to encompass organs, tissues, and cells.

[0085] According to the present invention, subjects given a diet containing the glycine or glycine tripeptide molecule of the present invention, or a pharmaceutically acceptable salt thereof, showed a significant decrease in liver weight, improvement in triglyceride lipid concentrations and total cholesterol in the blood and liver tissue, and a significant decrease in total visceral fat weight.

[0086] In a more preferred embodiment, the fats reduced by the present invention include triglycerides, cholesterol, and free fatty acids.

[0087] In a more preferred embodiment, the visceral fat reduced by the present invention includes epididymal fat, perirenal fat, mesenteric fat, and / or retroperitoneal fat.

[0088] 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 are enzymes that show elevated blood concentrations in the event of liver damage, serving as indicators of liver function.

[0089] Non-alcoholic fatty liver disease (NAFLD) is becoming increasingly common worldwide, especially in Western countries. In the United States, it is the most common form of chronic liver disease, affecting an estimated 80 to 100 million people. NAFLD is a general term for various liver conditions that affect people who drink little to no alcohol. As the name suggests, the main characteristic of NAFLD is the accumulation of large amounts of fat in liver cells. It is normal for the liver to contain fat. However, if more than 5% to 10% of the liver's weight is fat, the condition is called fatty liver (or steatosis).

[0090] NAFLD is strongly associated with the characteristics of metabolic syndrome, including obesity, insulin resistance, type 2 diabetes, and dyslipidemia; NAFLD is considered a hepatic manifestation of this syndrome.

[0091] Pediatric NAFLD is currently the leading form of pediatric liver disease. Studies suggest that abdominal obesity and insulin resistance are the primary causes of NAFLD development. As obesity becomes an increasingly common problem worldwide, the prevalence of NAFLD is simultaneously increasing. The only treatment shown to be truly effective in pediatric NAFLD is weight loss.

[0092] A more severe form of NAFLD is called non-alcoholic steatohepatitis (NASH). NASH causes the liver to swell and become damaged. NASH tends to develop in people who are overweight, i.e., obese, or who have diabetes, high cholesterol, high triglycerides, or inflammatory conditions. NASH, a potentially severe form of this disease, is characterized by hepatocyte ballooning and inflammation of the liver, which can progress to scarring and irreversible damage. This damage is similar to that caused by heavy alcohol use. Macroscopically and microscopically, NASH is characterized by inflammation of the lobules and / or portal vein, varying degrees of fibrosis, hepatocyte death, and pathological angiogenesis. In the most severe cases, NASH can progress to cirrhosis, hepatocellular carcinoma, and liver failure. Currently, NAFLD and NASH are treated with dietary changes, treatment of insulin resistance, or vitamin supplementation, such as vitamin E or D.

[0093] The NAFLD activity score (NAS) can be calculated according to Kleiner's criteria (Kleiner DE. et al., Hepatology, 2005;41:1313). NAS scores of 0–2 indicate no diagnosis of NASH, NAS scores of 3–4 indicate no diagnosis, borderline, or positive for NASH, and NAS scores of 5–8 indicate a primary diagnosis of NASH. Treatment outcomes for NASH include disease regression, stabilization, or a decrease in the rate of progression. Serial liver biopsies from patients suspected of having NASH can be used to assess changes in NAS scores and can be used as an indicator of disease progression. An increase in score suggests progression, no change suggests stabilization, while a decrease in score indicates a regression of NASH. In controlled clinical trials, the difference in NAS scores between placebo and test substance treatment groups is typically evaluated over a period of 6 months to 2 years, and this difference may indicate a treatment effect even if both groups are progressing. A specified score difference is usually required by regulatory authorities to demonstrate a meaningful change in NASH.

[0094] The present invention also provides a method for treating fatty liver, comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need thereof. In one embodiment, the tripeptide molecule reduced triglyceride levels in hepatic lipids and had no significant effect on leucine-negative controls. In another embodiment, the tripeptide molecule reduced total cholesterol levels in hepatic lipids and had no significant effect on leucine-negative controls. In any one of the above embodiments, the tripeptide molecule is Gly-Gly-Leu or Gly-Gly-dLeu.

[0095] The present invention also provides a method for increasing or enhancing hepatic lipid oxidation, decreasing triglyceride levels, or treating cholesterol accumulation, comprising administering glycine or one or more glycine tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need and measuring the effect it has on mRNA levels, in which case hepatic lipid oxidation, triglyceride levels, cholesterol accumulation, or any combination thereof is enhanced or treated.

[0096] A method for enhancing hepatic lipid oxidation, reducing triglyceride levels, or treating cholesterol accumulation, comprising administering a glycine tripeptide Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof to a subject in need and measuring the effect it has on mRNA levels, wherein hepatic lipid oxidation, triglyceride levels, cholesterol accumulation, or any combination thereof is enhanced or treated. A method in which the glycine-containing tripeptide molecule Gly-Gly-Leu or Gly-Gly-dLeu significantly induces the expression of hepatic lipid oxidation regulators, AMPKα1 or PPARα, without being affected by a leucine-negative control. A method in which the tripeptide molecule Gly-Gly-Leu or Gly-Gly-dLeu modulates triglyceride hydrolysis by significantly upregulating CPT1a, CACT, or ACADI (mitochondrial β-oxidation) or PNPLA2. A method by which the tripeptide molecule Gly-Gly-Leu or Gly-Gly-dLeu significantly upregulates the mitochondrial anion carrier UCP2, thereby regulating triglyceride hydrolysis.

[0097] A method by which the tripeptide molecule Gly-Gly-Leu or Gly-Gly-dLeu significantly increases the expression of ABCG5 and ABCG8, thereby regulating hepatic cholesterol homeostasis.

[0098] A method for treating the plasma lipid properties of a subject, comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need to reduce the subject's plasma triglycerides, plasma LDL levels, or atherosclerotic lesions.

[0099] A method for treating the plasma lipid properties of a subject, comprising administering it to a subject in need to reduce the subject's plasma triglycerides, plasma LDL levels, or atherosclerotic lesions, wherein the tripeptide molecule is Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof. A method in which Gly-Gly-Leu or Gly-Gly-dLeu reduces atherosclerotic lesions. A method for treating the plasma lipid properties of a subject, comprising administering the tripeptide molecule Gly-Gly-Leu to a subject in need, wherein it reduces plasma total cholesterol, plasma LDL, or a combination thereof.

[0100] In any embodiment of the method of the present invention, an additional lipid-lowering agent may be administered to the subject. The method wherein the additional lipid-lowering agent is a cholesterol absorption inhibitor, a PCSK9 inhibitor, a PPAR-α agonist, a 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 the administration of an additional cholesterol-lowering agent which is a cholesterol absorption inhibitor. The method wherein 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 PCSK9 inhibitor.

[0101] Atherosclerosis develops when the blood vessels (arteries) that carry oxygen and nutrients from the heart to the rest of the body become thickened and hardened (hardening of the arteries). Sometimes this restricts blood flow to organs and tissues. Atherosclerosis can lead to many complications, including myocardial infarction, coronary artery disease, carotid artery disease, peripheral artery disease, aneurysms, and chronic kidney disease.

[0102] 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 center or left side of the chest, shortness of breath, nausea, or heart failure, arrhythmia, cardiogenic shock, or cardiac arrest.

[0103] Coronary artery disease is a condition in which atherosclerosis narrows the arteries near the heart, potentially causing chest pain (angina), heart attack, or heart failure.

[0104] Carotid artery disease is a condition in which atherosclerosis narrows the arteries near the brain, potentially causing transient ischemic attacks (TIAs) or strokes. Symptoms may include sudden numbness or weakness in an arm or leg, temporary vision loss in one eye, or drooping of the facial muscles.

[0105] Peripheral artery disease occurs when atherosclerosis narrows the arteries in the arms or legs, and these circulatory problems are called peripheral artery disease. This reduces sensitivity to heat and cold, increasing the risk of burns and frostbite. Rarely, poor blood flow in the arms and legs can cause tissue death (necrosis). Symptoms include leg pain (claudication) when walking.

[0106] Aneurysms can develop when atherosclerosis causes serious complications, and they can occur anywhere in the body. An aneurysm is a bulge in the artery wall, and it can be an emergency situation; if it ruptures, it can be life-threatening.

[0107] Chronic kidney disease can be caused by atherosclerosis, which narrows the arteries and prevents the kidneys from delivering oxygenated blood. Over time, this can affect the kidney's ability to continuously eliminate waste products from the body. Symptoms include high blood pressure or kidney failure.

[0108] This disclosure provides a method for treating atherosclerosis by administering one or more glycine tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need thereof, in which case the administration of the glycine-containing tripeptide molecules treats atherosclerosis. In one embodiment, the method is a method for treating atherosclerosis by administering a glycine-containing tripeptide molecule, which is Gly-Gly-Leu and / or Gly-Gly-dLeu, to a subject. This disclosure also provides a method for treating complications of atherosclerosis by administering a glycine-containing tripeptide molecule to a subject having complications of atherosclerosis to treat myocardial infarction, coronary artery disease, carotid artery disease, peripheral artery disease, arteriosclerosis, or chronic kidney disease. In one embodiment, a method for treating complications of atherosclerosis involves administering a glycine-containing tripeptide molecule, such as Gly-Gly-Leu or Gly-Gly-dLeu, to subjects with complications of atherosclerosis, thereby treating myocardial infarction, coronary artery disease, carotid artery disease, peripheral artery disease, arteriosclerosis, or chronic kidney disease.

[0109] A method for treating inflammation in adipose tissue and circulation, comprising administering one or more glycine tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need thereof, wherein the administration of the tripeptide molecules reduces inflammation.

[0110] A method for treating inflammation in adipose tissue and circulating tissue, comprising administering Gly-Gly-Leu and / or Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein the circulating inflammation is reduced by reducing plasma MCP1 levels. A method for treating inflammation, wherein the inflammation in adipose tissue is present in epididymal adipose tissue (EAT) or subcutaneous adipose tissue (SAT), comprising administering Gly-Gly-Leu and / or Gly-Gly-dLeu to a subject in need thereof to reduce MCP1 mRNA levels.

[0111] A method for treating a subject to reduce plasma levels of glycine tripeptide molecules, comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need thereof, wherein the administration of the tripeptide molecules reduces the levels of plasma glycine tripeptide molecules.

[0112] A method for treating a subject to reduce plasma levels of a glycine tripeptide molecule, comprising administering the tripeptide Gly-Gly-Leu or Gly-Gly-dLeu to the subject in need.

[0113] A method for treating a subject to further reduce postprandial glucose, comprising administering one or more tripeptide molecules or pharmaceutically acceptable salts thereof to a subject in need. A method for treating a subject to further reduce postprandial glucose, comprising administering glycine tripeptides Gly-Gly-Leu and / or Gly-Gly-dLeu to a subject in need.

[0114] A method for treating a subject having liver disease, comprising administering Gly-Gly-Leu and / or Gly-Gly-dLeu to a subject in need thereof.

[0115] A method for treating a subject in need of the treatment, comprising administering Gly-Gly-Leu and / or Gly-Gly-dLeu to the subject, wherein the subject has a liver disease, and the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or alcoholic fatty liver.

[0116] A method for stabilizing or reducing the NAFDL activity score (NAS) in a subject, comprising administering a therapeutically effective amount of a composition containing Gly-Gly-Leu and / or Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, to the subject.

[0117] A method for stabilizing or reducing the NAFDL activity score (NAS) in a subject, comprising 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 comprises delaying, stabilizing, or reducing the progression of the fatty liver component of the NAS. A method comprising delaying, stabilizing, or reducing the progression of the lobular inflammatory component of the NAS. A method comprising delaying, stabilizing, or reducing the progression of the hepatocyte ballooning component of the NAS.

[0118] The method for stabilizing or reducing the NAFLD activity score, wherein the NAS differs by 1.5 points or more 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.

[0119] A method for alleviating hepatic fibrosis in a subject in need, comprising administering a therapeutically effective amount of a composition containing Gly-Gly-Leu and / or Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, to the subject.

[0120] A method for reducing plasma fibrinogen levels in a subject requiring treatment, comprising administering a therapeutically effective amount of a composition containing Gly-Gly-Leu and / or Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, to the subject.

[0121] A method for reducing plasma fibrinogen levels in subjects whose fibrinogen levels are greater than 300 mg / dL and require administration of a composition containing a therapeutically effective dose of Gly-Gly-Leu and / or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

[0122] One embodiment of the present invention is a kit for treating NAFLD, comprising DT-109, optionally a statin, and instructions for use. Another embodiment of the present invention is a kit for treating NASH, comprising DT-109, optionally a statin, and instructions for use. One embodiment of the present invention is a kit for treating NAFLD, comprising DT-110, optionally a statin, and instructions for use. Another embodiment of the present invention is a kit for treating NASH, comprising DT-110, optionally a statin, and instructions for use.

[0123] The present invention provides a method for alleviating hepatic fibrosis in a patient, comprising administering DT-109 or DT-110 or a pharmaceutically acceptable salt thereof. One embodiment is a method for alleviating hepatic fibrosis in a subject requiring it, comprising administering DT-109 or DT-110 to the subject. Another embodiment is a method for alleviating hepatic fibrosis in a subject requiring it, comprising administering DT-109 or DT-110 to the subject, in which case the subject has NASH.

[0124] Fibrinogen (factor I) is a mammalian glycoprotein involved in blood clot formation. During clot formation, fibrinogen is converted to fibrin by thrombin. Fibrinogen is synthesized in hepatocytes. Therefore, fibrinogen can be a prognostic indicator or blood marker for many diseases and may also be useful in influencing the onset and progression of disease.

[0125] In various embodiments, a method for treating at least one of the following conditions in a mammalian subject—hyperlipidemia, fatty liver, steatohepatitis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, hyperglycemia, metabolic syndrome, cardiovascular disease, and atherosclerosis—includes administering a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0126] In various embodiments, glycine-containing tripeptide molecules significantly reduce triglyceride levels in liver lipids without significant effect on leucine-negative controls.

[0127] In various embodiments, glycine-containing tripeptide molecules significantly reduce total cholesterol levels in hepatic lipids without significant effect on leucine-negative controls.

[0128] In various embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0129] This disclosure provides a method for enhancing hepatic lipid oxidation or utilization, reducing triglyceride levels, or treating hypercholesterolemia in subjects requiring such treatment. The method comprises administering a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, to a subject requiring such treatment, resulting in improvement of hepatic lipid oxidation, triglyceride levels, hypercholesterolemia, or any combination thereof. In relevant aspects of these embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0130] In relevant aspects of these embodiments, the glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof significantly induce the expression of hepatic lipid oxidation regulators AMPKα1 or PPARα, unaffected by leucine-negative controls.

[0131] In relevant aspects of these embodiments, the glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof modulate triglyceride hydrolysis by significantly upregulating CPT1a, CACT, or ACADI (mitochondrial β-oxidation) or PNPLA2.

[0132] In relevant aspects of these embodiments, the glycine-containing tripeptide molecules Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salts thereof modulate triglyceride hydrolysis by significantly upregulating the mitochondrial anion carrier UCP2.

[0133] In relevant aspects of these embodiments, the 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.

[0134] In relevant aspects of these embodiments, the plasma lipid properties of the subject involve administering a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, to a subject requiring such properties to reduce the subject's plasma triglycerides, plasma LDL levels, or atherosclerotic lesions. In relevant aspects of these embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0135] In relevant aspects of these embodiments, Gly-Gly-Leu, Gly-Gly-dLeu, glycine tripeptide molecules, or pharmaceutically acceptable salts thereof reduce atherosclerotic lesions.

[0136] In relevant aspects of these embodiments, Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof lowers plasma total cholesterol, plasma LDL, or a combination thereof.

[0137] This disclosure provides methods for treating adipose tissue and circulating inflammation in subjects requiring such treatment. The methods involve administering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to a subject requiring such treatment, wherein the administration of the glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof reduces the adipose tissue and circulating inflammation in the subject. In relevant aspects of these embodiments, circulating inflammation is reduced by lowering plasma MCP1 levels by administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to the subject. In relevant aspects of these embodiments, inflammation within adipose tissue is present in epididymal adipose tissue (EAT) or subcutaneous adipose tissue (SAT) and reduces MCP1 mRNA levels.

[0138] This disclosure provides a method for treating a subject to reduce plasma leptin levels in the subject in need. In some embodiments, the method comprises administering a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, to the subject in need, the administration of the glycine-containing tripeptide molecule reducing plasma leptin levels. In relevant aspects of these embodiments, the treatment is carried out using Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0139] This disclosure provides a method for treating a subject in need of such treatment, which involves reducing postprandial glucose in that subject. The method comprises administering a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof to the subject in need of such treatment.

[0140] In relevant aspects of these embodiments, the treatment of the subject involves administering a therapeutically effective amount of a glycine tripeptide molecule, such as Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0141] This disclosure provides a method for treating subjects who have or are at risk of developing liver disease, characterized in that the liver has excess cholesterol, triglycerides, or other lipids, such as NAFLD, NASH, or alcohol-related fatty liver, cirrhosis, or hepatitis. The method comprises treating or preventing liver disease by administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0142] In the relevant aspects of these embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or alcoholic fatty liver.

[0143] In relevant aspects of these embodiments, the disclosure provides a method for stabilizing or reducing the NAFDL activity score (NAS) in a subject, the method comprising 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 relevant aspects of these embodiments, the method comprises delaying, stabilizing, or reducing the progression of the fatty liver component of the NAS. In relevant aspects of these embodiments, the method comprises delaying, stabilizing, or reducing the progression of the lobular inflammation component of the NAS. In relevant aspects of these embodiments, the method comprises delaying, stabilizing, or reducing the progression of the hepatocyte ballooning component of the NAS. In relevant aspects of these embodiments, the NAS differs by at least 1.5 points after 6 months of treatment with Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0144] This disclosure provides a method for alleviating hepatic fibrosis in a subject requiring it, comprising administering a therapeutically effective amount of a glycine tripeptide molecule: Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to the subject.

[0145] This disclosure provides a method for treating atherosclerosis, the method comprising administering a therapeutically effective amount of a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof to a subject in need thereof. In relevant aspects of these embodiments, the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof.

[0146] This disclosure provides a method for treating complications of atherosclerosis, which involves administering a therapeutically effective amount of a glycine-containing tripeptide molecule to a subject with complications, for the treatment of complications selected from the group consisting of myocardial infarction, arteriosclerosis, coronary artery disease, carotid artery disease, peripheral artery disease, atherothrombotic stroke, aneurysm, or chronic kidney disease.

