FGF21 protein variants for the treatment of NASH

Abstract

The present disclosure relates to methods of treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject in need thereof by administering a human FGF21 protein variant, wherein the metabolic and / or cardiovascular disorder is selected from hypercholesterolemia, dyslipidemia, hypertriglyceridemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes, and obesity.

Description

[Technical Field]

[0001] The present disclosure relates to methods of treating, preventing, or managing cardiovascular and / or metabolic disorders in a human subject in need thereof by administering a human fibroblast growth factor (FGF21) protein variant that is an FGF21-Fc fusion protein.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 382,584, filed November 7, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted via the Patent Center in XML format. The contents of the XML file named "BPH-006PC_Sequence_Listing.xml," created on October 18, 2023, and having a size of 2,301 bytes, are incorporated herein by reference in their entirety. [Background technology]

[0004] background Fibroblast growth factor 21 (FGF21), a member of the fibroblast growth factor (FGF) family, regulates energy balance and glucose and lipid homeostasis through a heterodimeric receptor complex containing FGF receptor 1 (FGFR1) and β-Klotho. FGF21 is released primarily from hepatocytes and, to a lesser extent, from adipocytes and pancreatic β-cells. Preclinical animal studies have shown that FGF21 may have many beneficial pleiotropic effects, including reducing fat mass, improving glucose control, insulin resistance, dyslipidemia, and ameliorating nonalcoholic fatty liver disease (NAFLD) models.

[0005] Nonalcoholic fatty liver disease (NAFLD) is a term used to describe a broad range of conditions caused by the accumulation of fat within liver cells, resulting from excessive calorie intake due to poor diet and lack of exercise, or from complications involving an imbalance in glucose-lipid metabolism. NAFLD is prevalent, affecting one-third of the US population, and is associated with other metabolic diseases such as obesity, type 2 diabetes mellitus (T2DM), and hyperlipidemia. NAFLD can progress to nonalcoholic steatohepatitis (NASH), where excess liver fat leads to hepatocyte stress, thereby inducing local inflammation and, as the disease progresses, leading to fibrosis and ultimately cirrhosis. NASH can progress to cirrhosis and liver failure, which are the leading causes of liver transplantation, death, and the development of hepatocellular carcinoma (HCC). Furthermore, patients with NASH have a high incidence of cardiovascular events, such as stroke and heart attack, and cardiovascular disease is the leading cause of death in NASH patients. The prevalence of NASH in the United States is projected to increase from an estimated 17.3 million in 2016 to 27 million in 2030 (Estes C. et al. Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016-2030. J Hepatol. 2018;69(4):896-904).

[0006] Currently, there are no approved treatments for NASH, but numerous molecules are in development that represent many different mechanisms of action that target various aspects of NASH pathogenesis. 1 / 2To overcome this, several long-acting FGF21 analogs have been developed. Several FGF21 analogs and mimetics have progressed to early-stage clinical trials for obesity, T2DM, and NASH. These studies have demonstrated significant improvements in serum biomarkers of dyslipidemia, hepatic steatosis, and liver fibrosis in patients with NASH. More recently, phase 2a data for the FGF21 analog effluxifermin (EFX) suggest that improving hepatic fat translates to improved liver histology (Akero Therapeutics, Inc. Corporate Presentation: A Global Disease, A Pioneering Treatment. January 2021. Available online at https: / / ir.akerotx.com / static-files / d5a2bd98-8bad-412a-97d7-0b2bce1a0f13). Summary of the Invention

[0007] overview This disclosure is based, at least in part, on the protocol and results of a 12-week, Phase 2a, randomized, blinded, placebo-controlled study of BOS-580 (V103), an FGF21 protein variant, in obese subjects at risk for nonalcoholic steatohepatitis (NASH). The objective of this study was to evaluate the exposure-response relationship of BOS-580 based on dose and exposure, as well as biomarkers of tolerability and efficacy.

[0008] The human FGF21 protein variant BOS-580(V103) is as described in WO 2013 / 049247, WO 2015 / 138278, and WO 2019 / 123427, the contents of which are incorporated herein by reference. BOS-580(V103) is stabilized through the introduction of a novel disulfide bond and fused at its N-terminus to a human immunoglobulin G1 (IgG1) Fc fragment.

[0009] The amino acid sequence of BOS-580(V103) is shown in SEQ ID NO: 1. It comprises, from N- to C-terminus, an immunoglobulin Fc fragment, a linker GS, and a native mature human FGF21 variant representing amino acid positions 29-209 of the full-length FGF21 protein sequence (NCBI Reference SEQ ID NO: NP_061986.1) with the amino acid substitutions Q55C, R105K, G148C, K150R, P158S, S195A, P199G, G202A (numbering based on the full-length sequence).

[0010] A first aspect of the present disclosure relates to a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: for use in a method of treating, preventing, or managing a cardiovascular and / or metabolic disease in a human subject in need thereof, wherein the human FGF21 protein variant is provided at a dose of 75 mg for subcutaneous administration once every two weeks.

[0011] A further aspect of the present disclosure relates to the use of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for treating, preventing, and managing cardiovascular or metabolic disease in a human subject in need thereof, wherein the human FGF21 protein variant is provided at a dose of 75 mg for subcutaneous administration once every two weeks.

[0012] Yet another aspect of the present disclosure relates to a method of treating, preventing, or managing cardiovascular or metabolic disease in a human subject in need thereof, comprising subcutaneously administering to the human subject a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO:1 at a dose of 75 mg once every two weeks.

[0013] In some embodiments of any of the aforementioned aspects, the metabolic and / or cardiovascular disease is selected from hypercholesterolemia, dyslipidemia, hypertriglyceridemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes, and obesity.

[0014] In some embodiments of any of the aforementioned aspects, the metabolic and / or cardiovascular disease is non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH).

[0015] In some embodiments of any of the aforementioned aspects, the metabolic and / or cardiovascular disease is non-alcoholic steatohepatitis (NASH).

[0016] In one embodiment of any of the foregoing aspects, the human subject is obese.

[0017] In some embodiments of any of the aforementioned aspects, the human subject has a blood cholesterol level of at least 27.5 kg / m for a subject of Asian descent or Asian ancestry. 2 At least 30 kg / m with ethnic adjustment 2 Body mass index (BMI), especially 30 kg / m 2 ~45kg / m 2 have a BMI of.

[0018] In some embodiments of any of the aforementioned aspects, the human subject is suffering from and / or at risk of acquiring NASH.

[0019] In some embodiments of any of the foregoing aspects, the human subject has a liver fibrosis stage selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis), and / or F4 (cirrhosis), particularly F2 and / or F3.

[0020] In some embodiments of any of the aforementioned aspects, the human subject has a liver fat assessment based on a vibration-controlled transient elastography (VCTE) controlled attenuation parameter (CAP) score of greater than 300 dB / m, or greater than 400 dB / m.

[0021] In some embodiments of any of the aforementioned aspects, the human subject has a liver injury and fibrosis assessment based on a VCTE liver stiffness measurement (LSM) score of between 7-14 kPa, 7 kPa-12 kPa, or 7 kPa-9.9 kPa, and / or an aspartate aminotransferase (AST) activity of at least 20 U / L.

[0022] In some embodiments of any of the aforementioned aspects, the human subject further suffers from type 2 diabetes, hypertension, and / or hyperlipidemia.

