FGF21 protein variants for treatment of NASH

Abstract

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

Description

[0001] Field

[0002] The present disclosure relates to methods of treating, preventing, or managing cardiovascular and / or metabolic disorders in human subjects in need thereof by administering a human fibroblast growth factor (FGF21) protein variant that is an FGF21-Fc fusion protein.

[0003] Cross-reference to related applications

[0004] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 382,584, filed on November 7, 2022, the entire content of which is incorporated herein by reference in its entirety.

[0005] Sequence listing

[0006] This application contains a sequence listing, which has been submitted in XML format via Patent Center. The content of the XML file named "BPH-006PC_Sequence_Listing.xml", created on October 18, 2023, and having a size of 2,301 bytes, is incorporated herein by reference in its entirety.

[0007] Background

[0008] Fibroblast growth factor 21 (FGF21) is a member of the fibroblast growth factor (FGF) family and regulates energy balance as well as glucose and lipid homeostasis through a heterodimeric receptor complex comprising fibroblast growth factor receptor 1 (FGFR1) and β-Klotho. FGF21 is primarily released from hepatocytes and, to a lesser extent, from adipocytes and pancreatic β-cells. Preclinical studies in animals have demonstrated that FGF21 may have many beneficial pleiotropic effects, including reducing fat mass, improving glucose control and insulin resistance, improving dyslipidemia, and improving non-alcoholic fatty liver disease (NAFLD) models.

[0009] Non-alcoholic fatty liver disease (NAFLD) is a term for a series of conditions caused by an excessive calorie burden on the liver due to poor diet and lack of exercise, or a comorbid condition of lipid or glucose metabolism imbalance, resulting in fat accumulation in hepatocytes. NAFLD affects one-third of the US population and is associated with other metabolic diseases, including obesity, type 2 diabetes (T2DM), and hyperlipidemia. When excessive liver fat causes hepatocyte stress, NAFLD progresses to non-alcoholic steatohepatitis (NASH), which triggers local inflammation and, as the disease progresses, leads to fibrosis and ultimately cirrhosis. NASH can progress to cirrhosis and liver failure, which are becoming the leading causes of liver transplantation, death, and the development of hepatocellular carcinoma. In addition, people with NASH have a higher incidence of cardiovascular-related events, such as strokes and heart attacks, with cardiovascular disease being the leading cause of death in people with NASH. The prevalence of NASH in the United States is expected 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).

[0010] There are currently no approved NASH therapies, although many molecules are in development, representing many different mechanisms of action that target different aspects of the NASH pathogenesis. A variety of long-acting FGF21 analogs have been developed to overcome the short t that limits its clinical application of wild-type FGF21 1 / 2 . Several FGF21 analogs and mimetics have progressed to the early stages of clinical studies in obese, T2DM, and NASH subjects. These studies have demonstrated significant improvements in dyslipidemia, hepatic steatosis, and serum biomarkers of liver fibrosis in NASH subjects. Recently, phase 2a data for the FGF21 analog efruxifermin (EFX) showed that improvements in liver fat translated into improvements in 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

[0011] The present disclosure is at least partially based on the protocol and results of a 12-week Phase 2a randomized, blinded, placebo-controlled study of BOS-580 (V103), a variant of fibroblast growth factor 21 (FGF21) protein, in obese subjects at risk of developing non-alcoholic steatohepatitis (NASH). The aim of the study was to evaluate the exposure-response relationship of BOS-580 based on BOS-580 dose and exposure, as well as tolerance and efficacy biomarkers.

[0012] The human FGF21 protein variant BOS-580 (V103) is 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 by the introduction of a new disulfide bond and is fused to the Fc fragment of human immunoglobulin G1 (IgG1) at its N-terminus.

[0013] The amino acid sequence of BOS-580 (V103) is shown in SEQ ID NO: 1. It contains, from the N-terminus to the C-terminus, an immunoglobulin Fc fragment, a linker GS, and a native mature human FGF21 variant, which represents amino acid positions 29-209 of the full-length FGF21 protein sequence (NCBI reference sequence number NP_061986.1), with amino acid substitutions Q55C, R105K, G148C, K150R, P158S, S195A, P199G, G202A (numbering based on the full-length sequence).

[0014] A first aspect of the present disclosure relates to the use of a human FGF21 protein variant in a method for treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject in need thereof, wherein the human FGF21 protein variant comprises the amino acid sequence of SEQ ID NO: 1, and wherein the human FGF21 protein variant is provided by subcutaneous administration at a dose of 75 mg once every two weeks.

[0015] Another aspect of the present disclosure relates to the use of a human FGF21 protein variant in the preparation of a medicament for treating, preventing, or managing a cardiovascular or metabolic disorder in a human subject in need thereof, wherein the human FGF21 protein variant comprises the amino acid sequence of SEQ ID NO: 1, and wherein the human FGF21 protein variant is provided by subcutaneous administration at a dose of 75 mg once every two weeks.

[0016] Yet another aspect of the present disclosure relates to a method for treating, preventing, or managing a cardiovascular or metabolic disorder in a human subject in need thereof, the method 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.

[0017] In some embodiments of any of the foregoing aspects, the metabolic disorder 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.

[0018] In some embodiments of any of the foregoing aspects, the metabolic disorder and / or cardiovascular disorder is non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH).

[0019] In some embodiments of any of the foregoing aspects, the metabolic disorder and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH).

[0020] In some embodiments of any of the foregoing aspects, the human subject is obese.

[0021] In some embodiments of any of the foregoing aspects, the body mass index (BMI) of the human subject is at least 30 kg / m 2 , wherein the race adjustment for subjects of Asian or Asian descent is at least 27.5 kg / m 2 , particularly a BMI of 30 kg / m 2 to 45 kg / m 2 .

[0022] In some embodiments of any of the foregoing aspects, the human subject has NASH and / or is at risk of developing NASH.

[0023] In some embodiments of any of the foregoing aspects, the stage of liver fibrosis of the human subject is selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis), and / or F4 (cirrhosis), particularly selected from F2 and / or F3.