[0147] This disclosure also encompasses the treatment of such exemplified metabolic, cardiovascular, and inflammatory diseases by administration of glycine-containing tripeptide molecules in combination with second-line therapeutic agents for treating metabolic, cardiovascular, and inflammatory diseases. In some embodiments, the methods disclosed above further include administering a second therapeutic agent to a subject in need, selected from: cholesterol absorption inhibitors, PCSK9 inhibitors, PPARα agonists, ACE inhibitors, calcium channel blockers, ARBs, renin, GLP-1 or its synthetic variants, insulin or its synthetic variants, metformin, sulfonylurea compounds, thiazolidinediones (TZDs), PCSK9 inhibitors, SGLT2 inhibitors, DPP-IV inhibitors, HMGCoA reductase inhibitors, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ezetimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gemcapen (CI-1027), benpodic acid (ETC-1002), ACC inhibitors, ApoC-III inhibitors, ACL inhibitors, prescription fish oils, CETP inhibitors, antifibrotic agents, and combinations thereof.

[0148] Certain embodiments provide a kit for treating, preventing, or improving one or more metabolic and / or cardiovascular and / or inflammatory diseases as described herein, the kit comprising: a) a glycine-containing tripeptide molecule as described herein; and optionally b) further agents or therapies as described herein. The kit may further comprise instructions or labels for using the kit to treat, prevent, or improve one or more metabolic and / or cardiovascular and / or inflammatory diseases. In relevant aspects of these embodiments, the kit is a kit for treating a subject having NAFLD or NASH, comprising a selected tripeptide, optionally a statin, and instructions for use. In relevant aspects of these embodiments, the kit comprises DT-109 (Gly-Gly-Leu) and / or DT-110 (Gly-Gly-dLeu), optionally a statin, and instructions for use. In relevant aspects of these embodiments, the kit may further include cholesterol absorption inhibitors, PCSK9 inhibitors, PPARα 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 inhibitors (proprotein convertase subtilisin / kexin type 9), ezetimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gemcapen (CI-1027), benpodic acid (ETC-1002), ACC inhibitors, ApoC-III inhibitors, ACL inhibitors, formula fish oil, CETP inhibitors, antifibrotic agents, and combinations thereof. In relevant aspects of these embodiments, the kit may further include ezetimibe.

[0149] formulation

[0150] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority for use in animals such as humans, or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The term “carrier” means a diluent, adjuvant, excipient, stabilizer, or vehicle used to formulate a drug for administration. Pharmaceutical carriers may be sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. When pharmaceutical compositions are administered intravenously, water is a common carrier. Saline solutions and aqueous dextrose and glycerol solutions can be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, as needed. The pharmaceutical composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, or sustained-release formulation. The composition may also be formulated as a suppository using conventional binders and carriers such as triglycerides.

[0151] The pharmaceutical composition of the present invention comprises a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, mixed with at least one pharmaceutically acceptable excipient, carrier, or diluent. The pharmaceutically acceptable composition contains, for example, one or more formulation substances for modifying, maintaining, or preserving the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption or osmosis of the compound. Suitable formulations include amino acids (such as glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, sodium bisulfite); buffering agents (such as borates, bicarbonates, tris-HCl, citrates, phosphates, and other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing 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 immunoglobulin); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); and preservatives. Examples of substances include, but are not limited to, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide; solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic acid, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyroxapal); stability enhancers (sucrose or sorbitol); isotonic enhancers (alkali metal halides (in one embodiment, sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants).(Remington's Pharmaceutical Sciences, 18th Edition, ARGennaro, ed., Mack Publishing Company, 1990).

[0152] In one aspect of the present invention, a glycine-containing tripeptide molecule is supplied in a single-use glass vial containing a lyophilized cake prepared with a formulation buffer consisting of 10 mM glutamic acid, 2% glycine, 1% sucrose, and 0.01% polysorbate 20, pH 4.25. Reconstitution with a certain amount of sterile diluent, such as sterile isotonic saline or water, for example, 0.5 mL to about 10 mL, for example, 2.2 mL of sterile water, yields glycine tripeptide molecules at concentrations of 1 g / mL to about 100 g / mL from the cake. The glycine-containing tripeptide molecule is stored in a safe place under controlled conditions. Once the single-use vial is reconstituted, the drug is administered immediately (within 3 hours of reconstitution). Before injection, the test drug can be allowed to reach room temperature (15-30°C).

[0153] Dosage and administration

[0154] As used herein, “dose” generally refers to the amount of the active ingredient that can be administered once daily or several times daily (for example, a unit dose is a portion of the desired daily dose). In various embodiments, various doses and administration regimens are applied to the glycine or glycine-containing tripeptide molecules disclosed herein. For example, useful doses in the present invention are calculated based on the subject's body weight, ranging from 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. As used herein, the term “unit dose” may be used to indicate a discrete amount of a therapeutic composition containing a predetermined amount of the active compound. The amount of the active ingredient is generally equal to the amount of the active ingredient that can be administered once daily or several times daily (for example, a unit dose is a portion of the desired daily dose). A unit dose may also be understood as representing the total daily dose, which may be administered once daily or as a convenient fraction of such a dose (for example, a unit dose is the total daily dose, which may be given in fractional increments such as half or one-third of the dose).

[0155] As used herein, the term “start-day dose” refers to the amount of glycine-containing tripeptide molecules administered or prescribed per day to a patient who has not previously received a titration regimen of glycine tripeptide molecules and is initiating glycine-containing tripeptide molecule therapy. This amount may be administered in multiple unit doses or a single unit dose, at a single time during the day or in multiple doses during the day.

[0156] In some embodiments, the amount of the glycine-containing tripeptide molecule can 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, 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 can be from about 3 mg / kg / day to about 70 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule can be from about 7 mg / kg / day to about 40 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule can be from about 3 mg / kg / day to about 50 mg / kg / day.

[0157] In some embodiments, the dosage can 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 can preferably be 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, from about 700 mg to about 75 g. In some embodiments, the amount of the glycine-containing tripeptide molecule in the composition can 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 1000 mg to about 25 g.

[0158] In some embodiments, the therapeutically effective amount of the glycine-containing tripeptide molecule to be administered is from about 100 mg to about 200 g. This dosage may be administered as a once-daily dosage, or may be divided into several dosages administered throughout the day, for example, 1 to 5 dosages per day, preferably 2 or 3 dosages. 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 a solid oral dosage form.

[0159] 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% for the glycine-containing tripeptide molecule. In some embodiments, the chiral purity for the glycine-containing tripeptide molecule is 100%. In some embodiments, the composition has a chiral purity of 99.9% or more for the glycine-containing tripeptide molecule. In some embodiments, the composition has a chiral purity of 99.95% or more for the glycine-containing tripeptide molecule. In some embodiments, the composition has a chiral purity of 99.99% or more for the glycine-containing tripeptide molecule.

[0160] 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 solution or a parenteral solution.

[0161] For the sake of brevity, embodiments of the amount, chiral purity, and dosage form of the glycine-containing tripeptide molecule in the composition, which are described separately herein, can be combined in any suitable combination.

[0162] In another embodiment, the present invention relates to a composition comprising a glycine-containing tripeptide molecule that is chiralally pure to a glycine tripeptide molecule. In some embodiments, the amount of the glycine-containing tripeptide molecule may be about 5 mg / kg / day to 5000 mg / kg / day, or 5 mg / kg / day to about 2000 mg / kg / day, or 30 mg / kg / day to 1500 mg / kg / day, or 40 mg / kg / day to 1250 mg / kg / day, or 50 mg / kg / day to 1000 mg / kg / day, or 60 mg / kg / day to 500 mg / kg / day, calculated based on the body weight of the subject. In some embodiments, the amount of the glycine-containing tripeptide molecule may be about 10 mg / kg / day to about 1000 mg / kg / day. In some embodiments, the amount of the glycine-containing tripeptide molecule may be about 7 mg / kg / day to about 900 mg / kg / day. In some embodiments, the amount of glycine-containing tripeptide molecules may be about 5 mg / kg / day to about 800 mg / kg / day. In some embodiments, the dose may be 10 mg / day to 5,000 mg / day, more preferably 100 mg / day to 2,000 mg / day. In some embodiments, the composition is administered in doses of glycine tripeptide molecules 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. In some embodiments, the composition is administered in doses 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 2,500 mg to about 20 g. In some embodiments, the effective amount of glycine-containing tripeptide molecules to be administered to a subject in need to prevent or treat metabolic and / or cardiovascular and / or inflammatory diseases may be in the range of about 500 mg to about 200 g. This dose may be administered as a once-daily dose, or divided into several doses to be administered throughout the day, for example, 1 to 5 doses per day, preferably 2 to 3 doses per day.These doses of glycine-containing tripeptide molecules preferably have a chemical purity of 97% or higher and 99.6% or higher, 99.7% or higher, 99.8% or higher, 99.9% or higher, preferably 99.95% or higher, and more preferably 99.99% or higher. In a preferred embodiment, the composition containing the glycine-containing tripeptide molecule may have 100% chiral purity relative to the glycine-containing tripeptide molecule. The composition may further contain a carrier 5. The composition of the present invention may be administered orally, preferably as a solid oral dose, more preferably as a solid oral dose which may be a capsule or tablet. In a preferred embodiment, the composition of the present invention may be formulated as a tablet for oral administration.

[0163] In another embodiment, the present invention further provides a composition comprising a therapeutically effective amount of glycine tripeptide molecules. The composition may further contain a pharmaceutically acceptable carrier.

[0164] In some embodiments, the therapeutically effective dose of the glycine-containing tripeptide molecule may be approximately 1 mg / kg / day to approximately 10,000 mg / kg / day, approximately 5 mg / kg / day to approximately 5,000 mg / kg / day, approximately 20 mg / kg / day to approximately 1,000 mg / kg / day, approximately 30 mg / kg / day to approximately 1,000 mg / kg / day, approximately 50 mg / kg / day to approximately 10,000 mg / kg / day, or approximately 100 mg / kg / day to approximately 5,000 mg / kg / day.

[0165] In some embodiments, the therapeutically effective dose of the glycine-containing tripeptide molecule may be about 5 mg / kg / day to about 5,000 mg / kg / day. In some embodiments, the therapeutically effective dose of the glycine-containing tripeptide molecule may be about 10 mg / kg / day to about 4,000 mg / kg / day. In some embodiments, the therapeutically effective dose of the glycine-containing tripeptide molecule may be about 25 mg / kg / day to about 2,000 mg / kg / day. In some embodiments, the dose may be 10 mg / day to 1,000 g / day, more preferably 500 mg / day to 100 g / day. The therapeutically effective dose of the glycine-containing tripeptide molecule in the composition may preferably be about 250 mg to about 500 g, about 500 mg to about 400 g, about 750 mg to about 200 g, or about 1,000 mg to about 100 g. In some embodiments, the therapeutically effective amount of glycine-containing tripeptide molecules 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 glycine-containing tripeptide molecules is about 600 mg to about 300 g. This dose may be administered as a once-daily dose, or divided into several doses administered throughout the day, for example, 1 to 5 doses per day, preferably 2 to 3 doses per day. In some embodiments, the therapeutically effective amount of glycine-containing tripeptide molecules is about 500 mg to about 350 g. In some embodiments, the therapeutically effective amount of glycine-containing tripeptide molecules is about 750 mg to about 250 g. In some embodiments, the therapeutically effective dose of the glycine-containing tripeptide molecule is about 1,000 mg to about 150 g. In some embodiments, the therapeutically effective dose 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 in a solid oral dosage form. In some embodiments, the composition is in a liquid oral dosage form. In some embodiments, the composition is in a liquid parenteral dosage form.

[0166] In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is in solid oral dosage form. In some embodiments, the composition is in capsule form. In some embodiments, the composition is in tablet form.

[0167] In another embodiment, the beneficial effects of glycine tripeptide molecules or pharmaceutically acceptable salts thereof on the liver function of the target are observed in one embodiment in favorable changes in the expression of hepatic lipid oxidation, AMPKα1, glucokinase, 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 dehydrogenase or stearoyl-CoA desaturate. In a further embodiment, the beneficial effects of glycine tripeptide molecules or pharmaceutically acceptable salts thereof on the liver function of the target are observed in improvements in the expression of phosphoenoylpyruvate kinase, microsome transfer proteins, arylacetamine deacetylase, apolipoprotein C2, carnitine palmitoyltransferase II, or phospholipase D1.

[0168] The glycine tripeptide molecule of the present invention, or a pharmaceutically acceptable salt thereof, may be administered by any suitable route. For example, the compositions of the present invention can be administered orally, orally, intradermally, intraperitoneally, intranasally, subcutaneously, intramuscularly, or intravenously.

[0169] Formulations suitable for oral administration include, for example, solid, semi-solid, and liquid systems, such as tablets; soft or hard capsules containing multiparticles or nanoparticles, liquids, or powders; lozenges (including those filled with liquid); gums; gels; high-speed dispersibility formulations; films; suppositories; and sprays. In some embodiments, the peptides of the present invention are formulated for oral administration using delivery vehicles known in the art, including but not limited to microspheres, liposomes, enteric-coated dry emulsions, or nanoparticles.

[0170] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active peptide, the liquid dosage form may contain, for example, water or other solvents, commonly used in the art, such as inert diluents, solubilizers, and emulsifiers, e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid ester sorbitan, as well as mixtures thereof. In addition to inert diluents, the oral composition may also contain adjuvants such as wetting agents, emulsifiers, and suspension agents, sweeteners, flavoring agents, and fragrances.

[0171] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such a solid dosage form, the active peptide is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) wetting agents such as glycerol; d) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) dissolution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; 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 include a buffer. The active compound may also be in a microencapsulated form containing one or more of the excipients described above. Similar solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol, poloxamer, etc. Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules can be prepared using coatings and shells, e.g., enteric coatings and other coatings well known in the field of pharmaceutical formulation. Injectable formulations, e.g., sterile injectable aqueous or oily suspensions, may be formulated according to known techniques using appropriate dispersants or wetting agents and suspending agents. Sterile injectable formulations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, e.g., solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile fixative oils are conventionally used as solvents or suspension media.For this purpose, any brand of fixed oil containing synthetic monoglycerides or diglycerides can be used. Furthermore, fatty acids such as oleic acid are used in the preparation of the injectable formulation. The injectable formulation can be sterilized, for example, by filtration through a filter that retains bacteria, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0172] Those skilled in the art will understand that the amount of glycine-containing tripeptide molecular polypeptide administered for therapeutic use varies considerably. In one embodiment, a composition containing a glycine tripeptide molecule, or a pharmaceutically acceptable salt thereof, is substantially free of contaminants, with a contamination level of less than 0.02% (w / w). A glycine-containing tripeptide molecular composition suitable for injection into a patient is prepared, for example, by reconstituting a lyophilized sample containing purified glycine-containing tripeptide molecules and stabilized salts with a pharmaceutically acceptable diluent. Administration of a glycine-containing tripeptide molecular composition may be systemic or topical, as will be discussed in detail below herein, and may involve the administration of a therapeutically effective amount of a glycine-containing tripeptide molecular protein composition.

[0173] Combination Therapies and Compositions

[0174] In addition to treatment based solely on the delivery of compositions containing glycine tripeptide molecules or pharmaceutically acceptable salts thereof, combination therapy is particularly intended. With regard to the present invention, glycine-containing tripeptide molecular therapy is intended to be used in combination with other agents commonly used for the treatment of metabolic diseases, such as fatty liver disease.

[0175] In various embodiments, a second therapeutic agent(s) may be used in combination with a glycine tripeptide molecule, particularly for synergistic enhancement of activity. The co-administration of active ingredients can be carried out either by separate administration of the active ingredients to the patient, or in the form of a combination product in which multiple active ingredients are present in a single pharmaceutical formulation. When used as a monotherapy agent, the second therapeutic agent may be administered at an approved dose, or, when administered in combination with a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof, the second therapeutic agent may be administered first at or below a therapeutically effective concentration, such that the combination provides a therapeutic effect in the patient being treated.

[0176] To achieve suitable therapeutic outcomes using the methods and compositions of the present invention, compositions comprising a glycine-containing tripeptide molecule and at least one other therapeutic agent (a second therapeutic agent(s)) may be provided. In the present invention, the second therapeutic agent is intended to be selected from insulin-sensitizing agents including, but not limited to, pramlinitide, peptide YY (PYY), exenatide, or thiazolidinedione or metformin, or glimepiride, and any analogues of these compounds. Other second-line therapeutic agents used for the control of dyslipidemia, diabetes, and cardiovascular disease associated with atherosclerosis are well known in the art and may be used in combination with the glycine-containing tripeptide molecule of the present invention.

[0177] The combination therapy composition is provided in a combination amount effective to produce a desired therapeutic outcome in the treatment of metabolic disorders, e.g., glucose metabolism disorders, dyslipidemia, e.g., elevated cholesterol (pure or isolated hypercholesterolemia), and / or elevated triglycerides (TG) only (pure or isolated hypertriglyceridemia), and / or elevated cholesterol and TG (mixed or combined hyperlipidemia), NAFLD, NASH, fatty liver, hepatitis, peripheral artery disease, coronary artery disease, atherosclerosis, systemic inflammation, and fatty liver diseases, including ischemic stroke associated with plaque-associated thrombosis. The process involves simultaneously administering to a subject in need a therapeutically effective amount of the first therapeutic agent of the present invention (i.e., a composition comprising a therapeutically effective amount of a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof), and optionally a second therapeutic agent or factor(s). This is achieved by administering a single composition or pharmacological formulation containing both therapeutic agents, or by administering two separate compositions or formulations simultaneously, in which case one composition comprises a glycine-containing tripeptide molecular therapeutic composition and the other comprises a second therapeutic agent.

[0178] Useful antidiabetic drugs for use as a second-line therapy in combination with glycine-containing tripeptide molecules include insulin and insulin derivatives, e.g., Lantus® or HMR1964 or Levemir® (insulin detemir) or drugs described in WO2005005477 (Novo Nordisk), rapid-acting insulin (see U.S. Patent No. 6,221,633), inhaled insulin, e.g., Exubera® or oral insulin, e.g., IN-105 (Nobex) or Oral-lyn® (Generex Biotechnology), GLP-1 derivatives and GLP-1 agonists, e.g., exenatide, liraglutide or Novo Nordisk Examples include drugs disclosed in A / S WO98 / 08871, WO2005027978, WO2006037811 or WO2006037810, Zealand WO01 / 04156, Beaufour-Ipsen WO00 / 34331, plumlintide acetate (Symlin; Amylin Pharmaceuticals), BIM-51077, PC-DAC-exendin-4 (an exendin-4 analog covalently bound to recombinant human albumin), agonists such as those described in D. Chen et al., Proc. Natl. Acad. Sci. USA 104(2007)943, drugs described in WO2006124529, and orally effective hypoglycemic active ingredients.