[0023] In some embodiments of any of the aforementioned aspects, the administration is effective to improve at least one physiological parameter associated with NASH.

[0024] In some embodiments of any of the aforementioned aspects, the at least one physiological parameter is selected from liver fibrosis stage, liver fat assessment based on VCTE CAP, liver damage and fibrosis assessment based on VCTE, LSM score, and AST activity.

[0025] In some embodiments of any of the aforementioned aspects, the administration is effective to reduce liver fat, and is particularly effective to reduce liver fat content by at least 45%, at least 50%, or at least 60%.

[0026] In some embodiments of any of the foregoing aspects, the administration is effective to reduce liver damage, and in particular, is effective to reduce adiponectin deficiency, e.g., by increasing adiponectin levels by at least 50%, 75%, 100%, or 125%.

[0027] In some embodiments of any of the foregoing aspects, administration is effective in reducing liver fibrosis, and in particular is effective in reducing average alanine aminotransferase (ALT) by at least 20%, at least 30%, or at least 35%, and / or is effective in reducing aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or is effective in reducing average procollagen amino-terminal propeptide (ProC3) by at least 15% or at least 20%.

[0028] In some embodiments of any of the aforementioned aspects, the administration is effective to reduce HbA1c levels in a subject with type 2 diabetes by at least 0.6%.

[0029] In some embodiments of any of the aforementioned aspects, the administration has a Grade 1 or 2 gastrointestinal tolerability profile.

[0030] In some embodiments of any of the aforementioned aspects, the human FGF21 protein variant is administered in combination with one or more additional therapeutically active agents.

[0031] In some embodiments of any of the foregoing aspects, the one or more additional therapeutically active agents are selected from the group consisting of compounds useful in treating obesity, diuretics, beta-blockers, alpha-blockers, ACE inhibitors, angiotensin II receptor blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides such as GLP-1 agonists and dual GLP-1 and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics, statins, fibrates, aspirin, and anticoagulants, etc.

[0032] In some embodiments of any of the aforementioned aspects, the human FGF21 protein variant is aglycosylated.

[0033] In some embodiments of any of the aforementioned aspects, the human FGF21 protein variant is glycosylated.

[0034] In some embodiments of any of the aforementioned aspects, the human FGF21 protein variant consists of the amino acid sequence of SEQ ID NO:1. [Brief explanation of the drawings]

[0035] DESCRIPTION OF THE DRAWINGS [Figure 1A] 1 shows that administration of BOS-580 (SEQ ID NO: 1) did not result in adverse effects on lipid profiles. [Figure 1B] 1 shows that administration of BOS-580 (SEQ ID NO: 1) did not result in adverse effects on lipid profiles. [Figure 1C] 1 shows that administration of BOS-580 (SEQ ID NO: 1) did not result in adverse effects on lipid profiles. [Figure 1D] 1 shows that administration of BOS-580 (SEQ ID NO: 1) did not result in adverse effects on lipid profiles. [Figure 2] This indicates the occurrence of TEAEs in the gastrointestinal system. [Figure 3] Absolute change in HFF as determined by magnetic resonance imaging proton density fat fraction (MRI-PDFF) is shown. The normal reference range for MRI-PDFF is <5.6%. [Figure 4] The relative changes in HFF determined by MRI-PDFF are shown. [Figure 5] The percentage of patients who achieved a particular degree of fat loss is shown. [Figure 6] The levels of alanine aminotransferase (ALT), a biomarker of liver damage, are shown. [Figure 7]1 shows levels of aspartate aminotransferase (AST), a biomarker of liver damage. [Figure 8] 1 shows the levels of the amino-terminal propeptide of procollagen 3 (ProC3), a biomarker of liver fibrosis. [Figure 9] This indicates the degree of adiponectin deficiency. Adiponectin is an adipose-specific adipokine that reduces insulin resistance and suppresses liver inflammation / fibrosis. [Figure 10] HbA1c values ​​in diabetic NASH patients are shown. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description Disclosed herein are methods, compounds, and pharmaceutical compositions for use in treating, preventing, or managing (e.g., alleviating one or more signs and / or symptoms of) a metabolic and / or cardiovascular disorder, comprising subcutaneously administering to a human subject in need thereof a human FGF21 protein variant consisting of the amino acid sequence of SEQ ID NO: 1, or consisting of the amino acid sequence of SEQ ID NO: 1, at a dose of 75 mg once every two weeks.

[0037] Typically, human FGF21 protein variants comprising or consisting of the amino acid sequence of SEQ ID NO: 1 are obtained by expression in recombinant host cells transfected or transformed with a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. The recombinant host cells can be cultured in an appropriate culture medium, and the polypeptide can be isolated from the host cells or culture medium. The host cells can be prokaryotic or eukaryotic, such as insect or mammalian cells. Depending on the type of host cell, the human FGF21 protein variants can be glycosylated or aglycosylated / non-glycosylated.

[0038] The term "managing" is understood as the management and care of a patient with the goal of combating a disease, condition, or disorder, without resulting in a cure, but resulting in the alleviation of one or more symptoms of the disease, condition, or disorder and / or a reduction in hospital stay. In embodiments, "managing" or "management" with respect to a condition or disease includes the maintenance of symptomatology, including using one or more indexes or scores (e.g., as described herein), and / or the maintenance of one or more disease biomarkers (e.g., as described herein).

[0039] The term "preventing" refers to prophylactic administration to a healthy subject or a subject at risk of developing a disease, condition, or disorder described herein (e.g., a subject considered to be prediabetic or a subject with liver fibrosis at risk of developing NASH) to prevent the onset of one or more conditions referred to herein. Furthermore, the term "preventing" can also include prophylactic administration to a patient at a pre-stage of the condition being treated. In embodiments, "preventing" or "prevention" with respect to a condition or disease includes preventing the worsening of one or more symptoms and / or maintaining one or more disease biomarkers (e.g., as described herein), including using one or more indexes or scores (e.g., as described herein).

[0040] The term "treating" is understood as the management and care of a patient for the purpose of combating a disorder, such as, for example, reducing or ameliorating the progression, severity, and / or duration of a disease, disorder, or condition.

[0041] The term "metabolic disorder" refers to a disease, disorder, or condition associated with pathological metabolic parameters, such as elevated blood pressure, hyperglycemia, excess body and / or organ fat, abnormal cholesterol levels, abnormal triglyceride levels, and combinations thereof. Metabolic disorders may increase the risk of heart failure and stroke.

[0042] The term "cardiovascular disorder" refers to a disease, disorder, or condition of the heart and / or vascular system.

[0043] Non-limiting examples of metabolic and / or cardiovascular disorders that may be treated, prevented, or managed by the disclosure provided herein include hypertriglyceridemia, diabetes, e.g., type 2 diabetes, obesity, type 1 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease, acute myocardial infarction, atherosclerosis, peripheral artery disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, diabetic neuropathy, disorders associated with severe inactivating mutations in the insulin receptor (e.g., as described in Taylor et al., Diabetes Care 13 (1990), 257-279, the contents of which are incorporated herein by reference), and / or gastroparesis.

[0044] In certain embodiments, the metabolic and / or cardiovascular disorder is selected from hypercholesterolemia, dyslipidemia, hypertriglyceridemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes, and obesity.