[0024] In some embodiments of any of the foregoing aspects, the human subject has a liver fat assessment based on the controlled attenuation parameter (CAP) score of vibration-controlled transient elastography (VCTE) greater than 300 dB / m or greater than 400 dB / m.

[0025] In some embodiments of any of the foregoing aspects, the human subject has an assessment of liver injury and fibrosis based on the VCTE liver stiffness measurement (LSM) score of 7 to 14 kPa, 7 kPa to 12 kPa, or 7 kPa to 9.9 kPa and / or the aspartate aminotransferase (AST) activity of at least 20 U / L.

[0026] In some embodiments of any of the foregoing aspects, the human subject also has type 2 diabetes, hypertension, and / or hyperlipidemia.

[0027] In some embodiments of any of the foregoing aspects, administration is effective to improve at least one physiological parameter associated with NASH.

[0028] In some embodiments of any of the foregoing aspects, the at least one physiological parameter is selected from the stage of liver fibrosis, liver fat assessment based on VCTE CAP, liver injury and fibrosis assessment based on VCTE, LSM score, and AST activity.

[0029] In some embodiments of any of the foregoing aspects, administration is effective to reduce liver fat, particularly effectively reducing the liver fat fraction by at least 45%, at least 50%, or at least 60%.

[0030] In some embodiments of any of the foregoing aspects, administration is effective to reduce liver injury, particularly effective in reducing adiponectin deficiency, for example, by increasing the adiponectin level by at least 50%, 75%, 100%, or 125%.

[0031] In some embodiments of any of the foregoing aspects, administration is effective to reduce liver fibrosis, particularly effectively reducing the mean alanine aminotransferase (ALT) by at least 20%, at least 30%, or at least 35%, and / or effectively reducing the aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or effectively reducing the mean of procollagen type III amino-terminal propeptide (ProC3) by at least 15% or at least 20%.

[0032] In some embodiments of any of the foregoing aspects, administration effectively reduces the HbA1c level in a subject with type 2 diabetes by at least 0.6%.

[0033] In some embodiments of any of the foregoing aspects, administration has a gastrointestinal tolerance profile of grade 1 or 2.

[0034] In some embodiments of any of the foregoing aspects, the human FGF21 protein variant is administered in combination with one or more additional therapeutic active agents.

[0035] In some embodiments of any of the foregoing aspects, one or more additional therapeutic active agents are selected from compounds useful in obesity therapy, diuretics, β-blockers, α-blockers, ACE inhibitors, angiotensin II receptor blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, α-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 transporters (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylin mimetics, statins, fibrates, aspirin, and anticoagulants.

[0036] In some embodiments of any of the foregoing aspects, the human FGF21 protein variant is non-glycosylated.

[0037] In some embodiments of any of the foregoing aspects, the human FGF21 protein variant is glycosylated.

[0038] In some embodiments of any of the foregoing aspects, the human FGF21 protein variant consists of the amino acid sequence of SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figures 1A - 1D Showing that administration of BOS-580 (SEQ ID NO: 1) has no adverse effect on lipid profile.

[0040] Figure 2 Showing the incidence of TEAE of the gastrointestinal system.

[0041] Figure 3 Showing the absolute change in HFF determined by magnetic resonance imaging proton density fat fraction (MRI-PDFF). The normal reference range of MRI-PDFF < 5.6%.

[0042] Figure 4 Showing the relative change in HFF determined by MRI-PDFF.

[0043] Figure 5 Showing the proportion of patients achieving a specific degree of fat reduction.

[0044] Figure 6 Showing the level of alanine aminotransferase (ALT) as a biomarker of liver injury.

[0045] Figure 7 Showing the level of aspartate aminotransferase (AST) as a biomarker of liver injury.

[0046] Figure 8 To show the level of the amino-terminal propeptide of procollagen 3 (ProC3), which is a biomarker of liver fibrosis.

[0047] Figure 9 To show the degree of adiponectin deficiency. Adiponectin is an adipose-specific adipokine that reduces insulin resistance and alleviates hepatitis / fibrosis.

[0048] Figure 10 To show the HbA1c level in patients with diabetic NASH. Detailed implementation mode

[0049] Disclosed herein are methods, compounds, and pharmaceutical compositions for treating, preventing, or managing (e.g., alleviating one or more signs and / or symptoms of a disorder) metabolic disorders and / or cardiovascular disorders, including subcutaneously administering to a human subject in need a human FGF21 protein variant comprising or consisting of the amino acid sequence of SEQ ID NO: 1 at a dose of 75 mg once every two weeks.

[0050] Generally, the human FGF21 protein variant comprising or consisting of the amino acid sequence of SEQ ID NO: 1 is obtained by expression in a recombinant host cell, wherein the recombinant host cell is transfected or transformed with a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. The recombinant host cell can be cultured in a suitable medium, and the polypeptide can be isolated from the host cell or the medium. The host cell can be a prokaryotic host cell or a eukaryotic host cell, such as an insect or mammalian cell. Depending on the type of host cell, the human FGF21 protein variant can be glycosylated or deglycosylated / non-glycosylated.

[0051] The term "managing" refers to the management and care of a patient in order to combat a disease, condition, or disorder, which does not result in a cure but alleviates one or more symptoms of the disease, condition, or disorder and / or reduces the length of hospitalization. In an embodiment, "managing (managing or management)" of a condition or disease includes maintaining symptomatology, including using one or more indices or scores (e.g., as described herein), and / or maintaining one or more biomarkers of the disease (e.g., as described herein).

[0052] The term "prevention" refers to the prophylactic administration to healthy subjects or subjects at risk of developing a disease, condition or disorder described herein (e.g., subjects considered to be pre-diabetic or subjects with liver fibrosis and at risk of developing NASH) to prevent the development of one or more conditions mentioned herein. In addition, the term "prevention" may also include the prophylactic administration to patients in the pre-stage of a condition to be treated. In embodiments, "preventing" or "prevention" with respect to a condition or disease includes preventing the worsening of one or more symptoms, including using one or more indices or scores (e.g., as described herein), and / or maintaining one or more biomarkers of the disease (e.g., as described herein).

[0053] The term "treatment" is understood to mean the management and care of a patient to combat a condition, e.g., to reduce or ameliorate the progression, severity and / or duration of a disease, disorder or condition.