[0179] Antidiabetic drugs include, for example, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists as described in WO2006121860, as well as GLP-1 and GLP-2 and their synthetic variants and variants used to control blood glucose and treat diabetes, including type II diabetes.

[0180] Examples of second-line therapeutic agents useful in combination with the glycine-containing tripeptide molecules disclosed herein include orally effective hypoglycemic active ingredients, preferably sulfonylurea, biguanidine, meglitinide, oxadiazolidinedione, thiazolidinedione, glucosidase inhibitors, glycogen phosphorylase inhibitors, glucagon antagonists, glucokinase activators, fructose-1,6-bisphosphatase inhibitors, glucose transporter 4 (GLUT4) regulators, glutamine-fructose-6-phosphate amidetransferase (GFAT) inhibitors, GLP-1 agonists, potassium channel openers, such as pinacidil, chromalim, diazoxide, or RDTar et al., Diabetes 52, 2003, 2513, 2518, JBHansen et al., Current Medicinal Chemistry 11, 2004, 1595-1615, TMTagmose et al. See al., J.Med.Chem. 47, 2004, 3202-3211 or MJ Coghlan et al., J.Med.Chem. 44, 2001, 1627-1653, or Novo Nordisk Examples of compounds disclosed in A / S WO97 / 26265 and WO99 / 03861 include dipeptidyl peptidase IV (DPP-IV) inhibitors, insulin sensitizers, inhibitors of hepatic enzymes involved in stimulating gluconeogenesis and / or glycogenolysis, regulators of glucose uptake, glucose transport and glucose reabsorption, 11β-HSD1 inhibitors, protein tyrosine phosphatase 1B (PTP1B) inhibitors, sodium-dependent glucose transporter 1 or 2 (SGLT1, SGLT2) regulators, compounds that alter lipid metabolism such as antihyperlipidemic and anti-lipidemia 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.

[0181] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with an HMGCoA reductase inhibitor such as simvastatin, fluvastatin, pravastatin, lovastatin, atorvastatin, cerivastatin, rosuvastatin or L-659699.

[0182] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a cholesterol absorption inhibitor such as ezetimibe, tikeside, pamaqueside, FM-VP4 (sitostanol / campesterol phosphate ascorbyl; Forbes Medi-Tech, WO2005042692, WO2005005453), MD-0727 (Microbia Inc., WO2005021497, WO2005021495), or a compound described in WO2002066464, WO2005000353 (Kotobuki Pharmaceutical Co. Ltd.), or WO2005044256 or WO2005062824 (Merck & Co.) or WO2005061451 and WO2005061452 (AstraZeneca AB), and WO2006017257 (Phenomix) or WO2005033100 (Lipideon Biotechnology AG), or a compound described in WO2004097655, WO2004000805, WO2004000804, WO2004000803, WO2002050068, WO2002050060, WO2005047248, WO2006086562, WO2006102674, WO2006116499, WO2006121861, WO2006122186, WO2006122216, WO2006127893, WO2006137794, WO2006137796, WO2006137782, WO2006137793, WO2006137797, WO2006137795, WO2006137792, WO2006138163.

[0183] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with Vitrin™, which is a fixed combination of ezetimibe and simvastatin.

[0184] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of ezetimibe and atorvastatin.

[0185] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of ezetimibe and fenofibrate.

[0186] In a further embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of fenofibrate and rosuvastatin.

[0187] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with Synordia®, a fixed combination of fenofibrate and metformin.

[0188] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with ISIS-301012, an antisense oligonucleotide capable of regulating the apolipoprotein B gene.

[0189] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a PPARγ agonist such as rosiglitazone, pioglitazone, JTT-501, GI262570, R-483, or CS-011 (riboglitazone).

[0190] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with Competact®, which is a fixed combination of pioglitazone hydrochloride and metformin hydrochloride.

[0191] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with Tandemact®, which is a fixed combination of pioglitazone and glimepiride.

[0192] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a fixed combination of pioglitazone hydrochloride and an angiotensin II receptor antagonist such as TAK-536.

[0193] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a PPARα agonist, such as GW9578, GW-590735, K-111, LY-674, KRP-101, DRF-10945, LY-518674, or one of the agonists described in WO2001040207, WO2002096894, or WO2005097076.

[0194] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a mixed PPARα / γ agonist, such as naveglitazar, LY-510929, ONO-5129, E-3030, AVE8042, AVE8134, AVE0847, CKD-501 (robeglitazone sulfate), or WO00 / 64888, WO00 / 64876, WO03 / 020269, or one of those described in JPBerger et al., TRENDS in Pharmacological Sciences 28(5), 244-251, 2005.

[0195] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a PPARδ agonist, for example, GW-501516, or one of those described in WO2006059744, WO2006084176, WO2006029699, WO2007039172 to WO2007039178.

[0196] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with metaglidacene, MBX-2044, or another partial PPARγ agonist / antagonist.

[0197] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a fibrate drug such as fenofibrate, clofibrate, or bezafibrate.

[0198] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with an MTP inhibitor, such as impritapide, BMS-201038, R-103757, AS-1552133, or WO2005085226, WO2005121091, or WO2006010423.

[0199] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a CETP inhibitor, such as torcetrapib or JTT-705, or one of those described in WO2006002342, WO2006010422, WO2006012093, WO2006073973, WO2006072362, WO2006097169, or WO2007041494.

[0200] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a bile acid absorption inhibitor (see, for example, U.S. Patent No. 6,245,744, U.S. Patent No. 6,221,897, or WO00 / 61568), such as HMR1741, or those described in DE102005033099.1 and DE102005033100.9, WO2007009655-56.

[0201] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a high molecular weight bile acid adsorbent, such as cholestyramine or choleceveram.

[0202] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with an LDL receptor inducer (see U.S. Patent No. 6,342,512), such as HMR1171, HMR1586, or those described in WO2005097738.

[0203] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with, for example, the ABCA1 expression enhancer described in WO2006072393.

[0204] In further embodiments of the present invention, a glycine-containing tripeptide molecule is administered in combination with a PCSK9 (proprotein convertase subtilisin / kexin type 9) inhibitor, such as evolocumab, inclisiran, or an RNAi therapeutic agent against PCSK9. In further embodiments of the present invention, a glycine-containing tripeptide molecule is administered in combination with an antibody against PCSK9, such as alirocumab (Praluent) (75-150 mg every two weeks); and evolocumab (Repatha) (140 mg every two weeks, or 420 mg monthly).

[0205] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with Omacor® (ω-3 fatty acid; high-concentration ethyl ester of eicosapentaenoic acid and docosahexaenoic acid).

[0206] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with an ACAT inhibitor such as abasimibe or SMP-797.

[0207] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with an antioxidant, such as OPC-14117, probucol, tocopherol, ascorbic acid, β-carotene, or selenium.

[0208] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a vitamin, for example, vitamin B6 or vitamin B12.

[0209] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a lipoprotein lipase regulator, such as ibrolipim (NO-1886).

[0210] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with an ATP citrate lyase inhibitor, such as SB-204990.

[0211] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a squalene synthetase inhibitor, such as BMS-188494, TAK-475, or one described in WO2005077907 or JP2007022943.

[0212] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a lipoprotein (a) antagonist, such as gemcabene (CI-1027).

[0213] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a GPR109A agonist (HM74A receptor agonist; NAR agonist (nicotinic acid receptor agonist)), for example, sustained-release niacin combined with nicotinic acid or MK-0524A, or a compound described in WO2006045565, WO2006045564, WO2006069242, WO2006124490, WO2006113150, WO2007017261, WO2007017262, WO2007017265, WO2007015744, WO2007027532.

[0214] In another embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with, for example, a GPR116 agonist as described in WO2006067531 and WO2006067532.

[0215] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with a lipase inhibitor, such as orlistat or cetilistat (ATL-962).

[0216] In one embodiment of the present invention, a glycine-containing tripeptide molecule is administered in combination with insulin.

[0217] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a sulfonylurea such as tolbutamide, glibenclamide, glipizide, gliclazide, or glimepiride.

[0218] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a substance that enhances insulin secretion, such as KCP-265 (WO2003097064) or WO2007026761.

[0219] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glucose-dependent insulin secretory receptor (GDIR) agonist, such as APD-668.

[0220] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a biguanide, such as metformin.

[0221] In yet another embodiment, the glycine-containing tripeptide molecule is administered in combination with meglitinide, such as repaglinide, nateglinide, or mitiglinide.

[0222] In further embodiments, a glycine-containing tripeptide molecule is administered with mitiglinide in combination with glitazone, such as pioglitazone hydrochloride or rosiglitazone maleate, or with glitazone and glimepiride, or with metformin, or a combination thereof.

[0223] In a further embodiment, a glycine-containing tripeptide molecule is administered together with a combination of mitiglinide and an α-glucosidase inhibitor.

[0224] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a thiazolidinedione, such as troglitazone, siglitazone, pioglitazone, rosiglitazone, or a compound disclosed in Dr. Reddy's Research Foundation WO97 / 41097, particularly 5-[[4-[(3,4-dihydro-3-methyl-4-oxo-2-quinazolinylmethoxy]-phenyl]methyl]-2,4-thiazolidinedione.

[0225] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an α-glucosidase inhibitor, such as miglitol or acarbose.

[0226] 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.

[0227] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with two or more of the aforementioned compounds, for example, with sulfonylurea and metformin, sulfonylurea and acarbose, repaglinide and metformin, insulin and sulfonylurea, insulin and metformin, insulin and troglitazone, insulin and lovastatin, etc.

[0228] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glycogen phosphorylase inhibitor, such as PSN-357 or FR-258900, or one of those described in WO2003084922, WO2004007455, WO2005073229-31, or WO2005067932.

[0229] 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 one described in WO2004100875 or WO2005065680.

[0230] In one embodiment, the glycine-containing tripeptide molecule is replaced with a glucokinase activator, for example, LY-2121260 (WO2004063179), PSN-105, PSN-110, GKA-50, or for example, WO2004072031, WO2004072066, WO2005080360, WO2005044801, WO2006016194, WO2006058923, WO2006112549, WO2006125972, WO2007017549, WO2007017649, WO Administer in combination with the products listed in 2007007910, WO2007007040~42, WO2007006760~61, WO2007006814, WO2007007886, WO2007028135, WO2007031739, WO2007041365, WO2007041366, WO2007037534, WO2007043638, WO2007053345, WO2007051846, WO2007051845, WO2007053765, and WO2007051847.

[0231] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a gluconeogenesis inhibitor, such as FR-225654.

[0232] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a fructose-1,6-bisphosphatase (FBPase) inhibitor, such as CS-917 (MB-06322) or MB-07803, or one of those described in WO2006023515, WO2006104030, or WO2007014619.

[0233] 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)).

[0234] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glutamine-fructose-6-phosphate amide transferase (GFAT) inhibitor, for example, as described in WO2004101528.

[0235] In one embodiment, a glycine-containing tripeptide molecule is used as a dipeptidyl peptidase IV (DPP-IV) inhibitor, such as vildagliptin (LAF-237), sitagliptin (MK-0431), phosphate sitagliptin, 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 WO2003074500, WO2003106456, WO2004037169, WO200450658, WO2005058901, WO2005012312, WO2 005 / 012308, WO2006039325, WO2006058064, WO2006015691, WO2006015701, WO2006015699, WO2 006015700, WO2006018117, WO2006099943, WO2006099941, JP2006160733, WO2006071752, WO20 Administer in combination with the compounds listed in 06065826, WO2006078676, WO2006073167, WO2006068163, WO2006090915, WO2006104356, WO2006127530, WO2006111261, WO2007015767, WO2007024993, and WO2007029086.

[0236] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with Janumet®, a fixed combination of sitagliptin phosphate and metformin hydrochloride.

[0237] In one embodiment, a glycine-containing tripeptide molecule is used with 11-β-hydroxysteroid dehydrogenase 1 (11β-HSD1) inhibitors, such as BVT-2733, JNJ-25918646, INCB-13739, or, for example, WO200190090~94, WO200343999, WO2004112782, WO200344000, WO200344009, WO2004112779, WO2004113310, WO2004103980, WO20 04112784, WO2003065983, WO2003104207, WO2003104208, WO2004106294, WO2004011410, WO2004033427, WO2004041264, WO20040 37251, WO2004056744, WO2004058730, WO2004065351, WO2004089367, WO2004089380, WO2004089470~71, WO2004089896, WO20050 16877, WO2005097759, WO2006010546, WO2006012227, WO2006012173, WO2006017542, WO2006034804, WO2006040329, WO20060516 62, WO2006048750, WO2006049952, WO2006048331, WO2006050908, WO2006024627, WO2006040329, WO2006066109, WO2006074244, Administer in combination with the drugs listed in WO2006078006, WO2006106423, WO2006132436, WO2006134481, WO2006134467, WO2006135795, WO2006136502, WO2006138695, WO2006133926, WO2007003521, WO2007007688, US2007066584, WO2007047625, WO2007051811, and WO2007051810.

[0238] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a protein tyrosine phosphatase 1B (PTP1B) inhibitor, for example, as described in WO200119830~31, WO200117516, WO2004506446, WO2005012295, WO2005116003, WO2005116003, WO2006007959, DE102004060542.4, WO2007009911, WO2007028145, and WO2007081755.

[0239] In one embodiment, the glycine-containing tripeptide molecule is a sodium-dependent glucose transporter 1 or 2 (SGLT1, SGLT2) regulator, for example, KGA-2727, T-1095, SGL-0010, AVE2268, SAR7226 and cergliflozin, or for example, WO2004007517, WO200452903, WO200452902, PCT / EP2005 / 005959, WO2005 It should be administered in combination with 085237, JP2004359630, WO2005121161, WO2006018150, WO2006035796, WO2006062224, WO2006058597, WO2006073197, WO2006080577, WO2006087997, WO2006108842, WO2007000445, WO2007014895, WO2007080170, or ALHandlon in Expert Opin.Ther.Patents(2005)15(11),1531-1540.

[0240] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a GPR40 regulator, such as those described in WO2007013689 and WO2007033002.

[0241] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a GPR119b regulator, for example, one described in WO2004041274.

[0242] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a GPR119 regulator, such as those described in WO2005061489 (PSN-632408), WO2004065380, WO2007003960-62, and WO2007003964.

[0243] In a further embodiment, a glycine-containing tripeptide molecule is administered in combination with a GPR120 regulator.

[0244] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a hormone-sensitive lipase (HSL) inhibitor and / or phospholipase, for example, as described in WO2005073199, WO2006074957, WO2006087309, WO2006111321, and WO2007042178.

[0245] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an acetyl-CoA carboxylase (ACC) inhibitor, such as those described in WO199946262, WO200372197, WO2003072197, WO2005044814, WO2005108370, JP2006131559, WO2007011809, WO2007011811, and WO2007013691.

[0246] In a further embodiment, a glycine-containing tripeptide molecule is administered in combination with a xanthine oxidoreductase (XOR) regulator.

[0247] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, for example, one described in WO2004074288.

[0248] In one embodiment, the glycine-containing tripeptide molecules are, for example, US2005222220, WO2005085230, WO2005111018, WO2003078403, WO2004022544, WO2003106410, WO2005058908, US2005038023, WO2005009997, US2 It should be administered in combination with a glycogen synthase kinase 3β (GSK-3β) inhibitor, as described in 005026984, WO2005000836, WO2004106343, EP1460075, WO2004014910, WO2003076442, WO2005087727, or WO2004046117.

[0249] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a serum / glucocorticoid-regulated kinase (SGK) inhibitor, for example, as described in WO2006072354.

[0250] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a RUP3 receptor agonist, for example, as described in WO2007035355.

[0251] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a protein kinase Cβ (PKCβ) inhibitor, such as ruboxystaurin.

[0252] In another embodiment, a glycine-containing tripeptide molecule is administered in combination with an activator of the gene encoding ataxia vasodilator mutation (ATM) protein kinase, such as chloroquine.

[0253] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an endothelin A receptor antagonist, such as avosentan (SPP-301).

[0254] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with an "I-κB kinase" inhibitor (IKK inhibitor), as described in, for example, WO2001000610, WO2001030774, WO2004022553, or WO2005097129.

[0255] In one embodiment, a glycine-containing tripeptide molecule is administered in combination with a glucocorticoid receptor (GR) modulator, for example, as described in WO2005090336, WO2006071609, and WO2006135826.

[0256] In further embodiments, a 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).