[0045] In certain embodiments, the metabolic and / or cardiovascular disorder is selected from non-alcoholic steatohepatitis (NASH).

[0046] The term "NAFLD" or "non-alcoholic fatty liver disease" is defined as the accumulation of excess fat in the liver. This fat accumulation is not caused by heavy alcohol use. When heavy alcohol use causes fat accumulation in the liver, it is called alcoholic liver disease. There are usually two types of NAFLD: simple fatty liver and non-alcoholic steatohepatitis (NASH).

[0047] The term "NASH" or "nonalcoholic steatohepatitis" is defined as a form of nonalcoholic fatty liver disease (NAFLD) in which patients have hepatitis—inflammation of the liver—and liver cell damage in addition to intrahepatic fat. Inflammation and liver cell damage can lead to liver fibrosis, or scarring. In some cases, NASH can lead to cirrhosis or liver cancer. Complicated cirrhosis is called "decompensated" cirrhosis and is one of the leading reasons for liver transplantation.

[0048] The term "simple fatty liver," also known as nonalcoholic fatty liver (NAFL), describes a form of NAFLD in which there is fat in the liver but little or no inflammation or hepatocellular damage. Simple fatty liver can progress to NASH in a certain percentage of individuals.

[0049] In certain embodiments, the human subject is obese. A measure of obesity is the body mass index BMI = weight (kg) / [height (m)] 2 is a person's weight (kg) divided by the square of their height (m). Individuals with a BMI of 30 or greater are generally considered obese, but this guideline can be adjusted for ethnic differences. For example, after adjusting for ethnicity, a BMI of 27.5 can be considered obese for Asians (WHO Expert Consultation, 2004, Lancet, 363(9403):157-63). An Asian can be, for example, a subject of Asian descent or of Asian ancestry.

[0050] In certain embodiments, the human subject has a body mass of at least 27.5 kg / m, adjusted for ethnicity for subjects of Asian or Asian ancestry. 2 is at least 30kg / m 2 Body mass index (BMI), e.g., 30 kg / m 2 ~45kg / m 2 In embodiments, the BMI is about or at least about 20 kg / m 2 , or at least about 30 kg / m 2 , about or at least about 35 kg / m 2, about or at least about 40 kg / m 2 , about or at least about 50 kg / m 2 In embodiments, the BMI is about or at least about 20 kg / m 2 ~approximately or at least approximately 25 kg / m 2 , about or at least about 25 kg / m 2 ~approximately or at least approximately 30 kg / m 2 , about or at least about 30 kg / m 2 ~approximately or at least approximately 35 kg / m 2 , about or at least about 35 kg / m ) ~approximately or at least approximately 40 kg / m 2 , about or at least about 40 kg / m 2 ~approximately or at least approximately 45 kg / m 2 , or about or at least about 45 kg / m 2 ~approximately or at least approximately 50 kg / m 2 Includes the range.

[0051] In embodiments, the subject for treatment has been diagnosed with NASH, is suspected to be diagnosed with NASH, and / or is at risk of acquiring NASH.In embodiments, the subject diagnosed with NASH, is suspected to be diagnosed with NASH, and / or is at risk of NASH is prediabetic.In embodiments, the subject diagnosed with NASH, is suspected to be diagnosed with NASH, and / or is at risk of NASH shows one or more demographic and / or lifestyle factors that are used to select the subject for the treatment method herein, including: overweight, obesity, or associated comorbidities (for example, based on BMI, waist circumference, weight, prediabetes); age (for example, age 45 or above); African American, Pacific Islander American, or Hispanic / Latin American; have a family history of diabetes; have a sedentary lifestyle or lack of physical activity; have high blood pressure and / or abnormal cholesterol levels; have a personal history of gestational diabetes, heart disease, stroke, and / or polycystic ovarian syndrome (PCOS). In embodiments, the methods herein are intended to prevent the evolution of a disease or condition from an early stage (e.g., from prediabetes to diabetes, from NAFLD to NASH, etc.) and / or to alleviate the accumulation of damage due to lifestyle factors and / or risk factors. In embodiments, the prevention methods herein are designed for healthy or substantially healthy individuals to halt or slow the progression of disease, or prevent NASH to a more severe stage of NASH, etc. In embodiments, the accumulation of damage, the presence of lifestyle factors and / or risk factors, and any associated improvements thereto can be measured as described herein.

[0052] In embodiments, the diagnosis, suspected diagnosis, and / or risk of NASH is confirmed or assessed by the results of a liver biopsy. In embodiments, NASH is diagnosed when microscopic analysis of tissue shows fat accumulation, inflammation, and damage to liver cells. In embodiments, subjects with liver tissue showing fat without inflammation and damage are considered to have a diagnosis of simple fatty liver or NAFLD, and may also be at risk of developing NASH. In embodiments, the diagnosis of NASH and / or suspected NASH includes evaluating the results of blood tests, for example, using biomarkers such as liver enzymes alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST), platelet count, procollagen amino-terminal propeptide (ProC3), and / or hemoglobin A1C (HbA1c). In embodiments, diagnosing NASH, diagnosing suspected NASH, and / or assessing the risk of NASH involves using a scoring rubric / index such as, for example, the Fibrosis-4 (FIB-4) index of liver fibrosis, where the subject has an FIB-4 index of about or at least about 1.5, about or at least about 2.0, about or at least about 2.5, about or at least about 3.0, or about or at least about 3.5. In embodiments, diagnosing NASH, diagnosing suspected NASH, and / or assessing the risk of NASH involves using a scoring rubric / index such as, for example, the AST-to-Platelet Ratio Index (APRI), where the subject has an APRI of about or at least about 0.25, about or at least about 0.5, about or at least about 0.75, about or at least about 1.0, about or at least about 1.25, or about or at least about 1.5.

[0053] In embodiments, subjects diagnosed with NASH, suspected of NASH, and / or at risk for NASH have one or more of the following: enlarged liver, enlarged spleen, or ascites (e.g., as observed by imaging of internal organs and peripheral tissues), signs of insulin resistance (e.g., as observed by darkened skin patches on the extremities), signs of liver cirrhosis, and / or muscle loss.In embodiments, imaging techniques for determining liver fibrosis or inflammation, enlarged organs (liver, spleen), ascites or fluid accumulation, and / or muscle loss include ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and / or elastography imaging, such as vibration-controlled transient elastography, shear wave elastography, and / or magnetic resonance elastography. In embodiments, measurements of insulin resistance include a euglycemic hyperinsulinemic test, a fasting plasma glucose (FPG) test (e.g., glucose above 100-125 mg / dL), an A1C test (e.g., 5.7%-6.4% or above), and / or an oral glucose tolerance test (OGTT) (e.g., glucose above 140-199 mg / dL).

[0054] In certain embodiments, human subject suffers from NASH and / or is at risk of acquiring NASH.In such embodiments, human subject can have liver fibrosis stage (for example, using Batts-Ludwig criteria) selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), particularly F2 and / or F3.