[0054] The term "metabolic disorder" refers to a disease, disorder or condition associated with pathological metabolic parameters (e.g., elevated blood pressure, hyperglycemia, excess body and / or organ fat, abnormal cholesterol levels, abnormal triglyceride levels, and any combination thereof). Metabolic disorders may increase the risk of heart failure and / or stroke.

[0055] The term "cardiovascular disorder" refers to a disease, disorder or condition of the heart and / or vascular system.

[0056] Non-limiting examples of metabolic disorders and / or cardiovascular disorders to 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, glucose intolerance, hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease, acute myocardial infarction, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, diabetic neuropathy, conditions associated with severe inactivating mutations in the insulin receptor (e.g., as described in Taylor et al., Diabetes Care 13(1990), 257-279, the content of which is incorporated herein by reference) and / or gastroparesis.

[0057] In certain embodiments, the metabolic disorder 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.

[0058] In certain embodiments, the metabolic disorder and / or cardiovascular disorder is selected from non-alcoholic steatohepatitis (NASH).

[0059] The term “NAFLD” or “non-alcoholic fatty liver disease” is defined as a condition in which excessive fat is stored in the liver. This accumulation of fat is not caused by excessive alcohol consumption. When excessive alcohol consumption leads to fat accumulation in the liver, the condition is called alcoholic liver disease. Generally, there are two types of NAFLD: simple fatty liver and non-alcoholic steatohepatitis (NASH).

[0060] The term “NASH” or “non-alcoholic steatohepatitis” is defined as a form of non-alcoholic fatty liver disease (NAFLD) in which the patient has hepatitis - inflammation of the liver - and hepatocyte injury in addition to having fat in the liver. Inflammation and hepatocyte injury can lead to liver fibrosis or scarring. In some cases, NASH can lead to cirrhosis or liver cancer. Cirrhosis with complications is called “decompensated” cirrhosis, and it is one of the main reasons for liver transplantation.

[0061] The term “simple fatty liver”, also known as non-alcoholic fatty liver (NAFL), is a form of NAFLD in which the patient has fat in the liver but little or no inflammation or hepatocyte injury. In a certain percentage of individuals, simple fatty liver can progress to NASH.

[0062] In certain embodiments, the human subject is obese. Obesity is measured by the body mass index BMI = weight (kg) / [height (m)] 2 , the weight of a person in kilograms divided by the square of their height in meters. A person with a BMI of 30 or higher is generally considered obese, but the guidelines can be adjusted according to racial differences. For example, by racial adjustment, an Asian individual with a BMI of 27.5 can be considered obese (WHO Expert Consultation, 2004, Lancet, 363(9403):157 - 63). An Asian individual can be, for example, a subject of Asian or Asian descent.

[0063] In certain embodiments, the body mass index (BMI) of the human subject is at least 30 kg / m 2 , and for subjects of Asian or Asian descent, at least 27.5 kg / m after racial adjustment 2 , for example, a BMI of 30 kg / m 2 to 45 kg / m 2 . In an embodiment, the BMI is about or at least about 20 kg / m 2 , about or at least about 25 kg / m 2 , about 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 45 kg / m 2 , or about or at least about 50 kg / m 2 . In an embodiment, the BMI comprises about or at least about 20 kg / m 2 to about or at least about 25 kg / m 2 , about or at least about 25 kg / m 2 to about or at least about 30 kg / m 2 , about or at least about 30 kg / m 2 to about or at least about 35 kg / m 2 , about or at least about 35 kg / m 2 to about or at least about 40 kg / m 2 , about or at least about 40 kg / m 2 to about or at least about 45 kg / m 2 , or about or at least about 45 kg / m 2 to about or at least about 50 kg / m 2 range.

[0064] In an embodiment, the subject to be treated is diagnosed with NASH, suspected of having NASH, and / or at risk of developing NASH. In an embodiment, the subject diagnosed with NASH, suspected of having NASH, and / or at risk of developing NASH is prediabetic. In an embodiment, the subject diagnosed with NASH, suspected of having NASH, and / or at risk of developing NASH exhibits one or more demographic and / or lifestyle factors for selecting a subject for the methods herein, including being overweight, obese, or having a comorbidity associated therewith (e.g., being prediabetic based on BMI, waist circumference, weight); age (e.g., 45 years or older); being African American, Pacific Islander, or Hispanic / Latino; having a family history of diabetes; having a sedentary lifestyle or lack of physical activity; having high blood pressure and / or abnormal cholesterol levels; having a personal history of gestational diabetes, heart disease, stroke, and / or polycystic ovary syndrome (PCOS). In an embodiment, the methods herein are directed to preventing the progression of a disease or condition from its early stage (e.g., prediabetes to diabetes, NAFLD to NASH, etc.), and / or are directed to reducing the accumulation of damage due to lifestyle factors and / or risk factors. In an embodiment, the preventive methods herein are designed for healthy individuals or individuals who are substantially healthy to halt or slow the progression of the disease, or to prevent NASH from progressing to a more severe stage of NASH, etc. In an embodiment, the accumulation of damage, the presence of lifestyle factors and / or risk factors, and any improvements associated therewith are measurable as described herein.

[0065] In embodiments, the diagnosis, suspected diagnosis, and / or risk of NASH are determined or evaluated by liver biopsy results. In embodiments, NASH is diagnosed when microscopic analysis of tissue shows fat accumulation, inflammation, and / or hepatocyte injury. In embodiments, a subject with liver tissue showing fat without inflammation and injury is 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 the suspected diagnosis of NASH includes evaluating the results of blood tests, such as using biomarkers such as the liver enzymes alanine aminotransferase (ALT) and / or aspartic acid aminotransferase (AST), platelet count, procollagen type III amino-terminal propeptide (ProC3), and / or hemoglobin A1C (HbA1c). In embodiments, evaluating the diagnosis of NASH, the suspected diagnosis of NASH, and / or the risk of NASH includes using scoring criteria / indexes, such as the Fibrosis-4 (FIB-4) index for liver fibrosis, such as a subject having 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, evaluating the diagnosis of NASH, the suspected diagnosis of NASH, and / or the risk of NASH includes using scoring criteria / indexes, such as the aspartate aminotransferase-to-platelet ratio index (APRI), such as a subject having 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.