[0257] In further embodiments, the glycine-containing tripeptide molecule may be an NPY antagonist, such as an NPY-5 receptor antagonist such as naphthalene-1-sulfonic acid {4-[(4-aminoquinazoline-2-ylamino)methyl]cyclohexylmethyl}amide hydrochloride (CGP71683A); L-152804, or, for example, one described in WO2006001318; an NPY-4 receptor antagonist, such as one described in WO2007038942; an NPY-2 receptor antagonist, such as one described in WO2007038943; the peptide YY3-36 (PYY3-36) or a similar compound, such as CJC-1682 (PYY3-36 conjugated to human serum albumin via Cys34), CJC-1643 (in PYY3-36 derivatives that conjugate to serum albumin in vivo) or those described in WO2005080424, WO2006095166; peptide ovestatin derivatives described in WO2006096847; CB1R (cannabinoid receptor 1) antagonists (e.g., rimonabant, SR147778, SLV-319, AVE-16) 25, MK-0364 or its salts) or, for example, EP0656354, WO00 / 15609, WO2001 / 64632~64634, WO02 / 076949, WO2005080345, WO2005080328, WO2005080343, WO2005075450, WO2005080357, WO200170700, WO200302 6647-48, WO200302776, WO2003040107, WO2003007887, WO2003027069, US No. 6,509,367, WO200132663, WO2003086288, WO2003087037, WO2004048317, WO2004058145, WO2003084930, WO20030849 43, WO2004058744, WO2004013120, WO2004029204, WO2004035566, WO2004058249, WO2004058255, WO2004058727, WO2004069838, US20040214837, US20040214855, US20040214856, WO2004096209,WO2004096763, WO2004096794, WO2005000809, WO2004099157, US20040266845, WO2004110453, WO2004108728, WO2004000817, WO2005000820, US2005 0009870, WO200500974, WO2004111033-34, WO200411038~39, WO2005016286, WO2005007111, WO2005007628, US20050054679, WO2005027837, WO20050 28456, WO2005063761~62, WO2005061509, WO2005077897, WO2006047516, WO2006060461, WO2006067428, WO2006067443, WO2006087480, WO200608747 6, WO2006100208, WO2006106054, WO2006111849, WO2006113704, WO2007009705, WO2007017124, WO2007017126, WO2007018459, WO2007016460, WO200 Compounds listed in 7020502, WO2007026215, WO2007028849, WO2007031720, WO2007031721, WO2007036945, WO2007038045, WO2007039740, US20070015810, WO2007046548, WO2007047737, WO2007084319, WO2007084450; for example, cannabinoid receptor 1 / cannabinoid receptors as listed in WO2007001939, WO2007044215, WO2007047737. Id receptor 2 (CB1 / CB2) modulating compounds; MC4 agonists (e.g., 1-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid [2-(3a-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydropyrazolo[4,3-c]pyridine-5-yl)-1-(4-chlorophenyl)-2-oxoethyl]amide; (WO01 / 91752)) or LB53280, LB53279, LB53278 or THIQ, MB243, RY764, CHIR-785, PT-141 or WO2005060985,WO2005009950, WO2004087159, WO2004078717, WO2004078716, WO2004024720, US20050124652, WO2005051391, WO2004112793, WOUS2005022201 4, US20050176728, US20050164914, US20050124636, US20050130988, US20040167201, WO2004005324, WO2004037797, WO2005042516, WO2005040 As described in 109, WO2005030797, US20040224901, WO200501921, WO200509184, WO2005000339, EP1460069, WO2005047253, WO2005047251, WO2005118573, EP1538159, WO2004072076, WO2004072077, WO2006021655~57, WO2007009894, WO2007015162, WO2007041061, WO2007041052; orexin receptor Activators (e.g., 1-(2-methylbenzoxazole-6-yl)-3-[1,5]naphthyrizine-4-ylurea hydrochloride (SB-334867-A) or, for example, those listed 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]pyridine-5-yl-)propane- 1-Oxalate (WO00 / 63208) or as described in WO200064884, WO2005082893, WO2006107661, WO2007003804, WO2007016496, WO2007020213); histamine H1 / histamine H3 regulators, e.g., betahistine and its dihydrochloride; CRF antagonists (e.g., [2-methyl-9-(2,4,6-trimethylphenyl)-9H-1,3,9-triazafluoren-4-yl]dipropyramine (WO00 / 66585)); CRF BP antagonists (e.g.,Urocortin; urocortin agonists; β-3 adrenergic receptor agonists, e.g., 1-(4-chloro-3-methanesulfonylmethylphenyl)-2-[2-(2,3-dimethyl-1H-indole-6-yloxy)ethylamino]ethanol hydrochloride (WO01 / 83451); or Solabegron (GW-427353) or N-5984 (KRP-204) or those listed in JP2006111553, WO2002038543, WO2007048840~843; MSH (melanosynovitis) (Stimulating hormone) agonist; MCH (melanin-concentrating hormone) receptor antagonist (e.g., NBI-845, A-761, A-665798, A-798, ATC-0175, T-226296, T-71, GW-803430 or WO2005085200, WO2005019240, WO2004011438, WO2004012648, WO2003015769, WO2004072025, WO2005070898, WO2005070925, WO2004039780, WO2004092181, WO 2003033476, WO2002006245, WO2002089729, WO2002002744, WO2003004027, FR2868780, WO2006010446, WO2006038680, WO2006044293, WO200604 4174, JP2006176443, WO2006018280, WO2006018279, WO2006118320, WO2006130075, WO2007018248, WO2007012661, WO2007029847, WO200702400 4. Compounds as described in WO2007039462, WO2007042660, WO2007042668, WO2007042669, US2007093508, US2007093509, WO2007048802, JP2007091649); CCK-A agonists (e.g., {2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexylethyl)thiazole-2-ylcarbamoyl]-5,7-dimethylindole-1-yl}acetate trifluoroacetate (WO99 / 15525),Those described in SR-146131 (WO0244150) or SSR-125180 or WO2005116034); mixtures of serotonergic and noradrenergic compounds (e.g., WO00 / 71549); 5-HT receptor agonists, e.g., 1-(3-ethylbenzofuran-7-yl)piperazine oxalate (WO01 / 09111); mixed dopamine / norepinephrine / acetylcholine reuptake Reuptake inhibitors (e.g., tesofensin); 5-HT2C receptor agonists (e.g., lorcaserin hydrochloride (APD-356), BVT-933, or those listed in WO200077010, WO20077001~02, WO2005019180, WO2003064423, WO200242304, WO2005035533, WO2005082859, WO2006077025, WO2006103511); 5 -HT6 receptor antagonists, e.g., E-6837 or BVT-74316 or those described in WO2005058858, WO2007054257; bombesin receptor agonists (BRS-3 agonists); galanin receptor antagonists; growth hormone (e.g., human growth hormone or AOD-9604); growth hormone-releasing compounds (tertiary butyl 6-benzyloxy-1-(2-diisopropyl) (Lu-aminoethylcarbamoyl)-3,4-dihydro-1H-isoquinoline-2-carboxylate (WO01 / 85695); growth hormone secretagogue receptor antagonists (ghrelin antagonists), e.g., those described in A-778193 or WO2005030734; TRH agonists (e.g., see EP0462884); uncoupling protein 2 or 3 regulators; leptin agonists (e.g.,Lee,Daniel W.;Leinung,Matthew C.;Rozhayskaya-Arena,Marina;Grasso,Patricia. Leptin agonists as a potential approach to the treatment of obesity. Drugs of the See Future (2001), 26(9), 873-881); DA agonists (bromocriptine or doplexin); lipase / amylase inhibitors (e.g., WO00 / 40569); diacylglycerol O-acyltransferase (DGAT) inhibitors, e.g., BAY-74-4113, or e.g., US2004 / 0224997, WO2004094618, WO200058491, WO2005044250, WO2005072740, JP2005206492, WO2005013907, WO2006004200, WO2006019020, WO2, Those described in 006064189, WO2006082952, WO2006120125, WO2006113919, WO2006134317, WO2007016538; fatty acid synthase (FAS) inhibitors, e.g., C75 or those described in WO2004005277; e.g., stearoyl as described in WO2007009236, WO2007044085, WO2007046867, WO2007046868, WO20070501124 - CoAδ9 desaturate (SCD1) inhibitors; oxytomodulin; oleoylestrone or thyroid hormone receptor agonists or partial agonists, such as those listed in KB-2115 or WO20058279, WO200172692, WO200194293, WO2003084915, WO2004018421, WO2005092316, WO2007003419, WO2007009913, WO2007039125.

[0258] In a further embodiment, the glycine-containing tripeptide molecule is administered in combination with varenicline tartrate, a partial agonist of the α4-β2 nicotinic acetylcholine receptor.

[0259] In a further embodiment, a glycine-containing tripeptide molecule is administered in combination with trodasquemin.

[0260] In a further embodiment, a glycine-containing tripeptide molecule is administered in combination with a regulator of the SIRT1 enzyme.

[0261] In a further embodiment, the glycine-containing tripeptide molecule is administered in combination with dexamfetamine or amphetamine.

[0262] In further embodiments, a glycine-containing tripeptide molecule is administered in combination with fenfluramine or dexfenfluramine.

[0263] In a further embodiment, a glycine-containing tripeptide molecule is administered in combination with sibutramine.

[0264] In further embodiments, a glycine-containing tripeptide molecule is administered in combination with mazindol or phentermine.

[0265] Alternatively, secondary drug therapy is administered before or after polypeptide therapy with a glycine-containing tripeptide molecule, at intervals ranging from a few minutes to several weeks. In embodiments where the second drug and the glycine-containing tripeptide molecule are administered separately, it is generally considered that there should be no significant gap in the time between each delivery, and as a result, the second drug and the glycine-containing tripeptide molecule will still be able to exert a favorable combined effect. In such cases, it is considered most preferable to administer both modalities within approximately 12 to 24 hours of each other, and in one embodiment, within approximately 6 to 12 hours of each other, with a delay of only about 12 hours. In some situations, it is desirable to significantly extend the period for treatment, however, to allow several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration.

[0266] In some embodiments, a method for treating a metabolic disease, 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 prescribed fish oil, or a CETP inhibitor.

[0267] Systemic delivery of glycine-containing tripeptide molecule expression constructs or peptides to patients is a highly efficient method for delivering therapeutically effective compositions to counteract the immediate clinical symptoms of a disease. Alternatively, topical delivery of glycine-containing tripeptide molecules and / or a second therapeutic agent is appropriate in certain situations.

[0268] The present invention further provides a method for administering the composition of the present invention to a mammal. In one embodiment, the mammal is a human. The effective amount of the composition used therapeutically will depend, for example, on the circumstances and purpose of the treatment. Therefore, those skilled in the art will understand that the appropriate dose level for treatment will vary considerably, depending in part on the molecule being delivered, the indication for which the composition is used, the route of administration, and size (body weight, body surface or organ size) as well as the patient's condition (age and overall health). Accordingly, clinicians may titrate the dose and modify the route of administration to obtain the optimal therapeutic effect.

[0269] In one embodiment, a regimen for delivering a composition comprising a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof to a mammal would involve 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, or 50 mg / kg to 1000 mg / kg, or 60 mg / kg to 5000 mg / kg, given in daily doses or at longer or shorter intervals, e.g., every other day, twice a week, once a week, once a month, once every six months, or twice or three times a day, in equivalent doses. Administration may be by oral, intravenous, subcutaneous, intranasal, inhalation, transdermal, transmucosal, or any other route discussed herein.

[0270] In a further embodiment, the glycine-containing tripeptide molecule is administered by subcutaneous injection at doses specified herein. However, the glycine-containing tripeptide molecule may be administered by any of the methods discussed herein and known in the art. In a further embodiment, a single-site injection has a maximum permissible volume of 2.0 ml. If a higher concentration of the glycine-containing tripeptide molecule is required, it is conceivable that the glycine-containing tripeptide molecule may be injected at multiple sites or in larger doses. In another embodiment, the glycine-containing tripeptide molecule is administered at approximately the same time each day. However, alternative times for delivery are included in the present invention.

[0271] Pharmaceutical compositions may also be selected for parenteral delivery. Alternatively, compositions may be selected for inhalation or for delivery via the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art of those skilled in the art.

[0272] 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 with a propellant for aerosol delivery. In yet another embodiment, the solution may be sprayed. Lung administration is further described in PCT application PCT / US94 / 001875, which describes the pulmonary delivery of chemically modified proteins.

[0273] It is also intended that specific formulations may be administered orally. In one embodiment of the present invention, the glycine-containing tripeptide molecular composition administered by this method can be formulated with or without the use of carriers commonly used for formulating solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract when availability is maximized and prior systemic degradation is minimized. Additional agents may be included to enhance the absorption of the composition. Diluents, fragrances, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0274] Another glycine-containing tripeptide molecular composition may contain an effective amount of glycine-containing tripeptide molecules in a mixture with a non-toxic excipient suitable for the manufacture of tablets. The solution can be prepared in unit dosage form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0275] Additional glycine-containing tripeptide molecular compositions, including formulations containing compositions in sustained or controlled delivery formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained or controlled delivery means, such as liposomes or micelle carriers, bio-erosive microparticles or porous beads, and depot injections, are also known to those skilled in the art.

[0276] The frequency of administration will depend on the pharmacokinetic parameters of the glycine-containing tripeptide molecular composition in the formulation used. Typically, clinicians administer the composition until a dose is reached that achieves the desired effect. Therefore, the composition may be administered as a single dose, or as two or more doses over a period of time (with or without the same amount of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further refinement of the appropriate dose is routinely performed by those skilled in the art and is within the scope of tasks routinely carried out by those skilled in the art. The appropriate dose may be confirmed by using appropriate dose-response data.

[0277] In addition to the routes of administration disclosed herein, the compositions of the present invention can be introduced into mammals for therapeutic purposes in any manner, including but not limited to intravenous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intraarticular, intratumoral, cerebrospinal fluid, intrarectal and colonic, topical, subconjunctival, intrabladder, vaginal, epidural, intrarib, intracutaneous, inhalation, transdermal, transserous, intrachuccal, oral, intranasal, oral cavity or other body cavity dissolution, intravenous infusion into the airway, gas injection into the airway, injection into blood vessels, tumors, organs, etc., and injection or deposition into cavities within the mammalian body.

[0278] Alternatively, the composition may be administered topically by allowing the desired molecule to be absorbed or by implanting an encapsulated membrane, sponge, or other suitable material. When using an implantation device, the device may be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be carried out by diffusion, sustained-release bolus, or continuous administration.

[0279] In some cases, it may be desirable to use the composition ex vivo. In such cases, cells, tissues, or organs removed from the patient are exposed to the composition, and then the cells, tissues, and / or organs are transplanted back into the patient.

[0280] The composition of the present invention can be administered using one of several standard methods, including, for example, continuous infusion, bolus injection, intermittent infusion, inhalation, or a combination thereof. For example, one form of administration that can be used involves continuous intravenous infusion. In such an approach, the infusion dose of the composition of the present invention is, for example, 0.001 to 0.5 mg / kg body weight / hour, more preferably 0.01 to 0.2 mg / kg / hour, most preferably 0.03 to 0.1 mg / kg / hour, for example, infusing the drug over 1 to 100, 10 to 100, or about 12, 24, 48, 72, 84, or 96 hours. The infusion of the composition of the present invention may be preceded by a bolus injection if necessary. Such a bolus injection is administered in a dose in the range of about 0.001 to about 10 mg / kg. Variations in the dose and infusion duration of the composition of the present invention are also included in the present invention.

[0281] A single bolus injection may be administered, for example, by intravenous infusion via a central access line or peripheral venous line, or by direct injection using a syringe. Such administration may be preferable when the patient's risk of endotoxin exposure is short-term and therefore long-term administration of the drug is not required. For example, this mode of administration may be preferable, where appropriate, in surgical patients, for example, patients undergoing cardiac surgery, such as coronary artery bypass grafting and / or valve replacement surgery. In these patients, a single bolus injection of the drug may be administered over four hours prior to and / or during surgery. (Note that the amount of drug to be administered is based on the patient's weight and condition and is determined by a skilled physician.) Shorter or longer administration periods may be used as deemed appropriate by those skilled in the art.

[0282] If it is desirable to deliver the glycine tripeptide molecule of the present invention or a pharmaceutically acceptable salt thereof for a longer period of time, intermittent administration can be performed. In these methods, a loading dose is administered, followed by (i) a second loading dose and / or maintenance dose (or multiple doses), or (ii) a maintenance dose or multiple doses without a second loading dose, as deemed appropriate by those skilled in the art.

[0283] To achieve further delivery of the glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to the patient, a maintenance dose (or multiple doses) of the compound can be administered to maintain levels of the compound in the patient's blood. The maintenance dose may be administered at a lower level than the loading dose(s), for example, at about 1 / 6 of the loading dose. The specific amount to be administered in the maintenance dose may be determined by a healthcare professional with the aim of maintaining at least a minimum level of the compound. The maintenance dose may be administered, for example, every 12 hours for about 2 hours, starting at 24 hours and continuing at, for example, 36, 48, 60, 72, 84, 96, 108, and 120 hours. Naturally, the maintenance dose can be stopped at any point within this time frame that is deemed appropriate by a healthcare professional.

[0284] The above infusion methods can be performed using catheters (e.g., peripheral venous, central venous, or pulmonary artery catheters) and related products (e.g., infusion pumps and infusion tubing) that are widely available in the art. One important criterion to consider when selecting catheters and / or tubing for use in these methods is the effect that the material (or coating) of these products has on the size of the drug.

[0285] Additional catheter-related products that can be used in the method of the present invention can be identified by determining whether the material of the product alters the size of the compound under conditions consistent with those used for drug administration. Furthermore, if a patient already has a catheter in place that does not maintain the optimal drug size, a catheter insert made of a compatible material (e.g., polyamide polymer) or having a compatible coating can be used to prevent the drug solution from coming into contact with the surface of the unsuitable catheter. Such an insert, having an outer diameter small enough to be easily inserted into the existing catheter while maintaining an inner diameter large enough to adapt to the flow of the compound solution, is placed inside the existing catheter and connected to a tube or syringe through which the drug is delivered.

[0286] The appropriate frequency of administration can be determined by those skilled in the art, and it can be administered once to several times per day. The composition of the present invention may also be administered once a day or once every other day. In the case of acute administration, treatment is usually carried out over a period of several hours or several days, while chronic treatment can be carried out over several weeks, several months, or even several years.

[0287] For both chronic and acute administration, standard hepatic portal vein formulations, which can be prepared from formulations described elsewhere in this specification, can be used. Administration via this route offers several advantages, for example, by providing a rapid onset of action by delivering a glycine tripeptide molecule or a pharmaceutically acceptable salt thereof to the desired site of action at a higher local concentration. For the therapeutic portion to exert its desired effect, it must be in physical contact with its physiological target, such as a receptor present on hepatocytes. Site-specific drug delivery ensures that such interactions occur only at the desired anatomical location in the liver; therefore, the following criteria must be met: (i) it must be able to cross anatomical barriers such as the stomach and intestines; (ii) it should be selectively recognized by receptors present on hepatocytes, such as asialoglycoproteins; (iii) the exogenously delivered ligand for targeting should compete with endogenously produced ligands; and (iv) the created delivery system should be in vivo or in In vitro, the delivery system for the glycine tripeptide molecule or its pharmaceutically acceptable salt must be non-toxic, biocompatible, biodegradable, and physicochemically stable within hepatocytes; (v) it should have a uniform sinusoidal capillary distribution; (vi) it should have a controllable and predictable drug release rate so that only therapeutic doses of the glycine tripeptide molecule or its pharmaceutically acceptable salt are released into hepatocytes; (vii) drug release should not affect drug distribution; (viii) it should exhibit minimal drug leakage as it passes 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 causing signs of toxicity, and the carrier should not induce any modification of the disease; and (x) finally, the preparation of the delivery system for the glycine tripeptide molecule or its pharmaceutically acceptable salt should be easy, reasonably simple, reproducible, and cost-effective.

[0288] Glycine tripeptide molecules, or pharmaceutically acceptable salts thereof, can be administered as part of a drug delivery system intended for liver tissue delivery. Different methods for binding glycine-containing tripeptide molecules to liver-specific targeting moieties may include: (a) binding of the targeting moiety on a pre-formed nanocarrier, (b) binding of the targeting moiety by post-insertion method, (c) binding of the targeting moiety by an avidin / biotin complex, and (d) binding of the targeting moiety before nanocarrier formulation. Exemplary targeting moieties and carriers useful in the delivery of the 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, BioMed Research International, Volume 2013, the disclosure of which is incorporated herein by reference in its entirety.