[0055] In certain embodiments, a human subject, for example, a human subject suffering from and / or at risk of acquiring NASH, may be characterized by one or more of the following physiological parameters: Liver fat assessment score based on vibration-controlled transient elastography (VCTE) controlled attenuation parameters (CAP) above 300 dB / m, especially above 400 dB / m; liver injury and fibrosis assessment based on a VCTE liver stiffness measurement (LSM) score of at least 7 kPa, e.g., 7 to 14 kPa, 7 kPa to 12 kPa, or 7 kPa to 9.9 kPa; aspartate aminotransferase (AST) activity of at least 20 U / L; a NAFLD fibrosis (NFS) score of at least 1.455 indicating intermediate or high likelihood of fibrosis (calculated using age, AST / ALT ratio, platelet count, BMI, albumin (g / L), and impaired fasting glucose / diabetes (+1 if present)); A FIB-4 (fibrosis-4) score (age × AST) / platelet count (10) of at least 1.3 indicating advanced fibrosis 9 / L) × square root of ALT) an APRI index (ALT / platelet ratio index) greater than 0.5 indicating a moderate or high likelihood of advanced fibrosis; a FIBROSURE value of 0.31 to >0.58 (a serum test combining five biomarkers: haptoglobin, α2-macroglobulin, apolipoprotein A1, total bilirubin, and gamma-glutamyl-transferase) indicating moderate fibrosis, or a FibroSure value >0.58 indicating advanced fibrosis; an enhanced liver fibrosis (ELF) value of 7.7 to less than 9.8 indicating moderate fibrosis (mild to advanced fibrosis) or at least 9.8 indicating advanced fibrosis (combining three biomarkers of fibrosis: hyaluronic acid, tissue inhibitor of metalloproteinase 1, and amino-terminal peptide of procollagen 3); a magnetic resonance elastography (MRE) score of 2.97–3.62 indicating fibrosis stage F2, or greater than 3.62 indicating advanced fibrosis (F3); A FIBROSCAN-AST (FAST) score of greater than 0.67 indicating a high risk of progression or less than 0.35 indicating a low risk of progression.

[0056] In embodiments, methods for preventing or managing cardiovascular and / or metabolic disorders (e.g., NASH) include substantially maintaining the appearance of a liver biopsy result, substantially maintaining the concentration and / or activity of ASL, AST, ProC3, and / or HbA1C, as described herein; substantially maintaining a platelet count; substantially maintaining the FIB-4 index, NFS score, APRI, FIBROSURE value, ELF value, MRE score, and / or FAST score; substantially maintaining the Batts-Ludwig criteria, euglycemic-hyperinsulinemic test, FPG test, A1C test, or OGTT assessment or results; substantially maintaining a VCTE, CAP score, LSM score; and / or substantially maintaining the severity and / or frequency of one or more symptoms associated with a condition, disease, or comorbidity. In embodiments, "substantially maintaining" refers to a change or deviation of less than about 1%, 5%, or 10%, for example, in a qualitative or quantitative measure.

[0057] In certain embodiments, the human subject, e.g., a human subject suffering from and / or at risk of acquiring NASH, is further diagnosed with, suspected of being diagnosed with, and / or at risk of acquiring type 2 diabetes, hypertension, and / or hyperlipidemia. In certain embodiments, the human subject, e.g., a human subject suffering from and / or at risk of acquiring NASH, is further suffering from type 2 diabetes.

[0058] In certain embodiments, administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 is effective in improving at least one physiological parameter associated with a metabolic and / or cardiovascular disorder treated, prevented, or managed by the disclosure provided herein. In certain embodiments, administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 is effective in improving at least one physiological parameter associated with NASH.

[0059] In a human subject suffering from NASH and / or at risk of acquiring NASH, administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 may be effective in improving at least one physiological parameter selected from liver fibrosis stage, liver fat assessment based on VCTE CAP, liver damage and fibrosis assessment based on VCTE, LSM score, and AST activity.

[0060] In certain embodiments, administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a period of 12 weeks (starting from the first administration), is effective in reducing liver fat, particularly in reducing liver fat content by at least 45%, at least 50%, or at least 60%.

[0061] In certain embodiments, administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a period of 12 weeks (starting from the first administration), is effective in reducing liver damage, particularly reducing adiponectin deficiency, for example by increasing adiponectin levels by at least 50%, at least 75%, at least 100% or at least 125%.

[0062] In certain embodiments, administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a period of 12 weeks (starting from the first administration), is effective in reducing liver damage, particularly effective in reducing mean alanine aminotransferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or effective in reducing mean aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or effective in reducing mean procollagen amino-terminal propeptide (Pro-C3) levels by at least 15% or at least 20%.

[0063] In subjects suffering from and / or at risk of acquiring NASH, administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a period of 12 weeks (starting from the first administration), may be effective in reducing HbA1c levels in subjects suffering from type 2 diabetes, for example by at least 0.6%.

[0064] In certain embodiments, administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a period of 12 weeks (starting from the first administration), has a grade 1 or 2 gastrointestinal tolerability profile.

[0065] The human FGF21 protein variant of SEQ ID NO: 1 can be administered as a monotherapy or in combination with one or more additional therapeutically active agents. In some examples, the one or more additional therapeutically active agents are compounds useful in treating obesity (e.g., phentermine / topiramate, orlistat, lorcaserin, liraglutide, bupropion / naltrexone, and combinations thereof), compounds useful in treating hypertension (e.g., diuretics, beta-blockers, alpha-blockers, ACE inhibitors, angiotensin II receptor blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, and combinations thereof), compounds useful in treating diabetes (e.g., insulin, alpha ... -glucosidase inhibitors, biguanides, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides such as GLP-1 agonists and GLP-2 agonists such as semaglutide, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics and combinations thereof), compounds useful in NAFLD / NASH and cardiovascular treatment (e.g. statins, fibrates, aspirin, anticoagulants and / or combinations thereof).