[0066] In embodiments, a subject diagnosed with NASH, suspected of having NASH, and / or at risk of developing NASH has one or more of the following: an enlarged liver, an enlarged spleen, or ascites (e.g., as observed from imaging of visceral organs and surrounding tissues), signs of insulin resistance (e.g., observed darkening of skin patches on the extremities), signs of 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, such as vibration control transient elastography, shear wave elastography, and / or magnetic resonance elastography. In embodiments, measuring insulin resistance includes a euglycemic hyperinsulinemic clamp, a fasting plasma glucose (FPG) test (e.g., 100 to 125 mg / dL glucose or higher), an A1C test (e.g., 5.7% to 6.4% or higher), and / or an oral glucose tolerance test (OGTT) (e.g., 140 to 199 mg / dL glucose or higher).

[0067] In certain embodiments, a human subject has NASH and / or is at risk of developing NASH. In such embodiments, the stage of liver fibrosis in the human subject (e.g., using the Batts-Ludwig criteria) can be selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis), and / or F4 (cirrhosis), particularly selected from F2 and / or F3.

[0068] In certain embodiments, a human subject, such as a human subject having NASH and / or at risk of developing NASH, can be characterized by one or more of the following physiological parameters:

[0069] - Hepatic steatosis assessment based on the controlled attenuation parameter (CAP) score of vibration control transient elastography (VCTE) is greater than 300 dB / m, particularly greater than 400 dB / m;

[0070] - Hepatic injury and fibrosis assessment based on the liver stiffness measurement (LSM) score of VCTE is at least 7 kPa, e.g., 7 to 14 kPa, 7 kPa to 12 kPa, or 7 kPa to 9.9 kPa;

[0071] - Aspartate aminotransferase (AST) activity is at least 20 U / L;

[0072] - NAFLD fibrosis (NFS) score (calculated as age, AST / ALT ratio, platelet count, BMI, albumin (g / L), impaired fasting glucose / diabetes (if yes then +1)) is at least 1.455, indicating intermediate or high probability of fibrosis;

[0073] - FIB-4 (Fibrosis-4) score (calculated as (age × AST) / square root of platelet count (10 9 / L) × ALT) is at least 1.3, indicating advanced fibrosis,

[0074] - APRI index (ALT to platelet ratio index) is greater than 0.5, indicating intermediate or high probability of advanced fibrosis;

[0075] - FibroSure value (a serum test that combines five biomarkers: haptoglobin, α2-macroglobulin, apolipoprotein A1, total bilirubin, and gamma-glutamyl transferase) is greater than 0.31 to 0.58, indicating moderate fibrosis, or FibroSure value is greater than 0.58, indicating advanced fibrosis;

[0076] - The enhanced liver fibrosis (ELF) value (combining three fibrosis biomarkers: hyaluronic acid, tissue inhibitor of metalloproteinase 1, and amino-terminal propeptide of procollagen 3) ranges from 7.7 to less than 9.8, indicating moderate fibrosis (mild to advanced fibrosis), or at least 9.8, indicating advanced fibrosis;

[0077] - The magnetic resonance elastography (MRE) score ranges from 2.97 to 3.62, indicating fibrosis stage F2, or greater than 3.62, indicating advanced fibrosis (F3);

[0078] - The FIBROSCAN-AST (FAST) score greater than 0.67 indicates a high risk of progression, or less than 0.35 indicates a low risk of progression.

[0079] In embodiments, as described herein, methods for the prevention or management of cardiovascular and / or metabolic disorders (such as NASH) include substantially maintaining the appearance of liver biopsy results, substantially maintaining the concentration and / or activity of ASL, AST, ProC3, and / or HbA1C; substantially maintaining platelet count; substantially maintaining the scores of FIB-4 index, NFS score, APRI, FIBROSURE value, ELF value, MRE score, and / or FAST score; substantially maintaining the evaluations or results from Batts-Ludwig criteria, euglycemic hyperinsulinemic test, FPG test, A1C test, or OGTT; substantially maintaining VCTE, CAP score, LSM score; and / or substantially maintaining the severity and / or frequency of one or more symptoms associated with the condition, disease, or comorbidity. In embodiments, "substantially maintaining" means a change or deviation of less than about 1%, 5%, or 10%, such as in qualitative or quantitative measurements.

[0080] In certain embodiments, a human subject, such as a human subject with NASH and / or at risk of developing NASH, is further diagnosed with, suspected of having, and / or at risk of developing type 2 diabetes, hypertension, and / or hyperlipidemia. In certain embodiments, a human subject, such as a human subject with NASH and / or at risk of developing NASH, further has type 2 diabetes.

[0081] In certain embodiments, the administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 is effective for improving at least one physiological parameter associated with a metabolic disorder and / or cardiovascular disorder to be treated, prevented, or managed by the disclosure provided herein. In certain embodiments, the administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 is effective for improving at least one physiological parameter associated with NASH.

[0082] In human subjects with NASH and / or at risk of developing NASH, administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 can effectively improve at least one physiological parameter selected from the group consisting of stage of liver fibrosis, VCTE CAP-based hepatic steatosis assessment, VCTE-based hepatic injury and fibrosis assessment, LSM score, and AST activity.

[0083] 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 for reducing hepatic steatosis, particularly effective for reducing the hepatic steatosis fraction by at least 45%, at least 50%, or at least 60%.

[0084] 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 for reducing hepatic injury, particularly effective for reducing adiponectin deficiency, for example by increasing adiponectin levels by at least 50%, at least 75%, at least 100%, or at least 125%.

[0085] 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 for reducing hepatic injury, particularly effective for reducing alanine aminotransferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or for reducing aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or for reducing the mean value of procollagen type III N-terminal propeptide (Pro-C3) by at least 15% or at least 20%.

[0086] In subjects with NASH and / or at risk of developing 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), can effectively reduce the HbA1c level in subjects with type 2 diabetes, for example by at least 0.6%.