[0289] While specific glycine tripeptide molecules and their pharmaceutically acceptable salts, compositions, and methods have been described in this specification according to specific embodiments, the following examples are intended solely to illustrate the compounds described herein and are not intended to limit them. Each of the references cited herein is incorporated herein by reference in whole. [Examples]

[0290] method

[0291] Animal experiment procedures

[0292] All procedures for the animal experiment were approved by the Animal Experimentation Committee of the University of Michigan (PRO00008239) and were carried out in accordance with their guidelines. Eight-week-old male apolipoprotein E-deficient (apoE- / -) mice (B6.129P2-Apoetm1Unc / J, stock number: 002052) were obtained from Jackson Laboratory. The mice were fed a Western diet (WD, 42% fat calories, 0.2% cholesterol by weight, Envigo TD.88137). One week after WD feeding, blood was carefully collected from the facial vein to measure baseline plasma levels of total cholesterol (TC). Next, mice were maintained on a WD diet and randomly divided into five experimental groups. The following therapeutic agents were administered orally six times a week for 12 weeks: 1) WD + H2O (water was given as a vehicle control); 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 were maintained on a 12-hour light / dark cycle and given free access to food and water. Body weight and food intake were measured weekly and every three weeks, respectively.

[0293] OGTT and non-fasting blood glucose

[0294] At week 10, an oral glucose tolerance test (OGTT) was performed by force-administering 2 mg / g of glucose after an overnight fast. To evaluate 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 by force-administration. To evaluate the chronic effect of treatment, mice were administered glucose alone. Baseline blood glucose levels were measured after collecting blood from the tail tip using a blood glucose meter and test strips (NDC: 0193-7308-50, Contour Next). Mice were then administered glucose containing (acute) or not containing (chronic) the therapeutic agent, and blood glucose levels were measured every 30 minutes for up to 120 minutes. To evaluate the therapeutic effect on non-fasting blood glucose levels, blood samples were collected from the tail tip before and 30 minutes after the regular forced oral administration of the drug. Blood glucose levels were measured using a blood glucose meter and test strips (NDC: 0193-7308-50, Contour Next).

[0295] Plasma lipids, adipokines, and cytokines

[0296] Plasma levels of TC, triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) were measured using a Cobas Mira chemical analyzer (Roche Diagnostics) at the Chemistry Laboratory (MDRC) of the Michigan Diabetes Research Center, using assay reagents and protocols provided by the manufacturer. Plasma levels of glycine tripeptide molecules, resistin, interleukin-6 (IL-6), and monocyte chemotactic protein 1 (MCP1) were measured at the Chemistry Laboratory of the MDRC using a Luminex 200 chemical analyzer (Luminex Corporation), using assay reagents and protocols (Millipore Multiplex, MMHMAG-44K) provided by the manufacturer.

[0297] Histological analysis

[0298] Histological processing was performed at the In Vivo Animal Core (IVAC) Histology Laboratory within the University of Michigan Veterinary Medicine Laboratory (ULAM). Formalin-fixed tissues were graded with alcohol, removed with xylene, and then impregnated 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 to a thickness of 4 μm using an M355S rotary microtome (ThermoFisher Scientific) and mounted on glass slides. Following deparaffinization and hydration with xylene and stepwise alcohols, the slides were stained with Harris hematoxylin (ThermoFisher Scientific, catalog no. 842) for hematoxylin and eosin (H&E), differentiated with Clarifier (ThermoFisher Scientific, catalog no. 7401), stained with bluing reagent (ThermoFisher Scientific, catalog no. 7301), eosin Y, and alcohol (ThermoFisher Scientific, catalog no. 832), then dehydrated and removed stepwise with alcohol and xylene, and covered with coverslips using a Leica CV5030 automatic coverslipper and a micromount (Leica catalog no. 3801731). Immunohistochemical staining was performed on an IntelliPATH FLX automated immunohistochemical staining system (Biocare Medical, catalog number IPS0001US), and included blocking of endogenous peroxidase and nonspecific binding, detection using a commercially available detection system based on horseradish peroxidase biotin-free polymer, disclosure using diaminobenzidine pigment, and counterstaining of the nucleus with hematoxylin.A rat monoclonal primary antibody (clone CI: A3-1) specific to F4 / 80 (Bio-Rad ABD Serotec, catalog no. MCA497) was diluted 1:400 in DaVinci diluent (Biocare Medical, catalog no. PD900), incubated for 60 minutes, and then detected using a two-step probe-polymer incubation with Rat-on-Mouse HRP polymer (Biocare Medical, catalog no. RT517) for 10 minutes and 30 minutes, respectively.

[0299] RNA isolation, reverse transcription, and quantitative polymerase chain reaction (qPCR)

[0300] Total RNA was extracted from liver or adipose tissue samples using the QIAGEN RNeasy kit (QIAGEN). RNA was reverse transcribed to cDNA using SuperScript III and random primers (Invitrogen). Specific transcription levels were evaluated using a real-time PCR system (Bio-Rad) with iQ SYBR Green Supermix (Bio-Rad) and normalized ΔΔCt threshold cycling. Gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer pairs used for qPCR were obtained from Integrated DNA Technologies and are listed below: [Table 1]

[0301] Extraction of liver lipids

[0302] Liver was 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 its protein content was analyzed by the Bio-Rad Bradford assay. To assess the liver lipid composition, lipids were extracted from the supernatant using hexane (≧99%, 32293, Sigma-Aldrich) and isopropanol (≧99.5%, A426-4, Fisher Scientific) in a 3:2 ratio (v:v), and the hexane phase was evaporated for 48 hours. The amount of TG or TC in the liver was measured spectrophotometrically using a commercially available kit (Wako Chemicals). Data were normalized to protein levels and expressed as μgTG or TC / mg protein.

[0303] Liver protein extraction and Western blotting

[0304] Tissue extracts were prepared using radioimmunoprecipitation assay lysis buffer (RIPA buffer, Thermo Scientific) supplemented with a protease inhibitor cocktail (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 at room temperature for 1 hour in Tris-buffered saline-Tween20 (TBST) containing 5% nonfat milk, and incubated overnight at 4°C with primary antibodies. 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 membranes were incubated with secondary antibodies (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 visualized and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, version 2.1).

[0305] Analysis of arteriosclerotic lesions

[0306] After blood collection, mice were perfused with 20 mL of physiological saline through the heart, followed by 20 mL of 37% formalin. The 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 (0.2% Oil Red O (w / v) in methanol:1 N NaOH at a ratio of 3.5:1) for 50 minutes, followed by immersion in 70% ethanol for 30 minutes. The aortic tree was then maintained in DDW (Digestive Water). Subsequently, the attached fat and connective tissue were removed from the aortic tree, and it was opened longitudinally with Bennes scissors to expose the atherosclerotic lesions. Images of the entire aortic tree were acquired using a digital camera, and the ratio of the area of ​​the Oil Red O-stained sclerotic lesions to the total lumen surface area was quantified using ImageJ analysis software (http: / / imagej.nih.gov / ij / ).

[0307] statistical analysis

[0308] Data analysis was performed using SPSS 24.0 software (SPSS Inc., IBM). Data are presented as box plots or mean ± SEM. The number of animals used in each study is specified in the legend of each figure. One-way analysis of variance (ANOVA) followed by Tukey post-hoc tests was used for data analysis. Differences were considered statistically significant if p < 0.05.

[0309] result

[0310] Experiment 1: The effect of body weight on steatosis

[0311] The experimental design is shown in Figure 1A. After feeding a Western diet (WD) to apoE- / - mice for one week, blood was collected from the facial vein (FV) to measure baseline plasma levels of TC. The mice were then maintained on the WD diet and randomly divided into five experimental groups, to be administered the following therapeutic agents orally 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 observed throughout the study (Figure 1B). Endpoint measurements revealed a significant decrease in body weight in mice administered with glycine (12%, p<0.05, Figure 1C), while the total body weight gain from baseline to endpoint was significantly lower in mice treated with glycine or DT-109 (51%, p<0.01 or 28%, p<0.05, respectively, Figure 1D). Macroscopic examination of the abdominal cavity at the endpoint revealed yellowish discoloration of the liver and enlargement of the visceral fat pad in control and leucine-treated mice, which were attenuated by treatment with glycine, DT-109, or DT-110 (Figure 1E). Consequently, plasma levels of glycine-containing tripeptide molecules were significantly lower in mice administered with glycine, DT-109, or DT-110 (53%, 62%, or 56%, p<0.05, Figure 1F). In summary, these results demonstrate the protective effects of glycine, DT-109, and, to a lesser extent, DT-110 against WD-induced steatosis.

[0312] Experiment 2: Effects on glucose homeostasis

[0313] At 10 weeks of WD feeding, an OGTT was performed after an overnight fast. To determine the acute effect of the treatment on blood glucose levels, glycine (0.67 mg / g), leucine (0.33 mg / g), DT-109, or DT-110 (1 mg / g) were added to a glucose solution (2 mg / g) and administered by forced oral administration. As shown in Figure 2A, there was no significant difference in blood glucose levels between the groups at baseline. 30 minutes after glucose administration, blood glucose levels decreased significantly with all treatments, with glycine or DT-109 showing the most significant effect. At later time points (60-120 minutes), blood glucose levels decreased significantly in mice treated with glycine, DT-109, and DT-110, but not with leucine. To evaluate the chronic effects of the treatments, an OGTT was repeated at 11 weeks of WD feeding, but this time the treatment was not added to the glucose solution. No significant differences were observed between the groups after glucose loading without the addition of the therapeutic agent (Figure 2B). To evaluate the effect of the therapeutic agent on non-fasting blood glucose levels, blood was collected before (pre-oral administration) and 30 minutes after (post-oral administration) regular administration of the therapeutic agent by forced oral administration. Compared to control mice, blood glucose levels before forced oral administration were significantly lower in all groups. However, forced oral administration with water or leucine resulted in an upward trend in blood glucose levels, while forced oral administration with glycine, DT-109, or DT-110 significantly lowered blood glucose levels (10%, p<0.05, 21%, p<0.01, or 13%, p<0.05, respectively, Figure 2C). Finally, the endpoint glucose level (after 6 hours of fasting) was significantly lower in mice treated with glycine or DT-109 (41%, p<0.01, or 30%, p<0.05, respectively, Figure 2D). Analysis of hepatic gene expression revealed that genes regulating glucose uptake (GLUT1, GLUT2, and GLUT4) were upregulated by glycine treatment without causing significant changes in the expression of genes regulating gluconeogenesis (G6pc, PCK1, and FBP1). GLUT1 expression was also significantly induced in the livers of mice treated with DT-110 (Figure 2E). In summary, these results demonstrate the remarkable postprandial blood glucose-lowering properties of glycine, DT-109, and DT-110.

[0314] Experiment 3: Effects on hepatic lipid metabolism

[0315] H&E staining of liver samples revealed marked small-droplet and large-droplet fatty liver in control mice and leucine-treated mice. Importantly, treatment with glycine, DT-109, or DT-110 neutralized WD-induced fatty liver (Figure 3A). Therefore, liver lipid extraction and subsequent quantification of TG and TC content revealed that treatment with glycine, DT-109, or DT-110 significantly reduced liver TG (74%, 73%, or 68%, p<0.01, respectively, Figure 3B) and liver TC (76%, 71%, or 63%, p<0.01, respectively, Figure 3C), while leucine administration had no significant effect.

[0316] Experiment 4: Next, we evaluated the expression of genes that regulate the accumulation of TG, fatty acids, and cholesterol in the liver and explained the observed effects on fatty liver. Interestingly, treatment with glycine, DT-109, or DT-110 significantly induced the expression of AMPKα1 and PPARα, major regulators of hepatic lipid oxidation, but not with leucine (Figure 4A). Thus, major target genes that regulate TG hydrolysis (PNPLA2), mitochondrial β-oxidation (CPT1a, CACT, ACADI), and mitochondrial anion carrier UCP2 were significantly upregulated in the livers of mice treated with glycine, DT-109, or DT-110 (Figures 4A, 4B). Regarding genes that regulate hepatic cholesterol homeostasis, treatment with glycine, DT-109, or DT-110 significantly increased the expression of ABCG5 and ABCG8, major regulators of cholesterol bile excretion. Significant upregulation of LDLR was observed in the livers of mice treated with DT-110, and similar trends were observed with glycine and DT-109 treatment (Figure 4C). Overexpression of ABCG8 in the livers of mice treated with glycine, DT-109, or DT-110 was confirmed by Western blotting (Figures 4D and 4E). Overall, these results strongly suggest the potent protective effects of glycine, DT-109, and DT-110 against WD-induced fatty liver disease, related to the overexpression of genes that regulate lipid oxidation and cholesterol extraction into bile.

[0317] Experiment 5: Plasma lipid properties and their effects on atherosclerosis

[0318] Prior to randomization to the experimental group, blood was collected from mice one week after WD feeding to measure baseline plasma TC levels. Plasma TC was measured again at the endpoint 12 weeks after WD feeding, including therapeutic or water controls. As shown in Figure 5A, the time-dependent increase in plasma TC was significantly attenuated by treatment with glycine or DT-109, and a similar trend was observed for DT-110 treatment, but not for leucine treatment. Thus, endpoint plasma levels of TC and LDL were significantly reduced in mice treated with glycine or DT-109 (TC: 19%, p<0.05 or 25%, p<0.01; LDL: 30%, p<0.01 or 36%, p<0.01, respectively), and a similar trend was observed for DT-110 treatment (Figures 5B and 5C). Interestingly, plasma HDL levels were significantly lower in mice treated with glycine (35%, p<0.05), but were maintained in mice treated with DT-109 (Figure 5D). No significant changes were observed in plasma TG levels (Figure 5E). Finally, analysis of en-face aortic lesions using oil red O staining revealed a significant reduction in all atherosclerotic lesions in mice treated with glycine, DT-109, or DT-110 (48%, p<0.01, 62%, p<0.001, or 49%, p<0.01, respectively, Figures 5F and 5G), but not in mice treated with leucine.

[0319] Experiment 6: Effects on systemic, liver, and adipose tissue inflammation

[0320] Since inflammation plays a crucial role in the pathogenesis of both NAFLD and atherosclerosis, plasma levels of inflammatory cytokines and adipokines were next evaluated. While there were no significant differences between groups in plasma levels of IL-6 and resistin (Figures 6A and 6B), 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 with all treatments, with the most significant effect observed with DT-110 (Figure 6D). MCP1 expression in both EAT and SAT was significantly suppressed by treatment with glycine, DT-109, or DT-110, but not by leucine, according to lower MCP1 plasma levels (Figures 6D and 6E). Finally, glycine suppressed MCP1 expression in the liver (a similar trend was observed with DT-109, Figure 7A), but no significant changes were observed in tissue F4 / 80 as determined by immunohistochemistry (Figures 7B, 7C). Overall, these results suggest several anti-inflammatory effects of glycine, DT-109, and DT-110, which are mainly related to the suppression of MCP1 in adipose tissue and circulation.

[0321] Example x Glycine-based therapeutic agents for NAFLD

[0322] A. Preface

[0323] NAFLD, the most common chronic liver disease, affects 25% of the world's population. NAFLD encompasses a range of liver lesions, from fatty liver (HS) and non-alcoholic steatohepatitis (NASH), characterized by liver cell damage and lobular inflammation associated with fibrotic progression, to cirrhosis, which can lead to liver failure or hepatocellular carcinoma. Cardiovascular and metabolic comorbidities, including obesity, type 2 diabetes (T2D), metabolic syndrome (MetS), and dyslipidemia, are common in NAFLD patients. Apart from liver-specific mortality, cardiovascular disease is a leading cause of death in NAFLD patients, particularly those with NASH. Despite the global burden of NAFLD and the considerable efforts in drug development, there are currently no approved treatments.

[0324] Recent advances, particularly in metabolomics and gut microbiota, have deepened our understanding of the pathogenesis of NAFLD and suggested novel therapeutic targets. While abnormal lipid and carbohydrate metabolism are known to be involved in NAFLD, recent metabolomics-based studies have indicated that dysregulation of specific amino acid (AA) metabolism plays a role in the pathogenesis of NAFLD. In particular, circulating AA levels are elevated in most NAFLD patients, while glycine levels are decreased. Circulating glycine levels are negatively correlated with levels of HS, hepatocyte ballooning, and lobular inflammation. Plasma glycine, along with known biomarkers (e.g., aspartate aminotransferase (AST) and PNPLA3 genotype), has recently been included in predictive models for NASH. Low plasma glycine is associated with a higher prevalence of obesity, type 2 diabetes, MetS, coronary heart disease, and myocardial infarction, while high plasma glycine is associated with better lipid characteristics (summarized in Table S1).

[0325] [Table 2]

[0326] As a non-essential AA, glycine is primarily synthesized in the liver from several precursors. These reactions are catalyzed by key enzymes that promote glycine formation from serine (serine hydroxymethyltransferase, SHMT), from threonine (threonine dehydrogenase, TDH), from choline via sarcosine (choline dehydrogenase, CHDH; sarcosine dehydrogenase, SARDH), and from alanine to glyoxylate (AGXT). Glycine is utilized in multiple pathways to produce essential molecules such as nucleic acids, heme, and glutathione. In NAFLD or T2D, glutathione synthesis is reduced due to limited glycine availability, which recovers after nutrient supplementation. Beyond its antioxidant effects, glycine has dual benefits in lipid and glucose metabolism. Glycine intake lowers blood glucose levels, partly by stimulating insulin secretion from pancreatic β-cells. Genetic Obesity / Diabetes KK-A y In mice, dietary glycine improved glucose tolerance, reduced circulating triglycerides (TG) and alanine aminotransferase (ALT), while attenuating hemoglobin (HS) and inflammatory infiltration. In sucrose-fed rats, glycine reduced intraperitoneal fat and plasma TG, and increased hepatic mitochondrial FAO markers. Nevertheless, a comprehensive investigation of the role of glycine in NAFLD using models that fully mimic human disease and precise administration has not been pursued.

[0327] Given the burden of NAFLD, the lack of available treatments, and consistent reports linking decreased circulating glycine to NAFLD severity, there is strong rationale for a better understanding of glycine metabolism in NAFLD, which could lead to novel therapies. Herein, we identify the suppression of glycine biosynthesis genes, primarily AGXT1, in human and mouse NAFLD. A genetic approach to limiting glycine availability (AGXT1) - / -) and dietary therapy approaches resulted in exacerbation of hyperlipidemia and steatohepatitis. We searched for glycine compounds with dual lipid / glucose lowering properties and identified DT-109, which strongly protects mice from diet-induced NASH by regulating hepatic FAO.