[0066] In one aspect, provided herein is a combination therapy for treating, preventing, or managing metabolic and / or cardiovascular disorders comprising administering a human FGF21 protein variant of SEQ ID NO: 1 and one or more therapeutically active agents selected from the following: amiloride (Midamor), bumetanide (Bumex), chlorthalidone (Hygroton), chlorothiazide (Diuril), furosemide (Lasix), hydrochlorothiazide, or HCTZ. (Esidrix, Hydrodiuril, Microzide), indapamide (Lozol), metolazone (Mykrox, Zaroxolyn), spironolactone (Aldactone), triamterene (Dyrenium), acebutolol (Sectral), atenolol (Tenormin), betaxolol (Kerlone), bisoprolol (Zebeta), carteolol (Cartrol), metoprolol (Lopressor, Toprol) XL), nadolol (Corgard), nebivolol (Bystolic), penbutolol (Levatol), pindolol (Visken), propranolol (Inderal), sotalol (Betapace), timolol (Blocadren), doxazosin (Cardura), prazosin (Minipress), terazosin (Hytrin), benazepril (Lotensin), captopril (Capoten), enalapril (Vasotec), lisinopril (Monopril), lisinopril (Prinivil, Zestril), moexipril (Univasc), perindopril (Aceon), quinapril (Accupril), ramipril (Altace), trandolapril (Mavik), Norvasc (amlodipine), Plendil (felodipine), DynaCirc (isradipine), Cardene (nicardipine), Procardia XL, Adalat (nifedipine), Cardizem, Dilacor, Tiazac, DiltiaXL (diltiazem), Sular (nisoldipine), Isoptin, Calan, Verelan, Covera-HS (verapamil), Capoten (captopril), Vasotec (enalapril), Prinivil, Zestril (lisinopril), Lotensin (benazepril), Monopril (lisinopril), Altace (ramipril), Accupril (quinapril), Aceon (perindopril), Mav ik (trandolapril), Univasc (moexipril), Atacand (candesartan), Avapro (irbesartan), Benicar (olmesartan), Cozaar (losartan), Diovan (valsartan), Micardis (telmisartan), Teveten (eprosartan), chlorthalidone (Hygroton), chlorothiazide (Diuril), hydrochlorothiazide or HCTZ (Esidrix, Hydrodiuril, Microzide), indapamide (Lozol), metolazone (Mykrox,Zaroxolyn), amiloride (Midamor), bumetanide (Bumex), furosemide (Lasix), spironolactone (Aldactone), triamterene (Dyrenium), acebutolol (Sectral), atenolol (Tenormin), betaxolol (Kerlone), bisoprolol (Zebeta, Ziac), carteolol (Cartrol), carvedilol (Coreg), labetalol (Normodyne, Trandate), metoprolol (Lopressor, Toprol-XL), nadolol (Corgard), nebivolol (Bystolic), penbutolol (Levatol), pindolol (Visken), propranolol (Inderal), sotalol (Betapace), timolol (Blocadren), fibrinogen Phosphate derivatives, niacin, and omega-3 fatty acids, fenofibrate, gemfibrozil, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, pramlintide, acarbose (Precose), miglitol (Glyset), metformin, bromocriptine, alogliptin, linagliptin, saxagliptin, sitagliptin doxycycline, albiglutide (Tanzeum), dulaglutide (Trulicity), exenatide (Byetta), exenatide extended-release (Bydureon), liraglutide (Victoza), nateglinide (Starlix), repaglinide (Prandin), repaglinide-metformin (Prandimet), dapagliflozin (Farxiga), dapagliflozin-metformin (Xigduo)XR), canagliflozin (Invokana), canagliflozin-metformin (Invokamet), empagliflozin (Jardiance), empagliflozin-linagliptin (Glyxambi), empagliflozin-metformin (Synjardy), sotagliflozin, tofogliflozin, remogliflozin, luseogliflozin, ipragliflozin, atigliflozin, bexagliflozin, henagliflozin, lico gliflozin, glimepiride (Amaryl), glimepiride-pioglitazone (Duetact), glimepiride-rosiglitazone (Avandaryl), gliclazide, glipizide-metformin (Metaglip), glyburide (DiaBeta, Glynase, Micronase), glyburide-metformin (Glucovance), chlorpropamide (Diabinese), tolazamide (Tolinase), tolbutamide (Orinase, Tol-Tab), rosiglitazone (Avandia), rosiglitazone-glimepiride (Avandaryl), rosiglitazone-metformin (Amaryl M), pioglitazone (Actos), pioglitazone-alogliptin (Oseni), pioglitazone-glimepiride (Duetact), pioglitazone-metformin (Actoplus Met, Actoplus Met XR).

[0067] In some embodiments, the sodium glucose transporter (SGLT) inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, ertugliflozin, sotagliflozin, tofogliflozin, remogliflozin, luseogliflozin, ipragliflozin, atigliflozin, bexagliflozin, henagliflozin, licogliflozin, and pharmaceutically acceptable salts of any of these. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is dapagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is empagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is canagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is ertugliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is licogliflozin.

[0068] According to certain embodiments, administration of the human FGF21 protein variant of SEQ ID NO: 1 is accompanied by monitoring of FGF21 biological activity, for example, as described in WO 2013 / 049247, the entire contents of which are incorporated herein by reference.

[0069] In certain embodiments, at least one biomarker of liver function is evaluated to determine the success of treatment.Examples of biomarkers of liver function include but are not limited to alanine aminotransferase (ALT), aspartate aminotransferase (AST), amino-terminal propeptide of procollagen 3 (ProC3), GGT, and alkaline phosphatase.In further embodiments, at least one biomarker of glucose metabolism, such as HbA1c, is evaluated.

[0070] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a human FGF21 protein variant of SEQ ID NO: 1 for use in a method for treating, preventing and / or managing metabolic and / or cardiovascular disorders, e.g., hypertriglyceridemia and cardiac risk, insulin resistance, diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD) / non-alcoholic steatohepatitis (NASH), such as in patients with insulin receptor genetic mutations and lipodystrophy.

[0071] The pharmaceutical composition described herein comprises a therapeutically effective amount of the human FGF21 protein variant of SEQ ID NO: 1 and a pharmaceutically acceptable carrier suitable for subcutaneous administration. Usually, the pharmaceutical composition is in the form of a sterile, pyrogen-free parenterally acceptable composition. A particularly suitable excipient for parenteral injection is a sterile, isotonic solution that is properly preserved. The pharmaceutical composition can be in the form of a lyophilized product, such as a lyophilized cake. Suitable methods and ingredients for pharmaceutical formulation are well known (see, for example, Allen, Lloyd V. Ed., (2012) Remington's Pharmaceutical Sciences, 22nd Edition, which is incorporated by reference for this purpose).

[0072] In certain embodiments, the pharmaceutical composition comprises at least one unit dose of 75 mg of the human FGF21 protein variant of SEQ ID NO: 1 administered subcutaneously every two weeks.

[0073] The pharmaceutical compositions provided herein are for subcutaneous administration.Suitable formulation components and methods for subcutaneous administration of polypeptide therapeutics (such as antibodies, fusion proteins, etc.) are known in the art (see, for example, US 2011 / 0044977 A1, US 8,465,739 B2 and US 8,476,239 B2, each of which is incorporated by reference for this purpose).Typically, pharmaceutical compositions for subcutaneous administration include suitable stabilizers (such as amino acids such as methionine, and / or sugars such as sucrose), buffers, and / or tonicifiers.

[0074] In certain embodiments, the pharmaceutical composition comprises the human FGF21 protein variant of SEQ ID NO: 1 as a dry formulation, eg, a lyophilizate, that can be reconstituted as a solution prior to subcutaneous administration.

[0075] In certain embodiments, the pharmaceutical composition is in the form of a solution of about 50 mg / mL to about 150 mg / mL or about 100 mg / mL to about 150 mg / mL of an FGF21 protein variant (eg, after reconstitution of a lyophilized product).

[0076] In certain embodiments, the pharmaceutical composition comprises trometamol buffer at a concentration of, for example, 10 mM to 50 mM trometamol buffer, for example, 30 mM trometamol buffer (e.g., after reconstitution of a lyophilizate).

[0077] In certain embodiments, the pharmaceutical composition is in the form of a solution (eg, after reconstitution of a lyophilizate) comprising sucrose, eg, about 200 mM to about 300 mM sucrose, eg, 270 mM sucrose.

[0078] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) comprising a polysorbate, such as about 0.01% (w / w) to about 0.10% (w / w) polysorbate 20, e.g., 0.06% (w / w) polysorbate 20.

[0079] In certain embodiments, the pharmaceutical composition is in the form of a solution (eg, after reconstitution of a lyophilisate) at a pH in the range of 6.5 to 9, for example, a pH of about 8.0.

[0080] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilized product) comprising trometamol buffer, sucrose, and polysorbate 20, e.g., about 80 mg / mL to 100 mg / mL of an FGF21 protein variant, about 30 mM trometamol buffer, 270 mM sucrose, and 0.06% polysorbate 20, e.g., a pH of 8.0, or a pH of 7.5 to 8.5.