[0087] 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 gastrointestinal tolerance profile of grade 1 or 2.

[0088] The human FGF21 protein variant of SEQ ID NO: 1 can be administered alone or in combination with one or more additional therapeutic agents. In some instances, the one or more additional therapeutic agents can be selected from compounds useful in obesity therapy (e.g., phentermine / topiramate, orlistat, lorcaserin, liraglutide, bupropion / naltrexone, and combinations thereof), compounds useful in hypertension therapy (e.g., diuretics, β-blockers, α-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 diabetes therapy (e.g., insulin, α-glucosidase inhibitors, biguanides, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides such as GLP-1 agonists such as semaglutide and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylin mimetics, and combinations thereof), and compounds useful in NAFLD / NASH and cardiovascular therapy (e.g., statins, fibrates, aspirin, anticoagulants, and / or combinations thereof).

[0089] In one aspect, the present disclosure provides combination therapies for treating, preventing, or managing metabolic disorders and / or cardiovascular disorders, which include administering a human FGF21 protein variant of SEQ ID NO: 1 and one or more therapeutic 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), fosinopril (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, Diltia XL (diltiazem), Sular (nisoldipine), Isoptin, Calan, Verelan, Covera-HS (verapamil), Capoten (captopril), Vasotec (enalapril), Prinivil,Zestril (Lisinopril), Lotensin (Benazepril), Monopril (Fosinopril), Altace (Ramipril), Accupril (Quinapril), Aceon (Perindopril), Mavik (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), fibrate 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, 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, aleglitazar, bempagliflozin, hengagliflozin, linagliptin, glimepiride (Amaryl), glimepiride-pioglitazone (Duetact), glimepiride-rosiglitazone (Avandaryl), gliclazide; gliclazide-metformin (Metaglip), glibenclamide (DiaBeta, Glynase, Micronase), glibenclamide-metformin (Glucovance), chlorpropamide (Diabinese), tolazamide (Tolinase), tolbutamide (Orinase, Tol-Tab), rosiglitazone (Avandia), rosiglitazone-glimepiride (Avandaryl), rosiglitazone-metformin (AmarylM), pioglitazone (Actos), pioglitazone-alogliptin (Oseni), pioglitazone-glimepiride (Duetact), and pioglitazone-metformin (Actoplus Met, Actoplus Met XR).

[0090] In some embodiments, the sodium-glucose cotransporter (SGLT) inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, ertugliflozin, sotagliflozin, tofogliflozin, repagliflozin, luseogliflozin, ipragliflozin, alogliptin, begoogliptin, hengogliptin, linagliptin, and any pharmaceutically acceptable salts thereof. In some embodiments, the sodium-glucose cotransporter (SGLT) inhibitor is dapagliflozin. In some embodiments, the sodium-glucose cotransporter (SGLT) inhibitor is empagliflozin. In some embodiments, the sodium-glucose cotransporter (SGLT) inhibitor is canagliflozin. In some embodiments, the sodium-glucose cotransporter (SGLT) inhibitor is ertugliflozin. In some embodiments, the sodium-glucose cotransporter (SGLT) inhibitor is linagliptin.

[0091] According to certain embodiments, administration of the human FGF21 protein variant of SEQ ID NO:1 is accompanied by monitoring of FGF21 bioactivity, such as as described in WO 2013 / 049247, the entire content of which is incorporated herein by reference.

[0092] In certain embodiments, at least one biomarker of liver function is evaluated to determine treatment success. 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 is evaluated, such as HbA1c.

[0093] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising the human FGF21 protein variant of SEQ ID NO:1 for use in a method of treating, preventing, and / or managing metabolic and / or cardiovascular diseases such as hypertriglyceridemia and cardiac risk, insulin resistance (e.g., in patients with genetic mutations in the insulin receptor and lipodystrophy), diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD) / non-alcoholic steatohepatitis (NASH).

[0094] The pharmaceutical compositions described herein comprise a therapeutically effective amount of the human FGF21 protein variant of SEQ ID NO:1 and a pharmaceutically acceptable carrier suitable for subcutaneous administration. Generally, the pharmaceutical composition is in the form of a sterile, pyrogen-free, parenterally acceptable composition. Particularly suitable vehicles for parenteral injection are properly preserved sterile isotonic solutions. The pharmaceutical composition may be in the form of a lyophilized preparation, such as a lyophilized cake. Suitable methods of pharmaceutical formulation and components are well known (see, e.g., Allen, Lloyd V. Ed., (2012) Remington's Pharmaceutical Sciences, 22nd Edition, which is incorporated herein by reference for this purpose).

[0095] In certain embodiments, the pharmaceutical composition comprises at least one 75 mg unit dose of the human FGF21 protein variant of SEQ ID NO:1 to be administered subcutaneously every two weeks.

[0096] The pharmaceutical compositions provided herein are for subcutaneous administration. Formulation components and methods suitable for subcutaneous administration of polypeptide therapeutics (such as antibodies, fusion proteins, etc.) are known in the art (see, for example, US2011 / 0044977 A1, US 8,465,739 B2, and US 8,476,239 B2, all incorporated herein by reference for this purpose). Generally, pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (such as amino acids, such as methionine, and / or sugars, such as sucrose), buffers, and / or tonicifying agents.

[0097] In certain embodiments, the pharmaceutical composition comprises the human FGF21 protein variant of SEQ ID NO:1 as a dry formulation, such as a lyophilized formulation, which can be reconstituted into a solution prior to subcutaneous administration.

[0098] In certain embodiments, the pharmaceutical composition is in the form of a solution of the FGF21 protein variant at about 50 mg / mL to about 150 mg / mL or about 100 mg / mL to about 150 mg / mL (e.g., after reconstitution of the lyophilized formulation).

[0099] In certain embodiments, the pharmaceutical composition comprises (e.g., after reconstitution of the lyophilized formulation) a tromethamine buffer, such as at a concentration of 10 mM to 50 mM tromethamine buffer, such as 30 mM tromethamine buffer.

[0100] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of the lyophilized formulation) containing sucrose, such as about 200 mM to about 300 mM sucrose, such as 270 mM sucrose.