[0328] B. Abbreviation:

[0329] AA, amino acid; ACAA, acetyl-CoA acyltransferase; ACAD, acyl-CoA dehydrogenase; ACOT, acyl-CoA thioesterase; ACOX, acyl-CoA oxidase; ACSL, long-chain acyl-CoA synthetase; ACSM, medium-chain acyl-CoA synthetase; AGXT, alanine-glyoxylate aminotransferase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ApoE, apolipoprotein E; AST, aspartate aminotransferase; CACT, Cal Nitin / acylcarnitine translocase; Cas9, CRISPR-related protein 9; CCL, CC motif chemokine ligand; CCR, CC motif chemokine receptor; CD, solid feed; CHD, coronary heart disease; CLAMS, comprehensive laboratory animal monitoring system; CPT, carnitine palmitoyltransferase; CRISPR, clustered and regularly arranged short palindromic sequence repeat; DAG, diacylglycerol; DAO, D-amino acid oxidase; DEG, differential expression gene; ECI, enoyl-CoAδ isomerase; FA, Fatty acids; FAO, fatty acid β-oxidation; HADH, hydroxyacyl-coenzyme A dehydrogenase; HS, fatty liver; LDA, linear discriminant analysis; LEfSe, effect size of linear discriminant analysis; MCP-1, monocyte chemotactic protein 1; MetS, metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NF-κB, nuclear factor κ light chain enhancer of activated B cells; NMR, nuclear magnetic resonance; OGTT, oral glucose tolerance test; ORO, oil red O; PGC1α, PPARG coactivator 1α (PPARGC1α );PNPLA, patatin-like phospholipase domain-containing protein;PPAR, peroxisome proliferator-activated receptor;RER, respiratory exchange rate;SARDH, sarcosine dehydrogenase;SERPINE, serine peptidase inhibitor clade E;SHMT, serine hydroxymethyltransferase;T2D, type 2 diabetes;TC, total cholesterol;TDH, threonine dehydrogenase;TG, triglyceride;TGFBR, transforming growth factor β receptor;TGFβ, transforming growth factor β;TIMP, metalloproteinase tissue inhibitor;TLR (Toll-like receptor); TNF (tumor necrosis factor); TNFRSF (TNF receptor superfamily); WD (Western diet).

[0330] C. Transcript characteristics:

[0331] RNA sequencing data is registered with NCBI's SRA or GEO (registration number: PRJNA556537 or GSE126204). Metagenomics sequencing data is registered with NCBI's SRA (registration number: PRJNA544728).

[0332] D. Background and Objectives

[0333] The prevalence of non-alcoholic fatty liver disease (NAFLD), including steatohepatitis steatohepatitis (NASH), is increasing worldwide, yet there are no approved drug therapies. While decreased circulating glycine levels have been consistently reported in NAFLD patients, the causative role of glycine and its therapeutic potential remain unclear. To study glycine metabolism in NAFLD, we applied genetic and dietary strategies and evaluated glycine-based therapies.

[0334] E. Method:

[0335] Transcriptomics was performed in human and mouse livers using NAFLD to reveal the repression of glycine biosynthesis genes, mainly alanine-glyoxylate aminotransferase 1 (AGXT1). CRISPR / Cas9 was used to analyze AGXT1. - / - We generated mice and developed a glycine-modified diet. We searched for glycine 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 immunoblotting. Body composition and energy metabolism were assessed using NMR-based methods and indirect calorimetry. The gut microbiota was studied using 16S metagenomic sequencing.

[0336] F. Results:

[0337] A genetic approach to limit the availability of glycine (AGXT1 - / - Dietary approaches exacerbated diet-induced hyperlipidemia and steatohepatitis, suppressing mitochondrial / peroxisome fatty acid β-oxidation (FAO) as an underlying pathway and enhancing inflammation. A glycine-based tripeptide with potent glucose / lipid-reducing effects was identified (gly-gly-L-leu / DT-109). In mice with established NASH, DT-109 improved body composition and reduced circulating lipids, liver enzymes, and steatohepatitis by stimulating the FAO pathway. DT-109 reduced lobular / systemic inflammation and fibrosis by suppressing the NF-κB and TGFβ / SMAD pathways. The bacterium Clostridium sensu stricto was positively correlated with the severity of NAFLD and decreased with DT-109, while Alistipes showed an inverse correlation and increased with DT-109.

[0338] G. Method

[0339] (i) Animal experiment procedures

[0340] The animal experiment procedures were approved by the Animal Experimentation Committee of the University of Michigan (UM) (PRO00008239) and were carried out in accordance with their guidelines. Seven-week-old C57BL / 6J mice or apoE- / - mice (B6.129P2-Apoetm1Unc / J, stock:002052) were obtained from Jackson Laboratory. AGXT1- / - mice with a C57BL / 6J background were generated using CRISPR / Cas9 with a guide RNA: 5'-GGGTCCGGGGCCCTCCAACC-3' targeting exon 1 of AGXT1. Eight-week-old male C57BL / 6J, apoE- / - or AGXT1- / - mice were used throughout the study and given free-range diets: standard solid feed (CD, LabDiet 5L0D, 13% calories from fat); high-fat, high-cholesterol Western diet (WD, Envigo TD.88137, 42% fat); AA-specified WD with or without glycine (developed by Envigo-Teklad Custom Diets, Table S2): WDAA+Gly (TD.170525) or WDAA-Gly (TD.170526); high-fat, high-cholesterol, high-fructose NASH diet (Research Diets D17010103, 40% fat), which strongly induced NASH in the mice. If necessary, mice were orally administered glycine (Sigma-Aldrich G5417), leucine (Sigma-Aldrich L8912), or DT-109 (CSBio) at a dose of 0.125-1 mg / g body weight / day, or H2O as a control.

[0341] [Table 3]

[0342] (ii) Human data

[0343] Differentially expressed genes (DEGs) driving glycine biosynthesis were analyzed using two publicly available 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. A linear regression model with age, sex, and BMI as covariates was used to identify key glycine biosynthesis genes associated with NASH. A meta-analysis of the two studies was further performed using an equal-effects model to enhance statistical power. The correlation between gene expression levels and liver fat content was tested using previously published microarray data (GSE26106, n=206 liver transplant donors).

[0344] (iii) Histology and immunohistochemistry

[0345] All histological procedures were performed by the UM In Vivo Animal Core at the Institute of Histology. Technicians were blinded to the experimental group. NAFLD activity (NAS) or fibrosis was scored using H&E staining or Sirius Red staining.

[0346] (iv) Identification of glycine compounds

[0347] Compounds structurally similar to glycine were selected, and their structural, conformational, electronic, and steric equivalence modifications to the glycine skeleton were evaluated. A search was performed using SciFinder(C) to determine whether they were commercially usable. Oral LD 50 Water-soluble compounds containing >1 mg / g were defined as suitable for oral administration to mice.

[0348] (v) RNA sequencing

[0349] Library preparation and sequencing were performed using the UM DNA Sequencing Core on a NovaSeq6000 sequencing system (Illumina). RNA sequencing and pathway analysis were performed as described.

[0350] (vi) Comprehensive Laboratory Animal Monitoring System (CLAMS)

[0351] Body composition was assessed using a nuclear magnetic resonance (NMR)-based analyzer (Minispec LF90II; Bruker Optics) with the UM Animal Phenotyping Core. Oxygen consumption (VO2), carbon dioxide production (VCO2), and exercise capacity were measured using CLAMS (Columbus Instruments). Respiratory exchange rate (RER) is VCO2 / VO2.

[0352] (vii) Fecal microbiome analysis

[0353] As described above, we performed fecal DNA extraction, amplification using primers specific to the V4 region of 16S rRNA, characterization of the gut microbiota using linear discriminant analysis (LDA) effect size (LEfSe), and correlation analysis with disease parameters.

[0354] (viii) Statistical analysis

[0355] Statistical analysis was performed using GraphPad Prism 7.0. Unless otherwise specified, values ​​are expressed as the mean ± SD representing all scores. All data were tested for normality and equal variances. If normality and equal variances were confirmed, 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 more than two groups. Otherwise, a nonparametric test (Mann-Whitney U or Kruskal-Wallis) was used. A p-value < 0.05 was considered statistically significant.

[0356] (ix) Generation of AGNXT1- / - mice using CRISPR / Cas9

[0357] AGXT1- / - mice were generated in a C57BL / 6J background using CRISPR / Cas9. The guide RNA target site in exon 1 of the AGXT1 gene was 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 an A deletion 3 bases away from the protospacer adjacent motif (PAM, Figure 8A). A frameshift mutation after AA53 in the ORF introduced a stop codon midway, and Western blotting confirmed the absence of AGXT1 (Figure 8B). No CRISPR off-target effects were detected when evaluated using CRISPOR.

[0358] (x) Human data

[0359] We analyzed differentially expressed glycine biosynthesis genes in NASH patients using two publicly available datasets. The first study consisted of liver microarray data from 104 NASH patients and 44 healthy controls (GSE83452). Linear regression was used to test the correlation between gene expression levels and NASH, adjusting for age and sex as covariates. The second dataset included liver microarray data collected from 24 NASH patients and 24 healthy obese controls (GSE61260). Similarly, a linear regression model was used with age, sex, and BMI as covariates to identify significant glycine biosynthesis genes associated with NASH. To enhance statistical power and compare results, a further meta-analysis of the two studies was performed using an equal-effects model with the metafor R package. Genes with Benjamini-Hochberg adjusted p-values ​​< 0.05 and Cochran's Q heterogeneity test p-values ​​> 0.05 were considered significant.

[0360] We examined the correlation between gene expression levels and liver fat content using previously published microarray data (GSE26106) collected from liver transplant donors (n=206). Tissue dissection, sample exclusion, and microarray data generation were performed as described above. Liver fat content in donor livers was quantified using an organic solvent (hexane / isopropanol 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 liver fat correlated with glycine biosynthesis gene expression.

[0361] (xi) Histology, immunohistochemistry, and scoring of NAFLD activity and fibrosis

[0362] Histological procedures were performed by technicians blinded to the experimental group at the In Vivo Animal Core (IVAC) Histology Laboratory, University of Michigan. Formalin-fixed tissues were graded with alcohols, removed with xylene, and then impregnated with molten paraffin using an automated VIP5 or VIP6 tissue processor (TissueTek, Sakura-Americas). Using a Histostar Embedding Station (ThermoFisher Scientific), the tissues were sectioned to a thickness of 4 μm with an M355S rotary microtome (ThermoFisher Scientific) and mounted on glass slides. 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 minutes, transferred to 0.1% Sirius Red saturated with picric acid (Rowley Biochemical Inc.) for 90 minutes, and then transferred to 0.01N hydrochloric acid for 3 minutes.

[0363] NAFLD activity was scored using H&E staining. Fatty liver was scored on a scale of 0 to 3 (0: <5% fatty liver; 1: 5-33%; 2: 34-66%; 3: >67%). Hepatocyte ballooning was scored on a scale of 0 to 2 (0: normal hepatocytes; 1: normal size but thin cytoplasm; 2: thinly hypertrophied hepatocytes, at least 2x). Lobular inflammation was scored on a scale of 0 to 2 based on the number of inflammatory lesions counted 20x (0: none; 1: fewer than 2 lesions; 2: 2 or more lesions). The NAFLD activity score (NAS) was calculated as the sum of the fatty liver, hepatocyte ballooning, and lobular inflammation scores. Using Sirius Red staining, liver fibrosis was scored on a scale of 0 to 4 (0: no fibrosis; 1: perisinusoidal or portal venous fibrosis; 2: perisinusoidal and portal venous fibrosis; 3: bridging fibrosis; 4: cirrhosis).

[0364] For oil red O (ORO) staining, freeze-sectioning was used. Formalin-fixed liver samples were cryoprotected overnight in 20% sucrose at 4°C, blotted, and then rapidly frozen in liquid nitrogen in OCT compound (Tissue-Tek, catalog no. 4583). The samples were stored at -80°C until ready for freeze-sectioning. Before sectioning, the frozen blocks were raised to approximately -20°C and then sectioned to 5 μm on a Cryotome SME (Thermo-Shandon, catalog no. 77200227). The slides were stored at -80°C until staining. Before staining, the liver slides were thawed for 30 minutes to return to room temperature. The slides were fixed in 10% neutral buffered formalin for 20 minutes, rinsed with DDW, rinsed with 60% isopropanol, and then instilled in ORO-isopropanol stain (Rowley Biochemical Inc., H-503-1B) for 5 minutes. Next, the slides were rinsed with 60% isopropanol, followed by three changes of DDW. Then, the slides were counterstained with Harris hematoxylin and mounted in Aqua-Mount (Lerner Laboratories, catalog number 13800) aqueous mounting medium.

[0365] Immunohistochemical staining was performed on an IntelliPATH FLX automated immunohistochemical system (Biocare Medical) for blocking of endogenous peroxidase and nonspecific binding, followed by detection using a commercially available detection system based on horseradish peroxidase biotin-free polymer, disclosure using diaminobenzidine pigment, and counterstaining of the nucleus with hematoxylin. A rat monoclonal primary antibody (clone CI: A3-1) specific to F4 / 80 (Bio-Rad ABD Serotec, catalog no. MCA497) was diluted 1:400 in DaVinci diluent (Biocare Medical, catalog no. PD900), incubated for 60 minutes, and then detected using a two-step probe-polymer incubation of Rat-on-Mouse HRP polymer (Biocare Medical, catalog no. RT517) for 10 minutes and 30 minutes, respectively.

[0366] (xii) Plasma analysis

[0367] Complete plasma lipid characteristics (TC, TG, LDL, and HDL) were measured using a Cobas Mira chemical analyzer (Roche Diagnostics) and commercially available kits (Wako Chemicals 999-02601 and 994-02891) with manufacturer-provided reagents and protocols at the Michigan Diabetes Research Center (MDRC) or our laboratory. Plasma glycine-containing tripeptide molecules and resistins were measured using a Multiplex Assay (MMHMAG-44K, Millipore) on the MDRC's Luminex200 platform (Luminex). Plasma MCP-1 was measured using the Mouse CCL2 / JE / MCP-1 Quantikine ELISA Kit (R&D Systems). Clinical chemistry assays of ALT, AST, and ALP were performed at the University of Michigan IVAC using a Liasys330 chemical analyzer (AMS Diagnostics) with manufacturer-provided reagents and protocols. Plasma oxalates were measured using an oxalate assay kit (Abcam, ab196990). Plasma glucose was measured using a blood glucose meter and test strips (NDC:0193-7308-50, Contour Next). Plasma AA analysis was performed by the University of Michigan Metabolomics Core. 20 μL of plasma was derivatized and prepared for GC-MS analysis according to the instructions for the EZ Fast Amino Acids Analysis kit (Phenomenex). Briefly, the sample was subjected to column cleanup and transferred to a GC autosampler vial. The sample was then derivatized, dried at room temperature under a gentle nitrogen flow, and resuspended for GC analysis on an Agilent 69890N GC-5975 MS detector using the following parameters: 1 μL of sample was injected on a ZB-AAA 10m column (Phenomenex) with He gas at a flow rate of 1.1 mL / min at a split ratio of 1:15. The initial temperature of the GC oven was 110°C, and it was increased by 30°C per minute to 320°C. The pouring port temperature was 250°C, and the MS source temperature and MS quad temperature were 230°C and 150°C, respectively.Data were processed using MassHunter Quantitative Analysis version B.07.00. Metabolites were normalized to an internal standard labeled with the nearest isotope, quantified using two repeated injections of five standards, and a linear calibration curve with over 80% accuracy was created for each standard. Peak areas were used for difference analysis between groups.

[0368] (xiii) Quantitative real-time PCR analysis

[0369] Total RNA was extracted from mouse liver samples using the QIAGEN RNeasy kit (QIAGEN). RNA was reverse transcribed to cDNA using SuperScript III and random primers (Invitrogen). Specific transcription levels were evaluated using a real-time PCR system (Bio-Rad) with iQ SYBR Green Supermix (Bio-Rad) and normalized ΔΔCt threshold cycling. Gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer pairs used for qPCR were obtained from Integrated DNA Technologies and are listed in Table S3.

[0370] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0371] (xiv) RNA sequencing and data analysis

[0372] As described above, total RNA was extracted from mouse liver samples. Library preparation and sequencing were performed at the DNA Sequencing Core of the University of Michigan. RNA quality was evaluated using TapeStation (Agilent, Santa Clara, CA). The RNA integrity number (RIN) of all samples was >8.5. Samples were prepared using the Illumina NEBNext Ultra II Directional RNA Library Prep kit (NEB, E7760L) with a Poly(A) mRNA Magnetic Isolation module (NEB, E7490L) and the Illumina Unique dual NEBNext Multiplex Oligo (NEB, E6440L), and 10 ng to 1 μg of total RNA were subjected to mRNA poly(A) purification. The mRNA was then fragmented and copied to first-strand cDNA using reverse transcriptase and dUTP mix. The samples were repaired at the ends and ligated to the NEBNext adapter via the dA-tailing process. The products were purified and enriched by PCR to create the final cDNA libraries. The final libraries were quality and quantity checked by qPCR using TapeStation (Agilent) and the Kapa Library Quantification Kit for Illumina sequencing platforms (Kapa Biosystems, KK4835). The libraries were paired-end sequenced using the NovaSeq6000 sequencing system (Illumina).

[0373] The quality of the raw Fastq files was checked with FastQC v0.11.8 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Low-quality reads were trimmed using Trimmomatic v.0.35 with parameters SLIDINGWINDOW:4:20 MINLEN:25. 12 The resulting 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 Significant differentially expressed genes were then identified using the R package DESeq2. 15 Genes with an adjusted P-value less than 0.05 and an absolute digit change greater than 2 were considered significant differentially expressed genes (DEGs). Next, up-controlled and down-controlled DEGs were analyzed for KEGG pathways that were significantly enriched using the clusterProfiler package.16 The significance of enrichment was determined by the right-handed Fisher direct test followed by Benjamini-Hochberg multiple test adjustment.

[0374] (xv) Liver lipid analysis

[0375] Liver was 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 minutes). The supernatant was collected and its protein content was analyzed using the Bio-Rad Bradford assay. To assess the liver lipid composition, lipids were extracted from the supernatant using hexane (≧99%, Sigma-Aldrich 32293) and isopropanol (≧99.5%, Fisher Scientific A426-4) in a 3:2 ratio (v:v), and the hexane phase was evaporated for 48 hours. The amount of TG or TC in the liver was measured by spectrophotometric analysis using commercially available kits (Wako Chemicals 999-02601 and 994-02891). Liver diacylglycerol (DAG) levels were measured using an ELISA kit (Aviva Systems Biology, OKEH02607) according to the manufacturer's instructions. Liver TG, TC, and DAG data were normalized to protein levels.