[0081] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) comprising about 100 mg / mL of an FGF21 protein variant, about 30 mM trometamol buffer, 270 mM sucrose, and 0.06% polysorbate 20, at a pH of 8.0.

[0082] The present invention will now be described in more detail with reference to the following examples. [Example]

[0083] Example Example 1: Study Protocol: A 12-week, Phase 2a, randomized, blinded, placebo-controlled study of BOS-580 in obese subjects at risk for nonalcoholic steatohepatitis (NASH)

[0084] 1.1 Abbreviations and Definitions

[0085] [Table 1-1]

[0086] [Table 1-2]

[0087] [Table 1-3]

[0088] 1.2 Rationale The objective of this study was to evaluate the exposure-response relationship of BOS-580 (SEQ ID NO: 1) based on BOS-580 dose and exposure, as well as biomarkers of tolerability and efficacy. These data were used to confirm the benefit / risk profile and dosing rationale of BOS-580 to support its progression to late-stage development for the treatment of NASH.

[0089] 1.3 Objectives and Endpoints

[0090] [Table 2-1]

[0091] [Table 2-2]

[0092] 1.4 Overall design This is a two-part, multicenter, randomized, double-blind (sponsor open-label), placebo-controlled, safety study to evaluate subcutaneously administered BOS-580 over a 12-week repeated dose period. 2 We included up to 105 male and / or female subjects aged 18–75 years inclusive, with a body mass index (BMI) of ≥ 10%, an MRI-PDFF of ≥ 10%, a VCTE LSM of 7–9.9 kPa inclusive, and an AST of > 20 U / L.

[0093] Part A consisted of five cohorts (A1-A4, with A5 randomized separately) totaling approximately 75 subjects. Subjects were initially randomized into cohorts A1 to A4, with approximately 15 subjects per cohort. Subjects in cohorts A1 to A4 were then randomized within each cohort in a 4:1 ratio (BOS-580 vs. PBO). After cohorts A1 to A4 were randomized, approximately 15 subjects would be randomized in a 4:1 ratio (BOS-580 vs. PBO) to cohort A5.

[0094] Subjects returned to the study center periodically over a 12-week period for study drug administration, pharmacokinetic (PK), biomarker, and safety assessments.

[0095] In Part A, BOS-580 or PBO was administered subcutaneously as follows: Cohort A1: 300 mg once every 4 weeks (Q4W) (300 mg / month) or PBO. Cohort A3: 75 mg Q2W (150 mg / month) or PBO. Cohort A4: 75 mg Q4W (75 mg / month) or PBO.

[0096] Additional cohorts may be initiated in Part B to explore additional doses, dosing frequencies, or dose escalation, following data from the week 6 interim analysis of cohorts A1, A3, and A4.

[0097] In Parts A and B, subjects return to the study center for follow-up approximately 4 weeks after receiving their last study dose.

[0098] 1.5 Number of subjects Subjects were enrolled such that approximately 75 evaluable subjects completed Part A. Approximately 30 subjects may be enrolled in Part B (optional).

[0099] 1.6 Inclusion / Exclusion Criteria

[0100] [Table 3-1]

[0101] [Table 3-2]

[0102] [Table 3-3]

[0103] [Table 3-4]

[0104] [Table 3-5]

[0105] 1.7 Study duration and duration The study consisted of two screening periods. Subjects who met eligibility criteria at Screening I (days -42 to -10) proceeded to designated imaging evaluations at Screening II (days -28 to -7). The study treatment period consisted of biweekly or monthly doses (a weekly regimen could be considered for optional Part B). Post-treatment follow-up included a visit approximately 4 weeks after receiving the final study dose. The total duration of each subject's involvement was approximately 22 weeks.

[0106] 1.8 Test Analysis The following analyses were performed: The interim analysis was conducted when approximately 90% of subjects randomized to cohorts A1-A4 completed the week 6 visit. The data were used to guide sponsor-led decision-making regarding the clinical program. Based on the results of the week 6 interim analysis, an additional interim analysis may be conducted once all subjects in cohorts A1-A4 have completed week 12 of treatment.

[0107] Data from any subject enrolled in Part B may be included in the end-of-treatment 12-week analysis described above. Depending on the timing of when all subjects complete the End-of-Study Visit (Week 16) in Part A and when all subjects complete the End-of-Study Visit in Part B, the final analyses of Parts A and B may be completed simultaneously or separately. Additional analyses may also be performed if necessary.

[0108] Example 2 Preliminary test results 2.1 Demographics and baseline characteristics Average age 53.37 years old, approximately 75% Hispanic Median BMI: 34-36 kg / m 2 (30~45) ·40 women, 48 men 50% hypertension, 30% hyperlipidemia -40% T2DM; mean baseline HbA1c ~6 (5-9.9) Baseline liver fat content (HFF) was lowest in the placebo group (PBO), but was always above 10%. Vibration-controlled transient elastography (VCTE) showed a mean score of 8.1-8.7 (F2=7.1-9.9), excluding three subjects with an F2-VCTE of 7.0.

[0109] 2.2 Compliance At the time of the interim analysis, 85% of the A3 cohort and 44% of the PBO cohort reached 12 weeks Compliance with the study was good. The A2 and PBO cohorts had the most missed visits / late appointments / doses. "No longer in question" visits could have resulted in individual patients' study duration being longer than 16 weeks (especially in A3).

[0110] 2.3 Treatment-Emergent Adverse Events (TEAEs) The rate of any TEAE was lowest in the A4 cohort (50%) and highest in the A1 cohort (84.6%). The placebo TEAE rate was 35.3% Grade 1 events were the majority, with grade 2 being the rarest in A4, and A1 and A3 being similar. - The frequency of "related" TEAEs: A1 > A3, while A4 and PBO were similar (~25%). The main "related events" were gastrointestinal (A1>A3>PBO>A4) > metabolic and nutritional disorders > general disorders and injection site disorders. No significant dose-related changes in lipids, hematological parameters, or lipase levels were observed. No significant pruritic TEAEs were observed There was no weight gain A1 and A3 had the highest rates of study discontinuation. Adverse events of particular interest (nausea, vomiting, diarrhea) The rates were similar in A1 and A3; highest in A3 (57%) and <20% in A4 and PBO. Most common: Grade 2 nausea in A1 and A3, and Grade 2 vomiting in A1 Diarrhea in A3 is the most common

[0111] Figures 1A, 1B, 1C, and 1D show that administration of BOS-580 (SEQ ID NO: 1) did not adversely affect lipid profiles. BOS-580 treatment positively affected levels of HDL-cholesterol (increased) and triglycerides (decreased), which are known to reduce the risk of cardiovascular disease, at all dose schedules tested. LDL cholesterol did not change significantly over time in any direction, and in particular did not increase (increasing the risk of cardiovascular disease). Overall cholesterol levels did not change significantly in any direction with BOS-580 treatment.

[0112] Figure 2 shows the occurrence of gastrointestinal TEAEs. The most common TEAEs were adverse events of special interest (AESIs) from the gastrointestinal system category (e.g., nausea, vomiting, diarrhea, abdominal pain), followed by metabolic and nutritional conditions (e.g., increased appetite, hyperphagia, and food cravings). Increased appetite did not cause weight gain. Most of these events were mild and transient. The most frequent events occurred in cohorts A1 and A3.