[0101] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of the lyophilized formulation) containing polysorbate, such as polysorbate 20, such as about 0.01% (w / w) to about 0.10% (w / w) polysorbate 20, such as 0.06% (w / w) polysorbate 20.

[0102] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of the lyophilized formulation) having a pH in the range of 6.5 to 9, such as a pH of about 8.0.

[0103] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilized formulation) comprising a tromethamine buffer, sucrose, and polysorbate 20, e.g., a solution having a pH of 7.5 to 8.5, e.g., pH 8.0, comprising from about 80 mg / mL to 100 mg / mL of an FGF21 protein variant, about 30 mM tromethamine buffer, 270 mM sucrose, and 0.06% polysorbate 20.

[0104] In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilized formulation) having a pH of 8.0 and comprising about 100 mg / mL of an FGF21 protein variant, about 30 mM tromethamine buffer, 270 mM sucrose, and 0.06% polysorbate 20.

[0105] Furthermore, the present invention will be illustrated in more detail by the following examples.

[0106] Examples

[0107] Example 1:

[0108] Study Protocol: A 12-week Phase 2a randomized, blinded, placebo-controlled study of BOS-580 in obese subjects at risk of non-alcoholic steatohepatitis (NASH)

[0109] 1.1 Abbreviations and Definitions

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] 1.2 Rationale

[0116] The purpose 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 tolerance and efficacy biomarkers. These data were used to confirm the benefit / risk profile and dosing rationale of BOS-580 to support progression to late-stage development for the treatment of NASH.

[0117] 1.3 Objectives and Endpoints

[0118]

[0119]

[0120] 1.4 Overall Design

[0121] This is a two - part, multi - center, randomized, double - blind (sponsor - unblinded), placebo (PBO) - controlled safety study to evaluate subcutaneously administered BOS - 580 when dosed repeatedly over 12 weeks. The study includes up to 105 male and / or female subjects, aged 18 to 75 years (inclusive), body mass index (BMI) of 30 to 45 kg / m 2 (inclusive), MRI - PDFF ≥ 10%, VCTE LSM of 7 to 9.9 kPa (inclusive), and AST > 20 U / L.

[0122] Part A consists of 5 cohorts (A1 to A4, A5 is randomly assigned separately), with a total of approximately 75 subjects. Subjects are initially randomly assigned to cohorts A1 to A4, with approximately 15 subjects in each cohort. Then the subjects in cohorts A1 to A4 will be randomly assigned within each cohort at a ratio of 4:1 (BOS - 580 to PBO). Once cohorts A1 to A4 have been randomly assigned, approximately 15 subjects will be randomly assigned to cohort A5 at a ratio of 4:1 (BOS - 580 to PBO).

[0123] Subjects return to the study center for regular visits over 12 weeks for study drug administration, pharmacokinetics (PK), biomarker, and safety assessments.

[0124] In Part A, BOS - 580 or PBO is administered by subcutaneous (SC) injection as follows:

[0125] · Cohort A1: 300 mg every 4 weeks (Q4W) (300 mg / month) or PBO.

[0126] · Cohort A3: 75 mg Q2W (150 mg / month) or PBO.

[0127] · Cohort A4: 75 mg Q4W (75 mg / month) or PBO.

[0128] To explore additional doses, dosing frequencies, or dose - escalation titration in Part B, additional cohorts may be initiated, which will occur after the data from the mid - study analysis at week 6 in cohorts A1, A3, and A4.

[0129] In Parts A and B, subjects will return to the study center for follow - up visits approximately 4 weeks after receiving the last study dose.

[0130] 1.5 Number of Subjects

[0131] Enroll subjects such that approximately 75 evaluable subjects complete Part A. Approximately 30 subjects may be enrolled in Part B (optional).

[0132] 1.6 Inclusion / Exclusion Criteria

[0133]

[0134]

[0135]

[0136] 1.7 Study Phases and Durations

[0137] The study consisted of two screening phases. Subjects who met the eligibility criteria specified in Screening I (Day - 42 to Day - 10) proceeded to the imaging evaluations specified in Screening II (Day - 28 to Day - 7). The study treatment phase consisted of bi - weekly or monthly dosing (with a weekly regimen considered in optional Part B). Post - treatment follow - up included a visit approximately 4 weeks after receiving the last study dose. The total duration of participation for each subject was approximately 22 weeks.

[0138] 1.8 Study Analyses

[0139] The following analyses were conducted:

[0140] · An interim analysis was conducted once approximately 90% of the subjects randomized in cohorts A1 to A4 had completed their Week 6 visit. The data were used to guide sponsor - driven decisions regarding the clinical plan.

[0141] · Based on the results of the Week 6 interim analysis, an additional interim analysis could be conducted once all subjects in cohorts A1 to A4 had completed treatment through Week 12.

[0142] The available data for any subject enrolled in Part B could be included in the analysis of treatment through Week 12 listed above. Depending on the timing of all subjects in Part A completing their study - end visit (Week 16) relative to all subjects in Part B completing their study - end visit, the final analyses for Part A and Part B could be completed at the same time or separately. Additional analyses could also be conducted if needed.

[0143] Example 2

[0144] Preliminary Study Results

[0145] 2.1 Demographics and Baseline Characteristics

[0146] · Mean age 53.37, approximately 75% of the subjects were Hispanic

[0147] · Median BMI was approximately 34 - 36 kg / m 2 (30 - 45)

[0148] · 40 females, 48 males

[0149] · 50% hypertension, 30% hyperlipidemia

[0150] · Approximately 40% T2DM; mean baseline HbA1c ~6 (5 - 9.9)

[0151] · Baseline hepatic fat fraction (HFF), lowest in the placebo group (PBO), but all >10%

[0152] · Except for 3 subjects with vibration - controlled transient elastography (VCTE) of 7.0, F2 - mean score by VCTE 8.1 - 8.7 (F2 = 7.1 - 9.9)

[0153] 2.2 Adherence

[0154] · At the interim analysis, 85% of the A3 cohort and 44% of the PBO cohort reached 12 weeks

[0155] · Good study adherence. Missed / delayed visits / dosing highest in A2 and PBO cohorts

[0156] · Due to "out - of - window" visits (especially in A3), the study duration for individual patients may exceed 16 weeks

[0157] 2.3 Treatment - emergent adverse events (TEAE)

[0158] · Incidence of any TEAE lowest in A4 cohort (50%) to highest in A1 cohort (84.6%)

[0159] · Incidence of placebo TEAE was 35.3%

[0160] · Most events were grade 1, fewest grade 2 in A4; similar in A1 and A3

[0161] · Incidence of "related" TEAE by frequency: A1 > A3, while A4 and PBO were similar (approx. 25%)

[0162] o Most "related events" were gastrointestinal (A1 > A3 > PBO > A4) > metabolic and nutritional disorders > general disorders & injection site

[0163] · No significant dose - dependent changes were found in lipid, hematological parameters, or lipase levels.