[0376] (xvi) Western blot analysis

[0377] Liver samples were dissolved in radioimmunoprecipitation assay lysis buffer (RIPA buffer, Thermo Scientific) supplemented with a protease / phosphatase inhibitor cocktail (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 at room temperature for 1 hour in Tris-buffered saline-Tween20 (TBST) containing 5% nonfat 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), phospho-SMAD2 (Ser465 / 467, Cell Signaling Technology, #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). After washing the membrane with TBST, it was incubated with IRDye-labeled secondary antibody (LI-COR Biosciences, 1:10000) at room temperature for 1 hour. After TBST washing, the bands were visualized and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, version 2.1).

[0378] (xvii) Identification of glycine compounds

[0379] Compounds structurally similar to glycine were selected to evaluate structural, conformational, electronic, and steric equivalence modifications to the glycine skeleton (Figure 9A). A search was performed using SciFinder(C) to determine their commercial applicability. The degree of methylation of the amine moiety of glycine was investigated using N-methylglycine (Figure 9B), N,N-dimethylglycine (Figure 9C), and N,N,N-trimethylglycine (Figure 9D), and the substitution of amines to alcohols was evaluated using glycolic acid (Figure 9E). Modification of the acidic region Wx.

[0380] Before investigating glycinamide (Figure 9F), 2-amino-N-methylacetamide (Figure 9G), and ethanolamine (Figure 9H), conformationally restricted analogs were examined using 2-oxopiperazine (Figure 9I) and morpholine-2-one (Figure 9J). Acid equivalent substitution was investigated using (1H-tetrazole-5-yl)methaneamine (Figure 9K).

[0381] (xviii) Oral glucose tolerance test (OGTT)

[0382] An oral glucose tolerance test (OGTT) was performed after 12 hours of fasting. Blood samples were taken from the tail tip at 0, 15, 30, 60, and 120 minutes after forced oral administration of glucose (2 mg / g body weight) containing a specified dose (0.17–1 mg / g body weight) of leucine, glycine, DT-109, or other glycine compounds. Blood glucose levels were measured using a blood glucose meter and test strips (Contour Next).

[0383] (xix) Body composition and comprehensive laboratory animal monitoring system (CLAMS)

[0384] All measurements were performed by the Animal Phenotyping Core at the University of Michigan. 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 checked daily using the manufacturer's recommended reference sample. Mice were placed individually in measurement tubes with minimal restraint. Oxygen consumption (VO2), carbon dioxide production (VCO2), and motility were measured using a CLAMS (Columbus Instruments) integrated open-circuit calorimeter with an optical beam activity monitor. Before measurement, the weight of the mice was measured and they were individually placed in sealed chambers (7.9”×4”×5”) with free access to food and water. The study was conducted in a laboratory set to 20–23°C with a 12-hour–12-hour light-dark cycle (6:00 PM–6:00 AM). Measurements were carried out continuously for 48 hours. During this time, the animals were supplied with food and water via feeding and drinking devices located within the chambers. VO2 and VCO2 in each chamber were sampled continuously for 5 seconds at 10-minute intervals, and motility was recorded per second in the X and Z dimensions. The airflow velocity through the chambers was adjusted to maintain an oxygen difference of approximately 0.3% in a stationary state. The respiratory exchange rate (RER) was calculated as VCO2 / VO2. Total energy expenditure, glucose oxidation, and fatty acid oxidation were calculated based on the values ​​of VO2, VCO2, and protein degradation.

[0385] (xx) Fecal microbiome analysis

[0386] Whole-genome DNA of the gut microbiota was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (51540, Qiagen) according to the manufacturer's instructions. The DNA was prepared for community analysis as described above.17 Briefly, the DNA was amplified using barcoded dual-index primers specific to the V4 region of the 16S rRNA gene.18 The PCR reaction consisted of 5 μL of 4 μM isomolar primer set, 0.15 μL of AccuPrimeTaq DNA High Fidelity polymerase, 2 μL of 10× AccuPrime PCR Buffer II (Thermo Fisher Scientific 12346094), 11.85 μL of PCR-grade water, and 1 μL of DNA template. The PCR conditions were 2 minutes at 95°C, followed by 30 cycles of 20 seconds at 95°C, 15 seconds at 55°C, and 5 minutes at 72°C, followed by 10 minutes at 72°C. Each PCR reaction was normalized using the SequalPrep Normalization Plate kit (Thermo Fisher Scientific A1051001). The normalized reaction products were pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit (KK4873) for the Illumina platform. The amplicon library size was confirmed using the Agilent Bioanalyzer and the High Sensitivity DNA Analysis Kit (5067-4626) (approximately 399 bp). The pooled amplicon library was then sequenced according to a standard protocol on the Illumina MiSeq NANO platform (University of Michigan Laboratory of Microbial Systems Molecular Biology).

[0387] DNA sequencing data were processed using Mothur according to the SOP, focusing primarily on α and β diversity-based analysis. Sequences were trimmed to remove primers and barcodes, filtered for quality, and checked for chimeras as described above. A total of 3565 sequences from each sample were used for further statistical analysis. The trimmed DNA sequences were clustered using the mean adjacency approach to form operational taxa units (OTUs) with a 97% sequence similarity cutoff (3% sequence difference). Phylogenetic trees were constructed using the Clearcut program. An OTU-based approach was used to measure β diversity. A heatmap of the relative abundance of each OTU across all samples was generated using log2 scaling of the relative abundance (>1%) values ​​of the top 107 OTUs. Molecular AMOVA statistical analysis was performed to determine the significance of structural similarity between communities across the sampling groups. Weighted (WUnF) and unweighted (UWUnF) UniFrac metrics were estimated using UniFrac analysis. Constrained ordered RDA (redundancy analysis) was calculated, and the significance of environment variables was checked by forward selection analysis. Characterization of microbial features that distinguish gut microbiota was performed using the Linear Discriminant Analysis (LDA) Effect Size (LEfSe) method for biomarker discovery (http: / / huttenhower.sph.harvard.edu / lefse / ), which emphasizes both statistical significance and biological relevance. Accordingly, principal component analysis (PCA) was plotted using the Phyloseq package in R. The correlation between changes in altered bacterial genera and NAFLD-related parameters in liver or plasma was calculated using the non-parametric Spearman test.

[0388] (xxi) In vitro studies

[0389] HepG2 human hepatocellular carcinoma cell lines were obtained from the American Cell Culture and Cell Lineage Preservation Center (ATCC) and cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin (Pen-Strep, Gibco) at 37°C and 5% CO2. 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 ID s1190) and a non-targeting siRNA control (siCTL, siRNA ID AM4611) were obtained from Ambion. Using Lipofectamine RNAiMAX (Invitrogen), 20 nM siAGXT1 or siCTL was transfused into HepG2 cells in Opti-MEM reduced serum medium (Gibco) according to the manufacturer's protocol. RNA isolation and protein or lipid extraction were performed 48 hours after transfusion. Total RNA was purified from HepG2 cells using the QIAGEN RNeasy kit (QIAGEN). qPCR analysis was performed as described above using the human primer pairs listed in Table S3. Cells were lysed using RIPA buffer (Thermo Scientific) supplemented with a protease / phosphatase inhibitor cocktail (Roche Applied Science). The abundance of AGXT1 protein was assessed using Western blotting as described above. Cellular lipids were extracted from the cells using hexane:isopropanol in a 3:2 ratio, and the hexane phase was evaporated for 48 hours. The remaining cells in the plate were disrupted with 0.1 M NaOH for 24 hours, and aliquots were taken to measure cellular proteins using the Bradford protein assay (Bio-Rad). The TG or TC content of the cells was measured spectrophotometrically using a commercially available kit (Wako Chemicals). The TG and TC data of the cells were normalized to protein levels and expressed as μgTG or TC / mg protein.

[0390] H. Results

[0391] (i) Impaired glycine biosynthesis in mouse and human NAFLD

[0392] To investigate whether alterations in glycine metabolism contribute to the development of NAFLD, we first studied C57BL / 6J mice with HS induced by WD. After 12 weeks of WD feeding, we revealed hypercholesterolemia (Figure 10A) and HS as confirmed by H&E staining and oil red O (ORO) staining, as well as quantification of hepatic TG and total cholesterol (TC) (Figures 10B-D). Targeted metabolomics revealed that plasma glycine was most significantly decreased among all AAs, while its precursors, serine, threonine, and alanine, were significantly increased (Figure 10E), indicating impaired glycine biosynthesis. Therefore, we next studied the expression of genes that drive glycine formation. In mice with HS, we found that major glycine biosynthesis genes were downregulated, with AGXT1 being the most significantly suppressed (Figure 10F). Furthermore, AGXT1 was significantly downregulated in HepG2 cells by palmitic acid (PA)-induced TG accumulation (Figure 10G, H).

[0393] To test whether similar patterns are evident in more severe NAFLD, RNA sequencing was performed on livers from mice with progressive NASH and fibrosis induced by 24 weeks of NASH diet feeding (Figure 10I). Pathway analysis revealed alterations in known pathways involved in NASH, including upregulation of chemokine, NF-κB, Toll-like receptor (TLR), and transforming growth factor β (TGFβ) signaling, as well as downregulation of FA degradation and peroxisome proliferator-activated receptor (PPAR) signaling (Figure 10J). Interestingly, pathways regulating AA biosynthesis, including glycine, serine, threonine, and glyoxylic acid metabolism, were significantly downregulated in NASH, including AGXT1 suppression (P=0.0009, Figure 10K). AGXT1 suppression was confirmed by qPCR using an independent cohort of mice with diet-induced NASH (Figure 10L).

[0394] To investigate whether glycine biosynthesis genes are similarly suppressed in human NAFLD, a meta-analysis was conducted based on transcriptomics from the livers of NASH patients. In NASH, AGXT1 (β=-0.141, P=0.0041) and AGXT2 (β=-0.134, P=0.0135) were significantly downregulated, while D-amino acid oxidase (DAO), which catalyzes the breakdown of glycine to glyoxylic acid, was significantly upregulated (β=0.216, P=0.0025, Figure 10M). Among 206 samples obtained from liver transplant donors, AGXT1 expression was found to be inversely correlated with liver fat content (r=-0.199, P=0.0044, Figure 10N). Interestingly, a similar inverse correlation was observed between PPARα (a major regulator of hepatic FAO) expression and hepatic fat content (r=-0.154, P=0.0275), while the expression of CC motif chemokine ligand 5 (CCL5) and TGFβ, major players in steatohepatitis and fibrosis, was positively correlated with hepatic fat (r=0.185, P=0.0078 and r=0.284, P<0.0001, respectively, not shown). These results are consistent with recent reports on decreased circulating glycine in NAFLD in relation to the repression of glycine biosynthesis genes and suggest a role for AGXT1 in NAFLD.

[0395] (ii) Loss of AGXT1 exacerbates diet-induced hyperlipidemia and NASH.

[0396] To investigate the potential role of AGXT1, a liver-specific gene localized to peroxisomes or mitochondria in hepatocytes, in cellular lipid accumulation, AGXT1 was knocked down in HepG2 cells, which enhanced PA-induced TG accumulation (Figure 8C-E). To study the effects of AGXT1 loss in vivo, CRISPR / Cas9 was used to analyze AGXT1 - / - Mice were generated (Figure 8A, B). Under CD feeding, AGXT1 - / - Liver histology is AGXT1 + / +was equivalent to that, and furthermore, plasma liver enzymes were also found to be equivalent (Figs. 8F - K). However, after 12 weeks on a NASH diet, in AGXT1 - / - mice, plasma TG, TC, AST, and ALT were increased (Figs. 8L - O), but the glycine to oxalate ratio was significantly decreased (Fig. 8P). From the gross appearance of the abdomen, in AGXT1 - / - mice, a more hypertrophied and yellowish - colored liver was revealed (Fig. 11A). There was no significant difference in body weight, but liver weight was significantly increased (Figs. 11B, 11C, D). H&E and ORO staining combined with quantification of liver lipids revealed an increase in HS in AGXT1 - / - mice (Figs. 11A, E, F), and Sirius red staining revealed an increase in fibrosis (Fig. 11A). Further histological analysis confirmed high NAS and fibrosis scores in AGXT1 - / - mice (Figs. 11G, H, Fig. 11I).

[0397] AGXT1 - / - To understand the underlying mechanism of the acceleration of diet - induced NASH in AGXT1 + / + mice, RNA sequencing of the liver from AGXT1 - / - and subsequent qPCR validation were performed. Pathway analysis revealed suppression of energy metabolism and the FAO pathway in AGXT1 - / - mice, while inflammatory - inducing pathways were upregulated (Fig. 11J). Genes regulating peroxisomes (acyl - CoA thioesterase 3, ACOT3, acyl - CoA synthetase medium - chain 5, ACSM5, acyl - CoA synthetase long - chain 1, ACSL1) and mitochondrial FAO (hydroxyacyl - CoA dehydrogenase α, HADHA, and acetyl - CoA acyltransferase 2, ACAA2) were in AGXT1 - / -In mice, significant downregulation was observed (Figures 11K-M, although 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), fibrillation (TGFB1 / 2 and TGFBR2), and extracellular matrix (ECM) remodeling (COL1A2, COL4A2, and TIMP1) were significantly upregulated (Figures 11N, O). Therefore, AGXT1, a liver-specific glycine biosynthesis gene, is suppressed in NAFLD, and its deficiency accelerates diet-induced NASH.

[0398] (iii) Glycine deficiency worsens diet-induced hyperlipidemia and HS.

[0399] Decreased plasma glycine levels are associated with NAFLD and cardiovascular metabolic diseases, while higher levels are associated with favorable lipid characteristics. To investigate the role of dietary glycine in dyslipidemia, modified AA WD (WD) containing or without glycine was used. AA +Gly or WD AA -Gly, Table S2) was developed to treat hyperlipidemia. - / - Mouse CD, WD AA +Gly or WD AA -Feeding Gly for 10 weeks. WD AA -A significant decrease in plasma glycine was observed in mice fed Gly (Figure 12A). NMR-based body composition analysis and CLAMS analysis revealed that WD AA -In mice fed Gly, an increase in body weight and fat was observed, but WD AA This was not observed in mice fed +Gly (Figure 12B-D), and there were no significant changes in food intake, activity, or energy metabolism (Figure 12E-H). Therefore, plasma glycine-containing tripeptide molecules are WD AA In mice fed A-Gly, the levels increased (Figure 12I), and increased adipocyte hypertrophy was observed in epididymal adipose tissue and subcutaneous adipose tissue (EAT and SAT, Figure 12J). WD AA Compared to +Gly, WD AA-Mice fed Gly showed increased plasma TC and TG levels (Figure 13A-D). WD AA -In mice fed Gly, an increase in plasma glucose was observed, but WD AA This was not observed in mice fed +Gly (Figure 13E). Histological and lipid quantification confirmed that WD AA - An increase in HS after Gly feeding was observed (Figure 13F-H). Linear regression analysis showed a significant inverse correlation between plasma glycine and individual levels of plasma TC, glucose, or hepatic TG (Figure 13I-K). Therefore, dietary glycine deficiency exacerbates hyperlipidemia, hyperglycemia, and HS in hyperlipidemia mice.

[0400] (iv) DT-109: A glycine-based tripeptide with glucose / lipid bimodal properties.

[0401] Considering the above findings, we hypothesized that glycine-based compounds may have therapeutic potential through a glucose / lipid bimodal effect. Therefore, we explored compounds structurally similar to glycine by applying various chemical modifications to amine and carboxyl groups. Ten potential compounds were identified (Figure 9A-K), four of which were suitable for oral administration. Chronic glycine supplementation (1 mg / g / day) is associated with apoE - / -In mice, it was shown to reduce circulating triglycerides (TG), restore glucose tolerance in obese C57BL / 6 mice, and accelerate fat loss. In humans, ingesting glycine together with glucose attenuated the increase in plasma glucose by >50%. To test the glucose-lowering effect, oral glucose tolerance tests (OGTTs) were performed in C57BL / 6J mice administered glycine or a glycine-based compound (0.5 mg / g). None of the compounds tested attenuated the increase in blood glucose more efficiently than glycine (Figure 14A-D). Another AA reported to lower glucose in humans and mice is leucine. Previous research in the inventors' laboratory revealed the significant glucose-lowering effect of DT-109, a glycine tripeptide (gly-gly-L-leu) combined with leucine. DT-109 or its D-isomer (gly-gly-D-leu, DT-110) was tested using OGTTs. The glucose-lowering effect of DT-110 was similar to that of glycine, but DT-109 was the only compound that lowered glucose more efficiently than glycine, especially when compared to equivalent levels of its individual AAs (Figure 14G) (Figure 14E, F).

[0402] To test the lipid-lowering effect, apoE of hyperlipidemia - / -Mice were fed WD and orally administered DT-109 (1 mg / g / day), equivalent levels of leucine, glycine, or H2O (Figure 15A). After 10 weeks, DT-109 showed the most potent glucose-lowering effect in both OGTT and non-fasting glucose (Figures 15B, C). No significant differences were observed in food intake or body weight (Figures 15D, E). Lipid characteristics analysis showed that mice treated with DT-109 had the lowest TC and LDL without the HDL decrease observed in glycine-treated mice (Figures 15F-I), and also showed a significant difference in plasma TG (Figure 15J). Glycine or DT-109 reduced glycine-containing tripeptide molecules and adipocyte hypertrophy in EAT and SAT (Figures 15K, L). Histological and lipid quantification revealed a reduction in HS in the livers of mice treated with glycine or DT-109 but not with leucine (Figure 16A-C). qPCR analysis revealed upregulation of key FAO-regulating genes (PPARα, PNPLA2, carnitine / acylcarnitine translocase, CACT, carnitine palmitoyltransferase 1a, CPT1a, and acyl-CoA dehydrogenase long chain, ACADL) and downregulation of CCL2 without significant changes in TNFα in the livers of mice treated with glycine or DT-109 (Figure 16D). Therefore, DT-109 possesses dual glucose / lipid-reducing properties and prevents WD-induced HS in hyperlipidemia mice.

[0403] (v) DT-109 improves body composition and protects against diet-induced NASH.

[0404] To further investigate the therapeutic potential of DT-109 for NASH, an experimental approach was devised to model progressive NAFLD (Figure 17A). C57BL / 6J mice fed a NASH diet for 12 weeks showed increased plasma glucose, TC, AST, and ALT compared to mice fed CD (Figure 17B). A subset of mice were euthanized, and an increase in liver weight after feeding the NASH diet was confirmed (Figure 17C). H&E and ORO histology revealed HS, hepatocyte ballooning, and inflammatory cell infiltration, while Sirius red staining confirmed early fibrosis (Figure 17D). After confirming NASH (Figures 17D-F), the remaining mice were randomized and administered 0.125 mg / g / day or 0.5 mg / g / day of DT-109, equivalent levels of leucine, glycine, or H2O via forced oral administration for a further 12 weeks under a NASH diet. Mice were fed CD and given H2O as a control.