[0113] 2.4 Treatment efficacy Administration of BOS-580 resulted in a reduction in liver fat content (HFF) in cohorts A1 and A3, as shown in Figures 3 and 4.

[0114] Figure 3 shows the absolute change in HFF as determined by magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF). The normal reference range for MRI-PDFF is <5.6%. The absolute change in liver fat content was significant relative to that observed with placebo in cohorts A1 and A3. The absolute change went from a mean of >20% at baseline to <10% after only 12 weeks of treatment. This effect was significant relative to placebo in cohorts A1 and A3 but not in cohort A4. The majority of the effect was observed as soon as week 6 of treatment, with some additional gains by week 12.

[0115] Figure 4 shows the relative change in HFF by MRI-PDFF. The relative change in hepatic fraction was >60% in the A1 and A3 cohorts at week 12 and was significant compared to placebo. The majority of the effect was observed as soon as week 6, with some additional gains by week 12.

[0116] Figure 5 shows the percentage of patients achieving specific degrees of fat reduction. A relative reduction of ≥30% in HFF has been established as predictive of disease improvement based on histopathological assessment, and therefore the proportion of patients achieving this level of reduction (responders) was used as one measure of efficacy. More recently, a reduction of ≥50% has been considered predictive of fibrosis improvement, and therefore the proportion of patients achieving this level of reduction (super-responders) has been used as a more stringent criterion. In the interim analysis, 100% of patients were responders in the A1 cohort and >90% in the A3 cohort. The placebo rate was 28.6%. The A1 and A3 cohorts also had very high rates of super-responders (≥75%), while the placebo rate was 14.3%. During the 12-week treatment period, some patients (<=5%) normalized liver fat, but this was again relatively similar within cohorts A1 and A3.

[0117] 2.5 Biomarkers Figure 6 shows the levels of alanine aminotransferase (ALT), a biomarker of liver damage. Elevated liver enzymes are a sign of liver damage, and therefore normalization or a trend toward normal (decrease) is a sign of improved liver function. In the interim analysis, ALT decreased by more than 35% from a median baseline value of 40-55 U / L as early as week 6, and this decline persisted until the final evaluation at week 12 in cohorts A1 and A3, demonstrating a significant difference from placebo. Cohort A4 showed no significant difference from placebo-treated patients.

[0118] Figure 7 shows aspartate aminotransferase (AST) levels, a biomarker of liver damage. AST reduction from a median baseline of 23-34 U / L was greater than 40% at the final study measurement, achieved as early as week 6 in cohort A1. This change was significantly different from placebo in cohorts A1 and A3 at the final study assessments at weeks 6 and 12.

[0119] Figure 8 shows the levels of the amino-terminal propeptide of procollagen 3 (ProC3), a biomarker of liver fibrosis. Collagen 3 is the predominant collagen type deposited in the liver during the development of liver fibrosis. The N-terminal peptide of procollagen (Pro-C3) is enzymatically removed for collagen deposition in the extracellular space of liver tissue. This N-terminal peptide can be measured in the circulation, and its level correlates with the degree of fibrosis. A reduction of Pro-C3 of 15% or more or 20% or more predicts improvement in fibrosis. In cohort A1, treatment with 300 mg Q4W reduced Pro-C3 by at least 20% as early as week 6, and this effect persisted until the final study evaluation. This effect was significantly different from placebo. In cohorts A3 and A4, treatment with 75 mg Q2W or 75 mg Q4W reduced Pro-C3 levels by >15% at week 6, but the effect did not persist with longer treatment.

[0120] Figure 9 shows the degree of adiponectin deficiency. Adiponectin is an adipose-specific adipokine that reduces insulin resistance and attenuates liver inflammation / fibrosis. Adiponectin levels are known to be low in patients with NASH. Administration of FGF21 is expected to improve adiponectin levels because binding of FGF21 to its receptors on adipocytes directly stimulates adiponectin production. Treatment with all BOS-580 schedules demonstrated rapid efficacy as early as week 4, with >50% improvement observed by week 2. All treatment schedules remained significant compared to placebo through week 12 of treatment.

[0121] Figure 10 shows HbA1c values ​​in diabetic NASH patients in cohorts A1 and A3. In the subpopulation of patients with baseline HbA1c levels >6.5% (diabetic patients), decreases of -1.0% and -0.7% were observed in cohorts A1 and A3, respectively, compared with -0.5% in the placebo group. Of note, only one placebo patient reached week 12 at the time of analysis; therefore, the true median change in placebo patients over time cannot be assessed. However, metformin is known to be able to reduce HbA1c by 0.6% over weeks 12–24, which can be used as a benchmark. Treatment with BOS-580 demonstrated improvements in HbA1c levels that were better than this benchmark.

[0122] Overall, the data demonstrated superior efficacy, taking safety into account, particularly for cohort A3 75 mg Q2W (150 mg / month), compared with cohorts A1 300 mg Q4W (300 mg / month) and A4 75 mg Q4W (75 mg / month).

[0123] SEQ ID NO: 1 disclosed herein is defined as follows: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ VRQRYLYTDD ACQTEAHLEI REDGTVGGAA DQSPESLLQL KALKPGVIQI LGVKTSRFLC QKPDGALYGS LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGGSQAR SPSYAS

Claims

1. A method for treating, preventing, or managing cardiovascular and / or metabolic disorders in a human subject in need thereof, comprising subcutaneously administering a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 at a dose of 75 mg once every two weeks.

2. 2. The method of claim 1, wherein the metabolic and / or cardiovascular disorder is selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hypercholesterolemia, dyslipidemia, hypertriglyceridemia, type 2 diabetes, and obesity.

3. 3. The method of claim 2, wherein the metabolic and / or cardiovascular disorder is nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD).

4. 4. The method of claim 3, wherein the metabolic and / or cardiovascular disorder is nonalcoholic steatohepatitis (NASH).

5. The method of any one of claims 1 to 4, wherein the human subject is obese.

6. the human subject has a blood glucose level of at least 27.5 kg / m for subjects of Asian descent or Asian ancestry ２ At least 30 kg / m with ethnic adjustment ２ Body mass index (BMI), especially 30 kg / m ２ ~45 kg / m ２ The method according to any one of claims 1 to 5, wherein the subject has a BMI of:

7. 7. The method of any one of claims 1 to 6, wherein the human subject has been diagnosed with, is suspected of being diagnosed with, and / or is at risk of acquiring, NASH.

8. 8. The method according to any one of claims 1 to 7, wherein the human subject has a liver fibrosis stage selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), in particular F2 and / or F3.

9. 9. The method of any one of claims 1 to 8, wherein the human subject has a liver fat assessment based on vibration-controlled transient elastography (VCTE) or a controlled attenuation parameter (CAP) score of greater than 300 dB / m, or greater than 400 dB / m.

10. 10. The method of any one of claims 1 to 9, wherein the human subject has a liver injury and fibrosis assessment based on a VCTE Liver Stiffness Measurement (LSM) score between 7-14 kPa, 7-12 kPa, or 7-9.9 kPa, and / or an aspartate aminotransferase (AST) activity of at least 20 U / L.

11. 11. The method of any one of claims 1 to 10, wherein the human subject has been diagnosed with, is suspected of being diagnosed with, and / or is at risk of acquiring type 2 diabetes, hypertension, and / or hyperlipidemia.