[0164] · No significant pruritus TEAE

[0165] · No weight gain

[0166] · A1 and A3 had the highest study discontinuation rates.

[0167] ·Adverse events of special concern (nausea, vomiting, diarrhea)

[0168] o Incidence was similar in A1 and A3; highest in A3 (57%), while <20% in A4 and PBO

[0169] o Most frequent: Grade 2 nausea in A1 and A3, and Grade 2 vomiting in A1

[0170] o Diarrhea was most frequent in A3

[0171] Figure 1A , 1B , 1C and 1D showed that the administration of BOS-580 (SEQ ID NO: 1) had no adverse effects on lipid profiles. Treatment with BOS-580 had a positive effect on the levels of HDL-cholesterol (increase) and triglycerides (decrease) in all trial dose regimens, which are known to reduce the risk of cardiovascular disease. LDL cholesterol did not change significantly over time in either direction, and in particular it did not increase (which would increase the risk of cardiovascular disease). The total cholesterol level did not change significantly in either direction after treatment with BOS-580.

[0172] Figure 2 The occurrence of TEAEs in the gastrointestinal system was shown. The most frequently occurring TEAEs were adverse events of special concern (AESIs) from the organ class of the gastrointestinal system (e.g., nausea, vomiting, diarrhea, abdominal pain), followed by conditions from the metabolic and nutritional classes (e.g., increased appetite, hyperphagia, and food cravings). The increase in appetite did not lead to an increase in body weight. Most of these events were mild and transient. The most events occurred in cohorts A1 and A3.

[0173] 2.4 Treatment effects

[0174] As Figure 3 and 4 shown, the administration of BOS-580 led to a decrease in hepatic fat fraction (HFF) in cohorts A1 and A3.

[0175] Figure 3 The absolute change in HFF determined by magnetic resonance imaging proton density fat fraction (MRI-PDFF) was shown. The normal reference range for MRI-PDFF is <5.6%. The absolute change in hepatic fat fraction in cohorts A1 and A3 was significant relative to the change observed with placebo. The absolute change was from a mean >20% at baseline to <10% after only 12 weeks of treatment. For cohorts A1 and A3, this effect was significant relative to placebo, but not for cohort A4. Most of the effect was immediately observed after 6 weeks of treatment, and there was some additional increment at week 12.

[0176] Figure 4Shows the relative change in HFF determined by MRI-PDFF. The relative change in hepatic fat fraction at week 12 was >60% in cohorts A1 and A3 and was significant compared to placebo. Most of the effect was observed immediately at week 6, and there was some additional increment at week 12.

[0177] Figure 5 Shows the proportion of patients who achieved a specific degree of fat reduction. Based on histopathological evaluation, a relative reduction of ≥30% in HFF was determined to predict disease improvement, and thus the percentage of patients who achieved this level of reduction (responders) was used as one measure of efficacy. More recently, a reduction of ≥50% has been considered to predict fibrosis improvement, and thus the percentage of patients who achieved this level of reduction (super-responders) was used as a more stringent criterion. In the interim analysis, 100% of the patients in cohort A1 were responders, while more than 90% in cohort A3. The rate for placebo was 28.6%. In cohorts A1 and A3, the rate of super-responders was also very high (≥75%), while the rate for placebo was 14.3%. During the 12-week treatment period, there were also some patients who normalized hepatic fat (≤5%), again relatively similar within cohorts A1 and A3.

[0178] 2.5 Biomarkers

[0179] Figure 6 Shows the level of alanine aminotransferase (ALT) which is a biomarker of liver injury. Elevated liver enzymes are a sign of liver injury, and thus normalization or trending towards normal values (decrease) is a sign of improved liver function. In the interim analysis, ALT decreased by more than 35% as early as week 6 in cohorts A1 and A3 from a median of 40 - 55 U / L at baseline and continued until the last assessment at week 12, and was significantly different from placebo. Cohort A4 did not show a significantly different effect compared to patients treated with placebo.

[0180] Figure 7 Shows the level of aspartate aminotransferase (AST) which is a biomarker of liver injury. The decrease in AST from the baseline median level of 23 - 34 U / L was greater than 40% at the last study measurement and had been achieved as early as week 6 in cohort A1. For cohorts A1 and A3, this change was significantly different from placebo at the last study evaluations at weeks 6 and 12.

[0181] Figure 8Shows the level of the amino-terminal propeptide of procollagen 3 (ProC3), which is a biomarker for liver fibrosis. Type III collagen is the main type of collagen deposited in the liver during the pathogenesis of liver fibrosis. To deposit collagen in the extracellular space of liver tissue, the N-terminal peptide of procollagen (Pro-C3) is enzymatically removed. This N-terminal peptide can be measured in the circulation, and its level is correlated with the degree of fibrosis. A reduction of more than 15% or more than 20% in Pro-C3 predicts improvement in fibrosis. In cohort A1, treatment with 300 mg Q4W resulted in at least a 20% reduction in Pro-C3 as early as week 6, and this effect persisted until the last study assessment. This effect was significantly different from that of the placebo. In cohorts A3 and A4, treatment with 75 mg Q2W or 75 mg Q4W resulted in a reduction in ProC3 levels of >15% at week 6, but this effect did not persist with longer treatment.