[0405] At week 18, OGTT and non-fasting glucose measurements confirmed that DT-109 at 0.5 mg / g / day had the most potent glucose-lowering effect (Figure 17G, H). Body composition analysis revealed weight gain in all mice fed a NASH diet (Figure 17I), but mice treated with DT-109 at 0.5 mg / g / day showed reduced body fat and maintained lean body mass compared to the H2O control (Figure 17J, K), and further reduced adipocyte hypertrophy in EAT and SAT (Figure 17L), with no significant difference in food intake (Figure 17M). The metabolic response to diet-induced obesity involves a shift to high-fat versus low-carbohydrate utilization, reflecting a decrease in RER. CLAMS analysis revealed a decrease in RER in all groups fed a NASH diet, but no significant difference was observed in mice treated with DT-109 at 0.5 mg / g / day (Figure 17N~P). No significant differences were observed in energy consumption or activity (Figure 17Q, R).

[0406] At the endpoint, the increases in plasma AST, ALT, and alkaline phosphatase (ALP) observed in mice fed a NASH diet were attenuated by treatment with glycine or DT-109 (Figures 18A-C). Plasma TG decreased in mice treated with glycine or DT-109 (Figure 18D), and TC decreased with 0.5 mg / g / day of DT-109 (Figure 18E). Therefore, NASH diet-induced hepatomegaly and yellowish discoloration were significantly attenuated by treatment with glycine or DT-109, but not with leucine (Figures 18F, G, 18H), and NAS was significantly reduced with 0.5 mg / g / day of DT-109 (Figures 18I, 18J). Linear regression analysis showed a very significant positive correlation between individual levels of AST, ALT, or ALP and NAS (Figures 18K-M).

[0407] (vi) DT-109 reverses NASH-induced transcriptome changes: an important role of FAO

[0408] To investigate the mechanism by which glycine-based therapeutics protect against dietary NASH, RNA sequencing was performed on livers recovered at the endpoint. Principal component analysis (PCA) revealed that gene expression characteristics from mice fed a leucine-treated NASH diet clustered with H2O control, while glycine and 0.125 mg / g / day showed intermediate patterns. On the other hand, DT-109 at 0.5 mg / g / day clustered near CD mice (Figure 19A). Volcano plot analysis identified major DEG changes (3606 or 3145 DEG, respectively) in mice fed a NASH diet and treated with H2O or leucine compared to CD mice. These changes were significantly reduced by treatment with 0.125 mg / g / day or 0.5 mg / g / day of glycine or DT-109 (1300, 1093, or 642 DEG, respectively, Figure 19B). Analysis of the top 50 DEGs further demonstrated the similarity between DT-109 (0.5 mg / g / day) and the CD group (Figure 19C). Pathway analysis comparing NASH diet H2O control with CD confirmed suppression of pathways regulating glycine biosynthesis and glyoxylate metabolism, as well as downregulation of AGXT1, SHMT1, and SARDH, along with energy metabolic pathways and the FAO pathway. In contrast, known NASH-associated pro-inflammatory / fibrotic pathways were upregulated (Figures 19D, E). Comparative pathway analysis of NASH diet H2O control with DT-109 at 0.5 mg / g / day showed a similar pattern to CD, indicating that DT-109 reversed the NASH diet-induced changes in the underlying pathways (Figure 19F). Analysis of 50 genes involved in the major aspects of NASH pathogenesis (Figure 19G) revealed 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 overexpressed in CD mice and suppressed in NASH mice treated with H2O or leucine.As confirmed by qPCR and Western blot analysis, this was reversed by treatment with glycine or DT-109, particularly 0.5 mg / g / day (Figure 19H, I, Figure 19J). Thus, quantification of ORO and lipids revealed significant HS in the livers of NASH mice treated with H2O or leucine, which was significantly attenuated by glycine or DT-109 (Figure 19K-L, Figure 19M). In particular, diacylglycerol (DAG), known to promote liver damage and NASH, was significantly reduced by DT-109 at 0.5 mg / g / day (Figure 19N). Therefore, glycine-based therapeutics correct FAO impairment and reduce NASH diet-induced HS and lipotoxic lipids.

[0409] (vii) DT-109 reduces dietary inflammation and fibrosis of the liver in NASH.

[0410] RNA sequence analysis revealed the suppression of key inflammatory pathways / genes by glycine-based therapeutics (Figure 19F, G), suggesting an anti-inflammatory role. Indeed, immunohistochemistry for F4 / 80, a well-established marker of hepatic macrophages, was significantly increased in livers from mice treated with H2O or leucine under a NASH diet, but attenuated by glycine or DT-109 (Figure 20A, B). In plasma, monocyte chemotactic protein 1 (MCP-1 / CCL2) and resistin, known inflammatory markers in NASH patients, were lower in mice treated with glycine or DT-109 (Figure 20C, D). Therefore, RNA sequencing revealed that 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 in mice fed a NASH diet and treated with H2O or leucine, and attenuated with glycine or DT-109 (Figure 19G). This was confirmed by qPCR analysis, where NFKB2, RELB, and TNFα were significantly downregulated by either glycine or DT-109, while CCL2, CCR2, and CCR5 were downregulated only by DT-109 at 0.5 mg / g / day (Figure 20E).

[0411] RNA sequencing also showed that pathways / genes related to TGFβ signaling (TGFB1 / 2 and TGFBR1 / 2) and ECM remodeling (COL1A1 / 1A2 / 3A1 / 4A1 / 4A2, TIMP1 / 2, and SERPINE1) were upregulated by a NASH diet and attenuated by glycine or DT-109 (Figure 19F, G). Indeed, histological analysis based on Sirius Red and fibrosis scoring revealed that glycine or DT-109 had a protective effect against NASH diet-induced liver fibrosis, while leucine did not (Figure 20A, F, G). Linear regression analysis showed very significant positive correlations between individual levels of AST, ALT, or ALP and fibrosis scores, indicating that glycine or DT-109 mitigates NASH diet-induced liver injury (Figure 20H, I, J). To investigate whether glycine-based therapeutics alleviate TGFβ-mediated hepatic fibrosis, we then analyzed SMAD signaling and found a reduction in Ser465 / 467 phosphorylation of SMAD2, primarily by DT-109 (Figure 20K). qPCR analysis confirmed that TGFβ-related genes were significantly upregulated in the livers of NASH mice treated with H2O or leucine, and this regulation was attenuated by DT-109 (Figure 20L). Therefore, consistent with the reduction in HS and lipotoxicity, the two glycine-based therapeutics alleviate diet-induced steatohepatitis and fibrosis in NASH.

[0412] I. Discussion

[0413] While lipid and glucose metabolic abnormalities are known features of NAFLD, disruption of glycine metabolism has been suggested to be related to NASH. In particular, decreased circulating glycine is consistently reported in NAFLD patients, but the causes of glycine deficiency and potential treatments remain unclear. This specification provides evidence that glycine plays a causal role in the development of NAFLD using genetic and dietary approaches to limit glycine availability. In the search for potential glycine-based therapeutic agents for NAFLD, DT-109 was identified as having a dual glucose / lipid-lowering effect and strongly protecting mice from diet-induced NASH.

[0414] The results presented herein indicate that decreased glycine levels observed in NAFLD patients are associated with the suppression of hepatic glycine biosynthesis genes. In particular, in both mouse and human NASH, the inventors found significant suppression of AGXT1, which catalyzes the conversion of glyoxylic acid to glycine, and demonstrated that AGXT1 expression is inversely correlated with human hepatic fat content. While suppression of AGXT1 had been reported in NASH patients or mouse models, the inventors are the first to report that AGXT1 plays a causal role in NAFLD. Mutations in AGXT1 are the cause of primary hyperoxaluria type 1, which is caused by impaired conversion of glyoxylic acid to glycine and excessive hepatic oxalate production leading to renal failure. Interestingly, AGXT1 - / - Proteomics of mouse livers has shown significant changes in glucose and lipid metabolic pathways, but the role of AGXT1 in NASH has not been evaluated until now. Using CRISPR / Cas9, AGXT1 - / - Mice were generated, but these mice already showed worsened hyperlipidemia and NASH after 12 weeks on a NASH diet. AGXT1 - / - In mice, suppression of the FAO pathway was identified, and this suppression, in turn, promotes fatty liver disease and fibrosis.

[0415] Furthermore, we limited glycine availability through dietary therapy and compared the lipid characteristics and HS of hyperlipidemia mice fed with or without glycine-containing WD. The exacerbation of steatosis, hyperlipidemia, and HS observed in mice fed glycine-deficient WD is consistent with previous reports in various rodent models that dietary glycine accelerates lipid loss, improves glucose tolerance, and reduces plasma lipids or HS. Interestingly, despite the small sample size and short treatment duration, a decrease in HS and plasma liver enzymes was observed in NAFLD patients after supplementation with serine, a glycine precursor. In a thorough investigation using a progressive NAFLD model characterized by the coexistence of steatohepatitis and fibrosis, we report for the first time the protective effect of treating mice with a relatively low dose of glycine (0.33 mg / g / day).

[0416] During the search for glycine-based compounds, none of the identified compounds lowered plasma glucose more efficiently than glycine. Therefore, we tested combinations of glycine with leucine, another amino acid (AA) reported to lower glucose in humans and reduce HS in mice. In particular, by applying various T2D models, our laboratory revealed the potent glucose-lowering effect of the tripeptide gly-gly-L-leu, surpassing the effects of free glycine, leucine, or combinations thereof. We were the first to demonstrate that DT-109 also improves lipid characteristics, HS, and NASH using genetic and dietary models. While no significant effect was observed in mice treated with equivalent levels of leucine, metabolic benefits were evident after glycine treatment. Nevertheless, several outcomes, including robust glucose reduction, maintenance of HDL levels in hyperlipidemia mice, prevention of body composition changes due to the NASH diet, reduction of hepatic DAG, and a significant decrease in NAS, were demonstrated only with high doses of DT-109. It should be noted that significant benefits were also observed even at a low dose of DT-109 of 0.125 mg / g / day.

[0417] Lipid overload is central to the pathogenesis of NASH. When free fatty acids are supplied in excess to the liver and / or their excretion via FAO is impaired, they are used as substrates for lipotoxic species that induce pro-inflammatory / fibrogenic pathways that promote oxidative stress and steatohepatitis and fibrosis. Applying unbiased transcriptomics, we identified a suppressed major FAO pathway in the livers of NASH mice, which was reversed by DT-109, subsequently reducing HS and lipotoxic DAG. This suggests that glycine-based therapeutics may normalize hepatic FAO impairment, reduce HS and lipotoxicity, and thus attenuate the progression of NASH. Indeed, using a model characterized by steatohepatitis and fibrosis, glycine or DT-109 was found to attenuate NASH diet-induced hepatic / systemic inflammation and fibrosis, as revealed by histological, transcriptomics, and plasma analysis. In addition, previous studies have reported the anti-inflammatory and hepatoprotective effects of glycine in mice with endotoxemia. In T2D patients, glycine treatment (5g / day) for 3 months reduced hemoglobin A1c and plasma TNFR1.

[0418] In summary, by identifying glycine metabolism disorders in NAFLD, glycine-based therapeutic agents were identified, and these were proven effective in experimental NAFLD by regulating hepatic FAO. Furthermore, the present invention encompasses the following aspects. 1. A method for treating at least one of the following conditions in a mammal: hyperlipidemia, fatty liver, steatohepatitis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, obesity, hyperglycemia, metabolic syndrome, cardiovascular disease, and atherosclerosis, comprising administering a therapeutically effective amount of glycine, a glycine-containing tripeptide molecule, or a pharmaceutically acceptable salt thereof to a subject in need thereof. 2. The method according to item 1, wherein the glycine-containing tripeptide molecule significantly reduces liver triglyceride levels. 3. The method according to item 1, wherein the glycine-containing tripeptide molecule significantly reduces the total liver cholesterol level. 4. The method according to any one of claims 1 to 3, wherein the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. 5. A method for enhancing hepatic lipid oxidation or utilization, a method for lowering triglyceride levels in the blood of the subject, or a method for lowering cholesterol levels in the blood of the subject in a case of hypercholesterolemia requiring such reduction, wherein, as a result of the treatment, hepatic lipid oxidation increases and the triglyceride levels, the hypercholesterolemia, or any combination thereof are improved. 6. The method according to claim 5, wherein the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. 7. The method according to item 5 or 6, wherein the glycine-containing tripeptide molecule Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof significantly induces the expression of hepatic lipid oxidation regulators AMPKα1 or PPARα. 8. The method according to claim 5 or 6, wherein the glycine-containing tripeptide molecule Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof reduces liver triglyceride levels by significantly upregulating CPT1a, CACT, or ACADI (mitochondrial β-oxidation) or PNPLA2. 9. The method according to claim 5 or 6, wherein the glycine-containing tripeptide molecule Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof reduces liver triglyceride levels by significantly upregulating the mitochondrial anion carrier UCP2. 10. The method according to claim 5 or 6, wherein the glycine-containing tripeptide molecule Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof modulates cholesterol homeostasis in the liver by significantly increasing the expression of ABCG5 and ABCG8. 11. A method for treating plasma lipid characteristics of a subject requiring treatment of plasma lipid characteristics, comprising administering a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof to the subject to lower plasma triglyceride and plasma LDL levels, prevent further progression of atherosclerotic lesions, or regress atherosclerotic lesions present in the subject's arteries, thereby reducing the incidence of MACE, preventing, delaying, or reducing the severity of primary cardiovascular events. 12. The method according to claim 11, wherein the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. 13. The method according to paragraph 11 or 12, wherein the Gly-Gly-Leu, Gly-Gly-dLeu, or pharmaceutically acceptable salt thereof reduces lesions of atherosclerosis. 14. The method according to item 11 or 12, wherein the Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof lowers plasma total cholesterol, plasma LDL cholesterol, non-HDL cholesterol, VLDL cholesterol, or a combination thereof. 15. A method for treating inflammation in adipose tissue and a method for reducing the level of circulating inflammatory markers, comprising administering a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof to a subject in need thereof, wherein the administration of the glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof reduces the adipose tissue and the circulating inflammatory markers of the subject. 16. The method according to item 15, wherein the circulating inflammatory marker is reduced by lowering plasma MCP1 levels by administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to the subject. 17. The method according to item 15, wherein the inflammation in the adipose tissue is present in epididymal adipose tissue (EAT) or subcutaneous adipose tissue (SAT) and reduces the level of MCP1 mRNA. 18. A method for treating a subject in need of treatment to reduce plasma leptin levels, comprising administering a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof, wherein the administration of the glycine-containing tripeptide molecule reduces plasma leptin levels. 19. The method of item 18, wherein the treatment is carried out using Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. 20. A method for treating a subject in need of treatment, comprising administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, wherein the subject has liver disease. 21. The method according to item 20, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or alcoholic fatty liver. 22. A method for stabilizing or reducing the NAFLD activity score (NAS) in a subject, comprising administering Gly-Gly-Leu and / or Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, to the subject. 23. The method according to claim 22, wherein the method comprises delaying, stabilizing, or reducing the progression of fatty liver components of NAS. 24. The method according to any one of claims 22 to 23, wherein the method comprises delaying, stabilizing, or reducing the progression of inflammatory components in the lobules of the NAS. 25. The method according to any one of claims 22 to 24, wherein the method comprises delaying, stabilizing, or reducing the progression of the hepatic ballooning component of NAS. 26. NAS differs by at least 1.5 points after 6 months of treatment with Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof, according to any one of the methods in paragraphs 22 to 25. 27. A method for alleviating hepatic fibrosis in a subject requiring alleviation of hepatic fibrosis, comprising administering Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof to the subject. 28. A method for treating atherosclerosis, the method comprising administering a therapeutically effective amount of a glycine-containing tripeptide molecule or a pharmaceutically acceptable salt thereof to a subject in need thereof. 29. The method according to claim 28, wherein the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. 30. A method for treating complications of atherosclerosis, comprising administering a glycine-containing tripeptide molecule to a subject having the complication, selected from the group consisting of myocardial infarction, arteriosclerosis, coronary artery disease, carotid artery disease, peripheral artery disease, atherothrombotic stroke, aneurysm, or chronic kidney disease. 31. The method according to claim 30, wherein the glycine-containing tripeptide molecule is Gly-Gly-Leu, Gly-Gly-dLeu, or a pharmaceutically acceptable salt thereof. 32. The method according to any one of claims 1 to 31, further comprising administering a second therapeutic agent to a subject in need thereof, the second therapeutic agent comprising a cholesterol absorption inhibitor, a PCSK9 inhibitor, a PPARα agonist, an ACE inhibitor, a calcium channel blocker, an ARB, a diuretic, renin, GLP-1 or a synthetic variant thereof, insulin or a synthetic variant thereof, metformin, a sulfonylurea compound, thiazolidinedione (TZD), an SGLT2 inhibitor, a DPP-IV inhibitor, an HMGCoA reductase inhibitor, a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, ezetimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gemcapen (CI-1027), benpodic acid (ETC-1002), an ACC inhibitor, an ApoC-III inhibitor, an ACL inhibitor, a prescription fish oil, a CETP inhibitor, an antifibrotic agent, and a combination thereof. 33. A kit for treating subjects with NAFLD or NASH, comprising selected tripeptides, optionally statins, and instructions for use. 34. The kit described in item 30, wherein the kit includes DT-109 (Gly-Gly-Leu) and / or DT-110 (Gly-Gly-dLeu), optionally a statin, and instructions for use. 35. The kit according to item 32 or 33, wherein the kit optionally further comprises cholesterol absorption inhibitors, PCSK9 inhibitors, PPARα agonists, ACE inhibitors, calcium channel blockers, ARBs, diuretics, renin, GLP-1 or its synthetic variants, insulin or its synthetic variants, metformin, sulfonylurea compounds, thiazolidinedione (TZD), SGLT2 inhibitors, DPP-IV inhibitors, HMGCoA reductase inhibitors, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ezetimibe, gemfibrozil, fenofibrate, clofibrate, bezafibrate, pemafibrate, gemcapen (CI-1027), benpodic acid (ETC-1002), ACC inhibitors, ApoC-III inhibitors, ACL inhibitors, prescription fish oil, CETP inhibitors, antifibrotic agents, and combinations thereof. 36. The kit according to item 32 or 33, wherein the kit may include ezetimibe.

Claims

[Claim 1] A pharmaceutical composition for use in the treatment of hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD) or hepatic fibrosis in non-alcoholic steatohepatitis (NASH), comprising at least one tripeptide selected from Gly-Gly-Leu or Gly-Gly-dLeu or a pharmaceutically acceptable salt thereof.

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