12. 12. The method of any one of claims 1 to 11, wherein said administering is effective to improve at least one physiological parameter associated with NASH.

13. 13. The method of claim 12, wherein the at least one physiological parameter is selected from liver fibrosis stage, liver fat assessment based on VCTE or CAP, liver damage and / or fibrosis assessment based on VCTE, LSM score, and / or AST activity.

14. 14. The method of any one of claims 1 to 13, wherein said administering is effective to reduce liver fat, in particular to reduce liver fat content by at least 45%, at least 50% or at least 60%.

15. 15. The method of any one of claims 1 to 14, wherein said administration is effective to reduce liver damage, in particular to reduce adiponectin deficiency, optionally by increasing adiponectin levels by at least 50%, 75%, 100% or 125%.

16. 6. The method of any one of claims 1 to 5, wherein the administration is effective in reducing liver damage, and is effective in reducing mean alanine aminotransferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or is effective in reducing aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or is effective in reducing mean levels of procollagen amino-terminal propeptide (ProC3) by at least 15% or at least 20%.

17. 17. The method of any one of claims 1-16, wherein said administering is effective to reduce HbA1c levels in a subject with type 2 diabetes by at least 0.6%.

18. 18. The method of any one of claims 1 to 17, wherein said administration has a grade 1 or 2 gastrointestinal tolerability profile.

19. The method of any one of claims 1 to 18, wherein the human FGF21 protein variant is administered in combination with one or more additional therapeutically active agents.

20. 20. The method of any one of claims 1 to 19, wherein the one or more additional therapeutically active agents are selected from the group consisting of compounds useful in the treatment of obesity, diuretics, beta-blockers, alpha-blockers, ACE inhibitors, angiotensin II receptor blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides, GPL-1 agonists, dual GLP-1 and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics, statins, fibrates, aspirin, and anticoagulants.

21. The method according to any one of claims 1 to 20, wherein the human FGF21 protein mutant consists of the amino acid sequence of SEQ ID NO:

1.

22. The method of any one of claims 1 to 21, wherein the human FGF21 protein variant is glycosylated.

23. The method of any one of claims 1 to 22, wherein the human FGF21 protein variant is non-glycosylated.

24. A human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for use in a method for treating, preventing, or managing cardiovascular and / or metabolic disorders in a human subject, wherein the human FGF21 protein variant is provided for subcutaneous administration at a dose of 75 mg once every two weeks.

25. 25. The human FGF21 protein variant for use according to claim 24, wherein the metabolic and / or cardiovascular disorder is selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hypercholesterolemia, dyslipidemia, hypertriglyceridemia, type 2 diabetes, and obesity.

26. 26. The human FGF21 protein variant for use according to claim 24 or 25, wherein the metabolic and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

27. 26. The human FGF21 protein variant for use according to claim 24 or 25, wherein the metabolic and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH).

28. The human FGF21 protein variant for use according to any one of claims 24 to 27, wherein the human subject is obese.

29. the human subject has a blood glucose level of at least 27.5 kg / m for subjects of Asian descent or Asian ancestry ２の At least 30 kg / m with ethnic adjustment ２ Body mass index (BMI), especially 30 kg / m ２ ~45 kg / m ２ The human FGF21 protein variant for use according to any one of claims 24 to 28, having a BMI of

30. 30. The human FGF21 protein variant for use according to any one of claims 24 to 29, wherein the human subject has been diagnosed with NASH, is suspected of being diagnosed with NASH, and / or is at risk of acquiring NASH.

31. 31. The human FGF21 protein variant for use according to claim 30, wherein the human subject has a liver fibrosis stage selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), in particular F2 and / or F3.

32. 32. The human FGF21 protein variant for use according to any one of claims 24 to 31, wherein the human subject has a liver fat assessment based on a vibration-controlled transient elastography (VCTE) controlled attenuation parameter (CAP) score of greater than 300 dB / m, or greater than 400 dB / m.

33. The human FGF21 protein variant for use according to any one of claims 24 to 32, wherein the human subject has a liver injury and fibrosis assessment based on a VCTE liver stiffness measurement (LSM) score of between 7 and 14 kPa, 7 and 12 kPa, or 7 and 9.9 kPa, and / or an aspartate aminotransferase (AST) activity of at least 20 U / L.

34. The human FGF21 protein variant for use according to any one of claims 24 to 33, wherein the human subject has been diagnosed with, is suspected of being diagnosed with, and / or is at risk of acquiring type 2 diabetes, hypertension, and / or hyperlipidemia.

35. The human FGF21 protein variant for use according to any one of claims 24 to 34, wherein said administration is effective to improve at least one physiological parameter associated with NASH.

36. 36. The human FGF21 protein variant for use according to claim 35, wherein the at least one physiological parameter is selected from liver fibrosis stage, liver fat assessment based on VCTE CAP, liver damage and fibrosis assessment based on VCTE LSM score, and AST activity.

37. The human FGF21 protein variant for use according to any one of claims 24 to 36, wherein said administration is effective in reducing liver fat, in particular effective in reducing liver fat content by at least 45%, at least 50%, or at least 60%.

38. 38. The human FGF21 protein variant for use according to any one of claims 24 to 37, wherein said administration is effective in alleviating liver damage, in particular in alleviating adiponectin deficiency, optionally by increasing adiponectin levels by at least 50%, 75%, 100% or 125%.

39. 39. The human FGF21 protein variant for use according to any one of claims 24 to 38, wherein said administration is effective in reducing liver damage, is effective in reducing mean alanine aminotransferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or is effective in reducing aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or is effective in reducing mean levels of procollagen amino-terminal propeptide (ProC3) by at least 15% or at least 20%.

40. The human FGF21 protein variant for use according to any one of claims 24 to 39, wherein said administration is effective to reduce HbA1c levels in a subject with type 2 diabetes by at least 0.6%.

41. The human FGF21 protein variant for use according to any one of claims 24 to 40, wherein said administration has a grade 1 or 2 gastrointestinal tolerability profile.

42. The human FGF21 protein variant for use according to any one of claims 24 to 41, wherein said human FGF21 protein variant is administered in combination with one or more additional therapeutically active agents.

43. 43. The human FGF21 protein variant for use according to claim 42, wherein the one or more additional therapeutically active agents are selected from the group consisting of compounds useful in the treatment of obesity, diuretics, beta-blockers, alpha-blockers, ACE inhibitors, angiotensin II receptor blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides, GPL-1 agonists, dual GLP-1 and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics, statins, fibrates, aspirin, and anticoagulants.

44. A human FGF21 protein mutant for use according to any one of claims 24 to 43, consisting of the amino acid sequence of SEQ ID NO:

1.

45. The human FGF21 protein variant for use according to any one of claims 24 to 44, wherein said human FGF21 protein variant is glycosylated.

46. The human FGF21 protein variant for use according to any one of claims 24 to 44, wherein said human FGF21 protein variant is non-glycosylated.

47. Use of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for treating, preventing, and managing cardiovascular and / or metabolic disorders in a human subject, wherein the human FGF21 protein variant is administered subcutaneously at a dose of 75 mg once every two weeks.

48. The use according to claim 47, wherein the human FGF21 protein variant consists of the amino acid sequence of SEQ ID NO: 1.

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