[0182] Figure 9 Shows the degree of adiponectin deficiency. Adiponectin is an adipose-specific adipokine that reduces insulin resistance and alleviates liver inflammation / fibrosis. Low levels of adiponectin are known in patients with NASH. After administration of FGF21, the level of adiponectin is expected to improve because binding to the corresponding receptor of FGF21 on adipocytes directly stimulates the production of adiponectin. All BOS-580 treatment regimens caused rapid effects as early as week 4, with an improvement of >50% as early as week 2. All treatment regimens remained significantly superior to the placebo until week 12 of treatment.

[0183] Figure 10 Shows the HbA1c levels in diabetic NASH patient cohorts A1 and A3. In a subset of patients with baseline HbA1c > 6.5% (diabetic patients), a decrease of -1.0% and -0.7% was observed in cohorts A1 and A3, respectively, compared to -0.5% in the placebo group. Notably, only one placebo patient reached week 12 at the time of analysis, so the true median change over time in placebo patients could not be evaluated. However, it is known that metformin can reduce HbA1c by 0.6% within 12 - 24 weeks, which can be used as a benchmark. Treatment with BOS-580 showed a better improvement in HbA1c levels than this benchmark.

[0184] Overall, the data demonstrated that, among other things, compared to 300 mg Q4W (300 mg / month) in cohort A1 and 75 mg Q4W (75 mg / month) in cohort A4, 75 mg Q2W (150 mg / month) in cohort A3 showed better efficacy with respect to safety considerations.

[0185] As defined herein, SEQ ID NO: 1 is as follows:

[0186]

Claims

1. A method for treating, preventing, or managing a cardiovascular and / or metabolic disorder 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.

2. The method according to claim 1, wherein the metabolic disorder 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. The method according to claim 2, wherein the metabolic disorder and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

4. The method according to claim 3, wherein the metabolic disorder and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH).

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

6. The method according to any one of claims 1-5, wherein the body mass index (BMI) of the human subject is at least 30 kg / m 2 , wherein the race adjustment for subjects of Asian or Asian descent is at least 27.5 kg / m 2 , in particular a BMI of 30 kg / m 2 to 45 kg / m 2 .

7. The method according to any one of claims 1-6, wherein the human subject is diagnosed with NASH, suspected of having NASH, and / or at risk of developing NASH.

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

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

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

11. The method according to any one of claims 1-10, wherein the human subject is diagnosed with, suspected of having, and / or at risk of developing type 2 diabetes, hypertension, and / or hyperlipidemia.

12. The method according to any one of claims 1-11, wherein the administration is effective in improving at least one physiological parameter associated with NASH.

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

14. The method according to any one of claims 1-13, wherein the administration is effective in reducing liver fat, particularly effectively reducing the liver fat fraction by at least 45%, at least 50%, or at least 60%.

15. The method according to any one of claims 1-14, wherein the administration is effective for reducing liver injury, particularly effective for reducing adiponectin deficiency, optionally by increasing the adiponectin level by at least 50%, 75%, 100% or 125%.

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

17. The method according to any one of claims 1-16, wherein the administration effectively reduces the HbA1c level in a subject with type 2 diabetes by at least 0.6%.

18. The method according to any one of claims 1-17, wherein the administration has a gastrointestinal tolerance profile of grade 1 or 2.

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

20. The method according to any one of claims 1-19, wherein the one or more additional therapeutic active agents are selected from compounds useful in obesity therapy, diuretics, β-blockers, α-blockers, ACE inhibitors, angiotensin II receptor blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, α-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, amylin mimetics, statins, fibrates, aspirin and anticoagulants.

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

1.

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

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

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

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

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

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

29. The human FGF21 protein variant for use according to any one of claims 24 - 28, wherein the body mass index (BMI) of the human subject is at least 30 kg / m 2 , wherein the ethnic adjustment for subjects of Asian or Asian descent is at least 27.5 kg / m 2 , in particular a BMI of 30 kg / m 2 to 45 kg / m 2 .

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

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

32. The human FGF21 protein variant for use according to any one of claims 24-31, wherein the human subject has a liver fat assessment based on vibration-controlled transient elastography (VCTE) or controlled attenuation parameter (CAP) score 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 24-32, wherein the human subject has a liver injury and fibrosis assessment based on VCTE liver stiffness measurement (LSM) score of 7 to 14 kPa, 7 kPa to 12 kPa, or 7 kPa to 9.9 kPa and / or aspartate aminotransferase (AST) activity of at least 20 U / L.

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

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

36. The human FGF21 protein variant for use according to claim 35, wherein the at least one physiological parameter is selected from the stage of liver fibrosis, liver fat assessment based on VCTE CAP, liver injury 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 - 36, wherein administration is effective for reducing hepatic fat, particularly effectively reducing the hepatic fat fraction by at least 45%, at least 50% or at least 60%.

38. The human FGF21 protein variant for use according to any one of claims 24 - 37, wherein administration is effective for reducing liver injury, particularly effective for reducing adiponectin deficiency, optionally by increasing the adiponectin level by at least 50%, 75%, 100% or 125%.

39. The human FGF21 protein variant for use according to any one of claims 24 - 38, wherein administration is effective for reducing liver injury, effectively reducing the mean alanine aminotransferase (ALT) activity by at least 20%, at least 30% or at least 35%, and / or effectively reducing the aspartate aminotransferase (AST) activity by at least 20% or at least 30%, and / or effectively reducing the mean of procollagen type III N - terminal peptide (ProC3) by at least 15% or at least 20%.

40. The human FGF21 protein variant for use according to any one of claims 24 - 39, wherein administration effectively reduces the HbA1c level in a type 2 diabetic subject by at least 0.6%.

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

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

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

44. The human FGF21 protein variant for use according to any one of claims 24 - 43, which consists 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 - 44, wherein the human FGF21 protein variant is glycosylated.

46. The human FGF21 protein variant for use according to any one of claims 24 - 44, wherein the human FGF21 protein variant is non - glycosylated. Use of a human FGF21 protein variant in the preparation of a medicament for treating, preventing or managing cardiovascular and / or metabolic disorders in a human subject, said human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO:1, wherein the human FGF21 protein variant is provided by subcutaneous administration 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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